B5/T3
C.I
EPA-670/6-73-01
Physical
Chemical and
METHODS OF
SOLID WASTE TESTING
VS. ENVIRONMENTAL PROTECTION AGENCY
* OFFICE OF RESEARCH AND MONITORING
NATIONAL ENVIRONMENTAL RESEARCH CENTER, CINCINNATI
MAY 1973
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EPA -6700-73 -01
Physical
Chemical and
Microbiological
METHODS OF
SOLID WASTE TESTING
D.F. BENDER, M.L PETERSON,
AND H. STIERLI, EDITORS
US. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND MONITORING
NATIONAL ENVIRONMENTAL RESEARCH CENTER, CINCINNATI
MAY 1973
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REVIEW NOTICE
The Solid Waste Research Laboratory of the National Environmental
Research Center, Cincinnati, U.S. Environmental Protection Agency, has
reviewed this report and approved its publication. Mention of trade names
or commercial products does not constitute endorsement or recommendation
for use.
This manual Is drilled for use with a three.ring binder.
11
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FOREWORD
Man and his environment must be protected from the adverse effects
of pesticides, radiation, noise and other forms of pollution, and the unwise
management of solid waste. Efforts to protect the environment require a
focus that recognizes the interplay between the components of our physical
environment—air, water, and land. The National Environmental Research
Centers provide this multidisciplinary focus through programs engaged in
• studies on the effects of environmental contaminants on man and
the biosphere, and
• a search for ways to prevent contamination and to recycle valuable
resources.
This publication of the National Environmental Research Center,
Cincinnati, describes the physical, chemical, and microbiological methods
used by the Solid Waste Research Laboratory to analyze solid wastes and
solid waste related materials. The latter includes products and potential
pollutants resulting from the handling, processing, disposal, or recycling of
solid wastes. Five years of evaluating and applying available methods in
incinerator testing studies, municipal waste characterization studies, and
solid waste utilization schemes are represented. Although the results of these
studies have been published and are referenced throughout, the detailed
methodology is being made available in this single source for the first time.
A. W. Breidenbach, Ph. D.
Director
National Environmental Research Center,
Cincinnati
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PREFACE
This publication is a compilation of methods used by the Solid Waste
Research Laboratory of the National Environmental Research Center in
Cincinnati, Office of Research and Monitoring, U. S. Environmental
Protection Agency, to perform various physical, chemical, and microbiologi-
cal analyses in the field of solid waste management. It is not intended to be a
complete manual, but the first edition of a growing collection of methods
used by the Solid Waste Research Laboratory to test refuse, incinerator
residue, incinerator residue quench water, incinerator scrubber water, sani-
tary landfill leachate, and the products of laboratory and pilot studies
directed toward reclaiming waste through physical, chemical, and biological
transformation.
The preparation of this manual was under the general direction of
Mr. Harry Stierli, Chief, Support Services Branch, Solid Waste Research
Laboratory. Dr. Daniel F. Bender, Project Manager, Laboratory Support
Project, Support Services Branch, edited Part I, Physical Methods, and
Part II, Chemical Methods. Part III, Microbiological Methods, was prepared
by Dr. Mirdza L. Peterson, Senior Research Microbiologist, during her assign-
ment with the Support Services Branch.
Most methods previously reported in the literature have required some
modification for application to the analysis of solid wastes. The procedures
presented in this compilation were modified where necessary, evaluated, and
applied by the vanous authors.
Robert L. Stenburg, Director
Solid Waste Research Laboratory
Iv
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INTRODUCTION
Daniel F Bender and Harry Stierli
Because of the many requests for the information contained in this
publication, we decided to print this first edition in a form that is incomplete
from the standpoint of our overall plan. Waiting for a state of completion
would delay the release of the often requested information. The fact that we
have already planned for a second edition containing much more information
does not reduce the value of this edition. A great deal of the information
that will eventually be added is available in the literature, merely awaiting
compilation to produce it in a single source in the format we have chosen.
Other information to be added will result from current research, primarily on
samples of leachate from sanitary landfills.
In choosing the format for presenting these methods, we concentrated
on specific laboratory directions with adequate step-by-step explanations
and only enough discussion to provide a sound general background. The
evaluations, which employ more sophisticated statistical approaches, add
another dimension. Thus, we are attempting to create both a laboratory
manual for the technician who must produce the information and a sound,
though not in-depth, theoretical background for the analyst who must
evaluate the information. In addition, some of the more sophisticated
methods we have used (for example, those for analyzing reclamation
products) will have added value for persons involved in research rather than
in testing or monitoring.
V
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CONTENTS
Foreword
Preface
Introduction . Bender and Stierli
Part I Physical Methods
Laboratory Procedure for the Preparation
of Solid Waste Related Materials for Analysis Cohen
Part II Chemical Methods
Laboratory Procedure for Determining Total
Heat of Combustion in Solid Wastes Wilson
Laboratory Procedure for Determining Potential
Heat in Solid Wastes Wilson
Laboratory Procedure for Determining Percent
Ash and Percent Weight Loss of Solid Wastes
on Heating at 600 C Ulmer
Laboratory Procedure for the Gravimetric
Determination of Carbon and Hydrogen
in Solid Wastes Wilson
Laboratory Procedures to Determine the
Nitrogen Content of Solid Wastes Kaylor and Ulmer
Laboratory Procedure for the Gravimetric
Determination of Carbonate Carbon in
Solid Wastes Wilson
Extension of Carbon-Hydrogen Method to Include
Determination of Volatiles or Loss on Ignition
(L.O.I.) at 950 C Wilson
Mathematical Determination of Total Oxygen
in Solid Wastes Wilson
Mathematical Determination of Total Heat of
Combustion Content of Solid Wastes Wilson
The Alsterberg (Azide) Modification of the
Win kler Method for Determining the BOD of
Incinerator Quench Water and the Calibration of
the Weston & Stack Dissolved Oxygen Analyzer,
Model 300-B Wilson
vu
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The Dissolved Oxygen Analyzer (Weston & Stack, Inc.,
Model 300-B) Method for Determining the BOD of
Incinerator Quench Water Wilson
Methods for Determining Cellulose in Compost Lossin
Measurement of the Chemical Oxygen Demand
of Compost Lossin
Qualitative Determination for the Degree of
Decomposition of Compost by the
Starch-Iodine Method Lossin
Vacuum-Acid Hydrolysis of Fungal Protein and
Protein from other Sources Coleman
Laboratory Procedure for the Spectrophoto-
fluorometric Determination of Selenium in
Solid Waste Johnson
Part III Microbiological Methods
Methods for Bacteriological Examination of
Solid Waste and Waste Effluents Peterson
VII’
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PART I
PHYSICAL METHODS
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INTRODUCTION
Eventually this section will consist of (1) a practical, statistically based method of field sampling
for refuse, (2) sample handling and transporting characteristics, and (3) a discussion of laboratory
sample selection from the bulk field sample as well as the currently included method of sample
preparation for solid samples. When a general sample preparation procedure has been developed for
leachate samples, it will be included as well. One other physical method of importance involves the
application of a classification system to refuse samples (paper, glass, metal, etc.). A review of the
many schemes that have been developed is planned for future editions of this publication.
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LABORATORY PROCEDURE FOR THE
PREPARATION OF
SOLID WASTE RELATED MATERIALS FOR ANALYSIS
Israel R. Cohen*
DISCUSSION 2
APPARATUS 2
SAFETY PRECAUTIONS 2
PROCEDURE FOR COMBUSTIBLES 3
Drying 3
Grinding in Hammerrmll 3
Fine Grinding 5
Mixing 5
The Discard 6
PROCEDURE FOR NON-COMBUSTIBLES 6
Research Chemist, Solid Waste Research Laboratory, National Environmental Research
Center Cincinnati.
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METHODS OF SOLID WASTE TESTING
D ISCUSS ION
The term “solid waste related materials” as used here refers to (a) raw refuse that is delivered to an
mcinerator or landfill for disposal, (b) residues of the incineration process, and (c) refuse in various
stages of compostmg. It is assumed that major glass, ceramics, and metal components have been
removed before submission to the laboratory. The three basic operations required to prepare solid
waste related materials for detailed analysis are drymg, grinding or pulverizing, and mixing. The end
products of these operations must be so thoroughly homogenized that portions weighing as little as
100 to 200 mg may be extracted for analysis with full confidence that they are uniform and represent-
ative. Thus, any lack of precision or accuracy in the final results cannot be ascribed to the sample.
APPARATUS
Basically the apparatus needed to perform the above operations include ovens (preferably
mechanical convection), cutting, grinding, and pulverizing tools, balances, and a mixing device. The
following is a list of apparatus that has been found useful:
1. Ovens, Freas model 114, Thelco model 28, Blue M model POM-326 FXX
2. Pans for drying
3. Balance, Ohaus heavy-duty solution balance
4. Balance, top loading, 1 ,000-g capacity
5. Desiccator cabinet, large capacity
6. Shears, heavy duty
7. Hatchet
8. Saw
9. Plastic sheet
10. Sample splitter, Gilson, large capacity
11. Hammermill, W-W Grinder, Model F-21-P
12. Wileymill#3
13. Micromill, Weber Bros. laboratory pulverizing mill
14. Ore pulverizer, her
15. Sieves, 10 mesh and 60 mesh
16. Sieve shaker
17. Rotating mixer
18. Miscellaneous gloves, scoops, funnels, containers, brushes, etc.
19. Dust collector, Norblo, bag type size 48-3, type BA-2.3
SAFETY PRECAUTIONS
When handling refuse, the analyst should use gloves if possible (neoprene-coated canvas). He should
also wear some sort of face mask, such as a surgical mask, when preparing samples, especially when
they are in finely divided form. It is advisable to wear a transparent plastic faceshield while feedmg
matenal into the hammermill. The analyst should not use his hand to help push material into the
hammermill past the feed slot; a stick can be used if necessary. Do not open any grinding device while
it is running. If a mill clogs, turn off the motor before cleanng apparatus.
2
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Preparation for Analysis
PROCEDURE FOR COMBUSTIBLES
The procedures for all types of organic materials are essentially similar, if it is taken into account
that compost samples have already been coarsely ground either m a hammermill or rasping device.
It is preferable to dry the matenal before grmding. If the sample is small (3-5 lb of moist compost, or
less of a bulkier material) the drymg can be camed out conveniently in a large laboratory oven. If the
complete sample is dned, an industrial-size oven is most convenient. Where the latter is not available,
a prelimmary grinding step in a hammerm ill is usually necessary.
Drying
For a small sample, use a laboratory oven, for a large sample, use an industrial oven.
Procedure Comments
1. Weigh pan or pans.
2. Transfer material to pan and reweigh.
3. Note weight of sample.
4. Dry in oven at 70 to 75 C overnight or for
24 hr.
5. Remove sample and allow to cool, prefer- 5. If a desiccator is not available, allow sample
ably in desiccator, to cool covered with aluminum foil.
6. Reweigh.
7. Replace in oven for 1 to 2 hr.
8. Repeat steps 5 and 6.
9. If the weight loss is less than 1 percent
of total previous weight loss, calculate
percent of moisture from total weight lost.
10. If weight loss is over 1 percent of total 10. Example 0.10 percent in 10 percent mois-
previous loss, dry for an additional hour. ture
Continue intermittent drying until the 0.40 percent in 40 percent mois-
change in calculated moisture is within the ture
limit set in step 9.
Grinding in Hammermill
This procedure does not apply to compost or other previously ground material.
Dried sample.
Procedure Comments
I. Place sample collection box under mill.
2. Plug lead into power outlet.
3. Open cut-off in duct of dust-collecting
system.
4. Start blower.
5. Oil grinder beanngs with engine oil. 5. Motor bearings are permanently lubricated.
6. Put on personal safety equipment.
7. Start motor.
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35th s a flesearth Center
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METHODS OF SOLID WASTE TESTING
8. Feed sample into mill. 8. If components of sample have been segre-
gated by categories, mix these during feeding.
Tear thick magazines into thinner sections
and separate thick wads of paper. Remove
all metal objects (staples in magazines and
cartons, bottle caps, metal buttons or staples
on jeans and work clothes) and all other non-
combustibles (glass, ceramics, etc.) not pre-
viously removed from the sample. Place
these in a container marked “Discard.” Cut
articles of clothing into smaller sections
before feeding into mill and saw or chop
large pieces of wood or thick branches into
smaller pieces. Close metal inlet door after
inserting each batch of material.
9. Turn off grinder motor. 9. Remove electrical plug from outlet.
10. Turn off blower.
11. Clean out grinder and add material to 11. Remove clean-out plate at rear of grinder.
ground sample. Remove threads wound around shaft between
hammers and cut long strings or threads into
short pieces. Use electric light to illuminate
dark areas. Scrape off bits of material caught
in crevices.
12. If the sample weighs appreciably more
than 2 lb, reduce it to about this weight by
mixing on a plastic sheet and quartering or
passing it through a sample splitter. For
this purpose, the large-size splitter should
be used with apertures about in. wide.
Undried sample.
Procedure Comments
1. — 11. Steps 1 through 11 for the undried 1. — 11. More care must be exercised in
sample are the same as steps 1 through 11 cleaning the inside of the hammermil, since
for the dried sample. moist material will stick to the walls of the
mill more readily. It is advisable to line
sample collection box with plastic film or
bag.
12. Spread ground product on a sheet of 12. The sample may be mixed by manipulating
plastic and mix rapidly without compacting. the corners and sides of the sheet to move the
particles from one area to another. A spade
is often useful in this operation. Compaction
of the particles reduces their mobility.
13. Quarter the material down to manageable 13. About 3 lb is a convenient sample size;
size. store remainder in a labeled, plastic bag.
14. Dry sample as outlined under “Drying”.
4
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Preparation for Analysis
Compost.
A compost sample, as received in the laboratory, may appear to be quite wet or dry. In either case,
the sample should be dried before proceeding any further (unless assurances are received that the
sample has been dried). Drying is carried out as detailed in that section.
Compost usually contains varying quantities of glass and ferrous and non-ferrous metal. If the
sample has been relatively finely ground, it is not practical to remove the glass or non-ferrous metal.
The ferrous metal can be picked out by combing the sample with a strong magnet. If the sample is
quite coarse, much of the glass will be of a size that can be removed without too much difficulty by
judicious sieving and by the use of a current of air to scatter the lighter organic particles. If the
carbon/nitrogen ratio is to be determined, identifiable plastic should be removed because it is not
degradable. Removal of glass and metal reduces the wear on the Wiley mill knives. After following
these preliminary procedures, continue with those in “Fine Grinding.”
Fine Grinding
The material that has been reduced in a hammermill and dried is now to be ground in a Wiley mill
so that it will at least pass through a 2-mm sieve and preferably through a 1-mm sieve.
Procedure
1. Put a 2-mm sieve into a Wiley mill and close
mill.
2. Open cut-off in dust collection duct.
3. Position container under delivery spout.
4. Replace the 2-mm sieve with a 1-mm sieve
and regrind the sample.
5. Brush out all inside surfaces of the mill
into a separate container.
6. Put this material through a micro mill.
7 . Add the product to the main sample and
mix.
Comments
3. A standard Mason jar can be screwed directly
into the spout. If the ground sample is
caught in a different type of container, it is
advisable to provide one with a covering that
has a hole through which the spout may be
inserted.
4. The fmer the grind, the more uniform the
sample can be made.
5. This procedure includes the hopper, the
walls, the screen, and the spout. Remove as
much as possible of the material wedged
between the knives and their housing.
6. On the Weber’ mill, the 0.05-in, or 0.10-in.
screen may be used. For small samples,
substitute a long, narrow plastic bag for the
canvas bag provided.
Mixing
The final mixing or homogenization is accomplished by transferring the sample to a suitable con-
tamer that will be no more than half-filled by it. The container is closed tightly, positioned in the
5
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METHODS OF SOLID WASTE TESTING
Procedure
1. Weigh the dried sample.
2. Work the sample over with a strong magnet
to remove ferrous metal and magnetic iron
oxide.
3. Sieve the sample through a 1/4-inch mesh
sieve and pick out glass and ceramics.
4. Check the oil level in the pulverizer. The oil
cup should be quite full.
5. Adjust the movable pulverizer plate to give
a maximum size of about 10 mesh.
6. Put the sample through the pulverizer.
7. Screen the ground matenal through 10-mesh
and 60-mesh sieves. The material that has
been ground to pass through the 60 mesh
sieve is the fmal sample. The remainder is
reground with the plates placed together
with minimum clearance.
8. Mix as indicated under “Mixing.”
Comments
1. If the moisture content is to be determined,
this value is, of course, obtained in the
routine procedure.
2. If the magnet is enclosed in a piece of cloth,
it will be easy to remove the magnetic
material clinging to it by separating the cloth
from the magnet. The removal is otherwise
quite difficult to effect.
3. Though the pulverizer will grind this material,
the removal of ingredients not contributing
to the Btu value will enrich a sample whose
Btu value is low under the best conditions.
4. The gears run in a bath of regular engine oil.
Be careful not to get any oil into the sample
compartment.
5. This is accomplished by rotating the wheel
with the holes in the rim (just ahead of the
fly wheel) clockwise to give a coarse product
and counter-clockwise to give a fine product.
6. When restarting the motor after emptying
the product pan, it is advisable to empty the
grinding chamber. Starting the motor against
a load may cause burnout.
7. It may be necessary to make multiple passes
through the pulverizer to grind the whole
sample through the 60-mesh sieve. The coarse
material will often disclose bits of metal that
have been burnished during the first passage
through the pulverizer. These may be picked
out and added to the discard (see steps 2 and
3).
rotating mixer, and allowed to mix for not less than 1 hr, and preferably for 2 hr. The mixed sample
may then be reduced in size, if desired, by passing it through a sample splitter or by quartering.
The Discard
Weigh all metal, ceramics, plastic, and glass removed during processing. This information will be
used later in calculations.
PROCEDURE FOR NON-COMBUSTIBLES
Incinerator residues are usually wet when received and are dried at 100 to 105 C. The temperature
or time of drying is not critical. A 10-lb sample or smaller is usually enough to dry.
6
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PART II
CHEMICAL METHODS
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INTRODUCT ION
These methods have been modified, developed, evaluated, and/or applied to samples of
material from solid waste handling, processing, reclaiming, and disposal methods. They were
selected from many available methods used for water pollution, fuel, and fertilizer analyses,
as referenced in each manuscript.
These are the methods that worked best for us. In the absence of a thorough study, we do
not label these as recommended. Collaborative testing is necessary before such recommendations
are possible.
The first consideration in method selection was that it give the necessary information for
interpretation within a reasonable accuracy. We stressed simplicity, so that minimal training
would be required, and speed, so that the samples could be analyzed before further decomposi-
tion could occur. Some research objectives involved more sophisticated methods, which are
also included.
It is anticipated that in future editions this section will contain twice as many methods.
Thorough evaluation and collaborative testing of all the methods will have to wait, however,
until the urgency of finding methods that at least produce immediately needed information
has subsided.
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LABORATORY PROCEDURE FOR DETERMINING
TOTAL HEAT OF COMBUSTION
IN SOLID WASTES t
Donald L. Wilsont
DISCUSSION 2
AppAluTus 2
Requirements 2
Assembling
REAGENTS 4
Chemical Requirements 4
Preparation 4
SAFETY PRECAUTIONS 4
SAI%IPLE PREPARATION 4
PROCEDURE
STANDARDIZATION 9
CALCULATIONS 9
Standards 9
Samples 9
METHOD EVALUATION 10
ACKNOWLEDGMENTS 11
BIBUOGRAPHY 12
This method is meant to be used in conjunction with the Parr Instrument Company’s
Techmcal Manual No. 130, Operating the Adiabatic Calorimeter.
tResearch Chemist, Solid Waste Research Laboratory, National Environmental Research
Center, Cincinnati.
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METHODS OF SOLID WASTE TESTING
DISCUSSION
The heats evolved in the complete combustion of many compounds in oxygen have been
carefully determined. The method ordinarily used is to burn the substance in a combustion bomb
and to measure the heat evolved.
The term “heat of combustion” refers to the amount of heat liberated per unit of substance
burned. This process involves the change in enthalpy or heat content (H) of the system. The heat
of combustion or described in this method is expressed in calories per gram of sample or
British thermal units (Btu) per pound of sample.
The heat contents of various solid waste materials are important to some of the volume reduction
processes used to dispose of waste. In the incineration process, for example, the operating efficiency
of an incinerator can be measured by energy balance techniques, and analyzing heat contents of the
solid wastes before and after incineration is essential. Knowledge of the heat value of solid waste is
also necessary for incinerator design. In addition, heat content analyses need to be performed on
incinerator residue and compost used for landfill since the stability of these waste products is a
function of their heat contents.
To determine directly the heat change involved in a reaction, calorimeters are employed. A
calonmeter consists essentially of an insulated container of water in which the reaction chamber
is immersed. In an exothermic reaction, the heat generated is transferred to the water, and the
consequent temperature rise of the water is read from an accurate thermometer immersed in it.
The amount of heat evolved in the reaction may be calculated with data on the quantity of water
present, its specific heat, and the change in temperature. Special corrections must be applied for
radiation, rate of cooling of the calorimeter, temperature rise of the vessels, stirrer, etc. To com-
pensate for these corrections, the heat capacity of the calorimeter is determined by burning a
definite amount of a standard.
This method describes the capabilities and limitations of a Parr adiabatic calorimeter and furnishes
the instructions needed to obtain best results with this apparatus. Using this method, the heats of
formation of nitric acid and sulfuric acid are involved in the total heats of reaction, therefore, they
are determined and the data are adjusted.
APPARATUS
Requirements
I. Balance, analytical, 150-g capacity, 0.1-mg readability
2. Beakers, one 30-mi, one 100-ml, and one 250-mi
3. Bottle, aspirator, plastic, 1-gal capacity
4. Bottle, carboy, 5-gal capacity
5. Bottle, reagent, 1-liter capacity
6. Bottle wash, plastic, 125-mi capacity
7. Bulb, rubber, 30-mi capacity
8. Buret clamps
9. Buret, 50-ml capacity, three-way stopcock
10. Calorimeter, adiabatic, Parr No. 1221, with Techmcal Manual No. 130
2
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Laboratory—Total Heat of Combustion
11. Calorimeter bomb, double valve, Parr No. 1101 (Fisher #4-392-1)
12. Calorimeter fuel capsule, stainless steel, Parr No. 43AS, (Fisher #4-385-5); eight or more
13. Calorimeter ignition unit, transformer type, Parr Series 2900
14. Calorimeter mercunal thermometers, Parr No. 1601; two
15. Calorimeter oxygen filling connection, safety type, Parr Series 1823
16. Calorimeter thermometer reading lens, Parr No. 3003, two
17. Calorimeter water heater, automatic, electnc Parr Series 1500
18. Carver laboratory press, Model C or equivalent (Will #22562)
19. Carver test cylmder outfit or dies, specially made (internal diameter of cylinder is 3/4 in.
and length of internal cylinder chamber is 3-1/4 in.;* similar to outfit with 1-1/8-in internal
diameter of cylinder (Will #22591)
20. Clamp, pinchcock
21. Desiccator
22. Weighing dish with lid, eight or more
23. Drying oven
24. Flasks, volumetric, one 2-liter and one 1-liter
25. Funnels, filling, one 80-mm diameter and one 250-mm diameter
26. Jack, laboratory
27. Pipet, volumetnc, l-ml capacity
28. Ring, support, with clamp, 4-in, outside diameter (0. D.)
29. Scissors
30. Stopwatch
31. Support stand, rectangular base, 24-in, rod; two
32. Tubing, glass, 1/2-in, inside diameter (I. D.), about 3 ft
33. Tubing, plastic, 1/4-in. I.D , about 3 ft
34. Tubing, plastic, 1/2-in. I.D., about 6 ft
Assembling
The general arrangement of the calorimeter and accessories may be found in the Parr Company’s
Technical Manual No. 130, pages 17 to 21. The procedure section contains instructions on when to
plug in the water-heater cord.
For convenience, the distilled water is stored in a 5-gal carboy bottle with glass tubing extending
through the lid to near the bottom. A plastic tube is connected to the glass tubing and a pinchcock
clamp is used to prevent the flow of water. The sodium carbonate solution is stored in a 1-gal
plastic bottle with aspirator. Plastic tubing of 1/4-in. I. D., connects the aspirator to the automatic
filling buret. The plastic bottle rests on a laboratory jack atop a shelf, thus allowing the level of
reagent to be higher than the solution within the buret. A 250-mi beaker is put beneath the buret
to prevent spilling. A support ring is connected to a support stand about 1 ft above the base.
The l-ml pipet stored in an inverted filling funnel, 80 mm in diameter. For ease of pipeting, some
distilled water is stored in a 30-ml beaker.
*Although the smaller die is preferred, a larger die may be used with modifications in the procedure. The pellets are
made thicker and broken into four or more parts by using a sharp object or breaking by hand Handle the pellets
as little as possible.
3
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METHODS OF SOLID WASTE TESTING
REAGENTS
Chemical Requirements
1. Benzoic acid, primary standard with calorific value known (Fisher #A-68)
2. Methyl orange, ACS grade (Fisher #M-2 16)
3. Oxygen, 99.5 percent pure (the oxygen should be prepared from liquid air, since oxygen
prepared by electrolysis contains traces of hydrogen)
4. Sodium carbonate, Na 2 C0 3 , ACS grade, anhydrous
5. Steel wool
6. Wire, nickel-iron chromium alloy, No. 34 B. & S. gauge, wound on a card 10 cm long (Parr
45 ClO)
Preparation
1. Sodium carbonate solution,0.0725N: Dissolve 3.8421 g of Na 2 CO 3 in distilled water and dilute
to 1 liter (store in plastic bottle with aspirator).
2. Saturated methyl orange solution: Dissolve 0.5 g of methyl orange in distilled water and dilute to
1 liter (store in reagent bottle).
3. Wash solution: Pipet 1 ml of saturated methyl orange solution into a 1-liter flask and dilute to
the liter mark with distilled water.
4. Ignition wire: Using 10-cm marks on card provided with wire, measure and cut off 10 cm of wire
with scissors. Several 10-cm lengths may be made at one time and stored in a 100-mi beaker.
SAFETY PRECAUTIONS
See the Parr Company’s Technical Manual No. 130, page 9, “Hazards of Operation.”
SAM PLE PREPARATION
Total sample preparation procedures such as the drying and grinding techniques are described
in detail elsewhere. In general, since this method restricts the quantity of sample analyzed to
a weight of about 1.0 g or less that has been thoroughly dried, the entire sample (except glass,
metals, and ceramics) must be ground until the particle size is reduced to less than 2 mm, dried
until there is no more loss in weight, and then completely mixed before being analyzed.
Fluffy materials (standard benzoic acid, raw refuse, and compost) must be made into pellets
in order to fit the metal capsules. The procedure for pelleting is
Procedure Comments
1. Using Carver laboratory press with 3/4-in. 1. A 1-1/8-in. diameter punch and die may be
diameter punch and die, loosely fill the used, but the pellet will have to be broken.
chamber 1/2 to 3/4 full of sample. If at all possible, the 3/4-in, punch and die
should be used.
4
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Laboratory—Total Heat of Combustion
2. Compress the sample until the pressure is
5,000 to 7,000 lb, as read by the dial.
3. Transfer the pellet to a weighing dish
with lid.
4. Repeat for each determination.
3. Touch pellets as little as possible with
hands.
4. See “Method Evaluation.”
PROCEDURE
The following procedure applies to all solid waste matenals and the benzoic acid standard.
To ensure precision and to keep from exceeding the capacity of the instrument, it is advisable
that restrictions in Table 1 be followed.
TABLE 1
SAMPLE RESTRICTIONS
Sample type
Sampl
e wei
ght (g) Specifics of
analysis
Benzoic acid
1.0
±
0.2
pelleted
Raw refuse
0.4
to
0.8
pelleted
Residue (combustible)*
0.4
to
0.8
pelleted
Residue (fines)t
0.8
to
1.2
combustion
aid
added
Fly ash
0.8
to
1.2
combustion
aid
added
Compost
0.4
to
0.8
pelleted
Mostly readily combustible materials wiuch remained on a 1/2-in, sieve during manual sorting at the incinerator
site.
tMaterial remaimng after combustibles, metal, glass, and ceramics removed from incinerator residue.
Procedure
Weigh sample into tared capsule. If the
sample is in pellet form, it must be thin
enough to keep the weight within restric-
tions. If a combustion aid is added, sand-
wich the sample evenly between pellets of
the combustion aid, keeping the total
weight of the combustion aid in excess of
the sample weight.
Comments
a) Capsules should have been cleaned pre-
viously with steel wool and water, then
dried and stored in desiccator until weighed.
b) Use analytical balance and record to the
fourth decimal place.
c) Duplicate or triplicate samples are needed
(“Method Evaluation”).
d) Combustion aid is pelleted benzoic acid.
e) Remember: not more than 10,000 calories
should be liberated in any one test.
f) If the combustion aid is made with a die
5
-------
METHODS OF SOLID WASTE TESTING
2. Bind the fuse wire to 4A and 5A electrodes.
3. Place the capsule with sample in 5A elec-
trode.
4. Pipet one ml of distilled water into the No.
1101 bomb.
5. Place bomb head into the cylinder.
6. Screw cap down firmly by hand.
7. Close outlet needle valve.
8. Remove mlet valve thumb nut.
9. Attach filling tube with union nut firmly,
by hand.
10. Open the filling connection control valve
SLOWLY.
11. Allow pressure to nse slowly until gauge
reads 30 atmospheres.
12. Close connection control valve.
13. Push sideways on ball knob under relief
valve.
14. Detach connnecting tube.
15. Replace thumb nut.
16. Place bomb with its feet spanning the boss
in the bottom of the bucket.
17. Attach the thrust terminal to the bomb.
18. Lower bucket into the jacket with stirrer
at rear.
19. Place 2,000 ml (about 2,000 g) of distilled
water (at room temperature) into the
bucket.
20. Close the calonmeter by swinging the cover
to the nght and lowering it with the cam
lever.
larger than the recommended size, the pellet
must be broken to meet restnctions. Keep
the larger pellet under all of the sample.
2. See Parr Manual No. 130, page 28.
3. The fuse wire should be bent so that a loop
is just ABOVE the sample.
4. Use a rubber bulb for the pipeting. (Dis-
tilled water is stored in a 30-mI beaker.)
5. a) Make certain sealing ring is in good con-
dition.
b) ALWAYS keep cylinder in upnght posi-
tion and do not disturb the sample.
6. a) Keep outlet needle valve open.
b) Parr Manual No. 130, Figure 4, page 11,
may be helpful.
9. Use Parr, Series 1823. Consult the Parr
Manual No. 130, pages 20 and 21.
10. a) Oxygen tank valve must be open.
b) If the filling connection control valve
is opened quickly, sample may come out
of capsule, thus causing incomplete com-
bustion.
11. Although the pressure range is 25 to 35
atm., according to Parr Manual No. 130,
the same pressure must be used for the
entire test.
13. This step relieves gas pressure in connecting
tube.
17. Lead wire should not extend above the
bucket.
18. Lower handle to back of bucket.
19. a) Keep the amount of water constant
throughout all tests, once standardization
is completed.
b) A 2-liter volumetric flask should be used.
20. Parr Manual No. 130, figures 8 and 9, page
13, may be helpful.
6
-------
Laboratory—Total Heat of Combustion
21. Make SURE the pump and stirrer drive
shafts are seated properly by seemg to it
that the pump and stirrer pulleys are down
as far as possible and move freely.
22. Lower the thermometers into the calon-
meter
23. With all valves before and after the water
heater open, turn on the water slightly.
Note The two valves nearest the calori-
meter are always kept completely open.
NEVER close all four valves completely.
24. Wait until water runs from the discharge
tube, then close the hot water valve and
plug in the water heater.
25. Start the stir motor and, again, wait until
the water runs from the discharge tube.
(Occasionally the pulleys need to be tapped
down to maintain circulation of the water.)
26. Adjust the cold and hot control valves so
that the jacket temperature, “left” ther-
mometer, is the same or slightly lower than
the bucket temperature, “right” thermom-
eter.
27. If there is no change in the bucket tempera-
ture after 1 or 2 mm, record this tempera-
ture.
28. Press button on ignition unit and stopwatch
at the same time.
29. During the rapid rise in bucket temperature
(usually 5 mm), keep the jacket tempera-
ture about the same or slightly lower than
the bucket temperature.
30. When the system is approaching final equi-
librium temperature, keep the jacket tem-
perature within 0.1 degree of the bucket
tern perature.
21. If the water in the bucket is not being
stirred, its temperature will decrease soon
after the final increase because of the sample
combustion.
22. The thermometers should be submerged
about the same distance as during stand-
ardization.
23. a) If the water is turned on too much, leaks
may occur.
b) See ParrManualNo, 130, pages 18 and 19.
c) Counter-clockwise turns open all valves.
24. CAUTION: The water flow and heating of
the water continues throughout the use of
the instrument; however, the water heater
may become overheated if its electric cord is
not occasionally disconnected for a few
minutes.
26. a) The main water valve may need adjusting.
Lowering the flow rate increases the control
over water temperature.
b) Read thermometers to nearest 0.005 F.
c) See Parr Manual No. 130, pages 14 and
15.
d) Occasionally check the thermometers for
mercury separations and reunite the mer-
cury, if necessary, in accordance with the
thermometers’ certificates.
27. Use reading lenses and record to the nearest
0.005 of a degree F.
28. Keep switch closed for 4 to 5 sec only.
29. The rapid rise in bucket temperature begins
about 20 sec after button is pressed.
7
-------
METHODS OF SOLID WASTE TESTING
31. Record the final maximum bucket temper-
ature.
32. With the cold water valve slightly open,
close the hot water valve.
33. Stop the stir motor and wait for water to
drain out of the cover.
34. Raise the thermometer support with the
thermometers to its top position.
35. Using cam lever, lift the cover and swing to
the left.
36. Lift the bucket out of the jacket, discon-
nect the thrust terminal wire, and remove
the bomb.
37. With a filling funnel, pour the water from
the bucket into the 2-liter flask.
38. With a sponge, paper towel, or napkin,
remove excess water from top of bomb.
39. Slowly relieve the pressure from the bomb
by opening outlet needle valve.
40. Remove screw cap and bomb head.
41. Using a wash bottle with prepared solution,
wash the head and all interior surfaces of
the bomb; then collect the washings in a
250-ml beaker. Carefully lay the bomb
head aside.
42. Titrate the washings to a reagent color end
point, using 0.0725 N sodium carbonate
solution and record the milliliters of titrant.
43. Carefully remove all unburned pieces of
fuse wire from the bomb head and measure
their combined lengths with the card origi-
nally containing the wire.
44. Record the calorific value (to the nearest
tenth) of the wire used.
45. Repeats steps 1 through 24 for each
analysis.
31. a) The stable maximum temperature occurs
when the same temperature is observed in
three successive readings.
b) Use reading lenses and record to the
nearest 0.005 of a degree F.
32. This procedure will allow the jacket water to
cool to a starting temperature for the next
analysis.
33. Water has drained from the cover when the
flow from the discharge tube returns to
normal.
34. The thermometer support should remain at
all times while the cover is off.
37. A 250-mm diameter funnel is used.
39. This step
mm.
40. Discard test if there is definite evidence
of incomplete combustion.
41. The bomb head and all interior surfaces
should be rinsed with distilled water and
allowed to drain before the next analysis.
42. a) Record the milliliters of titrant as calories,
to the nearest tenth, involved in the heat
of formation of nitric acid.
b) Most often this value represents less than
5 percent of the final answer. If it represents
more, however, save the solution for deter-
mining sulfur content (see the Parr’manual
No. 130, pages 37, 48, and 49).
45. Steps 23 and 24 are not repeated in a con-
tinuous operation.
should require about 1 to 3
8
-------
Laboratory— Total Heat of Combustion
46. At the end of each continuous operating
cycle, disconnect the electric plug of the
water heater and then turn off the main
source of water.
STANDARDIZATION
The term “standardization” denotes the determination of the energy equivalent or water equivalent
factor (W) of the system (see Parr Manual No. 130, page 23). Benzoic acid, pnmary standard with
calorific value known, is the material used to determine the W factor. The benzoic acid is analyzed
in the manner previously described under “Procedure.”
CALCULATIONS
Standards
Compute the energy equivalent by substituting in the following equation:
- M1 M ÷ e 1 + e 3
w
where:
W = energy equivalent of calorimeter in calories per degree F
= heat of combustion of standard benzoic acid in calories per grain
M = mass of standard benzoic acid sample in grams
t = corrected temperature rise* in degrees F
= correction for heat of formation of nitric acid, in calories
e 3 = correction for heat of combustion of firing wire, in calories
Samples
Compute the calorific value per gram of sample by substituting in the following equation:
— tW - e 1 - e 2 - e 3 - e 4
Temperatures are corrected for thermometer variations by using graphs furnished by the Parr Company for each
thermometer. If the sample has a heat content above 100 or 200 calories per gram, however, this correction will have
little effect on the final answer.
9
-------
METHODS OF SOLID WASTE TESTING
where.
= heat of combustion of sample in calories per gram*
t = corrected temperature rise in degrees F
W = average energy equivalent of calorimeter found by standardization, in calories per degree F
e 1 = correction for heat of formation of nitric acid, m calories
= correctiont for heat of formation of sulfuric acid, in calones
e 3 = correction for heat of combustion of firing wire, in calories
e 4 = heat of combustion of combustion aid, in calories
M = mass of sample in grams, it should not include mass of combustion aid
METHOD EVALUATION
The accuracy of this method was established by analyzing sucrose, NBS #17. The average of three
determinations was 3,981 calones per gram, which is 46 calories per gram or 1.2 percent from the true
value.
This method can analyze solid waste materials to withm 40 Btu per pound, depending on the type
of sample4 therefore, this method should not be employed to analyze samples that contain less heat
content than the pooled standard deviation for that type of sample (Table 2). To ensure precision, the
particle size of the samples must be less than 2 mm or must pass through a 60-mesh sieve. The sample
must then be thoroughly mixed before being analyzed.
The standard deviation of the water equivalent factor (w) was 10 calories per degree F for one
analyst in 1968 and 6 calones per degree F for another analyst m 1970.
*(c /g) (1 8) = Btu/Ib
tCorrection is not necessary if considered in e 1 and if e 1 is not more than 5 percent of the final answer.
j Types of solid waste in this method refer to only solid samples (domestic ongin) such as raw refuse, incinerator
fly ash, incinerator residue, and compost
10
-------
Laboratory— Total Heat of Combustion
TABLE 2
STANDARD DEVIATION* OF THE Btu-PER-POUND DETERMINATION
Type of sample No. of samples Duplicates
Btu/lb
Range
(Btu/ Ib)
Triplicates
Raw refuse
Residue
10
57t
40
7,366 to 9,999
Finest
7
76
53
249 to 2,470
Combust ib1es
7
76
54
4,457 to 8,182
Fly ash
Compost.
Without sewage sludge
7
2
56
——
40
173
Zero to 601
5,661 to 6,11 8
With 6% sewage sludge
8
——
227
4,504 to 7,5 1 0
With 10-15% sewage sludge
2
——
110
3,915 to 4,174
*A vanance estimate can be calculated from the duplicate (or triplicate) set of observations for each sample
The pooled variance is essentially an average of all such estimates for samples of a given type It is assumed
that a single underlying variance exists for all samples of a given type The pooled variance is then the best
estimate of this underlying variance. The pooled standard deviation is the square root of the pooled variance
and is used to estimate the underlying standard deviation.
f The absolute value of the difference between duplicated observations should not exceed 1 96 (2) (s), confidence
interval, or 158 Btu/lb, more than 5% of the time. The covariance between the duplicated observations was ignored.
Fines are materials remaining after most of the readily combustible substances have been removed by manual
sorting The sorting was performed at the incinerator sites and 1/2-in sieve was employed to assist in the separation
§ Combustibles or organics are mostly the readily combustible materials Unlike the fines, these materials are
usually retained on a 1/2.in sieve
ACKNOWLEDGMENTS
The author gratefully acknowledges the assistance of Richard Carnes, Annella Johnson, Nancy
Ulmer, Donna Barnet, Israel Cohen, and James Doerger in developmg this method.
The author also wishes to thank the Division of Technical Operations, Office of Solid Waste
Management Programs, for providing samples from incinerators, and the PHS-TVA Compost Plant,
Johnson City, Tennessee, for supplying compost samples.
11
-------
METHODS OF SOLID WASTE TESTING
BIBLIOGRAPHY
1. Parr Instrument Company. Operating the adiabatic calorimeter. In: Oxygen bomb calorimetry
and combustion methods; technical manual No. 130. Moline, Illinois, Parr Instrument Com-
pany, 1966.
2. Cohen, Israel R. Laboratory procedure for the preparation of solid waste related materials for
analysis (included in this Manual).
3. Wilson, Donald L. Decomposition of calcium carbonate (CaCO 3 ) in the Parr adiabatic calorimeter
(series 1200). Unpublished memorandum to Chief, Chemical Studies Group, Solid Waste
Research Laboratories, Cincinnati, Sept. 14, 1970.
12
-------
LABORATORY PROCEDURE FOR DETERMINING
POTENTIAL HEAT IN SOLID WASTES S
Donald L. Wilsont
DISCUSSION
APPARATUS
REAGENTS
SAFETY PRECAUTIONS...
SAMPLE PREPARATION...
PROCEDURE
STANDARDIZATION
CALCULATIONS
Total Heat Content
Potential Heat Content.
Residual Heat Content
METHOD EVALUATION
ACKNOWLEDGMENTS
REFERENCES
2
2
3
3
3
3
4
4
5 This method is meant to be used in conjunction with “Laboratory Procedure for
Determining Total Heat of Combustion in Solid Wastes.”
tResearch Chemist, Solid Waste Research Laboratory, National Environmental Research
Center, Cincinnati.
2
2
2
2
3
-------
METHODS OF SOLID WASTE TESTING
D ISCUSS ION
Not all solid waste samples with similar total heat contents(enthaplies,or heat of combustion values)
are similarly igmtable or combustible. The readily available heat content of a solid waste sample, or its
potential heat, could be an important critena for evaluating the efficiency of an incinerator or for
measuring the usefulness of incinerator residue.
Potential heat is not a new concept. The construction industries have long been interested in
the potential release of heat of materials during fires (1). In the construction mdustries, potential
heat is defined as “the difference between the heat of combustion of a representative sample of the
matenal and the heat of combustion of any residue remaining after exposure to a simulated
standard fire, using combustion calorimetric techniques.” Conditions for a standard fire cannot
be simulated when dealing with incinerators. However, since a combustion aid is employed in the
method for total heat of combustion values (2), the ease with which solid waste materials ignite and
bum to completion can be measured by applying the same calonmetric techniques except omitting
the combustion aid and allowing only the flash heat of the ignition wire to ignite the sample.
The method presented here describes how to modify the total heat of combustion procedure
to obtain the potential heat of combustion values in incinerator residue and fly ash samples.
Other types* of solid waste materials usually do not have a potential heat value. Some incinerator
residue and fly ash samples have negative potential heat values. Such samples are high in carbonates,
which absorb heat upon decomposing (endothermic reaction).
With the total and potential heat values, the analyst can obtain the residual heat content of the
sample. Residual heat content is defined as the total heat content minus the potential heat content.
Residual heat value represents the heat content of a residue or fly ash sample that is not easily
obtainable and would probably exist regardless of incinerator efficiency.
APPARATUS
The apparatus for this method is the same as that descnbed in Reference 2.
REAGENTS
The chemical requirements and the preparation of reagents for this method are also the same as
those descnbed in Reference 2. Although benzoic acid is not used in this method as a combustion aid,
it is required in standardizing the calorimeter.
SAFETY PRECAUTIONS
See Parr Techmcal Manual No. 130, page 9, “Hazards of Operation” (3).
SAMPLE PREPARATION
The techniques of sample preparation are the same as those outlined in Reference 2.
PROCEDURE
This procedure applies to all solid waste materials that normally require a combustion aid to
determine total heat content. Since the mechanics are about the same as those outlined in Reference
The types of sohd waste used in this method include only solid samples (domestic origin) such as raw refuse,
incmerator fly ash, incinerator residue, and compost.
2
-------
Potential Heat
2 (except that there is no addition of a combustion aid), the details of the procedure are not repeated
here. Note, however, that duplicate results may not agree if the sample partially ignites one time and
not the next. This dispanty very seldom occurs; but if it does happen, the analysis should be repeated
until the sample ignites again. Failure to ignite can be detected by observing that the temperature rise
is very slight and the answer is around zero. Poor positioning of the ignition wire can cause a sample
to fail to ignite. The ignition wire must be installed, as mstructed, close to the sample but not
touching the sides of the sample container or the sample itself. The same restrictions on sample
portions apply for both methods. Since a combustion aid is not used in this method, the sample is
spread evenly on the bottom of the sample container.
STANDARDIZATION
The term “standardization” denotes the determination of the energy equivalent or water
equivalent factor (W) of the system (see Parr Manual No. 130, page 23). Benzoic acid, primary
standard with calorific value known, is the matenal used to determine the W factor. The benzoic acid
is analyzed in the manner described in the section on “Procedure” in Reference 2.
CALCULATIONS
Total Heat Content
The formula for computing the total heat content of a sample is described under “Calculations” in
Reference 2.
Potential Heat Content
The formula for computing potential heat content of a sample is the same as the formula for total
heat content, except that the “e 4 ” term, which is the heat content of the combustion aid, is omitted.
Residual Heat Content
Compute the residual heat content of a sample by substituting m the following equation
11 c(R) ‘ 1 c(t) ‘ 1 c(P)
where:
AHC(R) = Residual heat of combustion of sample in calories per gram or in Btu per pound t
= Total heat of combustion of sample as determined by the total heat of combustion
method used in conjunction with this method
c(P) = Potential heat of combustion of sample as determined by the method described
here
METHOD EVALUATION
This method can analyze solid waste materials with low or even negative potential heat contents.
Duplicate observations of the same sample will agree 95 percent of the time within about 25.8 to
144 Btu per pound, depending on the type of sample and whether or not some sample combustion
takes place (Table 1). Although a temperature rise was always observed in these tests, a lowering of
t Values must be expressed in the same units throughout the formula
3
-------
METHODS OF SOLID WASTE TESTING
initial temperature is very possible. For example, a sample high in carbonates, which decompose
easily, can produce a negative temperature change when the carbonates decompose upon being heated
by the ignition wire. To ensure precision, samples should be prepared in the manner described in
Reference 2.
TABLE 1
POOLED STANDARD DEVIATION* OF THE POTENTIAL
Btu-PER-POUND DETERMINATION
Type of
sample
Number of
samples
Duplicate observations
(Potential Btu/lb)
Range
(Btu/lb)
Residue (fines)t
9
52.0
597
to 2,580
Residue (fines)
8
13.8
—24.1
to 25.9
Fly ash
13
9.3
—35.3
to 12.3
*A variance estimate can be calculated from the duplicate (or triphcate) set of observations for each
sample. The pooled variance is essentially an average of all such estimates for samples of a given type.
It is assumed that a single underlying vanance exists for all samples of a given type. The pooled variance
is then the best estimate of this underlying variance. The pooled standard deviation is the square root of
the pooled variance and is used to estimate the underlying standard deviation.
f Fines are materials remaimng after most of the readily combustible substances have been removed by
manual sortmg. This material passed through a ½-inch sieve.
The absolute value of the difference between duplicated observations should not exceed 1.96 (y’ )(s),
confidence interval, or 144 Btu per pound, more than 5 percent of the time. The covariance between the
duplicated observations was ignored.
ACKNOWLEDGMENTS
The author wishes to express his appreciation to J. U. Doerger for performing the laboratory
analyses necessary to calculate the precision of this method. The author also thanks Betty Grupenhoff
of the Office of Solid Waste Management Programs for special computer assistance.
REFERENCES
1. Loftus, J. J., D. Gross, and A. F. Robertson. Potential heat;a method for measuring the heat
release of matenals in building fires. In: Proceedings; Sixty-Fourth Annual Meeting of the
Amencan Society for Testing and Materials, Philadelphia, June 25-30, 1961. The Society, p 61,
p. 1336-1348.
2. Wilson, Donald L. Laboratory procedure for determining the total heat of combustion in solid
wastes (included in this Manual).
3. Parr Instrument Company. Operating the adiabatic calorimeter. In: Oxygen bomb calorimetry
and combustion methods; technical manual No. 130. Moline, Illinois, Parr Instrument Company,
1966.
4
-------
LABORATORY PROCEDURE FOR DETERMINING
PERCENT ASH AND PERCENT WEIGHT LOSS
OF SOLID WASTES ON HEATING AT 600 C
Nancy S. U1mer
DISCUSSION 2
EQUIPMENT 2
REAGENTS 3
SAMPLE PREPARATION 3
SAFETY PRECAUTIONS 3
PROCEDURE 3
STANDARDIZATION 5
CALCULATIONS 5
METHOD EVALUATION 6
REFERENCES 8
Research Chemist, Solid Waste Research Laboratory, National Environmental Research
Center, Cincinnati.
-------
METHODS OF SOLID WASTE TESTING
DISCUSSION
Incineration has been employed since 1874 as a method of solid waste reduction( I). Although early
incinerator designs reflected the desire for recovering released heat for steam production and for
eliminating or reducing potentially hazardous materials, recent designs have been influenced by a need
for more efficient solid waste reduction and lower costs. Engineers and scientists have used various
parameters* (for example, the percent heat released, and the percent reduction in volume, weight, and
volatile solids) to evaluate the reduction efficiency of incinerator designs and to plan for the economic
disposal of the residue (2, 3).
A variety of analytical procedures have been employed for the determination of the volatile solids
in solid wastes. Researchers such as Kaiser(4) have adapted the ASTM standard procedure for coal(S)
to the analysis of refuse and residue. The heating of the sample in a closed crucible, as directed in the
ASTM technique, prevents the oxidation of elemental carbon, however, and results in a weight loss
attributable only to the volatilization of hydrocarbons. Other investigators, such as Schoenberger (6)
and Wiley and Spillane(7), have attained more complete oxidations by utilizing open-crucible
techniques.
A modification of the procedure proposed by the American Public Works Association(8) has been
used extensively by the research services laboratory staff for the characterization of refuse, residue,
and fly ash. Briefly, the technique involves (a) the introduction of two 2-g samples (each contained in
a porcelain crucible) into a cold muffle furnace, (b) the gradual increase in furnace temperature to
600 C, and (c) a 2-hr exposure of the samples to the latter temperature. While the crucibles are in the
furnace, the lids are either removed or tilted at an angle sufficiently large to insure the circulation of
air over the samples. After the 2-hr exposure to heat has been completed, the crucibles are removed
from the furnace, immediately covered with their respective lids, and cooled to room temperature in
a desiccator. The percent ash and the percent weight loss on heating at 600 C are calculatedt after
observing the weight lost by each sample.
A detailed description of the procedure is presented in the following sections. The details of a
Solid Waste Research Laboratory independent study on the applicability of this method for the
characterization of solid wastes is reported elsewhere(9).
EQUIPMENT
1. Balance, analytical, 0.1-mg readability
2. Boxes, sample storage, seamless aluminum, 3’/z-in. diameter, with tight-fitting lids (Fischer No.
3-485)
3. Crucibles, porcelain, Coors, high form, size 2, with lids
4. Desiccator, large, either Pyrex or stainless steel cabinet-type
5. Furnace, muffle, with indicating pyrometer and temperature controller (Lindberg Hevi-Duty
Muffle Furnace, Model 51333, with automatic Control Console, Model 59545, platinum,
platinum — 13 percent rhodium thermocouple, and six extra globar, silicon carbide heating
elements)
6. Gloves, asbestos, 11 in. long, provided with separate places for thumb and four fingers
*In this paper the term parameter means a variable or characteristic of interest.
tSince the observed decrease in sample weight reflects not only the weight lost by the vaporization of volatile
matenals and the combustion of fixed carbon, but also any weight gained (for example, by oxidation of metallic
components during heating), the term “weight loss on heating” appears more appropriate than the term “volatile
solids.” The term “ash” denotes, of course, the material remaining in the crucible after heating the sample.
2
-------
Percent Ash and Percent Weight Loss
7. Gloves, cotton, unlined (No. 2200 MUH, Wash-Rite, Inc., 1410 Cornell, Indianapolis, Indiana)
8. Ink, ceramic, marking, Coors, black
9. Mats, board, asbestos, 1/8 by 12 by 12 In.
10. Oven, drying, forced-air (or mechanically convected), capable of maintaining temperatures up to
ilOC
11. Pen, steel, for applying ceramic ink
12. Potentiometer, direct reading, with chromel-alumel thermocouple and scale, capable of reading
temperatures from 0 to 1,000 C (West Pyrotest, Model 9B, West Instrument Corp., Schiller Park,
Illinois)
13. Spatula (Scoopula, Fischer No. 14-357)
14. Tongs, crucible, nickel-plated, 20 in. long (Fischer No. 15-208)
REAGENTS
1. Inert standard (ground McDanel, high-temperature porcelain combustion tube fragments)
2. Combustible standard (A.C.S. sucrose or benzoic acid)
SAMPLE PREPARATION
A solid waste sample must undergo physical preparation before its charactenzation is initiated in
the laboratory. First it must be dried to constant weight, preferably in a forced-air (or mechanically
convected) oven. A temperature of 70 to 75 C should be used to dry municipal refuse (or compost),
incinerator residue and fly ash may be dried at 100 to 105 C. The particle size of the dried sample
should be reduced to 2 mm or less using a hammermill, pulverizer, or laboratory mill. To ensure
sample homogeneity, the ground samples should be thoroughly mixed. Finally, smce samples may
absorb moisture dunng the grinding and mixing processes, they should be redried for 3 hr at the
previously specified temperatures and then stored in a desiccator until the analyses are completed.
SAFETY PRECAUTIONS
The following suggestions apply to the analysis of samples.
1. Personal burns can be prevented by mtroducing the crucibles into a cool furnace and employing
asbestos gloves and long tongs while handling the hot crucibles and lids.
2. Bench-top damage can be prevented by using asbestos mats to support the hot crucibles and lids
before their introduction into the desiccator.
3. Breakage of desiccator glass can be prevented by (a) cooling the hot, covered crucibles a few
minutes before inserting them into the desiccator and (b) avoiding any direct contact of warm
crucibles with the glass.
PROCEDURE
The determination of the percent ash and the percent weight loss of each blank, standard, or solid
waste sample on heating at 600 C should be performed in duplicate as follows.
Procedure Comments
1. Transfer a numbered, clean, dry crucible and 1. (a) Using a steel pen, apply a Coors porcelain
lid from the desiccator to the pan of an ink number to the exterior surface of each
analytical balance. crucible and lid. Fire the dried ink either
with a gas flame or by heating in a muffle
3
-------
METHODS OF SOLID WASTE TESTING
furnace at 600 C for 1 hr. (The latter
technique also ensures low blank values for
new crucibles and lids.)
(b) Clean each crucible and lid in warm
detergent with a non-metallic brush; rinse
first with tap water, then with distilled
water; dry at 105 C for 1 to 2 hr; assemble
and then cool in a desiccator until needed.
(c) In steps 1 through 6, wear cotton gloves
to avoid finger printing the crucible and lid.
2. Weigh the crucible and lid to the nearest
0.0001 g.
3. Add approximately 2 g of the prepared 3. Do not add sample to the “blank.”
sample.
4. Weighr the crucible, lid, and sample to the
nearest 0.0001 g.
5. Transfer the covered crucible to a cool 5. (a) Use a tray or stack of asbestos mats to
muffle furnace, transport a number of crucibles simulta-
neously.
(b) WARNING: Introduction of samples into
a hot furnace may result in their sudden
ignition and loss, and in burns to the analyst.
(c) Space the crucibles to permit air circula-
tion around each. (Nine crucibles can be
arranged in five rows in the Lindberg furnace.)
6. Carefully tilt each crucible lid at an angle 6. Some analysts remove the crucible lids from
sufficiently large to insure the circulation of samples that do not sputter on ignition. Care
air over the sample. must then be exercised to return each lid to
the proper crucible after the heating is
completed.
7. Gradually heat the muffle furnace to 600 C. 7. (a) If using the Lindberg furnace, dial in the
desired setting on the digital set point after
turning the power and control switches to
the “on” position.
(b) It has been observed that higher digital
settings are required to achieve 600 C as the
elements of the Lindberg furnace age. It is
therefore recommended that the Lindberg
furnace temperature be monitored once a
week with an independent potentiometer and
a chromel-alumel thermocouple. The latter
may be introduced into the furnace through
the small space surrounding the door.
(c) A 30-minute period is required to heat
the Lindberg furnace to 600 C.
4
-------
Percent Ash and Percent Weight Loss
8. Maintain the furnace temperature at 600 C
for 2 hr.
9. Then turn off the furnace and immediately 9. (a) In steps 9 and 10, use an asbestos glove
transfer first a lid, then its corresponding and pair of tongs while handling the hot
crucible to a stack of at least three asbestos crucibles.
mats. Recover the crucible immediately. (b) Do not place the hot crucibles or lids
directly on table tops or metal trays.
10. Allow the covered crucibles to cool for 3 to 10. Do not permit the hot crucibles to touch the
5 mm., then transfer to a desiccator. glass of the desiccator.
11. After the crucible, lid, and sample have 11. (a) A hot crucible, lid, and sample usually
cooled to room temperature, weigh them to cool to room temperature in 1 to 2 hr.
the nearest 0.0001 g. (b)Wear a cotton glove to avoid finger-
printing the crucible and lid during their
transfer to the balance.
12. Calculate the initial and final weights, the 12. (a) See the section on calculations.
weight loss on heating, the percent ash, and (b) The means of the two determinations of
the percent weight loss on heating of each the percent ash and the percent weight loss
sample. on heating are usually reported.
STANDARDIZATION
Before initiating the characterization of solid waste samples, the analyst should evaluate his
technique by determining the percent ash or percent weight loss on heating a combustible standard
(for example, benzoic acid or sucrose), an inert standard (for example, ground McDanel high-
temperature combustion tube fragments), and three mixtures (for example, 3: 1, 1: 1, and l 3 parts
by weight) of a combustible and an inert standard. (See the discussion of accuracy in Method
Evaluation.)
Initially and periodically thereafter the analyst should also evaluate the applicability of his tech-
nique for cleaning and drying the crucibles and lids. The weight change on heating a properly cleaned
and dried, but empty, covered crucible (or blank) should not exceed 0.0004 g.
CALCULATIONS
The initial and final sample weights, the weight loss on heating (WLOH), the percent ash, and the
percent WLOH may be calculated as follows:
Initial sample weight (g) = B — A
Final sample weight (g) = C — A
Weight loss of the sample on heating (g) = B — C
°‘ h 0
,oas (B—A)
%WLOH= 100 (B-C )
= 100 — percent ash
Where A = the initial weight of the crucible and lid (g)
B = the initial weight of the crucible, lid, and sample (g)
C = the final weight of the crucible, lid, and sample (g)
5
-------
METHODS OF SOLID WASTE TESTING
METHOD EVALUATION
The mean observed percents WLOH of benzoic acid, sucrose, and mixtures of sucrose and McDanel
combustion tube fragments were always 99.92 percent of the theoretical percent WLOH (Table 1).
Although the mean observed percent WLOH of the 3. 1 (parts by weight) mixture of sucrose and
combustion tube fragments was 100.45 percent of the theoretical percent WLOH, no weight loss was
ever observed when the combustion tube fragments were heated alone.
TABLE I
ACCURACY OF THE METHOD
Type and identity of
sample
Theoretical
% WLOH
(T)
Mean observed
% WLOH
(M)
(100 M/T)
Combustible standards:
Benzoic acid
100
100.00
100.00
Sucrose
100
100.00
100.00
Sucrose-McDanel combustion
tube, parts by weight:
3:1
75
75.34
100.45
1:1
50
49.97
99.94
11
25
24.98
99.92
Inert standard:
McDanel combustion tube
0
0.00
—
The reproducibility of this procedure may be determined by calculating the standard deviations of
the determinations, the standard errors of the means of the determmations, and the coefficients of
variation. A review of our observations and calculations (Table 2) indicates that the standard devia-
tions of the determinations of the percents WLOH of the Delaware County, Pennsylvania, solid waste
samples and the standard errors of the corresponding mean percents WLOH often exceeded and varied
more than those of the standard samples; but the coefficients of vanation were always less than 0.05.
The nature or composition of the solid waste samples and the method of sample preparation may, of
course, influence the reproducibility.
Although sufficiently accurate and precise determinations of the percent ash and the percent weight
loss of solid wastes on heating at 600 C may be obtained with this procedure ui 4 hr, the applicability
of the two defined parameters may be limited. Research done by the Solid Waste Research
Laboratory has recently demonstrated that some residue and fly ash samples contain significant
quantities of carbonate( 10). Since the percent WLOH of Na 2 CO 3 and CaCO 3 on heating at 600 C is
very low, even in the presence of benzoic acid( 11), engineers and scientists may find it advisable to use
percent WLOH data collected at 960 C to evaluate the reduction efficiency of some incinerators.
6
-------
Percent Ash and Percent Weight Loss
TABLE 2
REPRODUCIBILITY OF THE METHOD
RSL
Sample identity No
Mean
observed
% WLOH
(M)
Standard
deviation of
the deter-
minations (S)
Standard
error of
the mean
(S/ /2)
Coefficient
of variation
(S/M)
Combustible standards
Benzoicacid lOU 000 000 000
Sucrose lOU 000 000 000
Sucrose-McDanel combus-
tion tube, parts by
weight
3 1 7534 006 004 000
II 4997 014 010 000
1 3 2498 001 001 000
Inert standard
McDanel combustion tube 0 00 0 00 0 00
Solid wastes *
Refuse 99 87.58 0.08 0 06 0 00
100 8903 010 007 000
101 8608 006 004 000
102 8676 101 071 001
103 9138 037 026 000
Residue
Combustible fractiont 105 52 04 0 52 037 001
107 5648 090 064 002
109 6296 065 046 001
Ill 4930 022 016 000
113 6180 221 156 0.04
Fines fractionl 104 1585 017 012 001
106 1263 037 026 003
108 1292 006 004 000
110 1182 016 011 001
112 25 63 047 0 33 002
lI2a 49.78 0.25 0.18 001
Fly ash 90 180 002 001 001
91 5 06 0 00 0.00 0.00
92 451 004 003 001
93 381 000 000 000
94 178 003 003 002
96 275 001 001 000
97 458 002 001 000
*These samples were collected at Incinerator No 3, Delaware County, Pennsylvania
tThe combustible fraction includes most of the residue that can be visually identified as containing food,
paper, plastics, rubber, leather, wood, textiles, or garden wastes
tThe fines fraction includes all the unidentified and some of the identifiable residue particles that pass
through a 0 5-in wire mesh sieve.
7
-------
METHODS OF SOLID WASTE TESTING
REFERENCES
1. American Public Works Association. Municipal refuse disposal. Chicago, Public Administration
Service, 1966, P. 140.
2. Schoenberger, R. J., and P. W. Purdom. Residue characterization according to furnace design.
Paper presented at the 1968 American Society of Civil Engineers Environmental Engineering
Conference, Chattanooga, Tennessee, May 12-17, 1968.
3. Achinger, W. C., and L. E. Daniels. An evaluation of seven incinerators. In: Proceedings; 1970
National Incinerator Conference, New York, May 17-20, 1970. American Society of Mechanical
Engineers, 1970, p. 52.
4. Kaiser, E. R. Chemical analysis of refuse components. In: Proceedings of the National Incinerator
Conference, New York, May 1-4, 1966. American Society of Mechanical Engineers, 1966, p. 85.
5. American Society of Testing Materials. Standard methods of laboratory sampling and analysis
of coal and coke; volatile matter. In: 1958 Book of ASTM Standards, including Tentatives.
pt. 8. D271-58. sect. 16-17. Philadelphia, 1958, P. 1006-1007.
6. Personal communication. R. J. Schoenberger, Drexel Institute, to J. Lechman, Bureau of Solid
Waste Management, Mar. 11, 1968.
7. Wiley, J., and J. T. Spillane. Methods for examination of raw and composted organic wastes.
Chemical Memorandum No. 4, Technical Development Laboratories, Communicable Disease
Center. Savannah, Georgia, U. S. Public Health Service, 1956, p. 8.
8. American Public Works Association, op. cit. p. 379-381.
9. Ulmer, N. S. Evaluation of a muffle furnace procedure for determining percent ash and percent
weight loss on heating of solid wastes; a Division of Research and Development open-file report.
Cincinnati, U. S. Environmental Protection Agency, 1971, p. 75.
10. Personal communication. D.L. Wilson, Solid Waste Research Laboratory to Author, Oct. 5,
1970.
11. Ulmer, N.S., op. cit.
8
-------
LABORATORY PROCEDURE FOR THE GRAVIMETRIC
DETERMINATION OF CARBON AND HYDROGEN IN
SOLID WASTES’
Donald L. Wilsont
DISCUSSION 2
APPARATUS 2
Requirements 2
Preparation. 6
Filling the Combustion Tube 6
Assembling the Combustion Train .. 7
Conditioning the Combustion Tube . 8
REAGENTS 8
SAFETY PRECAUTIONS.... 9
SAMPLE PREPARATION ... 10
PROCEDURE 10
Start-Up 10
Blanks 11
Standards 12
Samples 12
Shut-Down 14
CALCULATIONS 15
Standards 15
Samples 16
METHOD EVALUATION 17
ACKNOWLEDGMENTS 19
BIBUOGRAPHY 19
‘A description of this method appears in “Method for Macrodeternnnation of Carbon
and Hydrogen in Solid Wastes,” D. L. Wilson, Environmental Science & Technology,
5:609-614, July 1971.
tResearch Chemist, Solid Waste Research Laboratory, National Environmental Research
Center, Cincinnati.
-------
METHODS OF SOLID WASTE TESTING
DISCUSSION
Carbon and hydrogen are determined gravimetrically after combusting a weighed, dry, uniform
sample in an atmosphere of oxygen with a closed system and after fixing the combustion products
in an absorption train (Figure 1). The procedure (Figure 2)is designed to measure the total carbon
and hydrogen in dry solid wastes samples.
The 38-in. (1 1/2-rn. 0. D.) combustion tube, starting at a point 16 1/4 in. from the non-tapered
end contains the following matenals in the order named (Figure 3) Special mixture of asbestos, plati-
nized asbestos, and aluminum oxide; lead chromate; asbestos; copper dioxide, asbestos, lead chromate;
silver wool, lead dioxide; and silver wire. The plain asbestos is employed as a support between the
materials in the tube. The platinized asbestos located near the sample combustion area assists in the
combustion of condensed ring systems, particularly those containrng an angular methyl group that
may evolve as methane and thus escape complete combustion. Aluminum oxide is used to absorb
fluorine from the diffusing gaseous combustion products, and lead chromate oxidizes any SO 2 to
SO 3 and, finally, to the nonvolatile sulfate (PbSO 4 ). Copper oxide converts any carbon monoxide
to carbon dioxide, and lead dioxide retains the oxides of nitrogen as follows:
2N0 + 2PbO 2 Pb (NO 2 ) 2 ‘ PbO ÷ 1/2 02
2N0 2 + 2PbO 2 Pb (NO 3 ) 2 ÷ PbO + 1/2 02
The sections of silver wire and silver wool, located near the tapered end of the combustion tube,
absorb chlorine, bromine, and iodine from the gaseous combustion products before they diffuse
from the combustion tube into the absorption tubes where magnesium perchiorate and Indicarb
are employed to absorb the water vapor and carbon dioxide gas, respectively.
Samples containing (a) alkali or alkaline earths, as potash or calcium oxide, in the absence of
sulfur or phosphorus or (b) phosphorus in the absence of alkali or alkaline earths can only be
evaluated if an accelerator is added to the samples to ensure complete combustion and conversion
of all the carbon to carbon dioxide. Iron chips (carbon free) are used as the accelerator. The iron
ignites and starts the exothermic combustion reactions before the sample reaches the final induced
temperature of 950 C.
This method is applicable to raw garbage, compost, incinerator residue, and other dry, general
waste samples that can have a carbon content of 0.5 to 83.0 percent and a hydrogen content of
0.01 to 7.80 percent. Samples containing appreciable amounts of arsenic, antimony, bismuth, and
mercury should not be analyzed unless further modifications in the procedure are made, since these
elements would quickly detenorate the combustion tube packing.
Since hydrogen is analyzed as water, it is essential that all of the moisture be removed from the
samples before they are analyzed. All samples must be re-dned no more than a few days before
the analyses are performed.
APPARATUS
Requirements
1. Asbestos boards, 6 x 6 x 1/8 in., and 12 x 12 x 1/8 in.
2. Baffle, oxygen (Sargent #S-2 1787)
3. Balance, analytical, 200-g capacity, 0.1-mg readability
4. Barge, combustion, high punty nickel (Fisher #7-647)
2
-------
( )
Figure 1. The carbon-hydrogen train.
-------
METHODS OF SOLID WASTE TESTING
SAIPLE
COMBUST ION
ZONE
CARBON—
HYDROGEN
TRAIN
OXYGEN
[ CONCENTRATED H 2 SO 4
MAGNESIUM PERCNLORATEI
I
I ASCARITE 1 1TH
IACTIVATED ALUMINA
[ MAGNESIUM PERCNLORATEI
t
I MAGNESIUM PERCHLORATE I
Figure 2. General outline of carbon-hydrogen train.
MAIN SECTIONS TEMPERATURE
OXYGEN
ROOM
PURIFICATION
TEMPERATURE
SECTION
PURPOSE OF UNITS
REMOVES MATER AND SULFUR DIOXIDE
REMOVES MATER
REMOVES CARBON DIOXIDE
MAINTAINS OXYGEN FLOM
(250 cc./øIn.)
3 ,
[ FLOMMETER I
SAIPLE 11TH
COMBUSTION ACCELERATOR
COMBUSTION PRODUCTS
AND EXCESS OXYGEN
1
R DO°C
CONVERTS CONDENSED RINGS 11TH
PLATINIZED ASBESTOS ANGULAR METHYL GROUPS TO CARBON
AND DIOXIDE AND REMOVES FLUORD-
ALUMINUM OXIDE COMPOUNDS
REMOVES OXIDES OF SULFUR
CONVERTS CARBON MONOXIDE TO
CARBON DIOXIDE
REMOVES OXIDES OF SULFUR
PURIFICATION
OF
GASEOUS
COMBUSTION
PRODUCTS
ZONE
BDD°C
200°C
[ LEAD CHROMATE
[ COPPER OXIDE ]
LEAD CHROMATE
J r
[ SILVER MCDL
1
ILEAD DIOXIDE I
J r
ISILVER MIREJ
ABSORPTION
OF
ROOM
MATER AND
TEMPERATURE
CARBON DIOXIDE
SECTION
+
REMOVES HALOGENS AND CONVERTS
OXIDES OF NITROGEN TD FREE
NI TRDOEN
REMOVES OXIDES DF NITROGEN
REMOVES HALOGENS AND CONVERTS
OXIDES OF NITROGEN TO FREE
NITROGEN
ABSDRBS MATER
ABSORBS MATER
ABSORBS CARBON DIOXIDE
ABSORBS CARBON OIDXIDE
INDICATES CARBON DIOXIDE
IN EXIT GASES
I INDIEARB AND
IACTIVATED ALUMINA
INDICARB AND
ACTIVATED ALUMINA
IBARIUM HYDROXIDEI
ATMOSPHERE
4
-------
Carbon and Hydrogen
1” Space
12”
Furnace
(800°C)
½” Spac
4”
Furnace
(200°C)
Figure 3. Packed combustion tube.
1½” Special Mixture
1½” PbCrO 4 (12—20 mesh)
______ ½” Asbestos Plug
V’ Asbestos Plug
PbCr O 4
Sliver lool
(12 ,20 mesh)
Scale: ? “5lze
2” Sliver lre
( 34 B&S gauge)
2½” Space
Position Mark
Barge
8”
Furnace
(950°C)
9” CuO
PACKED
COMBUSTION
TUBE
5
-------
METHODS OF SOLID WASTE TESTING
5. Baskets, test tube, one size C, four size D (Fisher #14-966)
6. Boats, combustion, clay (Fisher #7-651) with covers (Fisher #7-652)
7. Boats, large, combustion, clay (Fisher #12-183N) with covers (Fisher #12-1835 N)
8. Bulbs, absorption, Nesbitt (for carbon dioxide), four (Fisher #7-51 5)
9. Bulbs, absorption, Stetser-Norton (for water), two (Fisher #7-56 5)
10. Charger-rake (Fisher #7-610)
11. Clamps, utility, three-prong grip (Fisher #5-768-10)
1 2. Desiccator cabinet (Fisher #8-645-5)
13. Erlenmeyer flask, two, 500-ml
14. Furnace, modified, combustion, organic, multiple unit, electric, tube type (Lindberg #123-T-S)
15. Furnace, muffle, operating temperature of 950 C
16. Glass tubing to fit two-hole stoppers
17. Gloves, lint-free
18. Inserter, sample (Dietert Co. #3452)
19. Jar, drying, 300mm m height (Fisher #9-210)
20. Oxygen cylinder with (a) pressure regulator, adjustable from 0 to 10 lb of pressure on the low
pressure side and (b) needle-valve control
21. Pan, enameled (Fisher #9-01 7)
22. Pan, stainless steel, two (Fisher #13-361, size D)
23. Rotameter, ends designed for tube connections, maintams an oxygen flow of 250 cc per mm
(Ace Glass, Inc., Rota-Kit, Tru-Taper #1, size lA-iS- I)
24. Stand, support, size A (Fisher #14-675)
25. Stopcock, glass, T-bore, 8mm 0. D. stems (Ace Glass, Inc. #8178)
26. Stopcock, hard rubber (Fisher #14-630)
27. Stoppers, two-hole, size #7
28. Tinier (Fisher #6-662)
29. Tube, combustion, Vycor, 38 in. long, 1 1/4 m. I. D., 1 1/2 in. 0. D., one end tapered to fit
3/16 in. 1. D. rubber tubing (may use McDanel tube, 36 in. long [ Fisher #CTT1 1436])
30. Tube cleaners, size A (Fisher #3-642)
31. Tubing, bubble, Argyle universal, plain, lumen 3/16-in, bore (Aloe Scientific #AR-500)
32. Tubmg, rubber, red, thin wall, 3/16-in, bore (Fisher #14-166)
33. Tubing, Tygon, 1/4-in. 1. D., 3/8-in. 0. D., 1/16-in, wall
34. Washers, Milligan gas, two, (Fisher #7-513)
35. Yardstick
Preparation
Filling the combustion tube.
Considerable care must be exercised in filling the combustion tube. The gaseous combustion
products diffusing through the tube must come in contact with large surface areas, but the materials
must not be so compact that the gas pressure heads will not afford the desired gas velocities through
the tube. The following procedure is recommended (see Figure 3 and the section on reagents).
a) Twist a number of strands (ten to fifteen, 6-8 in. long) of silver wire together and insert into
the tapered end of the combustion tube 1 (this occupies approximately 2 in. of the tube).
b) Hold the tube in a vertical position, that is, with the tapered end down.
6
-------
Carbon and Hydrogen
c) Add approximately 100 g lead dioxide to the tube. The layer should be 4 in. deep (a yardstick
may be helpful).
d) Insert 1 0 g silver wool to form a 1/2-in, layer.
e) Add approximately 50 g lead chromate to form a 2-in, layer (mix in a small amount of asbestos
to help prevent caking).
f) Insert asbestos loosely to form a 3/4-in, plug.
g) Add approximately 500 g cupric oxide to form a 9-in, layer.
h) Insert asbestos loosely to form a 1/2-in, plug.
i) Add approximately 37 g lead chromate to form a 1 1/2-in, layer (again mix in a small amount of
asbestos)
j) Add the special mixture consisting of 20 ml of platinized asbestos pIus 20 ml of asbestos plus 10
ml of aluminum oxide (approximate unpacked volume measurements). The layer should be
1 1/2 in deep
Assembling the combustion train.
The components of the apparatus are assembled in the following sequence:
a) Oxygen cylinder with attached regulator that will afford a delivery pressure of 10 (p. s. i. g.)
b) Hard rubber stopcock with on-off valve
c) Erlenmeyer flask, 500-ml, with two-hole, size #7 stopper (serves as a hquid-backflow trap)
d) Gas washer containing 150 ml concentrated sulfuric acid
e) Drying jar Magnesium perchlorate is placed in the lower half and ascarite, topped with
activated alumina, in the upper half. A small layer of glass wool is inserted beneath the
magnesium perchlorate and above the activated alumina. The oxygen flow into the jar is
through the bottom mlet.
Rotameter, with the stamless steel float at a level affording an oxygen flow of 250 cc per mm
g) Glass stopcock held by a utility clamp on a support stand
h) Combustion tube with sample inserter and oxygen baffle attached at the non-tapered end;
a position mark, etched on the outside of tube 2 1/2 in. from the non-tapered end to enable
the analyst to keep the tube in correct position within the furnace units; aluminum foil
placed around the tapered end of the tube to maintain heat inside the tube and prevent
water vapor from condensing
i) Two Stetser-Norton bulbs (for water absorption) set up in senes; each contains magnesium
perchiorate between two loose layers of glass wool
j) Two Nesbitt bulbs (for carbon dioxide absorption) assembled in series: a 1/2-in, layer of glass
wool is placed in the bottom of the bulb. Indicarb is then added to a point 3/4-in, from the
shoulder of the bulb. The Indicarb is covered by a 1/4-in, layer of activated alumina. Introduce
sufficient glass wool to reach the neck area and cotton in hollow stopper. (Note. For highly
combustible materials or materials with high carbon content, more than two Nesbitt bulbs
may be necessary )
k) Erlenmeyer flask, 500-mi, with two-hole, size #7 stopper (serves as a liquid-backflow trap)
1) Gas washer, containing 150 ml of barium hydroxidethymolphthalein solution
m) Tygon tubing as connections between the oxygen cylinder, stopcock, erlenmeyer flask, gas
washer, and drying jar
7
-------
METHODS OF SOLID WASTE TESTING
n) Argyle tubing, used between drying jar, rotameter, glass stopcock, and the sample inserter
o) Rubber tubing, between the tapered end of the combustion tube, absorption bulbs, second
Erlenmeyer flask trap, and final gas washer.
Conditioning the combustion tube
A freshly packed tube contains excess moisture and must be dried out for 2 hr as directed in
step 1 in the following “Procedure.” After the 2-hr penod, the analyst continues with the remaining
procedural steps.
If the combustion tube has been previously used but has remained idle for more than 1 day, the
analyst conditions the tube by performing only steps 2 and 3. Dunng this idle period, the glass
stopcock must be turned so that nothing can flow into the combustion tube. The rubber stop-
cock is likewise closed so that the sulfuric acid does not flow from the gas washer back into the
trap.
If the stopcocks are at their proper shut-down positions, no conditioning of the combustion
tube is necessary with an idle penod of I day or less.
A conditioned combustion tube in continuous use should last for about 6 months or approxi-
mately 700 analyses. A close estimate of the life span for a conditioned tube is impossible to deter-
mine because of the variation in the samples’ impurities and carbon-hydrogen concentrations.
Note: Before performing the following conditioning procedure, review the start-up
and shut-down procedures.
Procedure
With the 8-in, furnace at 950 C ± 20 C (con-
trol level about 8.9), the 1 2-in, furnace at
800 C ± 20 C (about 4.5), and the 4-in.
furnace at 200 C ± 10 C (about 2.0),
allow oxygen to flow through the combus-
tion train at 250 cc per mm, with the
rubber and glass stopcocks open. (Do not
connect absorption bulbs.)
NOTICE For newly packed tubes, wait
2 hr before starting the next step.
2. Follow the procedure outlined for analyz-
ing samples except use 0.2 to 0.5 g of
A. C. S. grade sucrose without a com-
bustion aid.
3. Repeat step 2 above if observed carbon
and hydrogen contents do not agree with
theoretical values.
Comments
1. a) Putting the temperature knobs of the
8-in, and the 12-in, furnaces on “high”
and the 4-in, furnace on 3 will allow the
heat-up time to be about only 35 mm.
b) CAUTION: Never allow the temperature
to exceed 1 ,000 C for the 8-in, furnace,
840 C for the 12-rn. furnace, and 220 C for
the 4-in, furnace.
c) The stainless steel float in the rotameter
should be approximately at the 8.2-cm level
(the setting established for oxygen gas).
2. a) Sucrose must be previously dried at 110
C for 1 hr.
b) See section on Samples.
c) CAUTION: Higher weights of sucrose
combust too violently.
3. This step is usually necessary only with a
freshly packed tube.
REAGENTS
All reagents are of ACS analytical reagent-grade quality. All solutions are prepared using distilled or
deionized water. Reagents are as follows:
8
-------
Carbon and Hydrogen
1. Oxygen, 99.5 percent pure (the oxygen should be prepared from liquid air since oxygen pre-
pared by electrolysis contains traces of hydrogen)
2. Sulfuric acid, concentrated
3. Dehydrite (anhydrous magnesium perchiorate)
4. Ascarite (sodium hydroxide on asbestos), 8-20 mesh.
5. Activated alumina, indicating, 8-14 mesh (Fisher #A-545)
6. Silver wire, 34 B & S gauge, pure grade (Fisher #20-272)
7. Lead dioxide, brown, 12-20 mesh
8. Silver wool
9. Lead chromate, 12-20 mesh; or powder which has fused at 820 C for 1 hr and ground to about
12-20 mesh
10. Asbestos, medium or long fiber, acid-washed
11. Cupnc oxide, black wire; before using, ignite at 800 C for 1 hr
12. Platinized asbestos, 5 percent (Fisher #P-l 52)
13. Aluminum oxide, anhydrous
14. Accelerator chips, Combax (iron) (Fisher #C-420)
15. Indicarb, 6-10 mesh (Fisher #1-181)
16. Absorbent cotton
17. Glass wool
18. Stopcock grease, KeI-F 90 (Fisher #14-635-10)
19. Barium hydroxide solution: dissolve 12.0 g Ba(OH) 2 in distilled water and dilute with same
to 1 liter
20. Thymolphthalein solution dissolve 10.0 g thymolphthalein in ethanol and dilute with same to
1 liter
21. Barium hydroxide-thymolphthalein solution. add 0.5 ml of the thymolphthalein solution to
150 ml of the barium hydroxide solution
SAFETY PRECAUTIONS
A wooden safety shield 2 ft x 2 ft x 3/8 In., or the equivalent, should be placed about 3 ft from
the right side of the furnace base, such that if the rubber stopper with the sample inserter should
blow out, the stopper and inserter would hit the shield.
The sample boats should never be inserted faster than the prescribed 1 in. each 5 mm after
the initial 5-mm wait, for the first 20 mm of total insertion time. Faster sample insertions may cause
the above mentioned blow-out or a more violent explosion. If the sample ignition is too violent,
the analyst should divert the oxygen flow by turning the glass stopcock and allowing the oxygen
to flow into the room.
Normally the oxygen flow rate is reduced dunng the initial ignition (which generally occurs
on th first or second I-in, insertion) and should not be adjusted during this period. The flow
rate of 250 cc per mm is sufficient to prevent the back flow of gases into the rotarneter. Always
keeping the sample inserter’s blunt end, while in initial position, approximately 1/4 in. away
from the baffle will help maintain the oxygen flow.
If the flow of exit gas stops, the oxygen inlet flow is immediately turned off by using the glass
stopcock, as mentioned above. This gas-flow stoppage may be due to an unopened absorption bulb
or too tight tube compaction.
9
-------
METHODS OF SOLID WASTE TESTING
The analyst should always be aware of the hot boats, the hot furnace parts, and the general
bubble flow-rate in the exit gas washer. He should not remain close to the combustion tube during
the sample’s initial ignition because possible explosions are anticipated mainly at this time.
SAMPLE PREPARATION
The details of sample preparation procedures that descnbe the drying and grinding techniques
are not discussed here. In general, raw refuse, residue (organics), and compost are dried at 70 C to a
constant weight and ground to a particle size of less than 1 to 2 mm. Residue (fines) and fly ash
samples are dried at 105 C to a constant weight and pulvenzed to pass through a #60 sieve.
All samples must be redried no more than one week before being weighed for the analyses.
Those samples originally dried at 70 C must be redried at 70 C for 4 hr; those samples onginally
dried at 105 C must be redried at 105 C for 1 hr. After redrying procedures, all samples must be
kept in tightly closed containers and in a desiccator until the analyses are performed.
PROCEDURE
Start- Up
The start-up procedure for the carbon-hydrogen analyses is always the same, except for the use
of a freshly packed tube, as previously mentioned. The following procedure outlines the routine steps
in preparing the carbon-hydrogen train for the analyses.
Procedure Comments
1. Set the temperature controls of the 8-in.
and 12-in, furnaces on high and that of
the 4-in, furnace on 3 for about 35 mm.
Final temperatures for the 8, 12, and
4-in, furnaces are 950 C ± 20 C, 800 C ±
20 C, and 200 C ± 10 C, respectively.
2. After each furnace has reached its appro-
priate temperature, set the 8-in, furnace
control on 8.9, the 12-in, furnace control
on 4.5, and the 4-in, furnace control on
2.0.
3. a) The glass stopcock should be open to
the room.
b) If the oxygen is not soon allowed to
flow, sulfuric acid will flow back into the
trap.
c) The stainless steel float in the rotameter
should be set and maintained at approxi-
mately the 8.2-cm level.
4. a) If the float drops drastically toward
the zero level, immediately return glass
stopcock to its former position. Then the
1 CAUTION: Never allow the temperature
to exceed 1,000 C for the 8-in, furnace,
840 C for the 12-in, furnace, and 220 C
for the 4-in, furnace.
2. These numbers are not absolute and may
change with the age of the elements.
3. Open the rubber stopcock and without
much delay allow the oxygen to flow
through at approximately 250 cc per mm.
The delivery pressure gauge is set at 10
(p. s. m. g.) Flow adjustments are made
with the needle-valve control.
4. Turn the glass stopcock to allow oxygen
to flow through the combustion tube.
10
-------
Carbon and Hydrogen
5. Attach at least two of each type of absorp-
tion bulbs and again notice the stainless
steel float and bubbles in the exit-gas
washer.
6. Adjust the oxygen flow to be approxi-
mately 250 cc per mm., the stainless steel
float should be at the 8.2-cm level.
7. With the train completely assembled, allow
the oxygen to flow through the system for
10 mm.
8. Remove the absorption bulbs from the
train and close each bulb to the atmos-
phere.
9. Determine and record the weight of each
CO 2 -absorbing bulb and each H 2 0-absorb-
ing bulb. These weights represent the initial
weights of the absorption bulbs.
10. After opening the absorption bulbs to per-
mit gas flow, quickly return the bulbs to
the train assembly.
11. When the gas starts through the exit-gas
washer, the train is ready for sample
analyses.
charger-rake rod may be used to loosen
the tube packing.
b) Absorption bulbs should not be attach-
ed.
5. a) The bubble flow rate in the exit-gas
washer is a good indication as to how the
oxygen is flowing through the train. If
the bubbles do not start, an absorption
bulb may be closed or packed too tightly.
b) If the samples to be analyzed are known
or suspected to contain more than 30 per-
cent carbon, and if sample weights of more
than 1 .5 g are used, attach three or more
absorption bulbs for the CO 2 collection.
6. The flat end of the sample inserter should
always be slightly away from the end of
the baffle to allow the free flow of
oxygen.
7. This step ensures the conditioning of the
absorption bulbs.
9. a) The bulbs should be near room tempera-
ture before being weighed. Normally, each
bulb will be near room temperature if the
order of weighing is started with the last
bulb in the train.
b) Before weighing, each bulb is momen-
tanly vented to the atmosphere and wiped
clean with a lint-free cloth.
c) Use an analytical balance with a 200-g
capacity and 0.1 mg readability.
d) Weights are recorded to the nearest
0.0001 g.
10. Start with the bulb furthest from the fur-
nace. The connection of the bulbs to
the furnace should be performed last
11. If the gas does not start through the exit-
gas washer, check bulbs, openings, and com-
paction of absorbing materials.
Blanks
Increase in weight of the absorption bulbs is due to I) sample ignition, 2) sample container’s
contamination, 3) atmospheric contamination during sample insertion, and 4) impure oxygen gas.
The blank analyses determine the effects of all the above factors except sample ignition on the weights
11
-------
METHODS OF SOLID WASTE TESTING
of absorption bulbs. These blank analyses are conducted like the regular sample analyses, except
that preignited, empty sample containers with covers and an unpacked combustion tube are
employed. The unpacked tube is needed for the blank analyses because lead dioxide is highly
hygroscopic.
The analyst is advised to perform triplicate observations and record the humidity (which may
be estimated) of the room during the analyses. Whenever the oxygen supply is changed or the room
humidity greatly changes, the blank analysis should be repeated. The carbon dioxide blank value
is usually zero and the water blank value normally varies with analysts
Standards
After conditionmg the combustion tube as previously described, the analyst should periodically
check the carbon-hydrogen train by analyzing standards such as sucrose, naphthol, urea, graphite,
calcium carbonate, and solid waste samples that have been previously analyzed. The procedure for
analyzing standards is the same as described for sample analyses. Analyze, however, only 0.2 to
0.5 g of sucrose, and without a combustion aid, since this standard burns quickly and easily. Benzoic
acid, although commonly used as a carbon standard, reacts too violently for this method.
Samples
At this point, all the necessary conditioning should have been performed so that the carbon-
hydrogen tram is now ready for sample analyses. The following procedure applies to all solid
waste materials, and also to blanks and standards with the previously mentioned changes. All
the sample containers and covers employed m the method must have been previously ignited at
950 C for 1 hr, then cooled and stored in a desiccator until used.
The analyst is advised to analyze samples of each particular type of solid waste material as a
unit. Switching back and forth between different types of materials (which usually vary greatly
in their carbon-hydrogen content) causes unnecessary reconditioning and rechecking of the com-
bustion tube. To ensure good results, the analyst should always analyze a standard before analyzing
a particular type of solid waste matenal. This standard should be of the same general character as
the type of material to be analyzed.
A combustion aid must be mixed with each sample after the sample has been weighed into
its container. Some samples combust vigorously and the sample injection procedure may have to
be slowed down; however, even vigorously reacting samples need a combustion aid.
The analyst must use his own judgment in the selection of the sample container, the number
of absorption bulbs in the train, the unscheduled removal of an absorption bulb, and the change
of the sample insertion procedure.
Procedure Comments
1. Transfer at least ito 2 g of a sample mto 1. a) Sample weights up to 10 g may be
each of two previously weighed combus- used if non-uniformity of sample warrants.
tion boats. Determme and record the b) Fluffy samples such as compost samples
weight of each sample to the nearest require the larger containers.
0.0001 g. c) Minimize handlmg of boats and lids
to prevent contammation.
d) Keeping the boats in a particular order,
or numbering them, will prevent mix-up
of samples.
12
-------
Carbon and Hydrogen
2. Sparsely sprinkle each sample with a few
iron chips. Using a spatula, mix the com-
bustion aid throughout the sample.
3. Store each boat (containing a sample) and
its lid in a desiccator until they are trans-
ferred to the combustion tube.
4. Cover each boat with its lid and transfer
them from the desiccator to insertion end
of combustion tube. IMMEDIATELY go to
the next step.
5. Remove stopper with attached sample In-
serter and place boat about half way into
tube. Then close the combustion tube by
moving the sample inserter, with baffle,
along the bottom of the tube; holding
the tube with the left hand, twist the
stopper tightly into the tube. IMMEDI-
ATELY go to next step.
6. Upon closing the combustion tube, set
a timer for 60 mm. Check the flow rate
to see if it is 250 cc per mm (stainless
steel float at 8.2-cm level) after the gases
are bubbling through the exit-gas washer.
e) DO NOT pellet any sample. If a I- to
2-g sample will not fit into a particular
boat, use a larger container.
2. a) Granular tin (Fisher #12-173) may be
used as an accelerator if iron chips are not
available.
b) REMINDER: DO NOT use a com-
bustion aid with sucrose.
3. a) It is convenient to use a stiff asbestos
pad to support the boats while in the
desiccator and during transfer from one
place to another.
b) The desiccator must contain CO 2 -ab-
sorbing materials.
4. REMINDER. Flat end of sample inserter
must be slightly away from the baffle to
permit free flow of oxygen.
5. a) Do this step quickly. The blank value
will only be applicable if the time required
for placing the boat in the combustion
tube is constant.
b) If the combustion tube contains the
boat and lid from the previous sample
analyses, use the charger-rake to remove
the boat and lid onto a stiff asbestos pad.
Then drop the boat and lid into an
enameled pan lined with asbestos pads.
After they have cooled, the boat and
lid are transferred to a stainless steel pan,
cleaned, and finally ignited at 950 C
for 1 hr before being reused.
6. a) The stainless steel float will drop mo-
mentarily and adjustment is needed if it
remains below the 7.1-cm level for more
than a few moments. DO NOT, however,
attempt to adjust the flow-rate if the
sample ignition h ’ started. Except when
using the large clay boats, sample ignition
usually starts after the sample is closer
to the 950 C zone.
b) SAFETY REMINDER: If bubbles in
exit-gas washer stop completely for more
than 5 to 10 min and the stainless steel
float begins dropping (caused by too vigor-
ous sample combustion), turn the glass stop-
cock, thus diverting the oxygen from the
combustion tube to the room. Whenever the
stopcock must be turned, the results of
that particular test must be discarded.
13
-------
METHODS OF SOLID WASTE TESTING
7. After 5 mm, move the sample boat 1 in.
toward the 950 C zone.
8. At the end of each of 3 successive 5-mm
intervals, move the sample boat 1-rn. to-
ward the 950 C zone.
9. After another 5 mm, move the sample boat
completely into the middle of the 950 C
zone.
10. Allow the oxygen to flow through the
train for the rest of the 60-mm period.
11. Remove the absorption bulbs from the
train and close each bulb to the atmos-
phere.
12. Determine and record the weight of each
CO 2 -absorbing bulb and each H 2 0-absorb-
ing bulb.
13. Return the sample inserter to the start
position.
14. If another sample, contained withm a boat,
is ready to be analyzed, repeat the pro-
cedure starting with step 4.
15. If the train is not to be used for more than
1/2 hr, see “Shut-Down” procedure.
7. If the sample has already ignited vigorously,
delay this step for another 5 mm.
8. a) DO NOT move the sample boat at a
faster rate, even if one of the 1-in, in-
sertion steps was forgotten. After some
experience, however, the analyst may ac-
celerate the insertion procedure when ana-
lyzing residue (fines) or fly ash samples.
b) During this step, the Indicarb may
indicate that another tared absorption bulb
should be added.
9. a) The entire penod of sample insertion
should not exceed 30 mm.
b) If the large clay boats were employed,
most of the boat will be beyond the center
of the 950 C zone.
11. The oxygen continues to flow through
the combustion tube while the absorp-
tion bulbs are removed. If more than 30
mm will elapse before another sample
is started, however, the oxygen flow must
be diverted away from the combustion
and mto the room by turning the glass
stopcock.
12. a) This weight represents the final weight
of each absorption bulb and is used as
the initial weight of each bulb for the
next sample.
b) The time required for weighmg should
not exceed 15 mm.
13. When the flat end of the sample inserter
is slightly away from the baffle, thus
permitting a free flow of oxygen through
the baffle, the sample inserter is in the
start position.
Shut-Down
As mentioned in the section on conditioning the combustion tube, the proper shut-down pro-
cedure prevents the necessity for reconditioning the combustion tube. If the train is not used for
14
-------
Carbon and Hydrogen
more than 1/2-hr and the oxygen is allowed to flow through the combustion tube, the packing
materials (especially the lead dioxide) will dry out and require reconditioning before the train can
be used again. If the train is to be used the same day but more than 30 mm will elapse, the furnaces’
temperatures may be maintained, but the oxygen flow must either be diverted from flowing through
the combustion tube or shut off completely. Whenever the oxygen flow is off, the glass and
rubber stopcocks must be closed as described below.
Procedure Comments
1. Disconnect the absorption bulbs from the 1. Any remaining boat and lid should be re-
combustion tube and close each one to the moved from the combustion tube.
atmosphere.
2. Having the combustion tube stoppered and 2. Furnaces need not be turned off if used
the sample inserter in the start position, later the same day.
turn each furnace off.
3. Turn off the oxygen flow first at the
main regulator valve on the oxygen cyl-
inder, then at the low pressure valve.
IMMEDIATELY go to the next step.
4. Turn the glass stopcock to divert any
oxygen flow from the combustion tube
to the room. IMMEDIATELY go to the
next step.
5. Close the rubber stopcock. 5. When the rubber stopcock is closed, the
backflow of sulfuric acid into the trap is
prevented.
CALCULATIONS
Standards
Formula.
Employ the following formula to calculate the theoretical concentration of either carbon or hydro-
gen in a standard sample:
E — ( N) (F) (100 )
— (S)(P)
where
% = the percent by weight
E = the element, either carbon or hydrogen
N = the number of atoms of the element in a molecule of the standard
F = a factor, derived by dividing the gram-atomic weight of the element by the gram-molecular
weight of the standard
S = the weight of the total sample
P = the decimal fraction representing the concentration of the standard compound in the total
analyzed sample. (Note. this decimal fraction is the only fraction containing the component
for which the sample is being analyzed.)
15
-------
METHODS OF SOLID WASTE TESTING
Example.
Pure Sucrose, C 12 H 22 (NBS Grade)
( 12.O1\
( 12) \342.3o/ (100 )
%C= =42.10
(1.0000) (1.00)
( 1.008 \
9’H— ( 22) \342.3o/ (100 ) —648
— (1.0000) (1.00) —
When the impurities listed on the bottle are considered, the calculated percents of carbon and
hydrogen in ACS grade sucrose are 42.09 and 6.48 respectively.
Samples
Carbon.
Employ the following formula to calculate the concentration of carbon in a solid waste sample:
( A-B) (X) (100 )
%C- (S)
where
% = the percent by weight
C = the element carbon
A = the sum total increase in the weight of the CO 2 -absorbing bulbs as determined in the unknown
analysis
B = the sum total increase in the weight of the CO 2 -absorbing bulbs as determined in the blank
analyses
X = a factor, derived by dividing the gram-atomic weight of carbon by the gram-molecular
weight of carbon dioxide (1. e., (12.01) ÷ (44.01) = 0.2729)
S = the weight of the total sample
Hydrogen.
Employ the following formula to calculate the concentration of hydrogen in a solid waste sample:
— ( A—B)(Y)(l00 )
%H- (S)
where
% = the percent by weight
H = the element hydrogen
A = the sum total increase in the weight of the H 2 0-absorbing bulbs as determined in the un-
known analysis
16
-------
Carbon and Hydrogen
B = the sum total increase in the weight of the H 2 0-absorbing bulbs as determined in the blank
analyses
Y = a factor, derived by dividing the gram-molecular weight of hydrogen by the gram-molecular
weight of water(i. e., (2.016) ÷ (18.02)0.1119)
S = the weight of the total sample
METHOD EVALUATION
The accuracy of this newly developed method is established in Table 1. This method can analyze
solid waste materials containing various forms of carbon to within one actual percent of the true
value. For these tests, sucrose, naphthol, and urea were selected to represent hydrocarbons.
Graphite was employed as an elemental form of carbon, and calcium carbonate as an inorganic
form. This method has accurately analyzed (100.00% recovery) specially prepared residue (fines)
samples calculated to contain only 0.46 percent carbon and 0.01 percent hydrogen. This method
should definitely not be employed to analyze samples that contain less than 0.1 percent carbon.
The precision of this method was determined by analyzing in triplicate a number of solid waste
samples of various types. The pooled standard deviation of the observations for each type of solid
waste was calculated using an Olivetti Underwood Programma 101. The calculations revealed that
in the analyses of each type of waste, the duplicate and triplicate determinations were equally
precise (Table 2). To ensure precision, the particle size of the samples must be less than 2 mm or
passed through a #60 sieve, then thoroughly mixed before analyzing.
With this macro method, the analyst normally uses a 1- to 2-g sample, but he is not restricted
to this amount. Because of the difficulties in preparing a very uniform sample, sample weights
below 1 g have been found inadequate when analyzing solid waste materials. But samples up to
10 g have been analyzed with no difficulty. The extra sample weights, however, add little to the
precision of this method.
TABLE 1
ACCURACY OF CARBON AND HYDROGEN DETERMINA-
TIONS OF STANDARD COMPOUNDS
Compound
Number of % Element
determinations C
calculated
% Element found
% Recovery
C H
H
C
H
Sucrose, NBS
6
42.10
6.48
42.07
6.39
99.93
98.61
Sucrose, ACS
6
42.09
6.48
42.02
6.39
99.83
98.61
1—Naphthol, ACS
3
83.31
5.59
82.72
5.86
99.29
104.83
Urea
3
19.99
6.71
19.38
6.66
96.95
99.25
Calcium carbonate
3
11.97
———
12.04
———
100.58
———
Graphite*
3
83.28
———
84.01
———
100.88
———
Since the graphite is not pure, the graphite, for this study, was ignited in air at 1,150 C to determine the
percent of ash impurities.
17
-------
METHODS OF SOLID WASTE TESTING
TABLE 2
STANDARD DEVIATION OF THE CARBON AND HYDROGEN
DETERMINATION ON SUCROSE AND SOLID WASTES
Type of
sample
Number of
samples
Standard
Carbon observations
deviations
Hydrogen
observations
Duplicates
Triplicates
Duplicates
Tnplicates
Sucrose, NBS
2
———
0.17
———
0.15
Sucrose, ACS
2
———
0.15
———
0.19
Compost
26
—--
0.29
--—
0.10
Compost
56
0.22
--—
0.14
-—-
Raw garbage
17
0.18
0.19
0.19
0.18
Residue:
Fines
16
0.04
0.06
0.04
0.03
Organics
8
0.21
0.23
0.22
0.18
Fly ash
9
0.04
0.08
0.06
0.04
A variance estimate can be calculated from the duplicate (or triplicate) set of observations for each sample.
The pooled variance is essentially an average of all such estimates for samples of a given type. It is assumed
that a single underlying variance exists for all samples of a given type. The pooled variance is then the best
estimate of this underlymg variance. The pooled standard deviation is the square root of the pooled variance
and is used to estimate the underlying standard deviation.
18
-------
Carbon and Hydrogen
ACKNOWLEDGMENTS
The author wishes to thank the Office of Solid Waste Management Programs for providmg samples
from incinerators, and Israel Cohen, Research Services Laboratory, Solid Waste Research Laboratory,
for preparing many of these samples.
The author gratefully acknowledges the contribution of personnel at the PHS-TVA Compost
Plant, Johnson City, Tennessee, who collected and prepared most of the compost samples used
in developing this method.
BIBLIOGRAPHY
1. American Public Works Association. Test for Hydrogen and Carbon. In: Municipal refuse
disposal. 2d. ed. Chicago, Public Administration Service, 1966. p. 398-399.
2. American Society for Testing Materials, Committee D-5, Coal and Coke. Sampling of Coal and
Coke, D-271-58. In: 1958 Book of ASTM standards, part 8. Philadelphia, American Society
for Testing Matenals, 1958. p. 1016-1020.
3. Furman, N. H., ed. Carbon. In: Scott’s standard methods of chemical analysis. 5th ed. V. 1.
Princeton, D. Van Nostrand Co., 1925. p. 2 18-228.
4. Horwitz, N., ed. Carbon and Hydrogen, 38.005-38.000. In. Official methods of analysis of
the Association of Official Agncultural Chemists. 10th ed. Washington, D. C., Association
of Official Agricultural Chemists, 1965. p. 741-743.
5. Steyermark, A. Microdetermination of Carbon and Hydrogen. In Quantitative organic
microanalysis. 2d. ed. New York, Academic Press, 1961. p. 221-275.
6. Roga, B., and L. Wnekowska. Carbon and Hydrogen. In Analysis of Solid Fuels, Chapter 3.
Katowice, Poland. Panstwowe Wydawnictwa Techniczne, 1952. p. 209-219. Available from
the U. S. Department of Commerce, National Technical Information Service, Springfield,
Virginia. TT61-31316.
19
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LABORATORY PROCEDURES TO DETERMINE
ThE NITROGEN CONTENT OF SOLID WASTES
W. H. Kaylor* and N. S. Ulmerf
INTRODUCTION 3
Discussion 3
References 3
KJELDAHL-WILFARTH.GUNNJNG-WJNKLER METHOD 3
DiscUSsiOn 3
Safety Precautions 4
Equipment 4
Reagents 4
Technique Evaluation s
Solid Waste Sample Preparation 5
Sample Analysis 5
Calculations 7
Accuracy and Precision 7
References 8
COMPREHENSIVE NITROGEN METHOD 9
Discussion 9
Safety Precautions 9
Equipment 9
Reagents 9
Technique Evaluation 10
Solid Waste Sample Preparation 10
Sample Analysis 10
Mr. Kaylor is nos i rving with the Pollution Source Control Program, Office of Water
Programs, Cincinnati.
f Research Chemist, Solid Waste Research Laboratory, National Environmental Research
Center, Cincinnati.
-------
METHODS OF SOLID WASTE TESTING
Calculations . 12
Accuracy and Precision . 12
References 13
AUTOMATED DUMAS METHOD 14
14
Safety Precautions 14
Equipment 14
Requirements 14
Assembly and Maintenance 16
Reagents 17
Requirements 17
Preparation, Maintenance and Storage 17
Technique Evaluation 17
Solid Waste Sample Preparation 17
Sample Analysis 17
Calculations 18
Accuracy and Precision 22
References 23
24
Bnef History of the Kjeldahl Method 24
Reference 25
2
-------
Nitrogen
INTRODUCTION
Discussion
Nitrogen becomes a significant solid waste parameter* when it is evaluated in conjunction with two
other parameters — carbon and hydrogen. The change in the C/N ratio of compost can be used to
determine (a) the degree and rate of biological decomposition of organic matter in the compost
(1,2) and (b) the suitability of the final product for use in agncultural soils (3). A knowledge of
the nitrogen, carbon, and hydrogen contents of an incinerator’s load and combustion products
can also enable the engineer and scientist to (a) calculate the theoretical air requirements and
combustion products, (b) formulate appropriate material and energy balance equations, and
(c) evaluate and control effectively the efficiency of an incinerator system (4).
To ensure the precise and accurate determination of the nitrogen content of solid wastes,
investigations of the applicability of existing analytical procedures were conducted in our laboratory.
Analyses of solid waste samples containing up to 8 percent nitrogen have demonstrated that the
Kjeldahl-Wilfarth-Gunning-Wmkler method, the comprehensive nitrogen method, and the automated
Dumas method (as employed in the Coleman Nitrogen Analyzer) may be used in the characterization
of solid wastes (5). Detailed descriptions of these three methods are presented in the following
sections.
References
1. University of California. Reclamation of municipal refuse by composting. Sanitary Engineering
Research Projects Techmcal Bulletin No. 9. Berkeley, June 1953. p. 48-5 8, 70, 78. (Senes 37).
2. Golueke, C. G., B. J. Card, and P. H. McGauhey. A critical evaluation of inoculums in com-
posting. Applied Microbiology, 1(2): 46. Jan. 1954.
3. University of California, op. cit., p. 65, 70.
4. Kaiser, E. R. Combustion and heat calculations for incinerators. Department of Chemical
Engineering Technical Report 1083-2. New York City, New York University, School of
Engineering and Science, Research Division, Dec. 1963. 23 p.
5. Ulmer, N. S., and W. H. Kaylor. An evaluation of the applicability of three methods for the
determination of nitrogen in solid wastes. Cincinnati, Solid Waste Research Laboratory
departmental report, 1971.
KJELDAHL-WILFARTH GUNNINGWINKLER METHOD
Discussion
The Kjeldahl-Wilfarth-Gunning-Wmkler methodt (1—5) may be employed to characterize solid
waste samples if their nitrogen content is primarily organic and/or ammoniacal. Since the method
will not recover nitrate nitrogen quantitatively, its applicability is presently limited to the analysis
of municipal refuse, unfortified compost, incmerator residue, and other samples with little, if any,
nitrate content. (Either the comprehensive nitrogen method or the automated Dumas method
should be employed to determine the total nitrogen content of samples containing significant
quantities of nitrate, e. g. fortified compost, or samples of unknown nitrate concentration.)
aIn this paper, the term parameter denotes a variable or characteristic of interest.
f See Appendix for a bnef history of the Kjeldahl Method
3
-------
METHODS OF SOLID WASTE TESTING
In the Kjeldahl-Wilfarth-Gunning-Winkler method, a sample is first digested with sulfuric acid.
To increase the speed of the reaction and ensure the digestion of substances whose decomposition
temperatures are above the boiling point of sulfuric acid, mercuric oxide and potassium sulfate
are added. After the digestion has been completed, the mixture is then treated with alkaline
sodium thiosulfate, which destroys the mercuro-ammonium complex and permits the distillation
of ammonia into 4.percent boric acid. A titration of the ammonium borate with standard sulfuric
acid then permits the calculation of the total organic and ammoniacal nitrogen content of the
sample.
Safety Precautions
1. Safety glasses should always be worn by the analyst while handling concentrated sulfuric acid
and digestion mixtures.
2. The analyst should exercise care m weighing, transferring, and disposing mercuric oxide, since
inhalation, ingestion, or contact with the compound may cause mercunal poisoning.
3. Exhaust hoods (or special traps) should be employed during sample digestion to minimize
the analyst’s exposure to the evolving fumes.
Equipment
1. Apparatus, Kjeldahl, digestion and distillation with (a) a 6-heater unit, either gas or electric,
capable of bringing 250 ml of water at 25 C to a rolling boil in 5 mm, (b) block tin condensers
enclosed in a copper coated cooling tank, and Cc) Pyrex delivery tubes (individual Pyrex
condensers and appropriate adapters may be substituted).
2. Balance, analytical, 0. 1-mg readability
3. Bottle, dropper, Pyrex, 250-mi
4. Bottles, reagent, Pyrex, 1-liter and 2-liter
5. Bulbs, connecting, cylindrical or spherical trap style, Pyrex, 5 to 6 cm in diameter
6. Buret,Pyrex, 25 ml, graduated in 0.l-ml divisions
7. Carrier designed to hold six 800-mi Kjeldahl digestion flasks(Fisher Scientific Co., Catalogue
#10-114)
8. Desiccator, large, either Pyrex or stainless steel cabinet type
9. Dispensers, delivery head with ‘1 24/40 joint, 25-mi and 40-ml (available from California
Laboratory Equipment Company, 1165 67th Street, Oakland, California 94608)
10. Flasks, Erlenmeyer, Pyrex, with 124/40 mouth, 500-ml
11. Flasks, Erlenmeyer, Pyrex, wide-mouth, 500-ml
12. Flasks, Kjeldahl, for digestion and distillation, Pyrex, 800-mi
13. Hood, exhaust and special stack to outside for venting fumes during the digestion of samples
14. Stoppers, rubber, size 7, for connecting bulbs to digestion flasks
15. Tubing, rubber, thin wall, for connectmg bulbs to condensers and cooling tank to cold water
supply and sink drain
Reagents
The chemicals in the following list should all be reagent grade and nitrogen-free.
1. Sucrose
2. Acetanilide
3. Disodium ethylenediaminetetraacetic acid (disodium EDTA)
4
-------
Nitrogen
4. Hengar granules, plain
5. Potassium nitrate
6. Mercunc oxide
7. Sulfuric acid, concentrated (95 to 98 percent)
8. Silicone antifoaming agent: General Electric No. 66 or Dow Corning Antifoam Q
9. Zinc metal, granulated
10. Alkaline thiosulfate solution: Dissolve 450 g sodium hydroxide in approximately 700 ml dis-
tilled water, cool, add 80 g sodium thiosulfate (Na 2 S 2 0 3 .51120), mix, and dilute with distilled
water to I liter.
11. Boric acid solution. Dissolve 40 g boric acid in distilled water and dilute with same to 1 liter.
12. Methyl purple solution (indicator): Dissolve 0.3 125 g methyl red and 0.2062 g methylene blue
in distilled water (or 0.1 percent ethyl alcohol) and dilute with same to 250 ml.
13. Sulfuric acid standard solution, 0.1 N: Dissolve approximately 3 ml of concentrated sulfuric
acid in 800 ml distilled water and dilute with same to I liter. Determine the normality of the
solution using a primary standard such as sodium borate.
Technique Evaluation
Before initiating the characterization of solid waste samples, the investigator should evaluate his
technique by analyzing compounds of known nitrogen content. Either 0.25 g acetaniide or 0.5 g
disodium EDTA may be used as a standard sample (see section on Accuracy and Precision).
Solid Waste Sample Preparation
A solid waste sample must undergo physical preparation before its characterization is initiated in
the laboratory. First it must be dried to constant weight, preferably in a forced-air or mechanically
convected oven. A temperature of 70 to 75 C should be used to dry municipal refuse or compost;
incinerator residue may be dried at 100 to 105 C. The particle size of the dried sample should then
be reduced to 2 mm or less using a hammermill, pulverizer, and/or laboratory mill. To ensure sample
homogeneity, the ground components should be thoroughly mixed. Finally, since samples may
absorb moisture during the gnnding and mixing processes, they should be redried for 4 hr at the
previously specified temperature and then stored in a desiccator until the analyses are completed.
Sample Analysis
The nitrogen contents of standard and solid waste samples should be determined in triplicate
by the following laboratory procedure. A reagent blank containing 2 g sucrose, previously dried
at 105 C for 1 hi, should also be analyzed with each set of samples. In this manner any nitrogen
present in the reagents will be detected, thus enabling the analyst to make appropriate corrections
in the calculation of the nitrogen contents of the standard and solid waste samples.
Procedure Comments
1. Weigh out approximately 2 g sucrose and 1. Glassine paper, used to support a sample
transfer to a 800-mi Kjeldahl digestion flask during weighing and transferring, must not
labelled “Blank.” be added to the digestion flask.
5
-------
METHODS OF SOLID WASTE TESTING
2. Accurately weigh out three 1.0— to 2.5—g
prepared solid waste samples and/or three
standard samples (quantities previously
specified) and transfer each to a labelled,
800-mi Kjeldahl digestion flask.
3. To each flask add I 6 g potassium sulfate,
0.7 g mercuric oxide, 25 ml concentrated
sulfuric acid, and a few plain Hengar
granules.
4. To the blank flask add three drops of sili-
cone antifoaming agent.
5. Place the flasks on the heating apparatus
in an inclined position.
6. Gently heat the contents of each flask
until frothing ceases; boil briskly until
the solution clears and for at least 30 mm
thereafter. (Samples containing organic
material will require a 2- to 3-hr digestion.)
7. While the digested samples are cooling, align
a 500-mi Erlenmeyer flask containing 50 ml
boric acid under each condenser to be em-
ployed during the distillation of ammonia
from the samples.
8. To each cooled sample, add 200 ml dis-
tilled water and mix. Then add 0.5 g zinc
and mix again.
9. Pour 75 ml alkaline thiosulfate solution
down the side of each flask and quickly
attach a trap-condenser apparatus. Mix the
contents of each flask and distill until
the total volume of the contents of the
receiving flask is 200 ml (50 ml boric acid
solution plus 150 ml distillate).
10. Add 4 drops of the methyl purple solution
to each Erlenmeyer flask and titrate the
contents with the standard 0.1 N sulfuric
acid solution to a light violet color. Record
the ml of sulfuric acid employed for each
sample.
11. Calculate the percent nitrogen in each
standard or solid waste sample as de-
scribed in the following section.
2. An analytical balance must be employed.
3. A larger volume of acid will be required
if the sample weight exceeds that recom-
mended.
5. The carrier may be used to support the
flasks dunng transfer.
6. The heaters should be previously adjusted
to bring 250 ml water at 25 C to a rolling
boil in 5 mm. Test heaters after preheating
(10 mm if gas, 30 mm if electric). Use
3 or 4 boiling chips to prevent superheating
of the water.
7. The tip of the condenser delivery tube
must be beneath the surface of the boric
acid solution to prevent the escape of
ammonia.
9. a) CAUTION: The heaters should be turned
on before connecting each flask to a
trap-condenser apparatus. This will mini-
mize the danger of liquid sucking back
through the condenser.
b) Immediately after mixing, lower the
Erlenmeyer flask containing the boric acid
solution so that the condenser delivery
tube will drain and the pressure in the
distillation flask will equalize.
10. The color of the solution will change from
green to light gray and finally to light
violet.
6
-------
Nitrogen
Calculations
The percent nitrogen in the samples should be calculated using the following formula:
%Nitrogen = ( A—B) (N)(14) (100 )
where
A = ml 0.1 N sulfuric acid employed in the titration of the standard or solid waste sample
B = ml 0.1 N sulfuric acid employed ‘in the titration of the blank
N = normality of the standard sulfunc acid solution
C = mg of standard or solid waste sample employed
Example:
( 12.61—0.30) (0.1040) (14) (100 )
%Nitrogen = 1016 = 1.76
Accuracy and Precision
Analyses of acetanilide or disodium EDTA samples containing 50 mg or less of nitrogen always
yielded 98.5 percent or more nitrogen recovery. If lower recovenes are observed, the analyst’s
technique should be suspected.
The reproducibility of this method has been determined by calculating the standard deviation
of replicate determinations of the nitrogen contents of various standard and solid waste samples.
The data are presented in Table 1.
7
-------
METHODS OF SOLID WASTE TESTING
TABLE 1
THE PRECISION OF THE METHOD
Number of
Observed
Type of sample
and source
Lab No
replicate
determinations
mean
percent
Standard
deviation
per sample
nitrogen
Standards
Acetanilide
6
10.20
003
Disodium EDTA
II
7.41
0.07
Solid wastes *
Municipal refuse
Cincinnati
It
12
1.07
0.05
Johnson City
6
6
1.01
0.05
New York
RSL208
8
2.36
0.02
Compost
Johnson City
W2IB-DO
4
0.52
0.02
WI6B-D7
WIIB-D14
W6B—D22
WIB—028
W3OA-D35
W25A-D42
X-D70
X—FP
W25D-SL-DL
4
4
4
4
4
4
4
8
6
0.83
0.70
0.78
0.72
1.02
0.83
0.71
0.96
0.88
005
0.03
0.03
0.00
0.04
0.03
003
0.12
0.02
Incinerator residue
Cincinnati
RB—I
RB-2
8
8
0.26
0.34
0.02
0.02
New York
RSL—205
RSL-205—80M
6
3
0.75
0.87
0.04
001
GIass and metals were removed from samples before analyses were initiated
f The sample consisted primarily of dirt, leaves, and wood.
The sample consisted primarily of food wastes.
References
1. American Public Works Association. Municipal refuse disposal. Chicago, Public Administration
Services, 1966. p. 390—391.
2. Gunning, J. W. Zietschrift für analytische Chemie, 28:188, 1889.
3. Horwitz, W., ed. Official methods of analysis of the Association of Agricultural Chemists.
10th ed. Section 2.044. Washington, D. C., Association of Agricultural Chemists, 1965, p. 16,
4. Wilfarth, H. Chemisches Zentrablatt, 16:17,113,1885.
5. Winkler, L. W. Zeitschrift fur angewandte Chemie, 26:23 1,1913.
8
-------
Nitrogen
THE COMPREHENSIVE NITROGEN METHOD
Discussion
In 1967 the comprehensive nitrogen method (CNM), a modification of the Kjeldahl method, was
proposed by Gehrke et a!. (1) for the determination of the nitrogen content of fertilizers, especially
those containing high chloride-nitrate ratios. Subsequent investigations in our laboratory have demon-
strated the applicability of the method in determining the total nitrogen (ammoniacal, organic,
and nitrate) contents of municipal refuse, compost, and incinerator residue (2).
A sample of solid waste is first heated with metallic chromium in an acid medium to reduce the
nitrates. The mixture is then digested with sulfuric acid. To increase the speed of the reaction and
ensure the digestion of substances whose decomposition temperature is above the boiling point
of sulfuric acid, mercuric oxide and potassium sulfate are added. After the digestion is completed,
the mixture is treated with alkaline sodium thiosulfate, which destroys the mercuro-ammonium
complex and permits the distillation of ammonia into 4-percent boric acid. A titration of the
ammonium borate with standard sulfuric acid then enables the analyst to calculate the total (ammoni-
acal, organic, and nitrate) nitrogen content of the sample.
Safety Precautions
1. Safety glasses should always be worn by the analyst while handling concentrated sulfuric acid
and digestion mixtures.
2. The analyst should exercise care in weighing, transferring, and disposing of mercuric oxide
since inhalation, ingestion, or contact with the compound may cause mercurial poisoning.
3. Exhaust hoods or special traps should be employed during sample digestion to minimize
the analyst’s exposure to the evolving fumes.
Equipment
The equipment required for determining nitrogen in solid wastes by the comprehensive nitrogen-
method is identical to that described in the preceding section on the Kjeldahl-Wilfarth-Gunning-
Winkler method.
Reagents
The chemicals in the following list should all be reagent grade and nitrogen-free:
1. Sucrose
2. Acetanilide
3. Disodium ethylenediamthetetraacetic acid (disodium EDTA)
4. Potassium nitrate
5. Chromium metal, powder, 100 mesh
6. Hydrochloric acid, concentrated
7. Potassium sulfate
8. Mercuric oxide
9. Norton Alundum, 14 x
9
-------
METHODS OF SOLID WASTE TESTING
10. Sulfuric acid, concentrated (95-98 percent)
11. Silicone antifoaming agent, General Electric No. 66 or Dow Corning Antifoam Q
12. Zinc metal, granulated
13. Alkaline thiosulfate solution: Dissolve 450 g sodium hydroxide in approximately 700 ml distilled
water, cool, add 32 g sodium thiosulfate (Na 2 S 2 03.5112 0), and dilute with distilled water to I
liter. (The concentration of sodium thiosulfate has been decreased from 160 g/liter to 32
g/liter to permit the addition of a larger volume of the solution, or quantity of alkali, without
increasing the quantity of thiosulfate used to precipitate the mercury.)
14. Boric acid solution: Dissolve 40 g of boric acid in distilled water and dilute with same to 1
liter.
15. Methyl purple solution (indicator): Dissolve 0.3125 g methyl red and 0.2062 g methylene
blue in distilled water (or 0.1 percent ethyl alcohol) and dilute with same to 250 ml.
16. Sulfuric acid standard solution, 0.1 N: Dissolve approximately 3 ml concentrated sulfunc
acid in 800 ml distilled water and dilute with same to 1 liter. Determine the normality of
the solution, using a primary standard such as sodium borate.
Technique Evaluation
Before beginning the characterization of solid waste samples, the investigator should evaluate his
technique by employing the procedure in the analyses of compounds of known nitrogen content. A
small quantity of an individual substance such as 0.25 g acetaniide, 0.5 g disodium EDTA, or 0.25
g potassium nitrate may be employed as a standard, but a mixture of either the aforementioned
quantities of acetaniide and potassium nitrate or disodium EDTA and potassium nitrate is preferable.
(See the section on accuracy and precision, p. 11.)
Solid Waste Sample Preparation
A solid waste sample should be prepared as described previously in the Kjeldahl-Wilfarth-Gunning-
Winkler method
Sample Analysis
The nitrogen contents of standard and solid waste samples should be determined in triplicate by
the following laboratory procedure. A reagent blank, containing 2 g sucrose previously dried at
105 C for 1 hr, should also be analyzed with each set of samples. In this manner, any nitrogen
present in the reagents will be detected, thus enabling the analyst to make appropriate corrections
in the calculation of the nitrogen contents of the standard and solid waste samples.
Procedure Comments
1. Weigh out approximately 2 g sucrose and 1. Glassine paper, employed to support a
transfer to a 800-mI Kjeldahl flask, labelled sample dunng weighing and transferring,
“Blank.” must not be added to the digestion flask.
2. Accurately weigh out three 0.75- to 1.0-g, 2. An analytical balance must be employed.
prepared solid waste samples and/or three
standard samples (quantities previously
10
-------
Nitrogen
specified) and transfer each to a labelled
800-ml Kjeldahl digestion flask.
3. To each flask add 1.2 g chromium and
35 ml distilled water. Let stand 10 mm
with occasional swirling.
4. Then add 7 ml concentrated hydrochloric
acid to each flask. Let stand until a
visible reaction occurs.
5. Transfer each flask to a burner and heat its
contents to a rolling boil (maximum heating
period is 5 mm). Remove from the burner
and cool.
6. Add 22 g potassium sulfate, 1.0 g mercuric
oxide, and 1.5 g alundum to each sample.
7. Transfer the flasks to the hood and add
25 ml concentrated sulfunc acid to each.
8. Add 3 drops of the antifoaming reagent
to the blank and any other sample con-
taining considerable organic material.
9. Place each flask on a preheated burner
adjusted to give a 5- to 7.5-mm boil test,
and digest the sample for I to 1 1/2 hr
with occasional gentle swirling.
10. While the digested samples are cooling,
align a 500-ml Erlenmeyer flask containing
50 ml boric acid under each condenser to
be employed during the distillation of
ammonia from the samples.
11. To each cooled sample, add 200 ml distilled
water and mix. Then add 0.5 g zinc and
mix again.
12. Pour 125 ml alkaline thiosulfate solution
down the side of the flask and quickly
attach a trap-condenser apparatus. Mix
3. Swirling will ensure solution of all the
nitrate present.
4. a) Add a few drops of antifoaming reagent
if foaming is anticipated.
b) Allow 1 to 5 mm for reaction.
5. a) The carner should be used to transport
the flasks.
b) The burners should be previously ad-
justed to bring 250 ml water at 25 C to a
rolling boil in 5 to 7.5 mm. Test heaters
after preheating (10 mm if gas, or 30 mm
if electnc). Use 3 or 4 boiling chips to pre-
vent superheating of the water.
7. Additional sulfuric acid will be required
if the quantity of sample exceeds that
recommended.
9. a) See the burner adjustment descnbed in
comment 5 b.
b) The use of a preheated burner mini-
mires foaming at this stage.
c) It should take 15 to 20 mm for the
copious white fumes to clear out of the
bulb of the flask. Most samples should then
be digested for an additional 30 mm. If
organic material containing refractory nitro-
gen is present, digest for a total of 60 mm
after the white fumes have evolved from
the flask.
10. The tip of the condenser delivery tube
must be beneath the surface of the boric
acid solution to prevent the escape of
ammonia.
12. a) The heaters should be turned on before
connecting each flask to a trap-con-
denser apparatus. This will minimize the
11
-------
METHODS OF SOLID WASTE TESTING
the contents of each flask and distill until
the total volume of the contents of the
receiving flask is 200 ml (50 ml boric acid
plus 150 ml distillate).
13. Add 4 drops of methyl purple to each
Erlenmeyer flask and titrate the contents
with the standard 0.1 N sulfuric acid
solution to a light violet color. Record
the milliliters of sulfuric acid employed
for each sample.
14. Calculate the percent nitrogen in each
standard or solid waste sample as described
in the following section.
danger of liquid sucking back through the
condenser.
b) Immediately after mixing, lower the
Erlenmeyer flask containing the boric acid
solution so that the condenser delivery tube
will drain and the pressure in the distillation
flask will equalize.
13. The color of the solution will change from
green to light gray, and finally to light
violet.
Calculations
The percent nitrogen in the samples should be calculated using the following formula:
where
%Nitrogen
_ (A-B) (N) (14) (100 )
C
A = ml 0.1 N sulfuric acid employed in the titration of the standard or solid waste sample
B = ml 0.1 N sulfuric acid employed in the titration of the blank
N = normality of the standard sulfuric acid solution
C = mg of standard or solid waste sample employed
Example.
% Nitrogen
— (12.61—0.30) (0.1040) (14) (100 )
1016
= 1.76
Accuracy and Precision
Analyses of acetanilide, disodium EDTA, and potassium nitrate samples containing less than 60
mg nitrogen always yielded 98.2 percent or more nitrogen recovery. If lower recoveries are observed,
the analyst’s technique should be suspected.
The reproducibility of the method has been determined by calculating the standard deviation of
replicate determinations of the nitrogen contents of various standard and solid waste samples. The
data are presented in Table 1.
12
-------
Nitrogen
TABLE 1
PRECISION OF THE METHOD
Number of
Observed
Type of sample
and source
Lab No.
replicate
determinations
mean
percent
Standard
deviation
per sample
nitrogen
Standards:
Acetamlide
6
10.27
0.04
Disodium EDTA
11
7.39
0.09
Potassium nitrate
11
13.64
0.09
Solid wastes: *
Municipal refuse
Cincinnati
Sf
8
0.32
0.01
Johnson City
6
6
0.94
0.04
New York
RSL—208
10
2.37
0.04
Memphis
RSL-294
8
0.87
0.04
Ogden
RSL-12
6
0.53
0.04
Compost
Johnson City
W2SD-SL-Dl
6
0.93
0.01
St. Petersburg
SPF
SPF-Fort.
6
6
1.03
8.02
0.05
0.14
Jamaica
JAM
6
0.52
0.04
Incinerator residue:
Cincinnati
RB-2
6
0.32
0.02
Memphis
RSL-292C
RSL—292 1
6
6
2.49
0.04
0.02
0.01
Ogden
RSL—17
5
0.08
0.01
New York
RSL-205
8
0.74
0.03
*Glass and metals were removed from samples before analyses were initiated.
tThis was a simulated sample.
*This sample consisted primarily of food wastes.
References
1. Gehrke, C. W., J. P. Ussary, C. H. Perrin, P. R. Rexroad, W. L. Spangler. A comprehensive
nitrogen method. Journal of the Association of Official Analytical Chemists, 50(4):965-975,
1967.
2. Ulmer, N. S. and W. H. Kaylor. An evaluation of the applicability of three methods for the
determination of nitrogen in solid wastes. Cincinnati, Solid Waste Research Laboratory de-
partmental report, 1971.
13
-------
METHODS OF SOLID WASTE TESTING
THE AUTOMATED DUMAS METHOD
Discussion
In 1960, G.M. Gustin described a simple automatic apparatus for the rapid determination of
nitrogen by the Dumas procedure (1). Shortly thereafter, the Coleman Instrument Corporation
manufactured an instrument capable of the automatic analysis of grain, fertilizers, soils, meat
products, feeds, biological materials, and other substances dissociable at temperatures under
1,100 C. Investigators in our laboratory have demonstrated that the Coleman Nitrogen Analyzer!!,
Model 29A, (see Figure 1) may also be employed successfully in the characterization of municipal
refuse, compost, and incinerator residue (2).
A 100-mg (or less), dry, homogeneous solid waste sample is first packed in a combustion tube.
After the tube has been inserted in the combustion train, high purity carbon dioxide is employed to
purge the system of entrapped air. The sample is then decomposed at 850 to 900 C in the presence
of oxidizing agents such as Cuprox (copper oxide) and Coboxide (cobalt oxide). The gaseous com-
bustion products (nitrogen, nitrogen oxides, methane, carbon monoxide, and carbon dioxide) are
swept along through the system by the flow of high-purity carbon dioxide. A platinum catalyst
mixed with the Cuprox packed in the combustion and post-heater tubes ensures the complete com-
bustion or oxidation of methane and carbon monoxide. The reduction of the nitrogen oxides to
gaseous nitrogen is effected by the Cupnn (reduced copper) in the post-heater tube. The gaseous
mixture flowing from the latter tube is then scrubbed thoroughly in a caustic solution to remove all
its carbon dioxide content. The remaining nitrogen is collected and measured in a SO,000-pl stainless
steel synnge linked to a digital counter. Since the purging, combusting, and sweeping actions are
entirely automatic, a sample analysis can be performed with ease in 1 5 mm.
Safety Precautions
1. The analyst should wear cotton gloves while handling the cold combustion or post-heater
tubes. (Observations in our laboratory have indicated that the tube durability, and hence pro-
cedural safety, may be decreased by repeated alternate exposures of the tube to perspiring
hands and furnace temperature.)
2. Dunng the analysis of substances that pyrolyze rapidly or detonate, a protective shield should
be attached to the side of the instrument and positioned in front of the combustion tube. If a
shield is temporarily unavailable, safety glasses should be worn to protect the eyes.
3. The temperature of the combustion tube furnaces should always be maintained below 1020 C
to prevent the sintermg of the Cuprox tube packing and the possible explosion of the com-
bustion tube.
4. The analyst should always remove a hot combustion tube from the train by carefully grasping
the cooler ends of the tube. NEVER grasp the hot center of the tube.
5. The analyst should wear an asbestos glove when inserting his hand into the muffle furnace to
mix or remove the Cuprox — platinum catalyst reagent being regenerated at 800 C.
Equipment
Requirements
1. Analyzer, Nitrogen II, Coleman Model 29A, complete with nitrometer (Coleman #29-3 12);
three Vycor combustion tubes (Coleman #29-3 28), Vycor post-heater tube (Coleman #29-337);
14
-------
- ii, ..‘
Figure 1. Coleman Nitrogen Analyzer II, Model 29A. (Courtesy of Coleman
Instruments Division of the Perkin-Elmer Corporation.)
Nitrogen
4 1r 00 E
E
*1
15
-------
METHODS OF SOLID WASTE TESTING
special scale thermometer (Coleman #29-340); 1 package of 100 disposable aluminum com-
bustion boats (Coleman #29-412); four Neoprene compression washers (Coleman #9584-V);
two combustion tube plugs (Coleman #9595V); copper gas-supply tubing, 60 in. long, with
compression fitting and bushing at one end to fit regulator outlet valve (Coleman #3825V);
two spring-loaded, joint-type pinch clamps (for mtrometer connections); a three-wire cord and
three-blade plug (for operation on 11 5 volts, 50/60 cycle, 750 watts); Operating Directions
D360-C (Coleman #29-904); and reagents 4, 5, 6, 8, and 10, listed in the next section
2. Balance, analytical, readability 0.000l-g
3. Barometer, accurate, readability 0.5-mm Hg
4 Desiccator, large, either Pyrex or stainless steel cabinet type
5 Dishes, evaporating, porcelain (These are used to support the Cuprox platinum catalyst reagent
while it is being regenerated. The size of the dishes will therefore depend on the inner dimensions
of the muffle furnace.)
6. Furnace, muffle, capable of maintaining 800 C for a 2-hr penod
7. Glass wool
8 Gloves, asbestos, 14 in. long
9 Gloves, cotton, large (These are available from either Davis Gloves, Springfield, Ohio, or Wash
Rite, Inc., 1410 Cornell, Indianapolis, Indiana.)
10. Mats, asbestos, 1/8 by 12 by 12 in., for supporting the hot evaporating dishes after they are
removed from the furnace.
11. Rack, aluminum (or cadmium-plated) for holding combustion tubes
12. Regulator, two stage, Coleman type for Coleman Nitrogen Analyzer, with CGA connection
#320 (Model 8C, Mathieson Scientific Company, #26285-05)
13. Rod, stirring, teflon-coated, 15 in. long, 1/4 in. diameter
14. Screwdriver, slot drive, blade 1/4 in. wide, 6 to 10 in. long
15. Shield, protective, for use in combustion of substances that pyrolyze rapidly or detonate
(Coleman #33-450)
16. Sieve, brass, 8-in., US #40
17. Sieve cover and receiver, brass, 8-in.
18. Spatula, stainless steel, rounded and pointed blades, micro
19. Support, gas cylinder
20. Tongs, crucible type, nickel plated steel, 18 in. long
21. Tweezers, 4-1/2 in. long, for grasping boats
22. Wrench, open end, 1 1/8 in., for installing pressure regulator on gas tank
23. Wrenches, 3/8-, 7/16-, and 1/2-in., for connecting copper tubing to output of gas regulator
and input fitting of instrument
Assembly and maintenance
The Coleman Nitrogen Analyzer and its accessories should be assembled, evaluated, and maintained
as described in the operating directions for the instrument.
16
-------
Nitrogen
Reagents
Requirements.
1. Acetanilide, ACS reagent grade, crystalline
2. Carbon dioxide, Coleman instrument grade, 99.99 percent pure (cylinder size 1)
3. Causticon, potassium hydroxide solution, 1 pint (Coleman #29-110)
4 Coboxide, cobalt oxide, 30-g bottle (Coleman #29-170)
5. Cupnn, metallic copper, 1-lb bottle (Coleman #29-120)
6. Cuprox, copper oxide, fines, 1-lb bottle (Coleman #29-140)
7. Cuprox — platinum catalyst reagent, 5-lb bottle (Coleman #29-165)
8. Disodium ethylenediaminetetraacetic acid, ACS reagent grade, powder (Minimum assay: 99
percent)
9. Mercury, reagent, redistilled, 50 ml
10. Potassium nitrate, ACS, reagent grade
11. Salicylic acid, ACS, reagent grade, nitrogen free
12. Silica gel, indicating, mesh size 6-16, 5 lb unit
13. Silver vandanate, 40-g bottle (Coleman #33-13 0)
Preparation, maintenance, and storage.
All fresh reagents should be employed as received. The replacement, regeneration, and/or main-
tenance of the reagents should be accomplished as outlined in the operating directions for the instru-
ment. All capped (but unsealed) reagent bottles containing solids should be stored in a desiccator to
minimize the sorption of moisture.
Technique Evaluation
Before initiating the analysis of solid waste samples, the investigator should evaluate his technique
by employing the procedure in the analyses of compounds of known nitrogen content. Either 0.05
g acetaniide or 0.10 g disodium EDTA should be used routinely as a standard sample. To evaluate
the recovery of nitrate nitrogen, the analyst should use a 0.1 -g potassium nitrate sample to which an
equal weight (i.e., 0.1 g) of nitrogen-free salicylic acid has been added. (See section on accuracy and
precision.)
Solid Waste Sample Preparation
The procedure for the preparation of a solid waste sample is the same as that previously outlined
in the Kjeldahl-Wilfarth-Gunning-Winkler method, except that the sample’s particle size should be
reduced to 0.5 mm or less before the mixing is begun.
Sample Analysis
The analyst should employ the following instructions in conjunction with the sample analysis
procedures outlined in the operating directions for the instrument:
17
-------
METHODS OF SOLID WASTE TESTING
1. All determinations (including blanks) should be performed in triplicate.
2. The sample should contain 100 mg (or less) organic matter and 40 mg (or less) nitrogen.
3. The combustion tube packing, employed in all analyses, should be modified by adding Cuprox
fines 1/4 in. below, 1/4 in. above, and completely around the aluminum boat. This packing, if
carefully performed, will prevent the fusion of the boat with the Vycor combustion tube.
4. The instrument should be operated on the normal 1 2-mm. cycle.
5. The furnace controls should be adjusted so that the post-heater tube furnace is maintained at
700 C and the lower and upper combustion tube furnaces attain 850 to 900 C during the final
portion of the combustion penod. (Occasionally, as in the analysis of the potassium nitrate-
salicylic acid standard mixture, the analyst may observe a sintenng of the Cuprox around the
sample in the combustion tube. Lower combustion furnace settings should then be employed
to minimize hazards and increase tube life.)
6. Daily furnace warm-up periods may be avoided by leaving the instrument in the standby
position overnight. (The nitrometer must be disconnected as usual.)
Calculations
The percent nitrogen in a standard or solid waste sample should be determined as follows.
1. Record V 0 , the observed volume of nitrogen (pi).
V 0 =R 2 -R 1
where
R 1 = the initial counter reading
R 2 the final counter reading
2. Determine V , the corrected nitrogen volume (p1)
V = V 0 - (Vb ÷ V 1 )
where
Vb = volume of blank (j 1)
Vt = volume correction for temperature (p1)
=Cf(t2 —t 1 )
where
C correction factor per degree Kelvin (Table 1)
t 1 = the initial syringe temperature in degrees Kelvin
t 2 = the final syringe temperature in degrees Kelvin
3. Determine P ’ the corrected barometnc pressure (mm Hg)
PC = P 0 - (Pb + P )
where
P 0 = the observed barometric pressure (mm Hg)
= the pressure correction for temperature (Table 2)
P , = the pressure correction for the vapor pressure of potassium hydroxide (Table 3)
18
-------
Nitrogen
TABLE 1
CORRECTION FACTOR (C 1 ) EMPLOYED IN THE CALCULATION OF THE VOLUME
CORRECTION FOR TEMPERATURE (V,)*t
Fmal counter
reading
(p1)
C 1 I:
(p1/K)
Final coun
reading
(pl)
ter
C 1 1
(p1/K)
0
12
24,000
92
1,000
15
25,000
95
2,000
19
26,000
98
3,000
22
27,000
102
4,000
26
28,000
105
5,000
29
29,000
109
6,000
32
30,000
112
7,000
35
31,000
115
8,000
39
32,000
119
9,000
42
33,000
122
10,000
45
34,000
127
11,000
48
35,000
129
12,000
52
36,000
132
13,000
55
37,000
135
14,000
59
38,000
139
15,000
62
39,000
142
16,000
65
40,000
145
17,000
69
41,000
148
18,000
72
42,000
152
19,000
76
43,000
155
20,000
79
44,000
159
21,000
82
45,000
162
22,000
85
46,000
165
23,000
89
47,000
169
D-360C Operating Directions
for the Coleman Model 29A Nitrogen
sBased on Coleman Instrument Corporation,
Analyzer II, Maywood, 111., 1966. p. 22.
tVt = Cf (t 2 — t 1 ) where t 1 and t 2 are the initial and final temperatures, expressed in degrees Kelvin, respectively.
Factor applicable only for measurements made with nitrometers having check valves.
19
-------
METHODS OF SOLID WASTE TESTING
TABLE 2
PRESSURE CORRECTION (Pb) FOR TEMPERATURE*
Room Temperature
(C)
Pb (m
m Hg)
p 0 t
= 700 to 749
P 0 = 750 to 780
10
1.2
1.3
15
1.8
1.9
20
2.3
2.5
21
2.4
2.6
22
2.5
2.7
23
2.7
2.9
24
2.8
3.0
25
2.9
3.1
26
3.0
3.2
27
3.1
3.3
28
3.3
3.5
29
3.4
3.6
30
3.5
3.7
31
3.6
3.8
32
3.7
3.9
33
3.9
4.1
34
4.0
4.2
35
4.1
4.3
*Based on Coleman Instrument Corporation, D-360C Operating Directions for the
Coleman Model 29A Nitrogen Analyzer II, Maywood, III. 1966, p. 22.
f P 0 = observed barometric pressure in mm Hg.
20
-------
Nitrogen
TABLE 3
PRESSURE CORRECTION (P ) FOR VAPOR PRESSURE
OF POTASSIUM HYDROXIDE*
Temperaturet Pv
(K) (mm Hg)
288 4.1
293 5.7
298 7.4
299 7.8
300 8.3
301 8.7
302 9.2
303 9.6
304 10.2
305 10.8
306 11.3
307 11.9
308 12.5
309 13.3
310 14.1
311 14.9
312 15.7
313 16.5
Based on Coleman Instrument Corporation, D-360C Operating Directions
for the Coleman Model 29A Nitrogen Analyzer II, Maywood, III., 1966, p. 22.
tFor practical purposes, the temperature of the potassium hydroxide is
the same as that of the syringe.
21
-------
METHODS OF SOLID WASTE TESTING
4. Determine percent nitrogen, using the following formula
P V
%N=- x— x 0.0449
where
T = t 2 , the final syringe temperature in degrees Kelvin
W = the sample weight in mg
Example:
If P 0 = 747.2 mm Hg at 24.9 C
W =39.7
R 1 (blank) = 5,219
R 2 (blank) = 5,270
R 1 (solid waste) = 5,270
R 2 (solid waste) = 5,620
t 1 = 303.1
t 2 (orT) = 303.3
Then Vb (blank) = 51
V 0 (solid waste) = 350
V (solid waste) 350 — [ 51 + (29) (303.3—303.1)]
= 293.2
= 747.2 — (2.9 + 9.6)
= 734.7
734.7 293.2
°/oN = 303.3 X X 0.0449
= 0.80
Accuracy and Precision
Analyses of acetanilide, disodium EDTA, or potassium nitrate samples containing less than 20 mg
nitrogen always yielded 99 percent or more nitrogen recovery. If lower recoveries are observed,
the analyst’s technique should be suspected.
The reproducibility of the method has been determined by calculating the standard deviation of
replicate determinations of various standards and solid waste samples. The data are presented in
Table 4.
22
-------
Nitrogen
TABLE 4
THE PRECISION OF THE METHOD
Type of
sample and
source
Lab No.
Number of
replicate
determinations
per sample
Observed
mean
percent
nitrogen
Standard
.
deviation
Standards.
Acetanilide
10
10.34
0.06
Disodium EDTA
14
7.56
0.02
Potassium nitrate
6
13.72
0.06
Solid wastes: *
Municipal refuse
Cincinnati
st
6
0.25
0.06
Johnson City
6
6
0.92
0.03
New York
RSL-2081
6
2.44
0.06
Memphis
RSL-294
6
0.72
0.07
Ogden
RSL-l2
10
0.49
0.06
Compost
Johnson City
Wi 1B-D14
W25D-SL-D1
6
12
0.78
0.82
0.08
0.06
St. Petersburg
SPF
SPF-Fort.
6
10
1.09
8.16
0.03
0.10
Jamaica
JAM
9
0.57
0.05
incinerator residue
Cincinnati
RB-2
5
0.28
0.03
Memphis
RSL-292C
RSL-292 1
6
6
2.55
0.03
0.06
0.01
Ogden
RSL-17
6
0.08
0.01
New York
RSL-205
3
0.84
0.00
*Glass and metals were removed from samples before analyses were initiated.
tThis was a simulated sample.
IThis sample consisted pnmarily of food wastes.
References
I. Gustin, G.M. A simple, rapid automatic micro-Dumas apparatus for nitrogen determination.
Microchemical Journal, 4:43-54, 1960.
2. Ulmer, N.S., and W.H. Kaylor. An evaluation of the applicability of three methods for the
determination of nitrogen in solid wastes. Cincinnati, Solid Waste Research Laboratory depart-
mental report, 1971.
23
-------
METHODS OF SOLID WASTE TESTING
APPENDIX
A Brief History of the Kjeldahl Method
The historical background and evolution of the Kjeldahl method have been thoroughly reviewed
by R.B. Bradstreet (1). The following brief resume will hopefully afford the analyst a greater appre-
ciation of the ingenuity and resourcefulness of the many scientists who contributed to the develop-
ment of the method.
In 1883 Johann Kjeldahl, a Danish chemist associated with the Carlsberg Laboratory in Copen-
hagen, published a new method for the determination of nitrogen. The procedure, developed
primarily to facilitate a study of the protein changes in grain, consisted of the following steps:
(a) Heating a sample for 2 hr in concentrated sulfuric acid to which fuming sulfuric acid and
phosphoric anhydnde had been added, (b) subsequent oxidation with powdered permanganate,
(c) dilution and transfer of the mixture to a distillation flask, (d) addition of alkali and then zinc,
(e) distillation of the ammonia into standard acid, (f) addition of potassium iodide and iodate to
the distillate, and finally (g) a titration of the liberated iodine with standard thiosulfate.
Shortly after the publication of this method, Kjeldahl’s contemporanes proposed numerous
modifications and improvements. In 1885, Wilfarth reported that the speed of the digestion could
be accelerated by the addition of a copper salt. He also described the catalytic effects of the oxides
of copper, iron, mercury, bismuth, manganese, zinc, and lead. Since mercuric oxide, the most
effective catalyst, tended to form a complex with ammonia and thus lower nitrogen recovery, its
use was discontinued until chemists discovered that the complex could be destroyed by the addition
of alkaline sulfide, thiosulfate, monosodium phosphate, potassium xanthanate, or potassium arsenate.
In the years following Wilfarth’s significant contribution, investigators have elucidated and established
many other substances capable of accelerating specific digestion reactions. The addition of a catalyst
has therefore become a routine and universally accepted step in the Kjeldahl procedure.
In 1889, J.W. Gunning reported that the addition of potassium sulfate to the digestion mixture
would raise its boiling point, increase the severity of the reaction, shorten the digestion period, and
hence permit the analyses of other types and sizes of samples. Although the applicability of many
other salts has been evaluated since Gunning’s observations were published, potassium sulfate is
still recommended, particularly for the analysis of refractory compounds. The lower acid-salt ratios
attainable with this salt have permitted digestion at higher temperatures without loss of nitrogen.
Although the suggestions of Wilfarth and Gunning greatly increased the scope of the Kjeldahl
method, research has since demonstrated that nitro, nitroso, azo, aminoazo, hydrazine, and other
compounds in which a nitrogen atom is linked to an oxygen atom (or atoms) or to a second nitrogen
atom must be reduced before their nitrogen contents can be recovered quantitatively. This reduction
has been accomplished in two general ways: (a) as a pretreatment of the sample with substances
such as powdered copper, zinc, chromium, titanous chloride, potassium iodide, and sodium hydro-
sulfite, and (b) by the addition of compounds such as sucrose, benzoic acid, phenol, and salicylic
acid directly to the acid and sample. The latter compounds supply a reducing effect during the
Kjeldahl digestion as they decompose to free carbon with subsequent reduction of the sulfuric acid
to sulfur dioxide.
Kjeldahl originally proposed that powdered potassium permanganate be added to the sample upon
completion of the acid-digestion. Although he believed this step necessary to ensure the complete
oxidation or conversion of any nitrogen not previously converted to ammonium sulfate by the
digestion, it was later discontinued when variations in nitrogen recovery were observed. During the
24
-------
Nitrogen
20th century, other oxidizing agents such as hydrogen peroxide, perchioric acid, and potassium
persulfate have occasionally been employed with more favorable results.
The procedure proposed by Kjeldahl for the recovery of ammonia and the fmal determination
of nitrogen has also been modified by numerous investigators. If the ammonia is distilled, a direct
heat distillation is usually employed; aeration and steam distillation techniques have been proposed,
however. Kjeldahl’s time-consuming titration has been replaced either by a back titration with a
known volume of standard acid or the direct titration of ammonium borate, as proposed by Winkler
in 1913. Other methods such as gravimetry, nesslerization, pH measurement, colorimetric reaction,
or neutralization of the digest followed by direct estimation of the ammonia have also been utilized
successfully in the analysis of samples.
References
1. Bradstreet, R.B. The Kjeldahl method for organic nitrogen. New York, Academic Press,
1965. 238 p.
25
-------
LABORATORY PROCEDURE FOR THE
GRAVIMETRIC DETERMINATION OF
CARBONATE CARBON IN SOLID WASTES
Donald L. Wilson*
DISCUSSION 2
APPAaA’FUS 2
Requirements 2
Preparation 6
Assembling the Train 6
Conditioning the Train 7
REAGENTS 7
Chemical Requirements 7
Preparation of Solutions 7
SAFETY PRECAUTIONS 8
SAMPLE PREPARATION 8
PROCEDURE 8
Blanks 8
Standard 8
Samples 8
CALCULATIONS 11
Standards 11
Samples 11
METhOD EVALUATION 12
ACKNOWLEDGMENTS 13
REFERENCES 13
*Research Chemist, Solid Waste Research Laboratory, National Environmental
Research Center, Cincinnati.
-------
METHODS OF SOLID WASTE TESTING
DISCUSSION
Inorganic carbon measurements on solid waste samples, in conjunction with total carbon measure-
ments, (1) serve as a criteria for evaluating incinerator efficiency to dispose of organic carbon
matter. High carbonate content in incinerator residue will cause hardness in leachate water.
The carbonate analysis is also necessary because the carbonate content affects the analysis of
other constituents and the analytical interpretation of other methods. For example, the precision
and accuracy of the ash-volatile determination (2) (loss-on-ignition or L.O.I. at 600 C) can vary
with the degree of decomposition of carbonates present (3). Our laboratory investigations showed
that during the calonfic determination, the degree of decomposition of calcium carbonate in incin-
erator residue samples depends upon the amount of combustion aid added to the sample (4). The
carbonate content of samples must be known in order to calculate their calorific values from ultimate
analyses. The oxygen content of samples is normally determined by subtracting from the volatile
portion of a sample the carbon, hydrogen, nitrogen, and sulfur contents. Such oxygen values may
be in great error because of not considering the oxygen combined with the carbonates and metal
oxides in the residue left from the volatile analysis (5).
An existing AOAC method (6) for determining carbonate carbon was found feasible for solid
waste samples. (7) Such samples with carbonate carbon contents from 0.05 to 8.00 percent have
been precisely and accurately analyzed.
Before the carbonate carbon content is determined, all solid waste samples must be dried, ground
to less than 2 mm, and thoroughly mixed. Employing between I to 5 g of sample in each determin-
ation produces data that are precise and accurate to a satisfactory degree.
Carbonate carbon is determined gravimetrically after (1) reacting a weighed, dry, uniform sample
with dilute hydrochloric acid inside a closed system, and (2) fixing the evolved gases in an absorption
train (Figure 1). The procedure (Figure 2) is designed to measure the total carbonate carbon in dry
solid waste samples.
APPARATUS
Requirements
1. Absorption bulb, Nesbitt, Pyrex; two or more (Fisher #7-517)
2. Balance, analytical, 200-g capacity, 0.1-mg readability (Sartonus, 2400 Series)
3. Basket, test tube (Fisher # l4-966D)
4. Beaker, Griffin low form, 250-ml
5. Bottle, gas-washing, Pyrex, with fritted cylinder, coarse porosity, 250-ml capacity; two
6. Bottle, narrow mouth, standard taper, 2,000 ml
7. Burner, gas
8. Clamp, utility, vinylized jaws, three-prong grip; five
9. Condenser, Kimble Modern Liebig, 400-mm jacket (Fisher #7-704 C)
2
-------
Figure 1. General schematic of carbonate-carbon train.
I—
0
-------
METHODS OF SOLID WASTE TESTING
CARBONATE—CARBON TRAIN PURPOSE OF UNITS
ROOM AIR
F ASCARITE 11TH 1 REMOVES CARBON DIOXIDE
J ACTIVATED ALUMINA I
F SA PLE 11TH 50 ML 1 CONVERTS CARBONATE CARBON
LOF I 4 PARTS HCI AND HEAT I TO CARBON DIOXIDE
REACTION PRODUCTS
AND MOISTURE
1
ITRAP1 COLLECTS WATER BACIFLOW FROM
CONDENSER
ICONDENSER ] REMOVES MOST OF WATER
1
ICONC. SULFURIC ACIDI REMOVES REMAINING WATER AND
SULFUR DIOXIDE
I SILVER SULFATE IN 1 REMOVES ACIDIC OASES (OTHER THAN
ICONC. SULFURIC ACIOI CARBON DIOXIDE). HYDROCHLORIC ACID
VAPORS. AND HYOROGEN SULFIDE VAPORS
IT AP I COLLECTS ACID OVERFLOW
IMAONESIUI LWCHLORATE ] COLLECTS WATER
4
[ FLOIWETER ] MAINTAINS GAS FLOW
(200—410 cc. un.)
I INDICARB AND 1 ABSORBS CARBON DIOXIDE
IACTIVATED ALUMINAI
1
I INDICARB AND j ABSORBS CARBON DIOXIDE
( ACTIVATED ALUMINAJ
[ T P ] REMOVES PARTICuLATES
VACUUM SOURCE
Figure 2. General outline of carbonate-carbon train.
4
-------
Carbonate Carbon
10. Cylinder, graduated, 50-mi
11. Cylinder, graduated, 100-mi
12. Cylinder, graduated, 500-mi
13. Drying bulb, unique type (absorption bulb may be used)
14. Erlenmeyer flask, wide-mouth, 125-mi
15. Erlenmeyer flask, wide mouth, 250 ml; four or more
16. Flask, filtering, side-arm, 50-mi
17. Flask, filtering, side-arm, 250-mi
18. Funnel, separatory, 60-mi
19. Gloves, lint-free
20. Hot plate (Fisher #1 1-494 or equivalent)
21. Rod, glass, about 6 in. long
22. Rotameter or flowrator meter, size 02, sapphire float, hose connections, tn-float tube #02F-
1/8-16-5 (Fischer and Porter # I OA- 1017 or equivalent)
23. Ring, support, with clamp, 4-in. O.D.
24. Stopcock, hard rubber (Fisher #14-630) (optional)
25. Stopper, one-hole, size 0
26. Stopper, one-hole, size 5; two
27. Stopper, one-hole, size 6; three
28. Stopper, two-hole, size 6; two
29. Stopper, two-hole, size 9
30. Support stand, rectangular base, 24-in, rod; two
31. Support stand, rectangular base, 36-in, rod
32. Timer (Fisher #6-662)
33. Tube, glass, about i-in, diameter, about 6 in. long
34. Tubing, bubble, Argyle universal, plain, lumen, 3/16-in, bore (Aloe Scientific #AR-500)
35. Tubing, glass, 3/16-in, bore, about 2 ft
36. Tubing, rubber, red, thin-wall, 3/16-in, bore (Fisher #14-166)
37. Tubing, Tygon, 1/4-in. I.D., 3/8-in. O.D., 1/16-in, wall
38. Tubing, vacuum-pressure, 3/8-in. ID.
39. Vacuum source and regulator valve or clamp
40. Wife square, asbestos center, 5-in sq
41. Alkalimeter, Knoor, carbon dioxide apparatus (Fisher #1-198)
42. Flask, Erlenineyer form, with standard taper joint-24/40, Pyrex, 250-mi. (Fisher #lO-047C)
(a corequisite of item 4l)
1f items 41 and 42 are purchased, then items 9, 15, 18, 25, 29, 33, and two of item 27 are unnecessary.
5
-------
METhODS OF SOLID WASTE TESTING
Preparation
Assembling the train.
The components of the apparatus are assembled in the following sequence:
a Glass tube: Activated alumina is placed in the lower fourth and ascarite topped with activated
alumina in the upper part. A small layer of glass wool is inserted beneath the lower activated alumina
and above the upper activated alumina. The glass tube is closed at each end with a one-hole, size 6
stopper. About 1 In. of glass tubing protrudes from the hole in the stopper. The room air-flow into
the tube is through the bottom inlet. The glass tube is attached to a 36-in.-rod support stand with a
utility clamp.
b. Separatory funnel (60-mi, with one-hole, size 0 stopper) is attached to 36-in.-rod support
stand with utility clamp.
c. Erlenmeyer flask, 250-mi, with two-hole, size 9 stopper and glass tubing inserted through
stopper holes. Bend one piece of glass tubing upward at the end just enough to keep it from hitting
the bottom of the flask; rest the flask on wire square on ring support, which is attached to the 36-
in -rod support stand.
d. Set gas burner under Erienmeyer flask (item c).
e. Erlenmeyer flask, 125-mi, with two-hole, size 6 stopper and glass tubing inserted through
stopper. This item serves as a liquid trap. If an alkalimeter is employed, the flask is placed after
the condenser of the alkaiimeter.
f. Condenser, 400-mm jacket, with one-hole, size 5 stopper at exit end and glass tubing inserted
through stopper. It is slanted at about a 30° angle (with exit end at top) and attached to a 24-in.-
rod support stand with utility clamp. Tygon tubing is attached to water source and drain, with water
entering lower part.
g. Alkalimeter (optional) replaces all items above except d and e.
h. Gas-washing bottle, containing 100 ml concentrated sulfuric acid.
i Stopcock, hard rubber (optional, helps prevent backflow of solutions).
j. Gas-washing bottle containing 100 ml silver sulfate solution.
k. Filtering flask, 50-mi, with one-hole, size S stopper and glass tubing inserted through stopper.
This item serves as a liquid trap.
I Drying bulb containing magnesium perchlorate; a small layer of glass wool is beneath and above
the magnesium perchiorate.
m.Rotameter with the sapphire float between the 4.6 and 6.8 levels to afford a flow of 200 to
410 cc per mm.
n. Two Nesbitt bulbs (for carbon dioxide absorption) assembled in series. These may be put in test
tube basket. A 1/2-in, layer of glass wool is placed in the bottom of the bulb. Indicarb is then added
to a point 3/4 in. from the shoulder of the bulb. The Indicarb is covered by a 1/4-in, layer of activated
alumina. Introduce sufficient glass wool to reach the neck area and cotton in hollow stopper. For
highly reactive materials or materials with high carbonate carbon content, more than two Nesbitt
bulbs may be necessary.
o. Filtering flask, 250-mi, with one-hole, size 6 stopper and glass tubing inserted through stopper.
This item serves as a particulate trap; it is connected to the vacuum source and regulator with vacuum-
pressure tubing.
6
-------
Carbonate Carbon
p. Argyle-bubble tubing, used between glass tube, funnel, 250-mi Erienmeyer flask, 125-mi
Erlenmeyer flask, condenser, gas-washing bottles, 50-mi filtering flask, drying bulb, and rotameter.
q. Rubber tubing used between rotameter, Nesbitt bulbs, and 250-mi filtering flask.
r. Kel-F # 90 stopcock grease is used for all ground glass connections.
Conditioning the train.
Unlike most trains, the carbonate-carbon train requires very little conditioning. If the train has
remained idle overnight, the analyst conditions the train by allowing room air to flow through the
system at 200 to 410 cc per mm for about 5 mm (see procedure).
REAGENTS
Chemical Requirements
The following chemicals are ACS reagent grade:
1. Activated alumina, indicatmg, 8-14 mesh (Fisher #A-545)
2. Ascarite (sodium hydroxide in asbestos) 8-20 mesh
3. Glass wool
4. Sulfuric acid, concentrated
5. Silver sulfate
6. Magnesium perchlorate
7. Indicarb, 6-10 mesh (Fisher #1-181) may be substituted by Ascarite
8. Stopcock grease, Kel-F #90 (Sargent #S-77346)
9. Hydrochloric acid, concentrated
10. Calcium carbonate
Preparation of Solutions
1. Silver sulfate solution: Dissolve about 20.0 g Ag 2 SO 4 m 100 ml (use graduate) of concentrated
H 2 SO 4 by putting both in a 250-ml beaker and heating on a hot plate. Use glass rod to break
up and mix Ag 2 SO 4 .
Note: Ag 2 SO 4 decomposes at 652 C. Allow solution to cool to near room temperature before
putting it into gas-washing bottle. Final volume should be about 100 ml.
2. Dilute hydrochloric acid solution (1:4). Using a 500-ml graduated cylinder, put 1,600 ml of
distilled water into a 2,000-ml bottle. Add 400 ml of concentrated HC1 and mix solutions by
shaking and inverting the bottle.
Note: Prepare new solutions whenever used solutions foam excessively or when analyses of
standard plus impurities indicate depletion of silver sulfate effectiveness.
7
-------
METHODS OF SOLID WASTE TESTING
SAFETY PRECAUTIONS
Follow general laboratory safety rules. This method has no pronounced safety hazards. Care must
be taken, however, when handling the concentrated acids.
SAMPLE PREPARATION
The details of sample preparation procedures that describe the drying and grinding techniques are
not discussed in this laboratory procedural report. In general, raw refuse, incinerator residue
(organics or combustibles), and compost are dried at 70 C to a constant weight and ground to a
particle size of less than 1 to 2 mm. Incinerator residue (fines or noncombustibles) and fly ash
samples are dried at 105 C to a constant weight and pulverized to pass through a #60 (0.25-cm)
sieve. Most of the metals, glass, and ceramics are removed before the samples are ground or pulverized.
All samples must be well mixed before aliquots are removed for analysis.
PROCEDURE
Blanks
Increase in weight of the absorption bulbs is due to 1) sample-acid reaction, 2) sample flask and/or
acid contamination, 3) atmospheric contamination during sample container’s connection to tram,
and 4) incomplete removal of carbon dioxide from the room air. The blank analyses determine the
effects of all the above factors, except sample-acid reaction, on the weights of absorption bulbs.
These blank analyses are conducted like the regular sample analyses, except that no sample is used.
The analyst is advised to perform triplicate tests and repeat the blank analyses with each new batch
of dilute hydrochlonc acid solution.
Standard
The analyst should periodically check the carbonate-carbon train by analyzing a standard (com-
monly calcium carbonate, previously dried at 105 C for 1 hr). The procedure for analyzing a standard
is the same as described for sample analyses. Analyze, however, only 0.2 to 1.0 g of calcium carbonate
smce this standard reacts quickly and easily with acid. A larger than 1 .0 sample of calcium carbonate
reacts too violently and causes gas to escape through the separatory funnel and into the room.
Samples
The following procedure applies to all solid waste materials as well as to blanks and standards
with the previously mentioned changes.
The analyst is advised to use 1.0- to 5 .0-g portions of samples for analysis. Sample weights up to
10 g may be used if nonuniformity of sample warrants. Since the density of raw refuse, compost,
and incinerator residue (organics or combustibles) samples is low, the analyst need weigh only 1.0-
to 3.0-g portions of such samples.
8
-------
Carbonate Carbon
Procedure
Comments
1. Transfer at least 1 to 5 g (or 1 to 3 g)
of a solid waste sample into each of two
250-mi Erlenmeyer flasks. Determine and
record the weight of each sample portion
to the nearest 0.0001 g.
2. With water going through the condenser,
and the stopcock of the separatory funnel
open, allow room air to flow through the
carbonate-carbon train at 200 to 410 cc
per mm. (If stopcock is employed between
gas-washing bottles, it must be open.)
3. Remove the absorption bulbs from the
train and close each bulb to the atmos-
phere.
Note: To prevent room air from entering
the bulbs, disconnect bulb nearest vacuum
source first.
4. Determine and record the weight of each
absorption bulb. These weights represent
the initial weights of the absorption bulbs.
5. After opening the absorption bulbs to
permit gas flow, quickly return the bulbs
to the train assembly.
6. After the rotameter mdicates that 200 to
410 cc per mm of gas is flowing through
the train, remove the empty 250-mi Erlen-
meyer flask and connect a similar flask
containing the weighed sample to be ana-
lyzed.
7. Close the stopcock of the separatory funnel.
1. a) Duplicate determinations are sufficient
for all solid waste samples. (See Method
Evaluation.)
b) The type of Erlenmeyer flask depends
on whether or not the Knoor alkali-
meter is employed.
2. a) The sapphire float in the rotameter
should be between the 4.6 and 6.8
levels.
b) If conditioning of the train is needed,
the air flow is maintained for about 5
mm.
c) An empty, 250-mi Erlenmeyer flask is
used whenever a sample is not being
analyzed.
3. A test tube basket is handy for carrying
absorption bulbs. Bulbs may be left in
basket while connected to the train.
4. a) The bulbs should be near room tempera-
ture before being weighed. The reaction
of CO 2 with the Indicarb produces heat.
b) Before weighing, each bulb is momen-
tarily vented to the atmosphere and
wiped clean with a lint-free cloth or
glove.
c) Use an analytical balance with a 200-g
capacity and 0.1-mg readability.
5. Start with the bulb furthest from the
vacuum source. The connection of the
bulbs to the vacuum source should be
performed last.
6. The sapphire float in the rotameter should
be between the 4.6 and 6.8 levels.
7. This stopcock cannot be closed for several
minutes.
9
-------
METHODS OF SOLID WASTE TESTING
8. With separatory funnel stopper removed, 8. If a Knorr apparatus is used, the drying
add 50 ml (using 50-mi graduated cylinder) tube atop the funnel must be removed to
of dilute HC1 solution (1:4). add the acid solution.
9. Replace separatory funnel stopper.
10. By turning stopcock, slowly add dilute 10. If the sample is calcium carbonate or very
acid to 250-mi Erlenmeyer flask. high in carbonate, addition of acid to the
sample is very, very slow.
11. As soon as all the acid has entered the 11. If stopcock is not closed immediately,
flask, close stopcock of funnel. there may be a loss of CO 2 .
12. Apply heat until sample and acid solution 12. Be sure all the acid solution comes into
starts to boil. contact with the sample. (Flask may need
swirling.)
13. With heat removed, slowly open stopcock 13. The flow rate should be maintained be-
of funnel, keeping rotameter float between tween 200 and 410 cc per mm.
the 4.6 and 6.8 levels.
14. With the stopcock completely open and 14. A 20-mm flush time is sufficient for every
the flow rate adjusted, set a timer for 20 type of sample.
mm.
15. After the 20-mm flush time, stop the flow 15. If another connection is separated first,
by removing the rubber tubing from the room air will enter the absorption bulbs.
last absorption bulb. Note: If a stopcock
is used between gas-washing bottles, it
must be closed.
16. Remove all the absorption bulbs from the 16. If necessary, the train may remain idle for
train and close each to the atmosphere. several hours.
17. As before, determine and record the weight 17. This weight represents the final weight of
of each absorption bulb. each absorption bulb and is used as the
initial weight of each bulb for the next
sample.
18. If another sample is to be analyzed, repeat
the procedure starting with step 5. (If stop-
cock is employed between gas-washing
bottles, it must be open.)
19. If the train is not to be used for the
remainder of the day, turn off water supply
to condenser, close vacuum source, shut
off gas, and close all stopcocks.
10
-------
Carbonate Carbon
CALCULATIONS
Standards
Employ the following formula to calculate the theoretical concentration of carbon in a standard
sample:
( N) (F) (100 )
%C= (S) (P)
where
% = The percent by weight
C = The element carbon
N = The number of atoms of the element in a molecule of the standard
F = A factor derived by dividing the gram-atomic weight of the element by the gram-
molecular weight of the standard
S = The weight of the total sample
P = The decimal fraction representing the concentration of the standard compound in
the total analyzed sample (Note: this decimal fraction is the only fraction contain-
ing the component for which the sample is being analyzed.)
Example:
Pure Calcium Carbonate CaCO 3
( 1)(12.01 ) (100 )
%C= 100.09 = 12.01
(1.0000) (1.00)
When the impurities listed on the bottle are considered, the calculated percent of carbon in ACS
grade calcium carbonate is still 12.01. (ACS grade is about 99.956 percent pure.)
Samples
Employ the following formula to calculate the concentration of carbon in a solid waste sample:
(S)
where
% = The percent by weight
C = The element carbon
A = The sum total increase in the weight of the absorbing bulbs as determined in the
unknown analyses
B = The sum total increase in the weight of the absorbing bulbs as determined in the
blank analyses
X = A factor derived by dividing the gram-atomic weight of carbon by the gram-
molecular weight of carbon dioxide; i.e., (12.01) ÷ (44.01) = 0.2729
S = The weight of the total sample
11
-------
METHODS OF SOLID WASTE TESTING
METHOD EVALUATION
The accuracy of this method was established by analyzing ACS grade calcium carbonate eight
times. The average percent of carbon found was 11 .97. (The calculated percent of carbon value is
12.01.) The standard deviation for these eight observations was 0.18. This method can analyze
solid waste materials containing various forms of carbon or excessive amounts of interferences to
within 0.02 to 0.50 of the actual percent of the established value, depending on the type of sample.*
For the forms of carbon, sucrose and urea were selected to represent organic carbon. Graphite was
employed as elemental form of carbon. Potassium fluoride, sodium sulfate, sodium chloride, and
sodium nitrite were employed as the interfering materials. This method should not be employed to
analyze samples that contain less than 0.01 percent carbonate carbon.
The precision (pooled standard deviation) of this method was determined by analyzing in tnplicate
a number of solid waste samples of various types. The pooled standard deviation of the observations
for each type of solid waste was calculated using an Olivetti Underwood Programma 101. The
calculations revealed that in the analyses of each type of waste, the duplicate and triplicate deter-
minations were about equally precise (Table 1). To ensure precision, the particle size of the samples
must be less than 2 mm (or pass through a #60 sieve) and thoroughly mixed before analyzing.
TABLE 1
STANDARD DEVIATION t OF THE CARBON (CARBONATE) DETERMINATION
ON CALCIUM CARBONATE AND SOLID WASTES
Type of
sample
Number of
samples
Carbon
Percent carbon
(range)
Duplicates
Triplicates
Calcium carbonatet
3
———
0.18
———
Residue.
Finest
Organics
12
4
0.01
0.18
0.02
0.15
0.05
0.59
to 0.69
to 8.00
Fly ash
6
0.13
0.15
0.70
to 1.39
Raw refuse
13
0.08
0.07
0.06
to 0.96
variance estimate can be calculated from the duplicate (or tnplicate) set of observations for each sample The
pooled variance is essentially an average of all such estimates for samples of a given type. It is assumed that a single,
underlying variance exists for all samples of a given type. The pooled variance is then the best estimate of this under.
lying variance. The pooled standard deviation is the square root of the pooled vanance and is used to estimate the
underlying standard deviation.
tACS grade.
Fines are materials remaining after most of the readily combustible substances have been removed by manual
sorting. The sorting was performed at the incinerator sites and a 1/2-in, sieve was employed to assist in the separation.
§Orgamcs or combustibles are mostly the readily combustible materials. Unlike fines, these materials are usually
retained on a 1/2-in, sieve.
t Types of solid wastes in this paper refer only to solid samples (domestic origin) such as raw refuse, incinerator
fly ash, incinerator residue, and compost.
12
-------
Carbonate Carbon
With this method, the analyst normally uses a I- to 5-g sample, but he is not restricted to this
amount. Because of the difficulties in preparing a very uniform sample, sample weights below 1 g
have been found inadequate when analyzing solid waste materials. But samples up to 10 g have been
analyzed with no difficulty. The extra sample weights, however, add little to the precision of this
method.
The performance of this method requires only periodic attention by the analyst. At best, eight
samples a day can be analyzed in duplicate (about 30 mm per determination), leaving the analyst
with approximately 2 hr of free time per day.
ACKNOWLEDGMENTS
The author wishes to thank the Division of Technical Operations, Office of Solid Waste Manage-
ment Programs, for providing samples from incinerators. The author also gratefully acknowledges
the contnbution of Israel Cohen, Solid Waste Research Laboratory, who prepared many of the
samples used in developing this method.
REFERENCES
1. Wilson, Donald L. Laboratory procedure for the gravimetric determination of carbon and
hydrogen in solid wastes (included in this Manual).
2. Ulmer, Nancy S. Laboratory procedure for the determination of volatiles in solid wastes
(included in this Manual).
3. Wilson, Donald L. Decomposition of carbonates in solid waste samples when volatilizing at
600 C or at 950 C. Unpublished memorandum to Chief, Chemical Studies Group, Solid Waste
Research Laboratory, Nov. 19, 1970.
4. Wilson, Donald L. Decomposition of calcium carb,nate (CaCO 3 ) in the Parr Adiabatic Calori-
meter (Series 1200). Unpublished memorandum to Chief, Chemical Studies Group, Solid
Waste Research Laboratory, Sept. 14, 1970.
5. Wilson, Donald L. The total oxygen content of solid samples collected at: (1) Atlanta,
(2) New Orleans, (3) Media, and (4) Greenwood Incinerators. Unpublished memorandum
to Chief, Facilities Section. Solid Wastes Research Laboratory, Jan. 27, 1971.
6. Horwitz, N. ed. Carbonate carbon, 2.107-2.108. In Official methods of analysis of the
Association of Official Analytical Chemists. 11th ed. Washington, D.C., Association of Official
Analytical Chemists, 1970. p. 25-26.
7. Wilson, Donald L. Evaluation of a method for the determination of inorganic carbon (carbon-
ates) in solid wastes. Cincinnati, Solid Wastes Research Laboratory, 1 970.
13
-------
EXTENSION OF CARBON-HYDROGEN METHOD
TO INCLUDE DETERMiNATION OF VOLATILES OR
LOSS ON IGNITION (L.O.I.) AT 950 C
Donald L. Wilsont
DISCUSSION 2
2
REAGENTS 2
SAFETY PRECAUTIONS 2
SAMPLE PREPARATION 2
PROCEDURE 3
STANDARDIZATION 4
CALCULATIONS 4
METHOD EVALUATION 4
ACKNOWLEDGMENT 5
REFERENCES 5
This method is intended to be used in conjunction with “Laboratory Procedure for
the Gravimetric Determination of Carbon and Hydrogen in Solid Wastes” included in
this Manual.
tResearch Chemist, Solid Waste Research Laboratory, National Environmental Research
Center, Cincinnati
-------
METHODS OF SOLID WASTE TESTING
DISCUSSION
The oxygen content of solid wastes samples is found indirectly from the volatile analysis (1)
The commonly used method (2, 3) of determining volatiles for solid waste samples presents an
awkward means of finding the oxygen content, however. The standard volatile-ash method for solid
wastes samples involves heating the sample to 600 C, but, at this temperature, the oxygen combined
in carbonates is partially lost because of decomposition of carbonates. Additional analyses (4) that
determine carbonate content of the samples and ash after volatilizing must therefore be performed
to determine the degree of decomposition of carbonates dunng volatilization.
A much easier approach to finding the oxygen content of solid wastes samples is to volatilize the
samples at a temperature at which almost all of the carbonates decompose. Although the temperature
in the regular volatile-ash method can be raised, a carbon-hydrogen method (5) for solid waste
samples provides a quick and easy means of determining volatiles at 950 C, a temperature at which
most carbonates, especially the most commonly present calcium carbonate, are decomposed.
The carbon-hydrogen method requires that a combustion aid (iron chips) be added to the sample.
This combustion aid oxidizes and increases in weight during the carbon-hydrogen analyses. The
percent of weight increase that is due to oxidation must be determined for each new batch (usually
5 Ib) of iron chips. The increase in weight of the iron chips is then subtracted from the weight of the
ash remaining after the carbon-hydrogen analyses to obtain the true weight of the material volatilized.
The terms volatile and ash are commonly used in solid waste management, but they are often
misunderstood and misleading. A more meaningful term, which will be used in this method, is loss-
on-ignition (L.O.I.) at a particular temperature (950 C in this procedure).
APPARATUS
The apparatus for this method is the same as that described in Reference 5. Note that in the
carbon-hydrogen procedure, samples are retained in clay or nickel boats; but since nickel boats may
undergo weight changes during the carbon-hydrogen tests, only clay boats may be used in this pro-
cedure.
REAGENTS
The chemical requirements and the preparation of reagents for this method are the same as those
described in Reference 5.
SAFETY PRECAUTIONS
The safety precautions are the same as those outlined in Reference 5. No additional hazards
are involved in this method.
SAM PLE PREPARATION
The techniques of sample preparation for this extension of the carbon-hydrogen method are
the same as those mentioned in Reference 5.
2
-------
Volatiles or Loss on Ignition
PROCEDURE
This procedure applies to all solid waste materials that are analyzed for their carbon-hydrogen
contents. Since the mechanics for this procedure are nearly the same as those outlined in the pro-
cedure for carbon-hydrogen contents, only the details pertinent to the analysis L. 0. 1. at 950 C
are discussed here.
Procedure
I. Transfer at least 0.2 to 0.5 gram of DRIED
iron chips into each of 10 previously
ignited, dry, clay combustion boats. Deter-
mine and record the total weight of each
sample plus boat to the nearest 0.0001 g.
2. Store each boat containing a sample in a
desiccator until it is transferred to the
combustion tube.
3. Analyze for blanks by the carbon-hydrogen
method, but put each boat with ash m a
desiccator after the analysis.
Note: Ash from samples high in carbonates
may easily absorb moisture from the atmos-
phere.
4. After each boat has cooled to room tem-
perature, determine and record the weight
of each boat plus contents to nearest
0.0001 g.
5. Analyze solid waste samples for their car-
bon-hydrogen contents by the regular meth-
od, but for each sample (a) determme and
record the weight of iron chips used to the
nearest 0.0001 g, and (b) determine and
record the weight of each boat plus con-
tents to the nearest 0.000 1 g. after each
analysis
Note: Use only clay boats.
Comments
1. a) This weight represents the normal
amount of combustion aid used in the
carbon-hydrogen method.
b) Minimize handling of boats to prevent
contamination.
c) Lids to boats are not needed.
d) Keeping the boats in a particular order
will prevent mix up of samples.
e) Boats should be kept in a desiccator
until used.
f) As needed, dry a small number of iron
chips at 105 C and store in a screw-top
bottle.
2. It is convenient to use a stiff asbestos pad
to support the boats while in the desic-
cator and durmg transfer from one place
to another.
3. a) Develop a technique of handling a boat
after the carbon-hydrogen analysis.
b) Care must be exercised not to spill the
ash content of each boat.
c) The carbon-hydrogen data obtained here
may be used as blank values for the
carbon-hydrogen analyses.
4. Data are now available for calculating the
percent of weight increase of the iron chips
(see Calculations).
5. a) Since the weight of the sample plus
clay boat is known, weight of the iron
chips may be determined by reweighing
the boat plus contents after iron chips
are added to and mixed with the sample.
b) Data are now available for calculating
L. 0. I. at 950 C for each sample analyzed
(see Calculations).
3
-------
METHODS OF SOLID WASTE TESTING
STANDARDIZATION
Standards are analyzed in the manner described under”Procedure” in Reference 5. Standards for
L. 0. 1. at 950 C may be established by repeated analysis of various solid waste samples or by pre-
paring a sample containing a defmite amount of an inert material (pulverized ceramics, for example).
CALCULATIONS
Formula for percent weight increase of iron chips:
w= ( A - 8) ( 100 )
B-X
where
W 1 = percent of weight increase of iron chips
A = total weight of each boat plus iron chips after combustion in the carbon-hydrogen
method
B = total weight of each boat plus iron chips before combustion in the carbon-hydrogen
method
X = weight of each boat
(B - X) = weight of iron chips before combustion
Note: The percent of weight increase for one case of 10 samples averaged 21.8 percent and ranged
from 17.7 to 24.6 percent.
Formula for L. 0. I. at 950 C:
[ (D—A)+(C)(W 1 )] (100 )
% L. 0. I. at 950 C =_____________________
where
D = total weight of each boat, sample, and iron chips before combustion in the carbon-
hydrogen method
A = total weight of each boat plus contents after combustion in the carbon-hydrogen
method
C = weight of iron chips (before combustion) used with each sample
Note: All data are on a dry basis.
METHOD EVALUATION
The data from this method are comparable with data obtained by muffling a solid waste sample at
950 C for 1 hr in air (3). Replicates generally show better agreement with this method than with
the muffling method, probably because of the greater temperature control in this method.
Once the percent of weight increase of the iron chips has been established, this method adds
about 5 to 10 mm extra time to each carbon-hydrogen test.
4
-------
Volatiles or Loss on Ignition
Metals not removed during sample preparation (7) could interfere with the accuracy of this
analysis. The L. 0. 1. at 950 C could be even less than that at 600 C since more metals could
oxidize at the higher temperature and with the pure oxygen atmosphere.
ACKNOW LEDGM ENT
The author wishes to thank James U. Doerger for performing the laboratory tests necessary to
establish this method.
REFERENCES
1. Wilson, Donald L. Mathematical Determination of Total Oxygen in Solid Wastes (included in
this Manual).
2. American Public Works Association. Test for Volatile Solids and Ash. In Municipal refuse
disposal. 3d. ed. Chicago, Public Administration Service, 1970. p. 393-395.
3. Ulmer, Nancy S. Laboratory Procedure for the Determination of Volatiles in Solid Wastes.
(In preparation.)
4. Wilson, Donald L. Laboratory Procedure for the Gravimetric Determination of Carbonate
Carbon in Solid Wastes (included in this Manual).
5. Wilson, Donald L. Laboratory Procedure for the Gravimetric Determination of Carbon and
Hydrogen in Solid Wastes (included in this Manual).
6. Wilson, Donald L. Mathematical Determination of Total Oxygen in Solid Waste (included in
this Manual).
7. Cohen, Israel R. Laboratory Procedure for the Preparation of Solid Waste Related Materials
for Analysis (included in this Manual).
5
-------
MATHEMATICAL DETERMINATION
OF TOTAL OXYGEN
IN SOLID WASTES
Donald L. Wilson*
DISCUSSION 2
APPARATUS 2
REAGENTS 3
SAFETY PRECAUTIONS 3
SAMPLE PREPARATION 3
PROCEDURE 3
Carbon-Hydrogen Analyses . . . 3
Carbonate Carbon Analysis . 3
Nitrogen Analysis 3
Sulfur Analysis 4
Chlorine Analysis 4
Volatile-Ash Analysis at 600 C 4
Volatile-Ash Analysis at 950 C 5
STANDARDIZATION AND CALIBRATION 5
CALCULATIONS
METHOD EVALUATION 7
REFERENCES 8
Research chemist, Solid Waste Research Laboratory, National Environmental Research
Center, Cincinnati.
-------
METHODS OF SOLID WASTE TESTING
DISCUSSION
The oxygen analysis of solid waste in one of the important ultimate analyses necessary to determine
the efficiency of operation of an incinerator, the design of furnaces for incineration, and a complete
materials balance of incoming and outgoing material. The oxygen content of solid waste samples must
be known if their calonfic values are to be calculated from ultimate analyses.
The direct approach of determining oxygen content in solid wastes would involve much time and
expense. Although a direct method is more exacting, an indirect approach (1) that employs a formula
with ultimate analyses and an ash value is presently being used for each coal or coke sample and
can be applied to solid waste samples. The formula must be modified, however, before it can be
applied to solid waste samples, since the oxygen formula applies to coal and coke samples and is
related to a particular method of ashing (2).
The modified method (3) is actually two oxygen formulas, each of which depends on the ashing
technique employed. One procedure of ashing is the standard method for solid waste samples (4).
It involves heating the sample to 600 C, but requires testing for the decomposition of carbonates.
The other ashing technique is to weigh the ash from the carbon-hydrogen method (5).Although
either ashing techmque may be used for this method, ashing as part of the carbon-hydrogen method
is recommended because the extension of the carbon-hydrogen procedure (weighing the residue and
correcting for oxidation of combustion aid) requires very little extra time or effort and eliminates
the need for determining the amount of carbonate decomposition.
Total oxygen content of a solid waste sample is defined either as all the oxygen contained in the
volatile-at-600-C portion plus the inorganically combined oxygen of carbonates in the sample, or
as all the oxygen contained in the volatile-at-950-C portion. This total oxygen value does not include
oxygen that is already combined with metals or silicon.
The recommended procedure for determining carbonate oxygen and organically bonded oxygen
is first to determine carbonate carbon, to multiply this answer by 4 for carbonate oxygen, and then
to obtain the organically bonded oxygen content by subtracting the carbonate oxygen value from
the total oxygen concentration.
The two new formulas for calculating total oxygen content in solid waste samples correlate data
from as many as eight different analyses. The eight components involved are: total carbon (5),
carbonate carbon in total sample (6), carbonate carbon in ash from volatile at 600 C, total hydrogen,
total nitrogen (7), total sulfur (8), total chlorine (10), and a value for volatile or ash at 600 C
or 950 C. The oxygen method descnbed here deals with the procedures for the eight related
components.
The new formulas for oxygen concentrations still have the errors inherent in the other analyses,
particularly the volatile analysis; however, they do take into account carbonate oxygen, which could
cause the greatest inaccuracy in total oxygen data for solid waste samples.
APPARATUS
This procedure requires no apparatus other than that needed to perform the eight different
analyses that provide the data for this method.
2
-------
Mathematical— Total Oxygen
REAGENTS
No reagents are necessary for this analysis. Reagents are needed, however, for the eight correlated
methods and are mentioned under the appropriate method.
SAFETY PRECAUTIONS
No safety hazards exist in this procedure. Safety precautions necessary in related methods are
discussed under those methods.
SAMPLE PREPARATION
Preparation of solid waste samples are discussed in Reference 11 and elsewhere.
PROCEDURE
Carbon-Hydrogen Analyses
Procedure
1. Determine total carbon and hydrogen con-
tents by the method developed particularly
for solid wastes samples, Reference (5).
Comments
1. Larger sample portions are analyzed with
this method than with other similar ones.
Carbonate Carbon Analysis
Procedure
1. Determine carbonate carbon content by
the method developed particularly for solid
waste samples, Reference (6).
Comments
1. a) This analysis is needed for the carbonate
oxygen content of the sample.
b) This same method is employed to find
carbonate carbon in ash from the volatile-
at-600 C analysis.
Nitrogen Analysis
Procedure
1. Nitrogen content of solid waste samples
may be determined by an AOAC method
(7).
2. Nitrogen content of incinerator raw refuse,
combustible incinerator residue, and com-
post samples may be estimated to be 0.75
percent (3).
3. Nitrogen content of noncombustible incin-
erator-residue samples and incinerator fly
ash samples may be estimated to be 0.15
percent (3).
Comments
I. This method is the most commonly used
for solid waste samples.
2. This estimate is the average nitrogen content
of 12 raw refuse samples. The nitrogen
contents ranged from 0.49 to 1.42 percent.
3. This estimate is the average nitrogen con-
tent of nine noncombustible-residue sam-
ples. The nitrogen concentrations ranged
from 0.04 to 0.31 percent.
3
-------
METHODS OF SOLID WASTE TESTING
Sulfur Analysis
Procedure
1. Sulfur content of solid waste samples may
be determined by an ASTM method (see
References 8 and 9).
2. Sulfur content of solid waste samples (raw
refuse, incinerator residue, incinerator fly
ash, and compost) may be estimated to be
0.20 percent (3).
Comments
1. a) This is the most commonly used method
for solid waste samples (see Reference
8).
b) The related bomb-combustion method
was developed especially for solid waste
samples (see Reference 9).
2. The exact average sulfur content of 21
samples (12 raw refuse and nine noncom-
bustible-residue samples) was 0.19 percent.
The sulfur concentrations ranged from 0.10
to 0.37 percent.
Chlorine Analysis
Procedure
1. Chlorine content of solid waste samples may
be determined by an AOAC method (10).
2. Chlorine content of incinerator raw refuse,
combustible incinerator residue, and com-
post samples may be estimated to be 0.50
percent (3).
3. Chlorine content of noncombustible incin-
erator residue samples and incmerator fly
ash samples may be estimated to be 0.10
percent (3).
Comments
1. This method is the most commonly used
for solid waste samples.
2. The exact average chlonne content of 12
raw refuse samples was 0.52 percent. The
chlorine concentrations ranged from 0.29
to 1.10 percent.
3. The exact average chlorine content of nine
noncombustible residue samples was 0. 11
percent. The chlorine concentrations ranged
from 0.06 to 0.1 5 percent.
Volatile-Ash Analysis at 600 C
Procedure
1. The volatile or ash content of solid waste
samples at 600 C may be determined by an
APWA method (3, 4).
Comments
1. a) This method is the most commonly used
for solid waste samples, but it is not
preferred
b) Although it is not commonly used in
solid waste management, the term L.O.I.
(loss-on-ignition) at 600 C is more mean-
ingful than the term volatiles.
c) Data obtained with this method do not
vary with the age of samples, which are
up to 2 years old.
d) Reproducibility of data is affected by
large amounts of carbonates present in
the sample.
4
-------
Mathematical— Total Oxygen
2. Determine carbonate carbon content of the
ash from the volatile analysis (see Carbon-
ate Carbon Analysis).
2. Carbonate carbon analysis is needed in
order to determine the degree of decom-
position of carbonate in the analysis of
volatile content at 600 C.
Volatile-Ash Analysis at 950 C
Procedure
1. The volatile or ash content of solid waste
samples at 950 C may be determined
either by muffling, as in the APWA method
for 600 C, or by weighing the ash from the
carbon-hydrogen method (3,4, 12).
2. If the ash data are obtained from the car-
bon-hydrogen method, the ash value must
be corrected for the increase in weight of
the combustion aid used (iron chips). The
average percent increase in weight of the
combustion aid is 21 8 percent.
Comments
1. a) This analysis (volatiles at 950 C) is not
the most commonly used for solid waste
samples, but it is preferred because it is
easily obtained with the carbon-hydrogen
method, and the decomposition of car-
bonates may be considered to be 100
percent at 950 C
b) Although it is not commonly used in
solid waste management, the term L.O.I.
(loss-on-ignition) at 950 C is more mean-
mgful than the term volatiles.
2. a) Since the ash may contain calcium oxide,
which easily absorbs moisture, the ash
from the carbon-hydrogen must be kept
in a desiccator until weighed.
b) This average percent of weight increase
was determined from 10 analyses with
iron chips. The weight of iron chips
varied from 0.1957 g to 0.4192 g The
percent of weight increase varied from
17.7 to 24.6 percent.
STANDARDIZATION AND CALIBRATION
No standardization and calibration are required for the total oxygen analysis. These techniques
are descnbed in the methods associated with this procedure for determining total oxygen content of
solid waste samples.
CALCULATIONS
1. Formula for total percent oxygen from volatiles at 600 C
where
44
% O=V 1 -(—)( j)(CId)-CO -H-N-S-Cl+(f)(C 1 )
% = the total percent by weight
0 = the element oxygen
= the percent of sample that volatilized at 600 C
5
-------
METHODS OF SOLID WASTE TESTING
= the molar ratio of carbon dioxide to carbon
C 1 = the percent of inorganically bonded (carbonate) elemental carbon
Cid = the decimal fraction of the amount of carbonate carbon that decomposed in the
volatile-at-600-C analysis
C 0 = the percent of organically bonded elemental carbon
H = the total percent of the element hydrogen
N = the total percent of the element nitrogen
S = the total percent of the element sulfur
Cl = the total percent of the element chlorine
f = the molar ratio of oxygen to carbon in carbonates
Note: N, S, and Cl may be estimated if not actually determined. All results are on a dry basis.
2. Formula for total percent oxygen from volatiles at 950 C:
%O=V 2 -Ce-H-N-S-Cl
where
% = the total percent by weight
0 = the element oxygen
V 2 the percent of sample that volatilized at 950 C
C = the total percent of the element carbon
Note: N, S, and Cl may be estimated if not actually determined. All results are on a dry basis.
3. Conversion of carbonate carbon analysis of ash from volatile at 600 C to original sample basis:
% = (Cia) (A)
where
% = the percent by weight
C = the carbonate carbon in ash at 600 C, on original sample basis (before ashing)
C ia = the percent of carbonate carbon in ash at 600 C
A = the decimal fraction of the amount of ash remaining after volatilizing the sample
at 600 C
Note: All results are on a dry basis.
4. Formula for the amount of decomposition of carbonates during the volatile-at-600-C analysis:
— ( C 1 - C 1 ) (100 )
% Cid - ___________
Ci
Note: All results are on a dry basis.
5. Conversion of data from a wet basis to a dry basis:
%Xd (l00)(%X )+(l00-m)
6
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Mathematical— Total Oxygen
where
% = the percent by weight
Xd = the ingredient on a dry basis, except volatiles
X = the ingredient on a wet basis, except volatiles
m = the percent of moisture (loss at 105 C)
Volatiles only.
%Vd =(V -m)÷(l00-m)
where
% = the percent by weight
Vd = the volatile portion of the sample, dry basis
V the percent volatile portion of the sample, wet basis
6. Formula for percent carbonate oxygen:
%0 ( )(Cj)
where
% = the percent by weight
= inorganically bonded (carbonate) elemental oxygen
7. Formula for percent organically bonded oxygen:
%0 =%0-%0
where
% = the percent by weight
00 = organically bonded elemental oxygen
METHOD EVALUATION
The accuracy of this method is dependent on the accuracy of the related analyses and the care
taken in removing metals during sample preparation.
The variance in the oxygen results is a composite of the ‘precision of the associated analyses. The
largest deviation from the average value exists usually in the volatile analysis; replication of the
volatile data is therefore the major factor in determining the precision of the oxygen data. Since the
carbonate content of a sample affects the replication of the volatile-at-600-C analysis, the oxygen
data of samples high in carbonate content can be expected to show poor precision if the data were
obtained with the volatile-at-600-C analysis.
An inspection of past data indicates that the oxygen value could, at most, vary I or 2 actual
percent from the average. This is well within limits of requirements for an estimated oxygen value
of solid waste samples.
The time required to perform the calculations for the oxygen value is trivial, but the related
laboratory analyses required are very time consuming. Such analyses are generally not performed
just for an oxygen value, however. The breakdown of total carbon data into organic and carbonate
7
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METHODS OF SOLID WASTE TESTING
carbon and the decomposition of carbonates in the volatile-at-600-C analysis are necessary informa-
tion for calculating oxygen values, but the concepts have also been useful in producing a better
understanding of the nature of solid waste samples and of the analytical tests performed on them.
REFERENCES
1. Amencan Society for Testing Materials. Oxygen. In 1969 Book of ASTM standards, including
tentatives. pt. 19. D 271-68, sect. 42. Philadelphia, 1969. p. 35.
2. American Society for Testing Matenals. Ash. In: 1969 Book of ASTM standards; including
tentatives. pt. 19. D 271-68, sect. 10-12. Philadelphia, 1969. p. 19-20.
3. Wilson, Donald L. Formulas (Incorporating Decomposition of Carbonates at 600 C) for the
Determination of Total Oxygen in Solid Wastes. Solid Waste Research Laboratory, National
Environmental Research Center, Cincinnati, October 22, 1971.
4. American Public Works Association. Test for Volatile Solids and Ash. In. Municipal refuse
disposal. 3d. ed. Chicago, Public Administration Service, 1970. p. 393-395.
5. Wilson, Donald L. Laboratory Procedure for the Gravimetric Determination of Carbon and
Hydrogen in Solid Wastes (included in this Manual).
6. Wilson, Donald L. Laboratory Procedure for the Gravimetric Determination of Carbonate
Carbon in Solid Wastes (mcluded in this Manual).
7. Horwitz, N. ed. Nitrogen, 2.048-2.075. In: Official methods of analysis of the Association of
Official Analytical Chemists. 11th ed. Washington, D C., Association of Official Analytical
Chemists, 1970. p. 16-20.
8. Amencan Society for Testing Matenals. Sulfur by the bomb washing method. 1n 1969 Book
of ASTM standards; including tentatives. pt. 19. D 271-68, sect. 22-23. Philadelphia, 1969.
p. 25-26.
9. Wilson, Donald L. Laboratory Procedure for Determining Total Heal of Combustion in Solid
Wastes (included in this Manual).
10. Horwitz, N. ed. Chlorine official final action, 34.110. In: Official methods of analysis of the
Association of Official Analytical Chemists. 11th ed. Washington, D. C., Association of Official
Analytical Chemists, 1970. p. 602.
11. Cohen, Israel R. Laboratory Procedure for the Preparation of Solid Waste Related Materials for
Analysis (included in this Manual).
12. Wilson, Donald L. Extension of Carbon-Hydrogen Method to Include Determination of
Volatiles or Loss on Ignition (L. 0. I.) at 950 C (included in this Manual).
8
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MATHEMATICAL DETERMINATION
OF TOTAL HEAT OF COMBUSTION CONTENT
OF SOLID WASTES
Donald L. Wilson
DISCUSSION 2
APPARATUS 2
REAGENTS 2
SAFETY PRECAUTIONS 2
SAMPLE PREPARATION 2
PROCEDURE 3
Carbon-Hydrogen Analyses 3
Oxygen Analysis 3
Nitrogen Analysis 3
Sulfur Analysis 3
Carbonate Carbon Analysis 4
STANDARDIZATION AND CALIBRATION 4
CALCULATIONS 4
METhOD EVALUATION 5
REFERENCES 6
Research Chemist, Solid Waste Research Laboratory, National Environmental Research
Center, Cincinnati.
-------
METHODS OF SOLID WASTE TESTING
DISCUSSION
The heat contents of various solid wastes materials (usually expressed as: British thermal units
(Btu) per pound of sample) are important for establishing an energy balance about an incinerator
and for determining its efficiency. The heat values of solid waste, and particularly of raw refuse, are
also considered when planning the design of an incinerator. The heat contents of incinerator residue
and compost used for landfill are essential data for determining the stability of these waste products.
The experimental method (1, 2) of determining heat contents of prepared solid waste samples (3)
is sometimes difficult to perform, and the accuracy of the data is at times questionable. The heat
contents of such samples may be determined mathematically by a modified Dulong formula (4)
with data from ultimate analysis. This mathematical approach can either eliminate the need for an
experimental test or verify the accuracy of such a test.
The formula for calculating the Btu-per-pound content of solid waste samples correlates data from
14 different analyses. The six directly employed components are (a) organic carbon (5, 6), (b) total
hydrogen (5), (c) organic oxygen (6, 7), (d) total nitrogen (8), (e) total sulfur (2, 9), and (f)
carbonate carbon (6). The eight related analyses are (a) total carbon (5), (b) carbonate carbon in ash
for volatile at 600 C (6, 7), (c) decomposition of carbonates at 600 C (6, 7), (d) volatile at 600 C
value (7, 10), (e) volatile at 950 C value (7, 11), (f) total oxygen (7), (g) carbonate oxygen (6, 7),
and (h) total chlorine (7, 12). The heat of combustion method described here deals with the
procedures for the six directly related components.
The modified Dulong formula still has the errors inherent in the other analysis; however, the
agreement between experimental and mathematical Btu-per-pound values is about equal to the agree-
ment between replicate experimental Btu-per-pound values (4).
Total heat of combustion content of a solid waste sample is defined as the oxidation of organic
carbon to carbon dioxide, hydrogen to water, nitrogen to nitrogen dioxide, sulfur to sulfur dioxide,
and the decomposition of carbonates present in the sample.
APPARATUS
This procedure requires no apparatus other than what is needed for the 14 different analyses that
must be performed to obtain the data for this method.
REAGENTS
No reagents are necessary for this analysis. The reagents are needed for the 14 correlated methods
and are mentioned in the appropriate method.
SAFETY PRECAUTIONS
No safety hazards exist in this procedure. Safety precautions necessary for related methods have
been discussed in those methods.
SAMPLE PREPARATION
Preparation of solid wastes samples are discussed in the carbon-hydrogen method (5) and elsewhere
(3). Therefore, sample preparation techniques are not repeated in this method.
2
-------
Mathematical—Total Heat of Combustion
PROCEDURE
Carbon-Hydrogen Analyses
Procedure
1. Determine total carbon and hydrogen con-
tents by the method developed particularly
for solid wastes samples (5).
Note: L.O.I. at 950 C may be determined at
the same time (7, 11).
Comments
1. a) Larger sample portions are analyzed with
this method than with other similar
methods.
b) The total carbon value will be used in
conjunction with the carbonate carbon
value to determine the organically bond-
ed carbon content.
Oxygen Analysis
Procedure
1. Determine total oxygen content by the
method developed particularly for solid
waste samples (7).
Note: Chlorine content is determined during
this analysis (7, 12).
Comments
1. a) This method overcomes difficulties when
applying an ASTM oxygen method to
solid waste samples.
b) The organically bonded oxygen content
of the sample is determined from the
total oxygen content and the carbonate
oxygen content. The carbonate oxygen
content is calculated from the carbonate
carbon content.
Nitrogen Analysis
Procedure
1. Nitrogen content of solid samples is deter-
mined by one of the recommended methods
for solid waste (8).
2. Nitrogen content of incinerator raw refuse,
combustible incinerator residue, and com-
post samples may be estimated to be 0.75
percent.
3. Nitrogen content of noncombustible in-
cinerator residue samples and incinerator
fly ash samples may be estimated to be
0.15 percent.
Comments
1. The Kjeldahl - Wilfarth - Gunning - Winkler
Method is the most commonly used meth-
od. This method does not, however, include
nitrate nitrogen in the total nitrogen data.
2. This estimate is the average nitrogen con-
tent of 12 raw refuse samples. These
nitrogen contents ranged from 0.49 to
1 .42 percent ’ -
3. This estimate is the average nitrogen con-
tent of nine noncombustible residue sam-
ples. The nitrogen concentrations ranged
from 0.04 to 0.31 percent.
Sulfur Analysis
Procedure
1. Sulfur content of solid waste samples may
be determined by an ASTM method (2, 9).
Comments
1. a) The ASTM method is the most com-
monly used method (9).
3
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METHODS OF SOLID WASTE TESTING
2. Sulfur content of solid waste samples (raw
refuse, incinerator residue, incinerator fly
ash, and compost) may be estimated to be
0.20 percent.
b) The related bomb-combustion method
was developed especially for solid waste
samples (2).
2. The exact average sulfur content of 21
samples (12 raw refuse and nine noncom-
bustible residue samples) was 0.19 percent.
The sulfur concentration ranged from 0.10
to 0.37 percent.
Procedure
Carbonate Carbon Analysis
Comments
I Determine carbonate carbon content by
the method developed particularly for solid
wastes samples (6).
where
1. This analysis is also needed to distinguish
organic carbon from total carbon, and
organic oxygen from total oxygen.
o 8
H-—
+ 8929(_ !)+4274( ).6382C 1
2
Btu per pound = Bntish thermal units per pound
14,096 = the heat of combustion (Btu per pound) of graphite carbon
C 0 = the decimal percent of organically bonded elemental carbon
60,214 = the heat of formation (Btu per pound) of liquid water (constant volume)
from hydrogen and oxygen gases
the total decimal percent of the element hydrogen
the decimal percent of organically bonded oxygen
the decimal percent of available hydrogen
the total decimal percent of the element nitrogen
the total decimal percent of the element sulfur
the decimal percent of inorganically bonded (carbonate) elemental carbon
Note: N and S may be estimated if not actually determined. All results are on a dry basis (prepared
samples).
STANDARDIZATION AND CALIBRATION
No standardization and calibration are required for this procedure. These techniques are disclosed
in the methods associated with this analysis.
CALCULATIONS
1. Formula for total heat or combustion content (Btu per pound) of solid wastes.
Btuperpound= 14,096 C 0 +60, 214 (H-p) + 1040N+3982S
11=
4
-------
Mathematical—Total Heat of Combustion
2. Formula for total percent of organically bonded carbon.
%C 0 =%C-%C 1
where
% = the decimal percent by weight
C = the element carbon (total)
3. Formula for percent of carbonate oxygen
%O =(- )(C 1 )
where
% = the percent by weight
= inorganically bonded (carbonate) elemental oxygen
4. Formula for percent of organically bonded oxygen.
%Oor%0 0 =%0 -%O
where
% = the percent by weight
0 or 00 = organically bonded elemental oxygen
= the element oxygen (total)
5. Conversion of data from a wet basis to a dry basis:
%Xd =(lOO)(%X )—(lOO-m)
where
% = the percent by weight
Xd = the ingredient on a dry basis, except volatiles
X = the ingredient on a wet basis, except volatiles
m = the percent of moisture (loss at 105 C)
Volatiles only
%Vd (V -m) 4 (l0O-m)
where
% = the percent by weight
Vd = the volatile portion of the sample, dry basis
V = the percent volatile portion of the sample, wet basis
METHOD EVALUATION
The accuracy of this method is dependent upon the accuracy of the related analyses and the care
shown in removing glass, ceramics, and metals during sample preparation.
When this procedure was applied to 61 samples from five incinerators (4), the data revealed that
5
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METHODS OF SOLID WASTE TESTING
the variation between calculated Btu-per-pound values and experimental Btu-per-pound values has
about the same magnitude as the variation between replicate experimental values. These calculated
Btu-per-pound values are well within limits of requirements for an estimated Btu-per-pound value of
solid waste samples.
The time required to perform the calculations for the Btu-per-pound value is short, but the related
laboratory analyses required are very time consuming; such analyses are generally not performed
for just a Btu-per-pound value, however. The same ultimate analysis data are used for establishing
a material balance about the same incinerator and also for determining the efficiency of that incin-
erator to reduce the volume of solid waste material.
REFERENCES
1. Parr Instrument Company. Operating the adiabatic calorimeter. In: Oxygen bomb calorimetry
and combustion methods. Technical Manual No. 130. Moline, Illinois, Parr Instrument Com-
pany, 1966.
2. Wilson, Donald L. Laboratory procedure for determining total heat of combustion in solid
wastes (included in this Manual).
3. Cohen, Israel R. Laboratory procedure for the preparation of solid waste related materials for
analysis (included in this Manual).
4. Wilson, Donald L. Prediction of heat of combustion of solid wastes from ultimate analysis
(submitted for publication).
5. Wilson, Donald L. Laboratory procedure for the gravimetric determination of carbon and
hydrogen in solid wastes (included in this Manual).
6. Wilson, Donald L. Laboratory procedure for the gravimetric determination of carbonate carbon
in solid wastes (included in this Manual).
7. Wilson, Donald L. Mathematical determination of total oxygen in solid wastes (included in this
Manual).
8. Kaylor, William H., and N.S. Ulmer. Laboratory procedures to determine the nitrogen content
of solid wastes (included in this Manual).
9. American Society for Testing Materials. Sulfur by the bomb washing method. In: 1969 Book of
ASTM standards; including tentatives. pt. 19. D 271-68, sect. 22-23. Philadelphia, 1969.
p. 25-26.
10. Ulmer, Nancy S. Laboratory procedure for determining percent ash and percent weight loss of
solid wastes on heating at 600 C (included in this Manual).
11. Wilson, Donald L. Extension of carbon-hydrogen method to include determination of volatiles
or loss on ignition (L.O.I.) at 950 C (included in this Manual).
12. Horwitz, W., ed. Chlorine-official fmal action. In: Official methods of analysis of the Association
of Official Analytical Chemists. 11th ed. sect. 34. 110. Washington, D.C., Association of Official
Analytical Chemists, 1970. p. 602.
6
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THE ALSTERBERG (AZ1DE) MODIFICATION
OF THE WINKLER METHOD FOR DETERMINING
THE BOD OF INCINERATOR QUENCH WATER AND
THE CALIBRATION OF THE WESTON & STACK DISSOLVED
OXYGEN ANALYZER MODEL 300-B
Donald L. Wilson
DISCUSSION 3
APPARATUS’ 4
REAGENTS 4
Chemical Requirements 4
Preparation of Solutions 5
SAFETY PRECAUTIONS 6
STANDARDIZATION 6
ANALYSIS OF SAMPLES 7
Sample Collection 7
Site Selection 7
Sample Size and Container 7
Sample Preservation and Shipment 7
Sample (and Blank) Preparation 7
Adjustment for Nitrification Process 7
Adjustment for Residual Chlorine 8
Dilution and Aeration 8
Determination of the DO Concentration 9
CALCULATIONS 9
Sample Volume to be Titrated 9
DO Content of Sample 10
BOD of Sample 10
Dilution Water Sample 10
Quench Water Sample 10
METHOD EVALUATION 11
Precision 11
Accuracy 12
Sensitivity 12
Research Chemist, Solid Waste Research Laboratory, National Environmental
Research Center, Cmcinnati.
-------
BIBLIOGRAPHY 12
APPENDIX • 13
INTRODUCTION 13
Principle of Tests 13
Test for Group A Substances (Sulfates) 13
Test of Group B Substances (Thiosulfates, Sulfites) . 13
Test for Group C Substances (Nitrites) 15
Test for Group D Substances (Ferrous Salts) .
Test for Group E Substances (Fernc Salts)
Test for Group F Substances (Residual Chlorine).
Test for Group G Substances (Chlorides)
Test for Group H Substances (Released Chlorine)
SENSITIVITY OF TESTS
Interferences with Tests
APPARATUS
REAGENTS
Introduction
Chemical Requirements
Preparation of Solutions
STANDARDIZATION
SAMPLE ANALYSIS
Test for Group A Substances
Test for Group B Substances
Test for Group C Substances
Test for Group D Substances
Test for Group B Substances
Test for Group F Substances
Test for Group G Substances
Test for Group H Substances
BIBLIOGRAPHY
METHODS OF SOLID WASTE TESTING
(Sulfates)
(Thiosulfates, Sulfites).
(Nitrites)
(Ferrous Salts)
(Ferric Salts)
(Residual Chlorine).
(Chlorides)
(Released Chlorine).
15
15
16
16
16
16
16
17
17
17
17
18
19
19
19
20
20
20
21
21
21
22
22
2
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Alsterberg Modification of Winkler Method for BOD
DISCUSSION
The Winkler method, developed in 1 888, is the routine chemical method for the determination of
dissolved oxygen (DO) The basic procedure involves the oxidation of manganous hydroxide (Mn )
by the oxygen dissolved in the water to manganic hydroxide
MnSO 4 +2KOH-* Mn (OH) 2 +K 2 S0 4 (1)
2Mn (OH) 2 ÷ 02 -÷ 2MnO (OH) 2 (2)
Manganous hydroxide is a white flocculant precipitate that changes to light brown when oxidized.
Since the reaction with the oxygen must occur on the surface of the floc particles, physical mixing at
this point is important.
When manganic hydroxide is acidified, manganic sulfate is formed
MnO (OH) 2 + 2H 2 SO 4 -÷ Mn (SO 4 )2 + 3H 2 0 (3)
In the presence of iodide, the manganic salt acts as an oxiding agent, releasing free iodine
Mn(S0 4 ) 2 +2K1—* MnSO 4 +K 2 SO 4 +12 (4)
The iodine, which is stoichiometncally equivalent to the DO of the sample, is titrated with thiosulfate
12 + 2Na 2 S 2 03 -+ Na 2 S 4 06 + 2NaI (5)
For convenience, the alkali (equation 1) and the iodide (equation 4) are combined into a single,
alkaline-iodide reagent.
The original Winkler method has been modified since oxidizing agents give a positive interference,
reducing agents a negative interference, and organic compounds a varied interference. The most
common interference is that caused by nitrites, commonly present in polluted waters and wastes.
The nitrite ion reacts with iodides as follows:
2NO 2 +2I+4H -3I 2 +2NO+2H 2 O (6)
2NO+ 1/202 +H 2 O- 2NO 2 +2H (7)
The Alsterberg (Azide) Modification uses sodium azide to reduce the nitrites in the following manners
NaN 3 +H -+HN 3 +Na (8)
HN 3 +NO 2 +H - N 2 +N 2 O+H 2 O (9)
The Alsterberg (Azide) Modification of the Winkler Method uses prepared dilution water and is
employed to standardize the Weston and Stack DO Analyzer. Before the method can be used to
determine the BOD of incinerator quench water, however, the samples must be analyzed for the
presence of mterfenng substances. If the final diluted sample contains more than 4 ppm nitrite,
I ppm ferrous iron, or 200 ppm ferric iron, or any sulfite or thiosulfate, or large amounts of chloride
(which may evolve as chlorine dunng the analysis), the Alsterberg Modification cannot be employed.
If, however, the final diluted sample contains less than 4 ppm nitrite, 1 ppm ferrous iion, or 50 ppm
fernc iron, valid results can be obtained using the procedure. In addition, samples that contain
residual chlonne or 50 to 200 ppm ferric iron, or that have a pH of less than 6.5 or greater than 8.3
can also be evaluated using certain alterations of the procedure.
The preliminary tests presented in the appendix can be employed to evaluate the applicability of
the Alsterberg (Azide) Modification in the determination of the BOD of a quench water sample. Since
the tests are qualitative and involve distinct color changes or precipitates, the concentration of the
reagents may be approximate. Standards should be employed, however, to verify the validity of the
preliminary analyses and to give the analyst some knowledge of the color intensity and hue.
3
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METHODS OF SOLID WASTE TESTING
Since the tests are not specific and hence give a positive reaction in the presence of several
substances, they are assigned group numbers. Each of the interfering substances mentioned earlier
does, however, fall into one of the groups.
APPARATUS
All glass and plastic apparatus must be cleaned thoroughly to ensure the removal of all materials
capable of exerting a BOD. Detergents may be used if cleaning is followed by thorough rinsing with
distilled water.
The apparatus requirements are as follows:
1. Incubation (BOD) bottles, 300-ml capacity
2. Air incubator or water bath, thermostatically controlled at 20 C ± I C
3. Buret, 50-mi
4. Volumetric flasks, 200-mi, specially marked for 203 ml
5. Graduates, 10-mi, 50-mi, i-liter, and 2-liter
6. Volumetric flasks, 1 00-ml, 500-ml, 1-liter
7. Mohr pipets, 10-mI with 1-mi divisions
8. Carboys, two 7-liter or more, polyethylene nalgene, wide-mouth
9. Reagent bottles, I-liter, narrow-mouth, ground glass stoppers
10. Erlenmeyer flasks, 500-mi
11. Beakers, 250-mi, 2-liter, and 3-liter
12. Siphon tubing
13. Balance, analytical (also trip, if available)
14. pH paper, range 2 to 9
15. Sample collection bottles, polyethylene (or similar unbreakable material), narrow mouth,
tightly fitting caps, about 1-liter, sterile (no material present that has a BOD value)
16. Magnetic stirrer with Teflon-coated stirring bar
17. Ice chest capable of holding several 1-liter, sample collection bottles and able to maintain a 5 C
temperature for 24 hr
REAGENTS
Chemical Requirements
The following chemicals are ACS, reagent grade:
1. Phosphate buffer solution, pH 7.2 (or prepared)
2. Potassium phosphate, monobasic
3. Potassium phosphate, dibasic
4. Sodium phosphate, dibasic, heptahedrate
5. Ammonium chloride
6. Magnesium sulfate, crystal
7. Calcium chloride, anhydrous
8. Ferric chloride, Jump
4
-------
Alsterberg Modification of Win kier Method for BOD
9. Sodium hydroxide or potassium hydroxide
10. Sodium iodide or potassium iodide
11. Sodium azide
12. Sulfuric acid, concentrated
13. Manganese(ous) sulfate, monohydrate (may be other hydrates)
14. Thyodene
15. Sodium thiosulfate, crystalline
16. Chloroform
17. Potassium biniodate, solid or 0.025N solution, or potassium dichromate
18. Potassium fluoride
Preparation of Solutions
All solutions are prepared with distilled water that (a) is distilled from a block-tin or all-glass still,
(b) contains less than 0.01 mg per liter copper, and (c) is free of chlorine, chloramines, caustic
alkalinity, organic materials, and acids. The solutions are prepared in the following manner.
1. Standard sodium thiosulfate solution, 0.025 N: Dissolve exactly 6.205 g Na 2 S 2 0 3 5H 2 0 in
distilled water and dilute to 1 liter. Preserve by adding 5 ml of chloroform. Note: [ a] TIus
solution is equivalent to 0.200 mg DO per 1.00 ml. When employed to titrate 203 ml of treated
sample (200 ml of original sample), each ml is then equivalent to 1 mg per liter (ppm) DO in
sample. [ bJ This solution is not stable more than 9 days (see Standardization).
2. Standard potassium biniodate solution, 0.025 N (if not purchased): Dissolve 0.81 24g KHIO 3 in
distilled water and dilute with same to I liter.
3. Standard potassium dichromate solution, 0.025 N (if standard potassium biniodate solution is
not available): Dissolve 1 .226g K 2 Cr 2 07 (previously dried for 2 hr at 110 C) in distilled water
and dilute with same to 1 liter.
4. Sodium hydroxide solution, approximately 1 N: Dissolve 41.6 g NaOH in distilled water and
dilute with same to 1 liter.
5. Sulfuric acid solution, approximately 1 N: Cautiously add 28 ml of concentrated H 2 SO 4 to
distilled water and dilute with same to 1 liter.
6. Phosphate buffer solution, pH 7.2 (if not purchased) Dissolve 8.5 g KH 2 P0 4 , 21.75 g K 2 HPO 4 ,
33.4 g Na 2 HPO 4 7H20, and 1.7 g NH 4 Clin 500 ml of distilled water and dilute with same to 1
liter. The pH of this buffer should be 7.2 without further adjustment.
7. Magnesium sulfate solution: Dissolve 22.5 g MgSO 4 7H 2 0 in distilled water and dilute with
same to 1 liter.
8. Calcium chloride solution: Dissolve 27.5 g anhydrous CaCl 2 in distilled water and dilute with
same to 1 liter.
9. Ferric chloride solution: Dissolve 0.25 g FeC I 3 6H 2 0 in distilled water and dilute with same to
1 liter.
10. Dilution water: Place 7 liters of distilled water in polyethylene, wide-mouth, carboys (other
volumes and containers may be used). Add 7 ml (I ml per liter of water) of each of the following
prepared solutions: Phosphate buffer, pH 7.2; magnesium sulfate; calcium chloride; and ferric
chloride. This water should be at 20 C before it is used.
5
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METHODS OF SOLID WASTE TESTING
11. Alkah-iodide-azide reagent: Dissolve 500 g NaOH (or 700 g KOH) and 135 g Nal (or 150 g KI)
in distilled water and dilute to 1 liter. To this solution, add 10 g NaN 3 dissolved in 40 ml of
distilled water, stirnng constantly. This reagent should not give a color with Thyodene indicator
when diluted and acidified.
12. Manganese sulfate solution: Dissolve 480 g MnSO 4 4H 2 0 (or 400 g MnSO 4 2H 2 0 or 364 g
MnSO 4 H 2 0) in distilled water, filter, and dilute to 1 liter.
13 Potassium fluoride solution. Dissolve 40 g KF 21120 in distilled water and dilute to 100 ml.
SAFETY PRECAUTIONS
Follow general laboratory safety rules. This method has no pronounced safety hazards. Care must
be taken, however, when handling the concentrated sulfuric acid. A pad of tissue held over the BOD
bottles when they are inverted will prevent any splattering of the treated sample that might leave
stains and harm the analyst’s hand.
STANDARDIZATION
With sodium thiosulfate prepared exactly, no standardization is needed. Standard sodium
thiosulfate solution, exactly 0.025 N, is equivalent to 0.200 mg DO per 1.00 ml, or when 203 ml of
the treated sample is titrated with 0.025 N thiosulfate
I ml 0.02 5 N thiosulfate = 1 mg per liter DO
A sodium thiosulfate solution is not stable, however, and solutions over 9 days old must be
standardized with a biniodate (preferred) or dichromate standard 0.025 N solution.
The procedure for standarization is as follows.
Procedure Comments
1. Dissolve approximately 2 g of KI in a 500-mi
Erlenmeyer flask with 150 ml of distilled
water.
2. Add 10 ml of dilute sulfuric acid.
3. Add 20 ml of standard solution.
4. Dilute to 300 ml with distilled water.
5. Titrate the liberated iodine with standardized
thiosuifate titrate to a pale straw color.
6. Add about 1/2 g of Thyodene indicator and
shake flask.
7. Continue titrating until blue color disappears.
8. Record the amount of titrant used.
9. Repeat steps 1 through 8 for a second test.
1. Trip balance may be used if available.
2. 1 part concentrated H 2 SO 4 and 9 parts
distilled water.
3. Biniodate or dichromate.
4. If dichromate standard solution is used, put
flask in the dark for 5 mm.
6. After Thyodene is added, a blue color appears
9. The absolute value of the difference between
duplicated readings should not exceed 1.96
or 0.36 ppm, more than 5 percent of the
time. See section on Precision.
6
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Alsterberg Modification of Win kier Method for BOD
ANALYSIS OF SAMPLES
Sample Collection
Site selection.
When the BOD of quench water is measured to determine the amount of oxidizable wastes that will
be discharged to a sewerage system serving an incinerator facility or to a drainage system associated
with the residue disposal area, the site of sample collection must be chosen with due consideration.
Settlement tanks, surface pools, sewers and other areas immediately adjacent to the sewerage or
drainage system are preferable collection sites.
Sample size and container.
Normally 50 ml of sample is needed to perfrom the BOD analysis, however, smce various dilutions
may be needed and a larger sample size may be more representative, it is recommended that a
1-liter sample of quench water be collected.
The samples should be collected m sterile, unbreakable bottles with narrow mouths and caps that
can be tightly fitted. The sample bottle should be completely filled. All containers must be thoroughly
nnsed, especially if cleaned with a detergent, before they can be reused.
Samples should not be collected on Monday or Tuesday unless the analysts are to work on Saturday
or Sunday (5-day BOD).
Sample Preservation and Shipment
If the sample analysis is to be initiated within 4 hr after collection, sample preservation measures
are not absolutely necessary. If the analysis will be started after 4 hr, however, samples should be
placed in an ice chest or similar container soon after collection so that they will be maintained in the
dark at 5 C. The bottle caps must be tightly fitted to prevent an increase in oxygen solubility with the
reduction in temperature.
Sample shipment to the laboratory should be immediate (via air freight if necessary) to ensure the
initiation of BOD analysis in the laboratory within 24 hr of sample collection. Samples received after
they are 24 hr old should not be analyzed. Because samples require some preparation before the actual
analysis and because the exact dilution requirements may not be known, samples (not at 5 C) should
be shipped to the laboratory so that they are received 2 to 4 hr before the end of the normal working
day, depending on the number of samples and laboratory personnel. Samples that have been shipped
in an ice chest at 5 C and kept under refrigeration may be analyzed the following day provided the
analysis can be initiated before the samples are 24 hr old.
Sample and Blank Preparation
Adjustment for nitrification process.
Before analysis, each sample and dilution-water blank is treated as follows to inhibit the nitrifica-
tion process:
Procedure Comments
I. Place 50 ml of a thoroughly mixed, quench 1. The exact volume of the quench water
water sample in a 250-ml beaker. sample depends on the dilution require-
ments. (See Dilution and Aeration.)
2. Using pH paper, check the pH of the sample. 2. Usually the pH is about 11.
3. Using 1 N NaOH or 1 N H 2 SO 4 , adjust 3. Omit if the sample already has a pH of 2 to
the pH of the sample to a range of 2 to 3.
3; maintain pH for 15 mm.
7
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METHODS OF SOLID WASTE TESTING
4. Then neutralize the sample
to 8.3.
to a pH of 6.5
4. Employ the same I N solutions as in step 3.
Adjustment for residual chlorine.
Since residual chlorine dissipates when samples either stand for 1 to 2 hr or are well aerated, no
adjustments are recommended.
Dilution and aeration.
Prepared samples must be diluted in order to obtain a measurable depletion of oxygen (2 ppm to
7 ppm) at the end of the 5-day incubation period. Since incinerator quench water usually has a BOD
of 100-300 ppm, a suitable or applicable dilution is 50 ml of sample diluted to 2 liters. If the analyst
suspects that the BOD of the quench water differs from the usual value, he should test various dilu-
tions since the analysis cannot be repeated on the same onginal sample after the 5-day waiting period.
To obtam more reliable results, 5 BOD bottles should be prepared: two for the initial DO (can be
immediately repeated if necessary) and three for the final DO (only two reasonable results are
needed). The final DO values should never be less than 1 .0 ppm.
Since the dilution water employed in the analysis of each quench water may contain a few
oxidizable materials capable of exerting a small BOD, each quench water analysis should mclude a
blank evaluation, i.e., a determination of the BOD of the dilution water. The observed BOD of the
quench water can then be corrected by subtracting the appropriate proportionate fraction of this
blank value.
The dilution and aeration procedures are as follows:
Procedure Comments
1. Pour the total prepared sample from the
250-ml beaker into a 2-liter graduate and
dilute to the mark with dilution water.
2. Aerate the sample by pouring it back and
forth from the graduate into a 3-liter beaker
at least 3 times.
3. Siphon the diluted, aerated sample or blank
from the beaker into 5 BOD bottles.
4. a) See Determination of the DO Concentra-
tion.
b) At least two reasonable DO results are
needed. See section on Precision.
5. a) During the incubation period, the samples
should not be exposed to the light.
b) See Determination of the DO Concentra-
tion.
c) Only two reasonable DO results are
needed. See section on Precision.
1. Solution still represents 50 ml of original
sample.
2. Dilution water blank is aerated in like
manner.
3. The sample should be stirred continuously
using a magnetic stirrer and a Teflon coated,
magnetic bar.
4. The DO concentration of the sample or
blank in 2 BOD bottles should be determined
immediately.
5. Then put the remaining 3 BOD bottles in an
incubator (or waterbath) and determine
their DO content after a 5-day incubation
period at 20 C.
8
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Alsterberg Modification of Win kier Method for BUD
Determination of the DO Concentration
Procedure
1. To each BOD sample bottle add 2 ml of
manganous sulfate solution and then 2 ml of
alkaline-iodide-azide reagent.
2. Replace the stopper, exclude air bubbles,
and mix by inverting 9 to 10 times.
3. When the precipitate settles, repeat the
inverting and settling.
4. After there is about 100 ml of clear supernate,
remove stopper and immediately add 2 ml of
concentrated H 2 SO 4 ABOVE the surface of
the sample.
5. Stopper the bottle and mix to dissolve the
precipitate.
6. By means of a modified, 200-mi volumetric
flask, transfer 203 ml of the treated sample
to a 500-mi Erienmeyer flask.
7. Titrate the treated aliquot with standardized
thiosulfate titrant to a pale straw color.
8. Add about 1/2 g of Thyodene indicator and
shake flask.
9. Continue titrating until blue color disappears.
10. Record the amount of titrant used.
11. Repeat steps 1 through 10 with each BOD
sample bottle.
Comments
1. a) When a sample is being evaluated for the
purpose of DO probe calibration, the
probe determination of the DO content
must precede the Winkler determination
of that same sample.
b) The tip of each pipette must be BELOW
the surface of the sample.
2. A Kimwipe pad held over the top of the
bottle helps prevent splattering.
4. If the diluted sample contains more than 50
ppm ferric iron, add 1 ml of KF solution
before the acid.
6. a) Volume adjusted for the 4 ml of reagents
added. (See Calculations.)
b) Transfer 204 ml if KF solution is used.
8. After Thyodene is added, a blue color
appears.
11. a) Titration values less than 1.0 ml should
be disregarded.
b) The absolute value of the difference
between duplicate readings should not
exceed 1.96 / i or 1.35 ppm, more
than 5 percent of the time (See Precision.)
CALCULATIONS
Sample Volume to be Titrated
Since a portion of the sample in the BOD bottle is displaced during the analysis because of the
addition of reagents, the volume of sample to be titrated must be adjusted to compensate for this loss.
The required volume is calculated as follows:
V 1 =200(V 2V)
9
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METHODS OF SOLID WASTE TESTING
where
V 1 = The volume of sample to be titrated
V 2 The volume of BOD bottle employed
V 3 = The volume of reagents added (The volume of the sulfuric acid added is not
included since the acid only displaces sample that has been deoxygenated.)
DO Content of Sample
The DO content of a sample is calculated as follows:
D=FV 4
where
D = The DO content of the sample being titrated
F = The correction factor-ratio of normality of standard (thiosulfate) to 0.025
(normality of biniodate or chromate)
V 4 = The volume of sodium thiosulfate used to titrate the sample
BOD of Sample
Dilution water sample.
The following formula should be employed to calculate the BOD of each individual sample of
dilution water.
BOD I =D 1 —D 2
where
BOD 1 = The biochemical oxygen demand of dilution water
= The DO content of initial (before incubation) dilution water
D 2 = The DO content of final (after incubation) dilution water
Quench water sample.
The initial DO concentration minus the final DO concentration equals the BOD of the diluted
sample. The BOD of the diluted sample times the dilution factor equals the BOD of the original sample.
The dilution factor is found by dividing the original amount of sample taken into the final dilution.
For example, 50 ml of sample diluted into 2 liters gives a factor of 40.
The following formula should be employed to calculate the BOD of each individual sample of
quench water.
BOD 2 =F [ (D 3 -D 4 )-P 1 (BOD 1 )]
where
BOD 2 = The BOD of quench water
F = The dilution factor
D 3 = The DO content of initial (before incubation) quench water
D 4 = The DO content of final (after incubation) quench water
P 1 The decimal fraction of dilution water used in the BOD analysis of the quench
water
10
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Alsterberg Modification of Wink/er Method for BOD
METHOD EVALUATION
Precision
After analyzing a number of quench water samples in duplicate (three final DO determinations
were performed to ensure reasonable duplicate results), the precision of the observations were
evaluated by calculating (with the Olivetti Programma 101) the pooled standard deviation of all
observations except those obtained on samples collected from dump truck drainage.
The results of these calculations are shown in Tables 1 and 2.
TABLE 1
PRECISION OF THE DO ANALYSIS
Type of
sample
No of
determinations 4
Pooled standard
deviation
(s)t
Confidence interval
± (I 96) / 2 (s)
Standards
(normality)
44
0.13
±036
Dilution water
(bldnk)
32
0.19
±0.53
Quench water
76
0.49
±1 35
Both dilution and
quench water
108
043
±1.19
* Includes initial and final determinations.
t A pooled standard deviation was computed for all determinations It was assumed that there was no statistically
significant difference between initial and final variances, that is, homogeneity of the variances was assumed
The absolute value of the difference between duplicate readings should not exceed 1.96 / j), or 036 ppm.
more than 5 percent of the time. The covariance between the duplicate readings was ignored
TABLE 2
PRECISION OF THE BOD ANALYSIS
Standard
Type of
sample
No. of
determinations
deviations
(S)*
Dilution
Factort
Con
± (
fidence interval
I 96) (40) (S)1
Dilution water
(blank)
8
0.27
40
±21 2
Quench water
19
0.69
40
±54.1
Both dilution and
quench water
27
0.61
40
±478
* The standard deviation of the difference between initial and final DO readings, (i.e., S = / i5) In this calcula-
tion it was assumed that the initial and final pooled variances were equal, and the covariance term between
initial and final readings was ignored.
t Dilution factor may vary, but for calculation purposes, the normal dilution factor is shown here
1 Ninety-five-percent confidence limits about a single BOO result, assuming a standard dilution factor of 40 or 2 5
percent dilution.
11
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METHODS OF SOLID WASTE TESTING
Accuracy
There is no standard against which the accuracy of the BOD test can be measured.
Sensitivity
This Alsterberg (Azide) Modification of the Winkler Method is not applicable to samples that have
a dilution factor of 40 and a 5-day BOD value of 54.1 ppm or less.
BIBLIOGRAPHY
1. American Public Health Association, American Water Works Association, and Water Pollution
Control Federation. Oxygen (dissolved). In: Standard methods for the examination of water and
wastewater. 12th ed. New York, American Public Health Association, Inc., 1965, p. 405-421.
2. American Society for Testing Materials, Committee D-19. Dissolved Oxygen in Industrial Waste
Water, Dl 5 89-60. In: Manual on industrial water and industrial waste water. 2nd ed. Philadelphia,
American Society for Testing Materials, 1966. p. 589-592.
3. Wilson, Donald L. Applicability of existing methods for the determination of the biochemical
oxygen demand (BOD) of incinerator quench water. Cincinnati, Solid Waste Research Laboratory,
Oct. 9, 1970.
4. Wilson, Donald L. The dissolved oxygen analyzer (Weston & Stack, Inc. Model 300-B) method for
the determination of the BOD of incinerator quench water (included in this Manual).
12
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Alsterberg Modification of Winkler Method for BOD
APPENDIX
QUALITATIVE TESTS FOR DETERMINING THE
PRESENCE OF INTERFERING SUBSTANCES IN QUENCH WATER
INTRODUCTION
Principle of Tests
These qualitative tests may be employed as a quick means of detecting the presence of oxidizing or
reducing substances (sulfites, nitrites, ferrous and ferric salts, and residual chlorine for example) that
will interfere with the Modified Winkler method for the determination of BOD (Figure 1).
Although the presence of sulfate does not affect the Modified Winkler method, the presence of
large quantities of it may indicate the possible presence of sulfite, which definitely affects the method.
Likewise, large amounts of chlorides may indirectly affect the method by releasing chlorine gas
during acidification.
The principle of each of these tests is discussed briefly in the following sections.
Test for Group A substances (sulfates).
When a barium chloride solution is added to a solution containing a sulfate-type material, an
insoluble white precipitate such as banum sulfate will form. This precipitate can be detected visually.
Test for Group B substances (thiosulfates, sulfites).
Thiosulfate will react with mineral acids to yield insoluble elemental sulfur.
S 2 0 3 2 +2H - ’SO +S+H 2 0
The presence of thiosulfates can be detected when a pale, whitish-yellow precipitate forms after the
addition of acid.
In the presence of an acid, dichromate will oxidize iodide to iodine, which will then impart a blue
color to the solution when Thyodene is present.
Cr 2 0 7 2 +6 1+ l4W-÷ 2Cr 3 +312 +7H 2 0
12 + Thyodene - Blue complex
Sulfite-like material may be detected by first treating a sample with an acid-iodide solution and a
small amount of Thyodene powder, and then performing a dropper titration using potassium
dichromate solution. If sulfite-like material is present in the sample, the volume of dichromate
solution required to obtain first a blue solution and then a yellow dichromate color will exceed that
required by a reagent blank solution.
Small amounts of sulfite can be detected in the presence of any amount of thiosulfate because, on
oxidizing a sulfite-thiosulfate mixture with iodine (neutral solution), the following reaction occurs:
SO 3 +12 +H 2 O- SO 4 ÷2I+2H
2S 2 03 -2 + 12 S 4 062 + 21 -
Hydrogen ions are formed only in the case of sulfite. The resulting acidity can be revealed by litmus
paper.
13
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METHODS OF SOLID WASTE TESTING
GROUP GROUP GROUP GROUP GROUP GROUP GROUP GROUP
1 1
Undiluted Undiluted Diluted, Diluted, Diluted, Undiluted Diluted, Undiluted
sample sample aerated aerated aerated sample aerated sample
sample sample sample sample
Group A Group B Group C Group D Group D Group F Group G H 2 SO 4 and
test soln. test soln. #1 test soin. test soin. test soln. test soln. test soln Group H
test paper
White ppt. Red soln. Orange-red Group E White ppt. Blue paper.
SULFATE NITRITES soln.. test soin. CHLORIDES RELEASED
present present FERROUS present CHLORINE
SALTS
_______ ______ present ________
Pale Clear Pale, Clear
whitish soin. whitish soln.
yellow ppt yellow
THIOSULFATES ppt:
present THIOSULFATES
present
Thyodene Deeper Thyodene
F orange-
Blue Group B red soln.. Blue soin.:
soln.. test soln. #2 FERRIC SALTS RESIDUAL
SULFITE present CHLORINE
absent present
Blue soln.
SULFITE
present
Figure 1. Flow chart of tests for Group A through H substances.
14
-------
Alsterberg Mothficatzon of Win kier Method for BOD
First reaction:
Second reaction. NH 2
HO 3 S - -- N N +
+H°
The presence of sulfite-type substances in a sample can produce false negative results with the tests
for interfering substances that are similar in effect to nitrite, ferrous iron, ferric iron, and residual
chlorine Since, however, no amount of sulfite-type material can be present m a sample, a positive test
for sulfite-type compounds eliminates the need for performing the other tests for interferences.
Test for Group C substances (nitrites).
The primary aromatic amine, sulfanilic acid, reacts in an acid solution with nitrites to form
diazonium cations that subsequently couple with l-naphthylamme (l-napthylamine hydrochloride or
N-(1-naphtyl)-ethylenediamine dthydrochloride may be substituted) to form the red p-benzene
sulfonic acid -azo -cx -naphthylamine The red color is then indicative of the presence of nitrite-type
substances.
NO 2 +HO 3 S— --NH 3 +H
(diazotization or condensation)
HO 3 S —N N+2H 2 0
coupling (electrophilic /aromatic
substitution occurs with phenols
in alkaline solutions)
HO 3 S— —--N= N
Test for Group D substances (ferrous salts). H 2 N
In the presence of an acid solution, ferrous salts will react with 1 ,10 phenanthroline to form a pale
orange-red complex; sometimes, however, the sample may have to be filtered to detect the color.
More expensive 1,10 dipyndyl can be substituted for the 1,10 phenanthroline. The organic base 1,10
dipyridyl reacts with ferrous iron to form a deep red, very stable, complex cation.
(I , 10 Phenanthroline) N + Fe 2
Femc salts do not react under these conditions, consequently, very small amounts of ferrous salts
can be detected in the presence of large proportions of ferric salts.
Test for Group E substances (ferric salts).
Ferric salts are reduced by hydroxylamine hydrochloride (NH 2 OH HCI) to ferrous salts and
allowed to react with 1,10 phenanthroline in the test for Group D. A more intense orange-red color
than is found in the test for Group D indicates the presence of ferric-type substances.
Red azo dye
15
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METHODS OF SOLID WASTE TESTING
Test for Group F substances (residual chlorine).
The presence of chlorine can be detected indirectly by its oxidation of iodide to iodine and the
subsequent formation of a blue color in the presence of Thyodene.
21 -+ 2H + Cl 2 - I + 2HC1
12 + Thyodene - Blue complex
Test for Group G substances (chlorides).
The presence of chlorides can be detected by the addition of silver nitrate to a portion of the
sample solution. If chloride-type substances are present, a white precipitate will form.
NaC1+AgNO 3 - AgCl+NaNO 3
Test for Group H substances (released chlorine).
The presence of chlorine gas that is liberated during analysis by the Alsterberg-Winkler procedure
can be detected by acidifying a portion of the sample and testing for the emission of chlorine-type gas
as described in the test for residual chlorine.
3NaCl + H + (oxidizing agent) -÷ Na 2 + HC1 + Cl 2
SENSITIVITY OF TESTS
These qualitative tests have the following sensitivities:
Sulfate > 0.5 ppm
Thiosulfate > 1.0
Sulfite > 1.0
Nitrite > 0.1
Ferrous salt > 1.0
Ferric salt > 1.0
Residual chlorine> 1.0
Chloride > 1.0
Released chlorine> 1.0
Interferences with Tests
The test for sulfates may be affected by the presence of sulfites and sulfides in the sample. Sulfites
and sulfides can be oxidized and precipitated with the sulfates to give a high result.
With the test for sulfites, other reducing agents such as sulfides and ferrous iron will cause high
results. Upon acidification, nitrite in the sample will oxidize all or part of the sulfite. Proteins in the
sample will tend to prevent the starch-iodine reaction, and cyanides will react with the iodine in the
sample.
The test for nitrites is affected by copper, which catalyzes the deomposition of the diazonium
salt. The azo dye reacts with peroxides to form azoxy compounds, with sodium hydroxide to form
hydrazo compounds, and with tin chloride to form amines. Also, chlorine bleaches the azo dye, thus
causing low results.
16
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Alsterberg Modification of Winkler Method for BUD
Copper, chromium, nickel, and zinc form complexes with 1,10 phenanthroline and thus consume
the reagent. Excess amounts of the 1,10 phenanthroline (about 10 times that of the iron concentra-
tion) may therefore have to be used for the ferrous (or ferric) salts test.
The tests for residual and released chlorine are not specific because other halogens are also detected.
Even ferric iron may oxidize the iodide ions in the test.
The test for chloride is not specific either, since bromide, iodide, and sulfice may precipitate with
the chloride to give a high result.
APPARATUS
All glassware must be thoroughly cleaned to ensure the removal of interfering substances.
Detergents may be used if cleaning is followed by thorough rinsing with distilled water.
The apparatus requirements are as follows:
1. Test tubes, 16 x 150 mm
2. Test tube rack
3. Reagent bottles, two 100-ml, one 200-ml, two 250-mi, five 500-ml (one of which is a dark
bottle), 1 one 1 ,000-ml
4. Dropper bottles
5. Volumetric flasks, one 100-mi, two 200-ml, one 250-ml, two 500-ml, seven 1 ,000-ml
6. Graduates, 10-mi, lOO-ml, and 500-mi
7. Balances, trip or triple beam and analytical
8. Filter paper
9. Pipets, five 1-ml, one 5-mi, one 50-ml
REAGENTS
Introduction
Since these preliminary tests are qualitative in nature, the concentrations of all reagents except the
standard solutions are approximate. The analyst should prepare the standards with care, however, as
they serve to familiarize him with the hue and density of color produced by the limit concentration
of impurities.
Chemical Requirements
The following chemicals are ACS, reagent grade:
1. Barium chloride
2. Sodium sulfite
3. Concentrated hydrochloric acid
4. Potassium iodide
5. Potassium dichromate
6. Thyodene
7. Sodium nitrite
8. Acetic acid, glacial
17
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METHODS OF SOLID WASTE TESTING
9. Sulfanilic acid
10. 1-Naphthyiamine (or l-naphthylamine hydrochloride or N-(1-naphthy) ethylenediamine di-
hydrochloride)
11. Ferrous sulfate, crystalline
12. 1,10 Phenanthroline (or 1,10 dipyridyl)
13. Femc chloride, lump
14. Hydroxylamine hydrochloride
15. Iodine
16. Concentrated sulfuric acid
17. Sodium chloride
18. Silver nitrate
Preparation of Solutions
All solutions are prepared with distilled water that (a) was distilled from a block-tin or all-glass still,
(b) contains less than 0.01 mg per liter copper, and (c) is free of chionne, chioramines, caustic
alkalinity, organic materials, and acids. The solutions are prepared in the following manner:
1. Group A—standard stock solution: Dissolve 0.3 697 g Na 2 SO 4 in a 1 ,000-mi flask with distilled
water and dilute to the mark. This stock solution contains 250 ppm sulfate ion and is stable for
at least 1 week.
2. Group A—standard working solution: Pipet 1 ml of stock solution into a 500-mi flask and dilute
to the mark. This working solution contains 0.5 ppm sulfate ion and should be prepared the
same day it is to be used.
3. Group A—test solution: Dissolve 5 g BaCi 2 in a 500-mi reagent bottle with distilled water and
dilute to about 500 ml. This solution is stable for at least 1 week.
4. Group B—standard stock solution: Dissolve 0.3936 g Na 2 SO 3 in a 1,000-mi flask with distilled
water and dilute to the mark. This stock solution contains 250 ppm sulfite ion and should be
prepared the same day it is to be used.
5. Group B—standard working solution: Pipet 1 ml of stock solution into a 250-mi flask and
dilute to the mark. This working solution contains 1.0 ppm sulfite ion and should be prepared
the same day it is to be used.
6. Group B—test solution No. 1: Prepare daily by dissolving 2 g KI in a 250-ml reagent bottle with
about 100 ml of distilled water. Add 10 ml conc. HC1 and dilute to about 250 ml.
7. Group B—test solution No. 2: Dissolve 2 g K 2 Cr 2 07 in a 500-ml reagent bottle with distilled
water and dilute to about 500 ml. This solution is stable for about 1 week.
8. Group C—standard stock solution: Dissolve 0.6000 g NaNO 2 m a 1,000-mi flask with distilled
water and dilute to the mark. This stock solution contains 400 ppm nitrite ion and should be
prepared the same day it is to be used; if stored in a refrigerator, however, the stock solution is
stable for 1 week.
9. Group C—standard working solution: Pipet 1 ml of stock solution into a 100-mi flask and dilute
to the mark. This working solution contains 4.0 ppm nitrite ion and should be prepared the
same day it is to be used.
10. Group C—test solution: Dissolve 10 ml acetic acid, 10 g suifanilic acid, and I g 1-naphthylamine
(or 1 -naphthylamine hydrochloride or N-( I -naphthy)ethylenediamine diliydrochloride) in a
I ,000-ml reagent bottle with distilled water and dilute to about 1,000 ml. Prepare daily.
ii. Group D—standard stock solution: Dissolve 0.9955 g FeSO 4 7H 2 0 in a 1,000-mi flask with
18
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Alsterberg Modification of Win kier Method for BUD
distilled water and dilute to the mark. This stock solution contains 200 ppm ferrous iron and
should be prepared the same day it is to be used.
12. Group D—standard working solution: Pipet 1 ml of stock solution into a 200-mi flask and dilute
to the mark. This working solution contains I ppm ferrous iron and should be prepared each day
it is to be used.
13. Group D—testsolution DissolveS g 1,10 phenanthroline (or 1,10 dipyridyl) in a 500-mi reagent
bottle with distilled water and dilute to about 500 ml. Prepare daily.
14. Group E—standard solution No. I: Dissolve 0.9676 g FeC I 3 ’ 6H 2 0 in a I ,000-ml flask with
distilled water and dilute to the mark. This No. I standard solution contains 200 ppm ferric iron.
Prepare daily.
15. Group E—standard solution No. 2 Pipet 50 ml of the above No. I standard solution into a
200-mi flask and dilute to the mark with distilled water. This No. 2 standard solution contains
50 ppm ferric iron. Prepare daily.
16. Group E—test solution: Dissolve 5 g hydroxylamine hydrochloride in a 500-ml reagent bottle
with distilled water and dilute to about 500 ml. Prepare about once each week.
17. Group F—standard solution. Dissolve about 1 g iodine in distilled water and dilute to 1,000 ml.
Prepare about once each week.
18. Group F—test solution Prepare daily by dissolving 2 g KI in a 250-ml reagent bottle with about
100 ml of distilled water. Add 5 ml conc. H 2 SO 4 and dilute to about 250 ml.
19. Group G—standard stock solution: Dissolve 0.8243 g NaCI in a 500-ml flask with distilled water
and dilute to mark. This stock solution contains 500 ppm chloride ion and is stable for about I
week.
20. Group G—standard working solution: Pipet 1 ml of stock solution into a 500-ml flask and dilute
to the mark. This working solution contains 1.0 ppm chloride ion and should be prepared the
same day it is to be used.
21. Group G—test solution: Dissolve 5 g AgNO 3 in a 500-mi, dark reagent bottle with distilled water
and dilute to about 500 ml. Prepare about once each week.
22. Group H—standard solution: Either the standard solution of Group F or the standard stock
solution of Group G may be used.
23. Group H—indicator solution. Dissolve 5 g Thyodene in a 100-mi reagent bottle with distilled
water and dilute to about 100 ml. This solution is stable for several weeks.
24. Group H—test paper: Impregnate l/4-in.-wide strips of ordinary filter paper with Group F test
solution and indicator solution immediately before use.
STANDARDIZATION
No special standardization is needed. The standard solutions mentioned under “Reagents” are
analyzed along with the unknown samples, however. This allows the analyst to compare the color of
an unknown to the color mtensity of the known impurity at maximum tolerable concentration.
SAMPLE ANALYSIS
Test for Group A substances (sulfates).
Procedure Comments
1. Pour equal volumes of sample (or standard 1. A cloudy white solution indicates Group A
working solution) and test solution into test and/or Group B.
tube.
19
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METHODS OF SOLID WASTE TESTING
Test for Group B substances (thiosulfates, sulfites).
Procedure
Procedure
Test for Group D substances (ferrous salts).
Comments
Procedure
Comments
1. Pour equal volumes of diluted, aerated sample
and test solution into test tube.
1. a) Test may first be performed with undiluted
sample.
b) Use dilution employed in DO determination.
c) Use Group D standard working solution as
a guide.
1. Pour equal volumes of sample and test solution
No. 1 into test tube. If sample turns a cloudy,
whitish yellow, stop at this point. If sample
remains clear, go on to step 2.
2. Add a small amount of Thyodene powder to
this clear mixture and to another test tube
with an equal volume of test solution No. 1.
If sample solution remains clear, go to step 3.
3. Perform a dropper titration with each solution,
using test solution No. 2 until each solution
has the yellow color of the test solution No. 2.
4. If only a slight amount of sulfite-type sub-
stance is present, repeat test with aerated,
diluted sample.
Test for Group C substances (nitrites).
1. Pour equal volumes of the diluted, aerated
sample and test solution into test tube.
2. After a few minutes, look for the red color of
an azo dye.
Comments
1. a) Use sulfite standard working solution as a
guide.
b)A cloudy, whitish yellow indicates thio-
sulfates or the like.
2. a) Test solution No. 1 may turn pale blue
because of some iodine created during
preparation of the solution.
b)If mixture with sample turns pale blue,
sulfite is not present.
3. a) The test solution No. 1 should turn yellow
with only I to 2 drops of titrant.
b)If the sample solution turns blue with ito
2 drops of titrant, and yellow after several
drops, sulfite-type substances are present.
The amount of titrant used for the sample
mixture must exceed the amount needed
for test solution No. I for sulfite-type
substances to be present.
4. Sulfite-type substances may react and dis-
appear upon aeration.
1. a) Test may first be performed with undiluted
sample.
b) Use dilution employed in DO determination.
2. a) Reagent reference blank may be needed.
b) Use nitrite standard working solution as a
guide for elimination of nitrite concentra-
tion.
c) If Group B is present, this test shows a
negative result.
20
-------
Alsterberg Modification of Win kier Method for BOD
2. After a few minutes, inspect the solution for
the formation of an orange-red color, indica-
tive of the presence of ferrous salts.
Test for Group E substances (ferric salts).
Procedure
1. Treat a portion of sample (standard solutions
No. 1 and No. 2 and diluted, aerated or
non-diluted quench water) as directed in steps
1 and 2 of test for Group D substances.
2. Add to each test tube an equal volume of
test solution.
3. After a few minutes, inspect the solution for
the formation of an orange-red color that is
deeper than the color formed in the Group
D test.
Test for Group F substances (residual chlorine).
Procedure
I. Pour equal volumes of undiluted sample (or
standard) and test solution into a test tube.
If the sample mixture turns a cloudy, whitish
yellow (as in Group B test), stop at this
point. If the solution is clear, go to step 2.
2. To the clear sample and reagent blank add a
small amount of Thyodene powder.
3. If a blue color forms that is deeper than the
blank, a chlorine-like substance is present.
4. If the test is positive, repeat with a diluted,
aerated sample.
Test for Group G substances (chlorides).
Procedure
I. Pour equal volumes of the diluted, aerated
sample (or standard) and test solution into
test tube.
2. a) Sample may need filtering to see to color.
b)If Group B is present, this test shows a
negative result.
c) The interference from nitrite ion may be
eliminated by adding an excess of test
solution.
Comments
1. May use same sample portion employed m the
test for Group D substances.
3. a) Comments for step 2 of the Group D test
apply here also.
b) If the intensity of the color is greater than
standard solution No. I but less than No.
2, the Modified Winkler Method can be
employed, provided the potassium fluoride
is used.
Comments
1. a) More readily available iodine may be used
as a standard instead of chlorine.
b) Use a reagent (test solution) blank.
c) Cloudy, whitish yellow is indicative of
thiosulfate.
2. Test solution may turn pale blue because of
some iodine created during preparation of the
solution.
4. Chlorine-like substances may dissipate upon
aeration.
Comments
I. Cloudy white solution indicates Group G. A
heavy white precipitation indicates possible
interference from Group H substances.
21
-------
METHODS OF SOLID WASTE TESTING
Test for Group H substances (released chlorine).
Procedure Comments
1. Fifi half a test tube with sample (or standard). I. This test need only be performed if the test
sample showed no Group F substances and
high amounts of Group G substances.
2. Pour 1 to 2 ml of concentrated H 2 SO 4 into a
test tube.
3. Immediately place test paper into upper half
of test tube.
4. After a few minutes, look for the paper to 4. Blue color indicates a free halogen, which
turn blue. interferes with the Modified Winkler method.
BIBLIOGRAPHY
1. American Public Health Association, Amencan Water Works Association, and Water Pollution
Control Federation. Nitrogen (Nitrite). 1w Standard methods for the examination of water and
wastewater. 12th ed. New York, Amencan Public Health Association, Inc., 1965. p. 205-208.
2. Amencan Public Health Association, American Water Works Association, and Water Pollution
Control Federation. Phenanthroline method. In: Standard methods for the exammation of water
and wastewater. 12th ed. New York, Amencan Public Health Association, Inc., 1965. p. 1 56-159.
3. Amencan Public Health Association, American Water Works Association, and Water Pollution
Control Federation. Sulfate. In: Standard methods for the examination of water and wastewater.
12th ed. New York, Amencan Public Health Association, Inc., 1965. p. 287-296.
4. Feigl, F. Free halogens. In: Qualitative analysis by spot tests. 3d. ed. New York, Elsevier
Publishmg Company, Inc., 1946. p. 276.
5. Feigl, F. Spot tests in inorganic analysis. 5th ed. New York, Elsevier Publishing Company, Inc.,
1958.
22
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THE DISSOLVED OXYGEN ANALYZER
(WESTON & STACK, INC., MODEL 300-B)
METHOD FOR DETERMINING THE
BOD OF INCINERATOR QUENCH WATER
Donald L. Wilson*
INTRODUCTION .. . 3
DISCUSSION 3
APPARATUS 4
Requirements 4
Preparation and Maintenance 5
Probe 5
Membrane Installation 5
Detection of Membrane Perforation 9
Servicing a Contaminated Probe 9
Glass and Plastic Apparatus 9
Recharging Batteries 10
REAGENTS 10
Chemical Requirements 10
Preparation of Solutions 11
SAFETY PRECAUTIONS 11
CALIBRATION 11
Zero Adjustment of Amplifier 11
Temperature Compensation of the Probe’s Output 12
Temperature Scale 12
Regular Adjustment of the Bridge Potential 12
Special Adjustment of the Bridge Potential 14
Probe 15
Various DO Saturation Levels 15
System of Known DO Depletion Capability 17
ANALYSIS OF SAMPLES 18
Sample Collection 18
Site of Collection 18
Sample Size and Container 18
SRe ch Chemist, Solid Waste Research Laboratory, National Environmental Research
Center, Cmcinnati.
-------
METHODS OF SOLID WASTE TESTING
Sample Preservation and Shipment . 18
Sample (and Blank) Preparation 19
Adjustment for Nitnfication Process 19
Adjustment for Residual Chlorine 19
Dilution and Aeration 19
Determination of the DO Concentration 20
CALCULATIONS 21
BOD of Dilution Water 21
BOD of Quench Water 21
METHOD EVALUATION 22
Precision 22
Accuracy 22
Sensitwity 22
BIBLIOGRAPHY 23
2
-------
Dissolved Oxygen Analyzer for BOD
INTRODUCTION
The analysis of an aerated, diluted sample for its BOD involves the determination of its dissolved
oxygen (DO) content before and after an incubation period. The difference between the initial DO
and the final oxygen content represents the oxygen demand of the sample.
The oxygen demand of incinerator quench water* (or similarly polluted water) is exerted by three
classes of materials: (a) carbonaceous organic material usable as a food source by aerobic organisms;
(b) oxidizable nitrogen derived from nitrite, ammonia, and organic nitrogen compounds that serve as
food for specific bacteria (e.g., Nitrosomonas and Nitrobacter); and (c) certain chemical reducing
compounds (e.g., ferrous iron, sulfite, and sulfide) that will react with molecularly dissolved oxygen.
Since the oxidation of nitrogenous materials may proceed at a variable rate, the nitrification process
is inhibited, thus restricting the BOD determination to the organic carbon present. The water sample
is acidified to pH 2 to 3 and subsequently neutralized to accomplish the inhibition of the nitrification.
Complete stabilization of a given sample may require an overly long incubation period for practical
purposes. The 5-day incubation period has been accepted as standard. For certain industrial wastes,
however, it may be advisable to determine the oxidation curve. Conversion of data from one incuba-
tion period to another can only be made if such special studies are carried out. Studies in recent years
have shown that the exponential rate of carbonaceous oxidation at 20 C rarely has a value of 0.1, but
may vary from less than one-half to more than twice this value. This fact usually makes it impossible
to calculate the ultimate carbonaceous demand of a sample from 5-day BOD values, unless the
exponential-rate value has been determined on the contaminated water under consideration.
Since incinerator quench water may contain many variables that affect the Winkler Method of
analysis, the Dissolved Oxygen Analyzer Method is recommended for BOD analysis of all quench
water samples. The Alsterberg (Azide) Modification of the Winkler Method is recommended for
standardization of the Analyzer using the relatively pure dilution water. Preliminary tests, which show
the validity of the Winkler DO value, are discussed in the chapter on the Winkler Method.
The sampling location at each site is very important in the evaluation of the data and should be
chosen on the basis of obtaining the most representative sample.
DISCUSSION
The Weston & Stack Dissolved Oxygen (DO) analyzer uses a specially designed probe to measure
accurately and quickly the amount of dissolved oxygen in gas streams and liquids. The probe is con-
structed of cast epoxy and is separated from the sample by a semipermeable membrane.
The analyzer is powered by AC or internal batteries and is ruggedly constructed and moisture-proof
to facilitate laboratory or field use. Interferences in water or gas samples are minimal with this
instrument. Hydrogen sulfide does not interfere, but will eventually corrode the lead anode. The
probe will then require cleaning. Dissolved or suspended solids will not affect the probe, provided the
analyzer is calibrated using a similar type of sample to account for partial pressure changes. Labora-
tory tests have reaffirmed that this probe is not affected by ferrous or ferric iron, sulfite,or nitrite.
Since temperature affects the rate of diffusion of dissolved oxygen through the Teflon membrane,
the probe output for a given concentration of dissolved oxygen is a function of the temperature. A
secondary resistance to oxygen diffusion exists at the Teflon-aqueous sample interface. The inter-
facial resistance is of minor significance when the sample is vigorously agitated to produce a high
degree of turbulence, a condition that is also necessary for the temperature compensation to function
satisfactorily.
Temperature compensation in the Weston & Stack analyzer is accomplished by an operational
amplifier. A thermistor (a resistor whose resistivity varies intensely with temperature) and a resistance
Quench water refers to water that has been used to cool the non-combustibles after emergence from the furnace.
3
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METHODS OF SOLID WASTE TESTING
network introduced into the feedback circuit for the amplifier provide suitable multiplication so
that temperature effect on the probe is limited to ± 2 percent over the temperature range of 0 to 50 C
when suitable turbulence is provided.
The probe must be calibrated by the Winkler Method to enable the analyst to read the true ppm
DO directly from the scale of the mstrument. This calibration, although normally needed only once a
month, preferably should be checked at the beginning and end of each 5-day incubation period.
Although a probe output can be obtained for any element or compound that diffuses through the
semipermeable (Teflon) membrane and is reduced at a potential of —0.578 volts or less, interferences
of this nature appear to be infrequent. Sulfite, nitrite, ferrous and ferric iron, and other reducing and
oxidizing substances that normally interfere with the Winkler Method apparently do not affect the
output of this probe. There are a few substances, however, that do affect the sensitivity of the probe
over a period of time. Hydrogen sulfide and chlorine, although not detected by the probe, will react
with the lead anode and cause a decline in sensitivity. Greases and oils will coat the semipermeable
membrane, increase the diffusion resistance, and decrease the probe output. Variations in dissolved
solids will alter the partial pressure of oxygen in the aqueous samples and hence the probe output.
The calibration and use of the instrument should be accomplished with these facts m mind.
APPARATUS
Requirements
1. Analyzer, Weston & Stack, Model 300-B, DO Ranges: 0-1.5 ppm and 0-15 ppm, temperature
compensation and temperature readout; AC powered with internal combination power-supply
and battery-charger
2. Probe, Weston & Stack, Model A-30 BOD agitator-thermistor assembly
3. Accessory kit containing membranes, electrolyte, syrmge, manual, recorder, and plug
4. Extra membranes, Teflon, ½ mm thick, 3 in. sq, 24 per package
5. Graduates, 50-mi, I-liter, and 2-liter
6. Beakers, 250-mi, 2-liter, and 3-liter
7. Siphon tubing
8. Rubberbands
9. BOD bottles, 300-mi capacity
10. Analytical balance
II. Volumetric flasks, 1 00-ml, 500-mi, and five 1-liter
12. pH paper, range 2 through 9
13. Air incubator or waterbath, thermostatically controlled at 20 C ± 1 C
14. Carboy, polyethylene nalgene, wide-mouth, two 2-gal (7 liters or more)
15. Test tube with 14 mm I.D.
16. Magnetic stirrer with Teflon-coated stirring bar
17. Stopwatch or accurate watch with second hand
18. Pipets, graduated or volumetric, two 2-mi
19. Reagent bottles, resistant glass, narrow-mouth with ground glass stoppers, 1-liter capacity, at
least one amber colored
20. Sample collection bottles, polyethylene (or similar unbreakable bottle) with narrow mouth and
tightly fitting caps, about 1-liter (or 1-qt) capacity, stenie
4
-------
Dissolved Oxygen Analyzer for BOD
Procedure
I. Remove the probe shield and thermistor
housing, electrolyte fill screw, and old mem-
brane from the probe.
2. Wrap a rubberband around a test tube,
having a 14-mm l.D.
3. Hold the test tube in an upright position by
encircling it with the fingers and thumb of
one hand.
4. Place one membrane sheet over the top of
the test tube and fist.
5. Gently depress the membrane about I m.
into the tube.
6. Pour electrolyte mto the depression.
7. Press the membrane down into the tube
with the probe.
8. Slip the rubber band off the tube and over
the membrane to a point ½ in. above the
holes near the tip of the probe.
9. Carefully draw up the membrane to provide
a snug fit.
10. Wrap a second rubber band as tightly as
possible just above the holes.
11. Remove the first (top) rubber band.
12. Trim membrane close to and above the
second rubber band.
13. Use the syringe to completely fill the cavity
in the probe with electrolyte.
14. Shake and tap the probe so that air bubbles
will escape from the fill hole.
15. Replace the fill screw and probe shield.
16. Connect the probe to the readout instrument
and turn the meter to “ON” and the selector
switch to “DO-MULl I.”
Comments
1. See Figure 1 for identification of probe
components.
2. See Figure 2 (a-f) for the diagrammatic
representation of steps 2, 6, 7, 9, 10 and 12.
3. The open end of the test tube should be
flush with the forefinger.
6. See preparation of electrolyte.
7. Do this carefully so the membrane is not
damaged. Keep air bubbles from being
trapped between the probe and membrane.
8. Remove the tube.
9. It is necessary to have a close, smooth fit
over the platinum electrode without stretch-
ing or tearing the membrane.
10. Apply securely to prevent leakage of elec-
trolyte from membrane.
13. The probe should be held upright.
14. Thumb is held over fill hole during shaking.
16. See Figure 3 for the location of components
of the instrument.
21. Regulator, 2-stage, with cylinder valve outlet for nitrogen (CGA No. 580)
22. Ice chest, capable of holding several 1-liter sample collection bottles and maintaining a 5 C
temperature for 24 hr.
Preparation and Maintenance
Probe
Membrane Installation. Since the performance of the probe is dependent upon a properly installed
membrane, the analyst should exercise great care in 1 performing the following instructions.
S
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METHODS OF SOLID WASTE TESTING
BODY
Figure 1. The probe. (Reproduced with permission of Weston & Stack, Inc.)
CORD RESTRAINER
SERVICE CAP
ELECTROLYTE
FILL SCREW
PIN HOUSING’
P1 NS
LEAD
PLATINUM CATHOO
AND
I NG
6
-------
Dissolved Oxygen Analyzer for BOD
a. Step #2: PosItion of the
rubber band on the test
tube.
c. Step #7: Pressing the mem-
brane down into the tube
with the probe.
Figure 2. Membrane installation
permission of Weston
d. Step #9: Drawing up the
membrane to provide a snug
fit across the face of the
platinum tip.
f. Step #12: The probe after
the removal of the upper
rubber band and the trim-
ming of the membrane.
procedure. (Drawings reproduced with
& Stack, Inc.)
b. Step #6: Pouring the elec-
trolyte into the depressed
membrane.
e. Step #10: Irapping a second
rubber band around the probe
Just above the small holes.
7
-------
FRONT
BACK
Figure 3. Front and back views of the Analyzer. (Reproduced with permission
Weston & Stack, Inc.)
00
INPUT
rr
0
0
0
t n
rn
-
C )
-------
Dissolved Oxygen Analyzer for BOD
17. Hold the probe with platinum electrode up
and shake it vigorously.
Procedure
I. Hold the membrane end of the probe in a
beaker containing clear water.
2. While looking through the water towards a
light source, search for a small stream (dif-
fused light) of electrolyte floating through a
hole in the membrane.
Procedure
1. Remove the probe shield and thermistor
housing, membrane, electrolyte fill screw,
and probe service cap screw (may use
quarter coin).
2. Turn probe upside down and shake out the
lead anode.
3. Clean the lead anode by immersing it in
warm, 10-percent NaOH (or HCI) solution;
then rinse thoroughly with distilled water.
4. Clean the platinum electrode and the inside
of the probe body with 6 N (1 1 )HC1, then
rinse thoroughly with distilled water.
5. Polish the platinum cathode with a soft
tissue.
6. Reassemble lead anode and probe service cap.
7. Install a new membrane as previously
directed.
17. If the meter needle oscillates, air bubbles
are present and steps 14 through 17 must be
repeated.
Comments
1. If the membrane was installed recently, the
probe should be nnsed thoroughly to remove
electrolyte that may be trapped in the folds
of the membrane.
2. If a hole is detected, a new membrane must
be installed.
Comments
4. The lead anode will not seat properly or
make contact if the lead ring inside the probe
is not completely clean.
6. Use a small amount of silicon grease on the
threads and the “0” ring.
7. See Membrane Installation.
Glass and plastic apparatus
Sampling bottles, tubing, containers, and the like must be thoroughly cleaned (sterile) toensure the
removal of materials capable of exerting a BOD. Detergents may be used if cleanmg is followed by
thorough nnsing with distilled water.
Detection of Membrane Perforation. When a hole develops in the membrane, the response rate of
the probe decreases as the electrolyte is diluted and the cathode is poisoned. To ensure the proper
performance of the probe, frequent inspections of the membrane should be performed.
Servicing a Contaminated Probe. After sitting for a few months with electrolyte in it, the inner
parts of the probe become contaminated and may not allow any calibration adjustment to be made or
cause the readout needle to drift downward. The procedure for cleaning a probe is as follows:
I. See Figure 1 for identification of probe parts.
3. All the yellow deposit must be removed.
9
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METHODS OF SOLID WASTE TESTING
Recharging batteries.
The Weston & Stack Dissolved Oxygen Analyzer, Model 300-B, is provided with an internally
combined AC power supply and battery charger. The instrument can be operated in the laboratory
directly on 110 AC or in the field, using the rechargeable nickel cadmium batteries. When the
instrument is employed in the field, a record of the hours of amplifier usage should be maintained.
Before each standardization of the instrument in the laboratory or utilization in the field, the analyst
should then check his record. If the amplifier usage 20 hr, the batteries should be recharged as
follows:
Procedure
1. While operating the instrument on 110 AC,
place the selector switch on “DO-Mult 1.”
2. Turn the power switch to “OFF/CHG.”
3. Determine if a charge is coming from the
batteries.
4. Place selector switch in the “Transit” posi-
tion and recharge the batteries for a maxi-
mum of 10 hr.
The following chemicals are ACS, reagent grade:
Comments
1. See Figure 3 for the location of the compo-
nents of the instrument.
3. The meter needle should respond and pos-
sibly oscillate. If no response occurs, check
the 12-volt batteries and connections. Re-
place components if necessary.
4. Rechargeable batteries last about 3 years.
Potassium iodide
Sodium sulfite
Sodium hydroxide
Hydrochloric acid, concentrated
Sulfuric acid, concentrated
Phosphate buffer solution, pH 7.2 (or prepared)
Potassium phosphate, monobasic
Potassium phosphate, dibasic
Sodium phosphate, dibasic, heptahedrate, crystalline
Ammonium chloride
Magnesium sulfate, crystalline
Calcium chloride, anhydrous
Ferric chloride, lumps
Manganese(ous) sulfate, monohydrate
Potassium hydroxide
Nitrogen, 99.9 percent pure
NOTE: The 30-volt battery is not rechargeable and lasts about 6 months.
REAGENTS
Chemical Requirements
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
10
-------
Dissolved Oxygen Analyzer for BOD
Preparation of Solutions
The water employed in the preparation of solutions must (a) be distilled from a block-tin or all-
glass still, (b) contain less than 0.01 mg copper per liter, and (c) be free of chlorine, chloramines,
caustic alkalinity, organic materials, and acids.
Solutions are prepared as follows:
1. Electrolyte solution: Dissolve 50.0 g of potassium iodide in distilled water and dilute with same
to 100 ml. Store solution in a dark brown bottle (8-oz bottles of this solution may be purchased
from either the Weston & Stack Company, 1426 Lewis Lane, West Chester, Pennsylvania,
19380, or their Ohio representative, Henry P. Thompson Co., 4866 Cooper Road, Cincinnati,
Ohio, 45242).
2. Sodium sulfite solution: Dissolve about 5 g of sodium sulfite in 500 ml of distilled water.
3. Sodium hydroxide solution, approximately 1 N: Dissolve 41.6 g NaOH in distilled water and
dilute with same to 1 liter.
4. Sulfuric acid solution, approximately 1 N: Cautiously add 28 ml of concentrated H 2 SO 4 to
distilled water and dilute with same to 1 liter.
5. Dilution Water: To 1 liter of distilled water at 20 C, add 1 ml of each of the following solutions:
phosphate buffer solution, pH 7.2; magnesium sulfate solution (22.5 g Mg SO 4 7112 0 per liter
of solution); calcium chloride solution (27.5 g anhydrous CaC1 2 per liter of solution); ferric
chloride solution (0.25 g FeC1 3 6H 2 0 per liter of solution).
6. Sodium hydroxide solution, 10 percent (w/v): Dissolve 10.0 g NaOH’in distilled water and dilute
with same to 100 ml.
7. Phosphate buffer solution, pH 7.2, may be purchased in prepared form or may be prepared as
follows: Dissolve 8.5 g KH 2 P0 4 , 21.75 g K 2 HPO 4 , 33.4 g Na 2 HPO 4 7H 0, and 1.7 g NH 4 Cl in
500 ml distilled water and dilute with same to 1 liter. The pH of this buffer should be 7.2
without further: adjustment.
8. Hydrochloric acid solution, 10 percent (v/v): Cautiously add 10 ml concentrated HCI (sp. gr.
1.19) to 75 ml distilled water and dilute with the latter to 100 ml.
9. Hydrochloric acid solution, 5 N: Cautiously add 42.8 ml concentrated HC1 (sp. gr. 1.19) to
40 ml distilled water and dilute with the latter to 100 ml.
10. Manganous sulfate solution, 0.25 M: Dissolve 21.13 g MnSO 4 H 2 0 in distilled water and dilute
with same to 500 ml.
11. Potassium hydroxide solution, 0.5 M: Dissolve 14.03 g KOH in distilled water and dilute with
same to 500 ml.
SAFETY PRECAUTIONS
Follow general laboratory safety rules. This method has no pronounced safety hazards.
CALIBRATION
Zero Adjustment of Amplifier
To ensure the proper amplification of the temperature-compensated signal from the probe, the
output of the amplifier should first be adjusted to zero while the probe is inserted into a solution
containing no dissolved oxygen. The procedure is as follows:
11
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METHODS OF SOLID WASTE TESTING
Procedure
I. Set the selector switch to “Transit” and
power switch to “OFF/CHG.”
2. Using the unmarked screw on the meter
face, set the meter needle to zero.
3. Place the probe in the sodium sulfite solution
for 2 mm or longer.
4. Set the selector switch to “DO-Mult 1” and
turn the Analyzer to “ON” for a period of
at least 2 mm.
5. Adjust the meter reading to zero using the
zero-adjustment screw (marked “zero”) on
the front of the instrument case.
6. Remove the probe from the sulfite solution
and rinse the membrane thoroughly with
distilled water.
Comments
1. See Figure 3 for the location of components
of the instrument.
2. Adjustment may not be necessary.
3. Use a regular BOD bottle to contain the
solution.
4. The agitator should be employed.
6. Keep the probe m BOD bottle of clean,
distilled water when not in use.
Temperature Compensation of the Probe’s Output
A thermistor (a resistor whose resistivity varies intensely with temperature) and a resistance net-
work introduced into the feedback circuit of an operational amplifier provide the temperature
compensation of the probe’s output. The compensation is accurate to ± 2 percent over a sample
temperature range of 0 to 50 C. An adjustment is necessary, however, if sample temperature varies
more than 5 C from the temperature of the probe. The adjustment is as follows:
Procedure Comments
1. If the probe and sample are not essentially
the same temperature (within 5 C), move
front-left switch (marked >5 & <5) to the
>5 position.
2. If the probe and sample are near the same
temperature (within 5 C), move above switch
to the <5 position.
Regular adjustment of the bridge potential.
Temperature Scale
The temperature scale of the instrument is calibrated at the factory and in normal operation should
not require re-calibration. The thermometer circuit is, however, designed as an unbalanced bridge
whose potential is supplied by a 30-volt battery. Regular (each day of use) adjustments of the bridge
potential should,therefore, be performed as follows:
Procedure
1. Turn the selector switch to Temperature.
Comments
1. See Figures 3 and 4 for the location of the
switch and other components of these
instructions.
1. a) See Figure 3 for the location of the switch.
b) Analyzer may be used to check tempera-
tures of room and sample (see Sample
Analysis).
2. Normally this step is applied.
12
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Dissolved Oxygen Analyzer for BOD
PROBE
INPUT
Figure 4. Side view of Analyzer. (Reproduced with permission of Weston &
Stack, Inc.)
LEFT SIDE
( fliW. Ab )
cHc. cA )
RIGHT SIDE
13
-------
METHODS OF SOLID WASTE TESTING
2. Press the Temp Test button.
3. While pressing the Temp Test button, adjust
the potentiometer by turning the Temp Adj.
screw until the needle indicates exactly 50 C.
4. Release the Temp Test button.
Special adjustment of the bridge potential.
2. The resistor’s activity is equivalent to the
thermistor’s resistance at 50 C.
3. If the needle cannot be adjusted to 50 C, the
probe may need cleaning or the 30-volt
battery may need replacement.
4. The Analyzer is now indicating temperature
correctly.
Procedure
1. With the power switch on OFF/CHG and the
selector switch on Transit, adjust the meter
to zero using the unlabeled screw on the
meter face.
2. Assemble probe and thermistor housing.
3. Connect the probe cable to connector.
4. Remove the back cover of the instrument by
removing all eight screws.
5. Locate the three potentiometer-adjustment
screws inside the unit.
6. Immerse the probe in a 0 C bath consisting
of distilled water and finely crushed ice;
then agitate the probe assembly.
7. While still agitating the probe, turn the
potentiometer screw that is farthest to the
right (facing the back of the Analyzer) until
a reading of 0 C is obtained on the meter.
8. Repeat the procedure with a water bath at
SOC except adjust meter with the Temp Adj
screw ONLY.
9. While pushing on the Temp Test button,
turn the potentiometer screw that is second
from the right (as you face the back of the
Analyzer) until the meter reads 50 C.
Comments
1. See Figures 3 and 4 for the location of the
components of the instrument.
5. Facing the back of the unit, four small
screws, aligned in a horizontal line, are
immediately beneath the handle attachment.
The multiplier adjustment screw is the one
on the far left; the others are the potentiom-
eter screws.
6. a) The temperature of the bath should be
checked with an accurate thermometer.
b) Equilibrium should be reached after 5 to
10 mm.
8. Temp Adj screw is located on the left side
(Figure 4) of the instrument case.
When a resistance component in the temperature bridge is replaced, the temperature scale must be
recalibrated. The special potentiometer adjustments are as follows:
3. Connector located on right side of instrument
case.
14
-------
Dissolved Oxygen Analyzer for BOD
10. Using a 25 C water bath, compare the meter 10. The third potentiometer adjustment screw,
temperature reading with that of an accurate which is located right of the multiplier
thermometer. If there is an error, readjust adjustment screw, should never be turned.
the potentiometer adjustment screw that is
farthest to the right (as you face the back of
the Analyzer).
Probe
Various DO saturation levels.
Before using the Analyzer, the analyst should calibrate the probe with samples whose DO concen-
trations have been determined by the Alsterberg (Azide) Modification of the Winkler Method.
Calibrate the probe at least once every 3 weeks, preferably each day the Analyzer is used.
The DO probe is a partial pressure device; this means that the transfer of DO through the semi-
permeable membrane is a function of the ratio of DO concentration to DO concentration at satura-
tion. For example, when a salt solution is saturated with oxygen at 20 C, it may contain only 3 ppm
of DO; water, however, when saturated with oxygen at 20 C will contain 9.2 ppm of DO. Since the
rate of oxygen transfer through the membrane would be the same in both cases, the probe output
would be the same. The probe must therefore be calibrated using a liquid similar to the sample to be
analyzed. Since quench water is greatly diluted with dilution water before analysis, the calibration of
the probe should be performed with dilution water before analyzing general quench water samples.
The probe must be calibrated each day it is used. If the membrane has been replaced, the calibration
of the probe will change slowly for a period of about 24 hr before it stabilizes. If the probe is used
during this period, frequent calibration checks will be required.
The probe must be calibrated to the specific turbulence of the system. Since the agitation is built
into the probe assembly of the Weston & Stack BOD Agitator, any calibration that is performed is
applicable to any container or use to which the BOD Agitator may be applied. (Note: The Modified
Winkler Method must be established before the probe can be calibrated.)
Procedure Comments
I. This may be done by pouring the water back
and forth from a graduate to a beaker at
least three times.
2. During the siphoning, the water should be
continuously stirred using a magnetic mixer
and a Teflon-coated magnetic bar.
3. See Determination of the DO Concentration.
5. Determine the DO of the same sample using 5. See Reference 5.
the modified Winkler Method.
6. Using the Analyzer and probe, determine
the DO of the second aerated sample.
1. Saturate a liter of dilution water with
oxygen.
2. Siphon the aerated water from the beaker
into each of 3 BOD bottles.
3. Using the Analyzer and probe, determine
the DO and temperature of one of these
aerated samples.
4. Adjust the calibration screw so that the
Analyzer reads the same ppm DO as observed
in Table 1 for the sample temperature.
4. a) See Figure 3 for the location of Cal screw.
b) An exact DO concentration at 20 C and
zero chloride concentration is 9.1 percent.
15
-------
METHODS OF SOLID WASTE TESTING
TABLE 1
SOLUBILITY OF OXYGEN IN WATER EXPOSED TO WATER-SATURATED AIR*t
Temperature
(C) 0
Chionde
concentration m water (mg/i)
Difference per
100 mg chloride
5,000
10,000 15,000 20,000
Dissolved oxygen (mg/i—ppm)
I
13 8
134
13.1
12 7
12.4
12 1
ii 8
11.5
11 2
11.0
10 7
10.5
10.3
10.1
9.9
9.7
95
9.3
91
89
8.7
8.6
84
83
8.1
8.0
78
77
7.5
7.4
7.3
13.0
12 6
12.3
120
11.7
11.4
11.1
10.9
106
104
10.1
9.9
9.7
9.5
9.3
9.1
90
88
8.6
8.5
83
81
80
7.9
7.7
7.6
74
7.3
7.1
7.0
6.9
12.1
11.8
115
11.2
110
10.7
10.5
10.2
100
98
96
9.4
9.2
9.0
8.8
86
85
83
82
80
7.9
77
7.6
74
73
7.2
7.0
69
6.8
6.6
65
11.3
11.0
10.8
10.5
10 3
10.0
9.8
9.6
94
92
90
8.8
86
85
8.3
8.1
80
78
77
76
7.4
73
7.1
70
6.9
6.7
6.6
6.5
6.4
6.3
6.1
0017
0016
0015
0015
0.0 14
0 014
0.0 14
0013
0013
0.012
0012
0011
0.0 11
0011
0010
0.0 10
0010
0010
0 009
0.009
0.009
0 009
0 008
0 008
0 008
0.008
0008
0 008
0.008
0008
0 008
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
14.6
14 2
13.8
13 5
13.1
12 8
12.5
12.2
11 9
11.6
11.3
11.1
10.8
10 6
10.4
10.2
100
9.7
9.5
9.4
9.2
9.0
88
8.7
85
8.4
8.2
8.1
7.9
7.8
7.6
75
7.4
7.3
72
7.1
7.0
6.9
68
6.7
66
6.5
6.4
63
6.2
6.1
6.0
59
5.8
57
5.6
Source Standard Methods for the Examination of Wastes and Wasrewater 12th ed.
Published, 1965 p. 409 Reproduced by permission, American Public Health Association,
Inc., American Water Works Association, and Water Pollution Control Federation.
tAt a total pressure of 760 mm Hg Under any other barometric pressure,P (mm, orP’,
in.), the solubihty, S’ (mg/i), can be obtained from the corresponding value in the table
by the equationS
S’-S
— 760-p
in which S is the solubthty at 760 mm (29 92 in.) and p is the pressure (mm) of saturated
water vapor at the temperature of the water For elevations less than 3,000 ft and
temperatures belowi25 C, p’can be ignored The equation then becomes.
P - P ’
S —S 760 —S 2992
Dry air is assumed to contain 20.90 percent oxygen (Calculations made by Whipple
and Whipple)
16
-------
Dissolved Oxygen Analyzer for BOD
7. Considering the difference between the
Analyzer and the Modified Winkler Method
with the first aerated sample, adjust the
calibration screw.
8. Determine the DO of the same sample using
the Modified Winkler Method.
9. The third aerated sample is used as a re-check
of the calibration point If the calibration
screw needs more adjustment, then more
aerated sample should be prepared and
analyzed until the calibration screw no
longer needs adjustment.
9. After calibration, the probe should always be
kept in a BOD bottle filled with distilled
water to prevent air bubbles from entering
the probe.
Having performed the above calibration procedure, the probe has now been calibrated at the upper
and lower (zero adjustment of amplifier) DO saturation limits. The Analyzer’s DO measurements are
linear between these limits; however, the analyst may wish to venfy the DO readmgs between these
limits. This may be done by performing the probe calibration as just described except that pure
nitrogen should be allowed to bubble for about 1 5 to 30 mm through the aerated samples before
the DO analysis. This treatment with nitrogen gas will lower the saturated oxygen concentration to
about 3 to 5 ppm. The nitrogen gas will not interfere with the Modified Winkler Method’s DO
analysis.
System of known DO depletion capability.
The calibration procedures described thus far are usually employed only in the laboratory. The
following method is applicable both in the laboratory and in the field where regular calibration
procedures would be difficult to perform. This method uses a system of known oxygen depletion
capability to evaluate indirectly the probe’s response below the saturation level. It is not a true probe
calibration method. The probe or Analyzer must first be calibrated by comparing its values with
those obtained by the Modified Winkler Method, then, while it is being operated in the field, it can
easily be checked for performance by the following method.
Procedure
1. Prepare 3 BOD bottles of aerated water
samples as previously described in the probe
calit ration procedures.
2. Using the analyzer and probe, determine the
DO of one of these aerated samples.
3. Remove the probe and nnse it with distilled
water.
4. Using a volumetric pipet, add 2 ml of 0.25
M manganous sulfate solution to the same
BOD bottle.
Comments
2. See Determination of the DO Concentration.
3. When the probe is not being used, keep it in
a BOD bottle that is filled with distilled
water.
4. The tip of the pipet should be placed
beneath the surface of the sample.
17
-------
METHODS OF SOLID WASTE TESTING
5. Immediately initiate the addition of 2 ml of
0.5 M potassium hydroxide solution; record
the exact time of initiation.
6. After the KOH has been added, stopper the
bottle and mix the contents by repeated
inversions.
7. After not more than 8 mm have elapsed,
insert the probe into the BOD bottle.
8. After exactly 10 mm have elapsed since the
addition of the KOH solution, record the DO
concentration.
9. Repeat steps 2 through 8 with the other two
prepared BOD bottles.
10. Determine the depletion of DO for each
sample by subtracting the final ppm DO from
the initial observation.
5. a) Use a volumetric pipet.
b) Use a stopwatch or other accurate device
to measure exactly the 10-mm reaction
period.
7. Equilibrium is attained in about 2 min.
10. The average depletion should be 3.60 ± 0.25
ppm. If the depletion is not this value, re-
check calibrations; the instrument may have
to be returned to the manufacturer.
Site of collection.
ANALYSIS OF SAMPLES
Sample Collection
When the BOD of quench water is measured to determine the amount of oxidizable wastes that
will be discharged to a sewerage system serving an incinerator facility or to a drainage system
associated with the residue disposal area, the site of sample collection must be chosen with due
consideration. Settlement tanks, surface pools, sewers, and other areas immediately adjacent to the
sewage or drainage system are preferable collection sites.
Sample size and container.
Normally, 50 ml of sample is needed to perform the BOD analysis; however, since various dilutions
may be needed, and a larger sample size may be more representative, it is recommended to collect 1
liter of quench water sample.
The samples should be collected in sterile, unbreakable bottles with narrow mouths and caps that
can be tightly fitted. The sample bottle should be completely filled. All containers must be thoroughly
rinsed, especially if cleaned with a detergent, before they can be reused.
Samples should not be collected on Monday or Tuesday unless the analysts are to work on Saturday
or Sunday (5-day BOD).
Sample Preservation and Shipment
If the sample analysis is to be initiated within 4 hr after collection, sample preservation measures
are not absolutely necessary. Samples should otherwise be placed in an ice chest (or similar container)
18
-------
Dissolved Oxygen Analyzer for BOD
soon after collection so that the samples are maintained in the dark at 5 C. The bottle caps must be
tightly fitted to prevent an increase in oxygen solubility with the reduction in temperature.
Sample shipment to the laboratory should be immediate (via air freight if necessary) to ensure the
initiation of BOD analysis in the laboratory within 24 hr of sample collection. Samples that are more
than 24 hr old should not be analyzed.
Since the Analyzer requires calibration, the laboratory personnel should be informed at least 2 days
before the arrival of samples. Because samples require some preparation before the actual analysis and
since the exact dilution requirements may not be known, samples not at 5 C should be shipped to
the laboratory so that they are received at least 2 hr before the end of the normal working day.
Samples shipped in an ice chest at 5 C and then refrigerated may be analyzed the following day.
Sample and Blank Preparation
Adjustment for nitrification process.
Before analysis, each sample and dilution water blank is treated as follows to inhibit the nitrification
process:
Procedure Comments
1. Place 50 ml of a thoroughly mixed quench 1. The exact volume of quench water sample
water sample in a 250-ml beaker. depends upon the dilution requirements. See
Dilution and Aeration.
2. Using pH paper, check the pH of the sample. 2. Usually the pH is about 11.
3. Using 1 N NaOH or 1 N H 2 SO 4 , adjust the 3. Omit if the sample already has a pH of 2 or 3.
pH of the sample to a range of 2 to 3; main-
tain pH forl5 min.
4. Then neutralize the sample to a pH of 6.5 to 4. Employ the same 1 N solutions as in step 3.
8.3.
Adjustment for residual chlorine.
Chlorine, in concentrations normally found in chlorinated water and sewage effluent, does not
influence the probe output or the determination of oxygen. Chlorine will react with lead and hence
cause the probe sensitivity to decrease after long exposure. Since residual chlorine dissipates when
samples stand for 1 to 2 hr or are well aerated, no adjustments are recommended.
Dilution and aeration.
Prepared samples must be diluted in order to obtain a measurable depletion (2 ppm to 7 ppm) of
oxygen at the end of the 5-day incubation period. Since incinerator quench water usually has a BOD
of 100 to 300 ppm, a suitable or applicable dilution is 50 ml of sample diluted to 2 liters. If the
analyst suspects that the BOD of the quench water differs from the usual value, he should test various
dilutions since the analysis cannot be repeated on the same original sample after the 5-day waiting
period. To obtain more reliable results, 3 BOD bottles should be prepared: 3 for the initial DO, and
the same 3 for the final DO. The final DO values should never be less than 1.0 ppm.
Since the dilution water used in the analysis of each quench water sample may contain a few
oxidizable materials capable of exerting a small BOD, each quench water analysis should include a
19
-------
METHODS OF SOLID WASTE TESTING
blank evaluation, i.e., a determination of the BOD of the dilution water. The observed BOD of the
quench water can then be corrected by subtracting the appropriate proportionate fraction of this
blank value.
The dilution and aeration procedures are as follows:
Procedure Comments
1. Pour the total prepared sample from the 1. Solution still represents 50 ml of original
250-mi beaker into a 2-liter graduate and sample.
dilute to the mark with dilution water.
2. Aerate the sample by pouring it back and 2. The dilution water blank is aerated in a like
forth from the graduate into a 3-liter beaker manner.
at least 3 times.
3. Siphon the diluted, aerated sample or blank 3. The sample should be stirred contmuously,
from the beaker and into 3 BOD bottles. using a magnetic stirrer and a Teflon-coated
magnetic bar.
4. The DO concentration of the sample or 4. a) See Determination of the DO Concentra-
blank in the 3 BOD bottles should be tion.
determined immediately. b) Only two reasonable DO results are needed.
See Precision.
5. Then put the same 3 BOD bottles in an 5. a) During the incubation period, the samples
incubator (or waterbath) and determine should not be exposed to the light.
their DO content after a 5-day incubation b) See Determination of the DO Concentra-
period at 20 C. tion.
c) Only two reasonable DO results are needed.
See Precision.
Determination of the DO Concentration
The Analyzer has the components necessary for operation when shipped. Before attempting
to place the analyzer into service, check to ascertain that a) the membrane is free of holes, b) the
needle zeroes correctly, c) the temperature reading is correct, and d) the probe is free of air bubbles.
Procedure Comments
1. Turn the toggle switch located in the lower 1. In most cases the entire body of the probe is
left-hand corner of the front panel to “<5.” essentially at the temperature of the sample.
If the probe body temperature varies more
than 5 C from the sample temperature, turn
the toggle switch to “<5.”
2. Turn the selector switch to DO-Mult 1.
3. Turn toggle switch located in front lower
right hand corner of case to ON.
20
-------
Dissolved Oxygen Analyzer for BOD
4. Insert probe into BOD bottle.
5. Wait 2 mm for equilibrium and then read the
ppm dissolved oxygen directly on top of
scale.
6. If temperature of the sample is desired, turn
selector switch to Temp and read tempera-
ture (C) directly on the bottom scale.
7. Turn toggle switch (right-hand corner) to
OFF and selector switch to TRANSIT.
4. The agitator should be operating.
5. a) Record value.
b)The absolute value of the difference
between duplicated readings should not
exceed l.96 /2 s,or 0.58 ppm, more than
5 percent of the time. See Precision.
6. a) Record value.
b) Accuracy is ± 1 C.
7. Perform this step if the meter is not to be
used for several hours.
CALCULATIONS
BOD of Dilution Water
The following formula should be employed to calculate the BOD of each individual sample of
dilution water.
where
BODI =D -D 2
BOD 1 = The BOD of dilution water
D 1 = The DO content of initial (before incubation) dilution water
D 2 = The DO content of final (after incubation) dilution water
BOD of Quench Water
The initial DO concentration minus the final DO concentration equals BOD of the diluted sample.
The BOD of the diluted sample times the dilution factor equals the BOD of the original sample.
The dilution factor is found by dividing the original amount of sample taken into the final dilution;
for example, 50 ml of sample diluted into 2 liters gives a factor of 40.
The following formula should be used to calculate the BOD of each individual sample of quench
water.
where
BOD 2 =F [ (D 3 -D 4 )-P 1 (BOD 1 )1
BOD 2 The BOD of quench water
F = The dilution factor
D 3 The DO content of initial (before incubation) quench water
D 4 = The DO content of final (after incubation) quench water
P 1
= The decimal fraction of dilution water used in the BOD analysis
of the quench water
21
-------
METHODS OF SOLID WASTE TESTING
METHOD EVALUATION
Precision
After analyzing a number of quench water samples in duplicate (three determinations were
performed to ensure reasonable duplicate results), the precision of the observations were evaluated by
calculating (using the Olivetti Programma 101) the pooled standard deviation of all observations
except those obtained on samples collected from dump truck drainage. The results of these calcula-
tions are shown as follows:
Precision of the DO Analysis: *
Number of determinationst 82
Pooled standard deviation (s) 1: 0.21
Confidence interval ± 1.96 .. ,/ (s) § ±0.5 8
Precision of the BOD Analysis:
Number of determinations 20
Standard deviation (s) ¶ 0.30
Dilution factor # 40
Confidence interval ± 1.96 (40) S ±23.5
Assistance in the statistical analysis was provided by the Statistical Section of the Office of Solid Waste
Management Programs.
tNormally, at least two initial and three final determinations were made for each sample.
IA pooled standard deviation was computed for all determinations. It was assumed that there was no
statistically significant difference between initial and final variances; i.e., homogeneity of the variances
was assumed.
§ The absolute value of the difference between duplicated readings should not exceed 1 .96 / (s), or
0.58 ppm, more than 5 percent of the time. The covariancebetween the duplicated readings was ignored.
¶The formula for the standard deviation of the difference between Initial and final DO readings is
S = V’(s2 + s ). In this calculation it was assumed that the initial and final pooled variances were equal,
and the covariance term between initial and final readings was ignored.
#The dilution factor may vary, but for calculation purposes, the normal dilution factor is shown here.
**The confidence linuts for a single BOD result are 95 percent, assuming a standard dilution factor of 40,
or 2.5 percent dilution.
Accuracy
There is no standard with which the accuracy of the determination can be measured. The accuracy
of the instrument is 1 percent of the reading and is better than ± 0.1 ppm.
Sensitivity
This DO Analyzer method is not applicable to samples with a dilution factor of 40 that have a
5-day BOD value of 23.5 ppm or less.
22
-------
Dissolved Oxygen Analyzer for BOD
BIBLIOGRAPHY
1. American Public Health Association, American Water Works Association, and Water Pollution
Control Federation. Oxygen (dissolved). In: Standard methods for the examination of water and
wastewater. 12th ed. New York, American Public Health Association, Inc., 1965. p. 405-421.
2. Whipple, G. C., and M. C. Whipple. Journal of the American Chemical Society, 33:362, 1911.
3. Weston & Stack, Incorporated. Operation manual, Dissolved Oxygen Analyzer, Model 300. West
Chester, Pennsylvania, 1968.
4. Wilson, Donald L. Applicability of existing methods for the determination of the biochemical
oxygen demand (BOD) of incinerator quench water. Cincinnati, Solid Waste Research Labora-
tory. October 9, 1970.
5. Wilson, Donald L. The Alsterberg (Azide) Modification of the Winkler Method for determining
the BOD of incinerator quench water and the calibration of the Weston & Stack Dissolved
Oxygen Analyzer, Model 300-B (included in this Manual).
23
-------
METHODS FOR DETERMINING
CELLULOSE IN COMPOST*
Richard D. Lossint
INTRODUCTION 2
ANTHRONE COLORIMETRIC METHOD 2
Reagents 2
Procedure 2
Calculations 2
GRAVIMETRIC METHOD 3
Reagents 3
Procedure
Calculations
DISCUSSION 3
REFERENCES 4
*Repnnted from COMPOST SCIENCE Journal of Waste Recycling, 12 (1) . 12-13,
January-February 1971
tAt the time this study was performed, Mr. Lossin was a Research Chemist with the
now Solid Waste Research Laboratory, National Environmental Research Center,
Cincinnati.
-------
METHODS OF SOLID WASTE TESTING
INTRODUCFION
The major constituent of municipal refuse is cellulose, which is degraded by microorganisms in the
composting process to yield a humus-like, stable product usable as a soil conditioner or mulch. It was
necessary, therefore, to develop methods for the determination of cellulose as part of the in-house
research on the characterization of compost and the composting process at the U. S. Public Health
Service—Tennessee Valley Authority Composting Project at Johnson City, Tennessee. The two
methods presented here are modifications of two methods previously reported (1, 2) and were found
to be accurate and reproducible for all analyses performed on compost.
ANTHRONE COLORIMETRIC METHOD
Reagents
1. Diluted H 2 SO 4 , reagent grade
(760 ml concentrated H 2 SO 4 + 300 ml water)
2. Anthrone reagent: 1 g anthrone in 500 ml cold, 96-percent H 2 SO 4 ;
let stand at room temperature 4 hr before use
3. Benzene, reagent grade
4. Pure cellulose standard (Whatman No. 1)
Procedure
Weigh out a finely ground (2-mm mesh) and redned compost sample to the nearest milligram,
place in a Soxhiet extractor, and extract with benzene for 8 hr. Dry the sample and weigh it in order
to compute the percent material extracted with benzene. Extract this sample again with hot water for
8 hr. Dry the sample and weigh it in order to compute the percent material extracted with water. Take
between 0.5 to 1.0 g of the dried sample weighed to the nearest milligram and place it in a 250-mi
beaker; wet it with a few drops of 95-percent ethanol, methanol, or acetone. Pipette in 10.0 ml
water, then 60.0 ml diluted sulfuric acid, and stir to dissolve the cellulose. After about 5 mm,
pipette exactly 10.0 ml of the cellulose solution mto a 500-mi volumetric flask and dilute to 500 ml.
Pipette 1 ml of this into a test tube, add exactly 10.0 ml anthrone reagent, mix, cap the tube to
prevent the steam from condensing on the inside of the tube, and heat in a 100 C bath for 15 mm.
Cool to room temperature and read the absorbance at 630 mp. Run cellulose standards to bracket
the sample concentration and a blank with each series of samples. A blank and standards must be
included with each group of samples heated in the 100 C bath.
Calculations
— ( g cellulose found) x [ 100— (% benzene extract + % water extract) ]
% cellulose — g of extracted sample
If all the sample can be recovered and used after the benzene and water extractions, it is unneces-
sary to compute the percent of extracted material. Simply know the initial weight of the sample, the
number of grams found (from the anthrone standard curve), and compute the percent cellulose from
this. This is easily accomplished by placing 0.5 to 1.0 g of compost in a porcelain thimble with an
asbestos filter, capping with glass wool, extracting, and then washing all the sample into a beaker and
proceeding with the determination as before.
2
-------
Cellulose in Compost
GRAVIMETRIC METHOD
Reagents
All of the following should be reagent grade:
1. Concentrated nitric acid
2. Glacial acetic acid
3. Benzene
4. Ether
5. Methanol
6. Acetone
Procedure
Weigh out about 1 g of redried, finely ground compost to the nearest milligram, place in a 1 25-ml
Erlenmeyer flask, and add 6 ml water, 24 ml glacial acetic acid, and 2 ml concentrated nitric acid.
Bring to a gentle boil on a hotplate for 20 mm, cool to about 80 C, add 50 ml benzene, and swirl
vigorously for about 2 mm to extract materials soluble in benzene. Set up a Gooch crucible with an
asbestos filter on a suction flask; decant as much of the benzene layer as possible into the filter (with
suction), taking care not to let the bottom layer spill over. Then add 50 ml of ether to the flask, swirl
vigorously, let settle, and decant all the liquid into the crucible. Wash all the solid material into the
crucible with acetone, taking care not to leave any behind. Wash the filter cake thoroughly with
successive 1 00-ml portions of hot benzene, hot methanol, and ether. After washing, clean the outside
of the crucible, place in an oven to dry, cool in a desiccator, weigh to the nearest milligram, and
ignite at 625 C in a muffle furnace for 1 hr. Cool, weigh, and report the loss after ignition.
Calculations
loss after igmtion x 100
initial sample weight = percent cellulose
DISCUSSION
Duplicate samples should always be run to ensure precision.
Swirling the benzene with the hot reaction mixture is necessary to ensure rapid filtering of the
successive solvents used. Compost contains a tar-like material that plugs the filter, and most of this is
soluble in benzene.
An alternate approach is provided by combining the two methods. Complete the solvent washings
as in the gravimetric method, then dissolve the entire sample, as in the anthrone method, and deter-
mine the cellulose colorimetrically. This was done three times with a 0-day composite compost
sample, and the results were 49.4, 49.4, and 49.6 percent, respectively. The gravimetric method on
the same sample yielded 50.2, 50.0, and 50.3 percent, respectively. The higher results indicate either
that (a) some substances were not removed by the extractions (0.7 percent) and were not cellulose,
but were lost after ignition, or that (b) there was some interfering material in the sample that
suppressed the anthrone reaction slightly.
Analyses were performed (Table 1) on samples to which known amounts of cellulose had been added
to check the gravimetric method further. The graph in Figure 1 shows the standard addition results
for the 0-day compost.
3
-------
METHODS OF SOLID WASTE TESTING
TABLE 1
ANALYSES OF SAMPLES CONTAINING KNOWN AMOUNTS OF CELLULOSE
Cellulose
Type of sample (%)
0-day compost:
Found amount
50.3
55.1
60.6
65.4
Theoretical amount
55.2
60.5
65.4
56-day compost:
Found amount
24.3
37.6
29.4
60.9
Theoretical amount
36.8
28.7
61.5
1-year compost:
Found amount
19.1
28.0
38.3
48.1
60.8
Theoretical amount
28.2
39.1
49.2
61 .1
The gravimetric method is recommended because it is more rapid and easier to perform than the
anthrone colorimetric method.
It is often difficult to obtain a representative sample from compost because of its nonhomogeneity.
For an analysis to be valid, it is necessary to work with a representative sample; that is, the overall
characteristics of the sample must be similar to the part being sampled. To this end, the following
sampling procedure is outlined:
From the digestor or windrow, select small grab samples (50-100 g) randomly to make a total
composite sample of at least 1 kg—the larger the sample, the better. This sample should be rough-
ground (by W-W grinder, haminermill, etc.) and thoroughly mixed; duplicate samples are taken from
this for testing. In general, the representative character and homogeneity of the sample are improved
by the following: (1) increasing the sample size, (2) taking larger numbers of composites, (3) grinding
fmer, and (4) mixing better.
The reliability of the testing procedure can be checked by taking several different samples from the
same sample population (compost pile) and performing the same test on all. If they are not all nearly
the same, then the sampling technique should be modified to correct any gross fluctuations.
REFERENCES
1. Viles, F. J., and L. S. Silverman. Determination of starch and cellulose with anthrone. Analytical
Chemistry, 21:950-953, 1949.
2. Crampton, E. W., and L. A. Maynard. The relation of cellulose and lignin content to the
nutritive value of animal feeds. Journal of Nutrition. 1 5(4):383-395, 1938.
4
-------
MEASUREMENT OF THE
CHEMICAL OXYGEN DEMAND OF COMPOST*
Richard D. Lossint
INTRODUCTION 2
REAGENTS 2
PROCEDURE 2
CALCULATIONS 2
DISCUSSION 3
ACKNOWLEDGMENT 4
*Repnnted from COMPOST SCIENCE Journal of Waste Recycling, 12 (2) 31-32,
March-April 1971.
fAt the time this study was performed, Mr. Lossm was a Research Chemist with the now
Solid Waste Research Laboratory, National Environmental Research Center, Cincinnati.
-------
METHODS OF SOLID WASTE TESTING
INTRODUCTION
Microorganisms are able to utilize municipal refuse as a growth substrate, eventually degrading it to
the humus-like substance we call compost. During this process the refuse is oxidized by the micro-
organisms to yield carbon dioxide, water, and heat as the primary products of their metabolism. We
can measure the extent of biological oxidation in the system by measuring the extent to which the
refuse (compost) is oxidized by chemical means. This ability to be oxidized is called the chemical
oxygen demand or COD.
Currently there is no published standard method for the determination of the COD of compost.
The procedure presented here was developed after extensive research at the U. S. Public Health
Service—Tennessee Valley Authority Composting Project at Johnson City, Tennessee, as part of the
in-house research on the characterization of compost and the-composting process.
REAGENTS
1. 1.000 N potassium dichromate, reagent grade
2. Standardized ferrous ammonium sulfate, reagent grade
3. Concentrated sulfuric acid, reagent grade
4. Ferroin indicator (orthophenanthroline)
PROCEDURE
Weigh out 0.2 to 0.3 g of finely ground, dry compost (2-mm mesh) to the nearest milligram and
place the sample in a 250-mi flask fitted with a reflux condenser. Pipette in exactly 50.00 ml 1.000
N K 2 Cr 2 07 and add 30 ml distilled water and 20 ml concentrated 112 804. Reflux this mixture
gently for 1 hr. (Equally good results were obtained with a 250-ml Erlenmeyer flask and a hotplate
instead of a reflux apparatus, the water lost by evaporation being periodical1y replaced.) Cool the
reflux mixture, dilute to 250 ml in a volumetric flask, mix well, pipette 10.00 ml into a 500-ml
Erlenmeyer flask, and add about 100 ml water, and 20 ml of concentrated sulfuric acid. Titrate with
standardized ferrous ammonium sulfate (about 0.1 to 0.2 N) using ferroin as an indicator. Standard
Methods for the Examination of Water and Wastewater (12th ed., 1965, p. 510-514) should be
consulted for techniques and standardization procedures.
CALCULAT IONS
(inmg/g)
g of sample
where
A = ml of titrant used for blank
B = ml of titrant used for sample
C = normality of the titrant
The constant 200 is derived as follows:
Standard Methods defines COD as follows:
COD = ( A — B) x C x 8,000 (in mg/liter)
ml sample
2
-------
Chemical Oxygen Demand of Compost
Here 10 ml of sample is used, and the total sample size is 250 ml, or ‘/4 liter. The expression now
becomes:
COD = ( A — B) x C x 8,000 x % (in mg/250 ml)
10 ml
or COD = (A - B) x C x 200 (mg/250 ml)
Since the entire sample has been diluted to 250 ml, we simply substitute the grams of sample used
to obtain the answer in milligrams per gram.
DISCUSSION
It is advisable to run a duplicate of the original sample and a duplicate of each dilution. This
investigator found very good precision in duplicate assays, and this indicates that the method is stable
and reproducible for a given sample of compost. This observation is further substantiated by compar-
ing the COD (in mg per g of randomly selected windrows) with the age of the compost (Figure 1).
Johnson City compost is considered stable and ready for use at 8 weeks, which corresponds to a COD
of less than 700 mg per g as opposed to about 900 mg per g for fresh refuse.
E
0
0
U
90
800
700
600
500
400
300
AGE OF COMPOST ,weeks
Figure 1. COD of randomly selected windrows versus age.
0 4 8 12 16 20 24 28
3
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METHODS OF SOLID WASTE TESTING
It is often difficult to obtain a representative sample from compost because of its nonhomogeneity.
For an analysis to be valid, it is necessary to work with a representative sample; that is, the overall
characteristics of the sample must be similar to the part being sampled. To this end, the following
sampling procedure is outlined:
From the digestor or windrow, select small grab samples (50-100 g) randomly to make a total
composite sample of at least 1 kg; the larger the sample, the better. This sample should be rough
ground (by W-W grinder, hammenmil, etc.) and thoroughly mixed; duplicate samples are taken from
this for testing. In general, the representative character and homogeneity of the sample are improved
by the following: (1) increasing the sample size, (2) taking a larger number of composites, (3)
grinding finer, (4) mixing better.
The reliability of the testing procedure can be checked by taking several different samples from
the same sample population (compost pile) and performing the same test on all of them. If they are
not all nearly the same, then the sampling technique should be modified to correct any gross
fluctuations.
ACKNOWLEDGMENT
The author wishes to express his gratitude to Donald J. Dunsmore for his assistance in the initial
work in developing this procedure.
4
-------
QUALiTATIVE DETERMINATION FOR THE
DEGREE OF DECOMPOSITION OF COMPOST BY THE
STARCH-IODINE METHOD *
Richard D. Lossint
INTRODUCTION 2
THEORY 2
PROCEDURE 2
DISCUSSION 9
REFERENCES 10
BIBUOGRAPHY 10
*Repnnted from COMPOST SCIENCE Journal of Waste Recycling, 11 (6) : 16-17,
November 1970
tAt the time this study was performed, Mr. Lossin was a Research Chemist with the
now Solid Waste Research Laboratory, National Environmental Research Center,
Cincinnati.
-------
METHODS OF SOLID WASTE TESTING
INTRODUCTION
A s imple and rapid test has long been needed for determining the degree of decomposition of
compost. Many tests have been proposed and used, including measurement of the reduction in total
carbon or in the carbon-nitrogen ratio, increase in percent ash, decrease in cellulose, and decrease in
lipids. These methods are adequate for determining the completeness of composting on the assump-
tion that a representative sample has been taken. All have, however, obvious disadvantages: each
requires complicated sample preparation, considerable time, and, in most cases, expensive equipment.
This investigator has always found starch in measurable quantities (2 to 6 percent) in municipal
refuse. Since starch is in the class of easily degraded substrates that must be broken down and
metabolized before the refuse becomes a microbiologically stable product, testing for its presence
will indicate whether or not the compost is stabilized. The rationale of the test rests on the hypoth-
esis that all refuse contains a measurable quantity of starch and that the starch must be degraded
before the compost can be considered acceptable, no matter how much was initially present. The
test discussed here is based on the formation of the starch-iodine complex in an acidic extract of
compost and has the following advantages: It is rapid and easily performed, it is specific for starch,
and the equipment needed is very simple.
Although the method has not been tested on different composts, it was valid for compost from
the Joint Public Health Service—Tennessee Valley Authority Composting Project in Johnson City,
Tennessee. The method is presented here to aid other workers in the field.
THEORY
Three types of carbohydrates are found in compost: sugars, starch, and cellulose (Figure 1). These
constituents decrease with age of the compost (Figures 2-4). Sugars are metabolized first, being almost
completely consumed within a week; starches are next, followed by cellulose. By the fourth to fifth
week of windrow composting, the starch has passed through its maximum rate of degradation. This
is the stage in the process where the compost first has an acceptable appearance and odor, has gone
through its maximum temperature phase, and has reached its maximum pH (Figures 5 and 6). In
other composting processes with different types of refuse, these events may occur at different time
intervals, but the relationships can be expected to be approximately the same.
Certain polysaccharides (starches: amylose, amylopectin, glycogen, and dextrans) form charac-
teristic color complexes when combined with molecular iodine (1-3). Amylose (straight-chain starch)
gives an intense blue-black color; amylopectin (moderately branched) gives from light blue to purple
to red colors, depending on the degree of branching; and glycogen (highly branched) gives reddish-
brown colors. Dextrans, which are partial degradation products of starch, give reddish-brown to
yellow colors. The hydrolysis of starch may be followed by a gradual change in. the iodine complex
color (blue-black to light blue to purple to red to yellow to no color).
In the present study, a test was run to determine whether all of the starch found in the compost
was complexing with iodine. The weight of starch-iodine complex recovered from lO-g compost
samples varied with different compost ages. This corresponded very well with determinations of the
amount of starch in the compost.
PROCEDURE
The iodine reagent is prepared by dissolving 2.00 g KI in 500 ml H 3 0, then dissolving 0.80 g 13.
The other reagent is perchioric acid (36 percent).
Place about 1 g compost (see Discussion) in a 100-ml beaker, wet with a few drops of ethanol if
dry, add 20 ml perchloric acid (36 percent), and stir. Filter through open-texture filter paper (What-
man No. 90). Add 2 ml iodine reagent to the filtrate and stir. Place a few drops on a spot plate or
2
-------
Degree of Decomposition of Compost
Figure 1. Amylose (A), amylopectin (B), and glycogen (C).
AGE OF WINDROW (WEEKS)
Figure 2. Decrease of sugar versus age of compost.
B
C
0
I-
z
uJ
V
w
0.80
0.60
0.40
0.20
0.10
0
1 2 4 6 8
10
3
-------
1.0
METHODS OF SOLID WASTE TESTING
4.0
U
43.0
z
w2.0
U
U.’
a-
50
0
-a
D
-a
-J
U
I.-
z
‘ La
V
a.
30 -
I I
a a
4 6 8
AGE OF WINDROW (WEEKS)
Figure 3. Decrease of starch versus age of compost.
I I
I
10
0 1 2 4 6 8 10
AGE OF WINDROW (WEEKS)
Figure 4. Decrease of cellulose versus age of compost.
I I
0 1 2
4
-------
160 -
Degree of Decomposition of Compost
3 4 5
AGE OF WINDROW (WEEKS)
Figure 6. pH versus age of compost.
140 -
I ’
w
I-
4
U.’
U . ’
I-
120 -
100
80 -
0
I I I I I
1 6
2 3 4 5
AGE OF WINDROW (WEEKS)
Figure 5. Temperature versus age of compost.
9
8.
7.
6.
pH
0 1 2
6
7
5
-------
METHODS OF SOLID WASTE TESTING
other white background, and then note the color and precipitate. Finished compost will give yellow
color and very little precipitate. Poor or unfinished compost will give dark blue color and heavy blue
precipitate. The color sequence normally found as the age of compost increases is blue-black
light blue -÷ grey -+ green -+ yellow. A reddish color may be found with partially degraded compost.
It is possible that either little or no color will be produced by the test described or, more Likely,
tests on all samples for the entire compost cycle will be too dark. There are five variables present in
this test, one or more of which are responsible for these deviations: (1) starch concentration and
type, (2) particle size of compost, (3) sample size, (4) iodine concentration, (5) perchioric acid
concentration.
The method has been checked on a variety of samples by altering all these variables, and the
following comments reflect the results.
The iodine concentration must be in excess of that needed so as not to become a limiting factor.
If all the ages of compost give a color reaction so dark that it is difficult to differentiate between
them, one or more of the following steps may be taken: (1) Reduce the perchloric acid concentration.
Concentrations down to 5 percent were found to give positive tests with unfinished compost. (2)
Dilute the filtrate before adding the iodine reagent. (3) Reduce the sample size.
Step 3 is not advisable since a reduction in sample size will increase the chance of a nonrepresenta-
tive sample. It is best to have the sample as large and as finely ground as possible. Step 1 is preferable,
Step 2 being next. If the color is too light, then the best policy is to increase the sample size or grind
the sample more finely. Do not increase the perchlonc acid concentration. When standardizing the
test, be sure to use finished and unfinished compost as standards, making certain that the variables
are adjusted to give a blue color with unfinished compost and a yellow color with finished compost.
Once these variables are established, they must not be altered. Do every test in exactly the same
fashion.
DISCUSSION
One must remember that this is only a qualitative spot test, designed simply to show a relative
change; nothing is being quantitatively determined. The test is to be used only as a control measure
for checking the completeness of composting in one particular process. After one has checked satis-
factory and unsatisfactory compost with this test, it will become apparent that finished compost
always gives a characteristic color reaction (yellow), whereas unfinished compost does not. Finished
compost has never given a positive starch reaction with this test.
It is difficult to obtain a representative sample from compost because of its nonhomogeneity. For
the test to be valid, it is necessary to work with a representative sample; that is, the overall charac-
teristics of the sample must be similar to the part being sampled. To this end, the following sampling
procedure is outlined:
From the digestor or windrow, select small grab samples (50-100 g) randomly to make a total
composite sample of at least 1 kg; the larger the sample, the better. This sample should be rough-
ground (by W-W grinder, hammermill, etc.) and thoroughly mixed; duplicate samples are taken from
this for testing. In general, the representative character and homogeneity of the sample are improved
by the following: (I) increasing the sample size, (2) taking a larger number of composites, (3) grinding
finer, (4) mixing better.
There is always a finite probability, however small, of selecting a sample at any stage of the corn-
posting cycle that will contain excessive starch or no starch at all. The reliability of the testing
procedure can be checked by taking several different samples from the same sample population
(compost pile) and performing the same test on all of them. If they are not all nearly the same, then
the sampling technique should be modified to correct any gross fluctuations.
6
-------
Degree of Decomposition of Compost
REFERENCES
1. Jirgensons, B. Natural organic macromolecules. New York, Pergamon Press, Inc., 1962. 464 p.
2. Ravve, A. Organic chemistry of macromolecules; an introduction. New York, Marcel Dekker,
Inc., 1967. 498 p.
3. West, E. S., and W. R. Todd. Textbook of biochemistry. 3d ed. New York, Macmillan Company,
1961. 1,4.23 p.
4. McCready, R. M., J. Guggolz, V. Silviera, and H. S. Owens. Determination of starch and
and amylose in vegetables. Analytical Chemistry, 22(9):! 156-1158, 1950.
BIBLIOGRAPHY
Clark, J. M., Jr., ed. Experimental biochemistry. London, W. H. Freeman & Co., 1964. 228 p.
Conn, E. E., and P. K. Stumpf. Outlines of biochemistry. 2d ed. New York, John Wiley & Sons, Inc.,
1966. 468 p.
Davidson, E. A. Carbohydrate chemistry. New York, Holt, Rinehart and Winston, Inc., 1967. 441 p.
Fogg, G. E. The growth of plants. Baltimore, Md., Penguin Books, Inc., 1963. 288 p.
Levene, P. A., and L. C. Kreider. Oxidation and hydrolysis of polygalacturonide methyl ester to
levo-tartaric acid. Journal of Biological Chemistry, 120(2): 591-595, Sept. 1937.
Meyer, B. S., D. B. Anderson, and R. H. Bohning. Introduction to plant physiology. Princeton, N. J.,
D. Van Nostrand Company, Inc., 1960. 541 p.
Pigman, W. W., ed. Carbohydrates; chemistry, biochemistry, physiology. New York, Academic Press,
Inc., 1957. 902 p.
Robin, M. B. Optical spectra benzamide-triiodide ion complexes: a model of the starch-iodine
complex. Journal of Chemical Physics, 40(1 1):3369-3377, June 1, 1964.
Rundle, R. E., and R. R. Baldwin. The configuration of starch and the starch-iodine complex. 1. The
dichroism of flow of starch-iodine solutions. Journal of the American Chemical Society,
65(4):554-558, Apr. 1943.
White, A., P. Handler, and E. L. Smith. Principles of biochemistry. 4th ed. New York, McGraw-Hill
Book Company, Inc., 1968. 1187 p.
7
-------
VACUUM-ACID HYDROLYSIS OF FUNGAL PROTEIN
AND PROTEIN FROM OTHER SOURCES
W. Emile Coleman
DISCUSSION 2
APPARATUS 2
REAGENTS 3
Preparation of Solution 3
SAFETY PRECAUTIONS 3
SAMPLE PREPARATION 3
DEAERATION AND HYDROLYSIS 4
METHOD EVALUATION 6
REFERENCES 7
Research Chemist, Solid Waste Research Laboratory, National Environmental Research
Center, Cincinnati.
-------
METHODS OF SOLID WASTE TESTING
DISCUSSION
All proteins yield amino acids when hydrolyzed, since a protein molecule may contain hundreds
or thousands of amino acid groups. The amino acids are united through an acid amide type of bond
called a peptide linkage ¶ 1 t • The nature of the pep tide linkage between the amino acids of the
protein is shown below. (C4)
NH 2 9 H H H H ¶?
R— C—C —N —C—C—N—C—C ---etc.
H R R
Just as is the case with acid amides, the peptide linkages in proteins are resistant to hydrolysis and
require prolonged boiling with relatively strong acids or alkalies for complete liberation of each
amino acid. The acids most commonly used are hydrochloric (6 N), sulfuric (8 N), and hydriotic
(57 percent). Among bases, 5 N NaOH and 14 percent Ba(OH) 2 are commonly used. Acid hydrolysis
of proteins is generally preferred. Alkaline hydrolysis, although efficient in liberating the amino
acids, partly converts the pure, optically active forms that exist in proteins into racemic mixtures
(mixtures of D and L forms) (1). Acid hydrolysis is essentially free from this objection. Both acid
and alkaline hydrolysis lead to some decomposition and loss of the more unstable amino acids.
The developed technology for producing fungal protein in the Solid Waste Research Laboratory
is the result of research efforts directed toward recycling both starchy and cellulosic wastes (2, 3).
The quality of the protein produced by fermentation depends on the amino acid profile that was
determined by a quantitative amino acid analysis with an Automatic Amino Acid Analyzer (4).
Before an analysis, however, hydrolysis of the protein must take place to produce individual amino
acids.
The subject procedure uses acid hydrolysis under vacuum conditions. The use of a vacuum elim-
inates the oxidizing atmosphere, which would result in oxidation or loss of the amino acids. This
hydrolysis procedure can be applied to any protein-bearing material, plant, or animal.
APPARATUS
1. Vacuum pump, capable of obtaining 5O-lOO ,t of pressure
2. Pressure gauge, range of 5 0 p to 1 atm.
3. Cold trap
4. Dewar flask containing dry ice with acetone
5. Evaporating dishes (one per sample)
6. Glass funnels (one per sample)
7. Filter paper, #40
8. Constant temperature oven, thermostatically controlled at 110 C .± 1 C
9. All-glass pressure system as shown in Figure 1
10. Analytical balance
11. Pipet, 10-ml, one
12. Vacuum hydrolysis tubes (one per sample) (4)
2
-------
Fungal Protein
13. Hi-Vacuum grease
14. Filtering flasks, 50-mi, one per sample
REAGENTS
1. Hydrochloric acid, reagent grade
2. Prepurified nitrogen, one cylinder
Preparation of Solutions
Hydrochloric acid, 6 N: Add equal volumes of distilled water and concentrated HCI. One liter
of solution is sufficient.
SAFETY PRECAUTIONS
When presented to the analyst for protein analysis, the fungal growths are usually still active or
“alive.” Before attempting analysis, the molds (fungal growths) should be autoclaved for 15 to 20
minat 120 C.
SAMPLE PREPARATION
The fungal protein is usually grown in a 500-ml flask containing 100 ml of growth media. Before
analysis, the fungal mass is treated as follows:
Proceduic Comments
1. After the fungal mass has been autoclaved, 1. The growth media can be discarded.
decant the growth media and retain the
mold in the flask.
2. Add approximately 200 ml of distilled 2, 3, 4. This washing procedure is vital because
water to the flask. it ensures removal of all salts remaining
from the growth media. If not removed, the
3. Wash the mold in distilled water by shaking metal ions in the growth media become
for about 5 mm, then decant the liquid, bonded to the cation exchange resin in the
chromatographic column and thus interfere
4. Repeat steps 2 and 3 twice. with the analysis. Such a condition ad-
versely affects the capacity of the resin to
attract amino acid molecules, thus resulting
in loss of resolution. In addition, if the
ammonium ion (an ingredient of the media)
is not removed by washing, it produces a
huge peak that covers up several of the
amino acids on the chromatogram.
5. After the last washings are decanted, pour 5. Whatman 40 filter paper will suffice.
the mold onto a funnel containing coarse
filter paper.
3
-------
METHODS OF SOLID WASTE TESTING
6. Transfer the mold from the filter paper
into an evaporating dish.
7. Place evaporating dish in an oven thermo-
statically controlled at 75 C for 24 hr or
freeze dry the material.
8. At the end of 24 hr obtain and record the
dry weight of the fungal protein.
9. With mortar and pestle, grind the dried
mold until uniform m size.
6. Shaking the paper will dislodge the mold.
7. The analyst should plan his time so that
this step can be accomplished by 9:00
am.
DEAERATION AND HYDROLYSIS
Procedure
1. Weigh out 10 - 20 mg of the dried protem
material into the bottom section of a re-
usable vacuum hydrolysis tube.
2. Pipet 10 ml of the 6 N HC I into the
hydrolysis tube.
3. Lightly coat the ground-glass ball joint
and stopcock of the hydrolysis tube with
high vacuum grease; then clamp securely.
4. Close stopcock on top section of vacuum
hydrolysis tube and connect to pressure
system as shown in Figure 1.
5. Start the vacuum pump and evacuate pres-
sure system below 50 i of Hg pressure.
6. SLOWLY open the stopcock on the hydrol-
ysis tube to deaerate the mixture. Observe
when pressure gauge reaches 50 - lOOiz
of Hg pressure.
7. Close the stopcock on the hydrolysis tube
and shut off the vacuum from the pressure
system.
8. Flush the system with nitrogen for about
3 mm.
9. Open the stopcock on the hydrolysis tube
and allow tube to fill with nitrogen.
10. Close stopcock on vacuum hydrolysis tube
and shut off nitrogen supply.
Comments
1. The two-section hydrolysis tube can be
seen in Figure 1.
3. Use Dow — Corning or Apiezon H.
6. If the stopcock is opened too suddenly,
the sample will be pulled into the vacuum
lines. A slight tap on the side of the tube
will help to dislodge air bubbles when the
vacuum is applied.
7. A large stopcock is installed in the system
to isolate the vacuum pump.
8. Nitrogen pressure at the point of entry to
the system is 2-3 PSI.
9. Two miii is sufficient.
4
-------
S
Pressurized Tank
(Prepurified Nitrogen)
t
Cold Trap
Vacuum
Pump
Hydrolysis Tube
C lai
I
Figure 1. Pressure system for deaerating samples.
-------
METHODS OF SOLID WASTE TESTING
11. Evacuate system below 5Op, then open
stopcock on vacuum hydrolysis tube and
evacuate sample down to 50 to lOOp.
12. Repeat steps 7 to 12 twice.
13. While vacuum pump is running, close stop-
cock on vacuum hydrolysis tube, then
remove tube from vacuum system.
14. Place vacuum hydrolysis tube in an up-
right position in a constant temperature
oven maintained at 110 C ± 1 C for 22 hr.
15. At the end of 22 hr, remove sample from
oven and cool to room temperature.
16. Slowly open stopcock on hydrolysis tube,
then remove top section.
17. Filter contents of tube through a medium
porosity, sintered glass filter under a slight
vacuum.
18. Pour the filtrate from the filtering flask 18. Try to maintain the total volume between
into an evaporating dish; then wash flask 20 and 30 cc.
3 tunes with distilled water and transfer
washings into the same evaporatmg dish.
19. Place the evaporating dish in a freeze drier
and allow to remain there until residue is
dry.
20. Add 5 ml of citrate buffer, pH 2.2 to the 20. If undissolved material remains after stir-
freeze-dried material in the evaporating ring, filter through Whatman 40 filter
dish; stir with rubber policeman until paper.
residue is dissolved.
21. Store hydrolyzate in a stoppered bottle. 21. If an analysis of the protein hydrolyzate is
not made immediately, store in a freezer.
METHOD EVALUATION
Aliquots of a calibration mixture containing 18 amino acids, O.5p moles each, were put through
the same hydrolysis procedure. Recovery of all the amino acids was quantitative. The recovery for
all amino acids ranged from 95 to 101 percent.
To test the accuracy of the method, three different samples from the same protein source were
hydrolyzed using the subject procedure. Three amino acid analyses were run on the protein hydrol-
yzates, and the protein content of each was determined by the summation of the individual amino
acids present in each hydrolyzate. The percent protein for the three samples were: 13.30, 13.39,
and 13.15.
The interferences most likely to reduce the yield of amino acids in the hydrolyzate have been
pointed out in the Comments. Any mineral salts growth media remaining with the mold will greatly
6
-------
Fungal Protein
reduce the accuracy of an analysis. The presence of undigested starchy and cellulosic substrates in
the hydrolysis mixture causes oxidation of the amino acids, thus producing lower yields.
The washing and drying process normally requires a 24-hr period, and another 24-hr are required
for the degassing and hydrolysis process. The analyst could save a day’s work if the laboratory sub-
mitting the protein sample were instructed as to the washing and drying techniques. The degassing
and hydrolysis procedures could be started immediately if the sample were dried and ground on
receipt.
REFERENCES
1. West, E.S., and W.R. Todd. Textbook of biochemistry. 2nd ed. New York, Macmillan Company,
1955. 1312 p.
2. Rogers, C.J., P.V. Scarpino, W.E. Coleman, D.F. Spino, and T.C. Purcell. Production of fungal
protein from cellulosic and waste cellulosics. Environmental Science & Technology, 6: 71 5-719,
August 1972.
3. Rogers, C.J., W.E. Coleman, D.F. Spino, and T.C. Purcell. Fungal biosynthesis of protein from
potato waste. Presented at the American Chemical Society National Meeting in Chicago, Illinois
September 1970.
4. Phoenix Precision Instrument Company. Liquid chromatography handbook. Philadelphia, Pa.,
1968. 185 p.
7
-------
LABORATORY PROCEDURE FOR THE
SPECfROPHOTOFLUOROMETRIC DETERMINATION OF
SELENIUM IN SOLID WASTES
Henry Johnsont
DISCUSSION 2
APPARATUS 2
REAGENTS 2
SAFETY PRECAUTIONS 3
SAMPLE PREPARATION 4
PROCEDURE 4
STANDARDIZATION AND CALIBRATION 4
CALCULATIONS 5
METHOD EVALUATION S
BIBUOGRAPHY 5
*A descnption of this method appears in “Determination of Selenium in Solid Waste,”
Henry Johnson, Environmental Science & Technology, 4:850-853, Oct. 1970.
tResearch Chemist, Solid Waste Research Laboratory, National Environmental Research
Center, Cincinnati.
-------
METHODS OF SOLID WASTE TESTING
DISCUSSION
The determination of selenium in solid waste and in the effluents of solid waste treatment systems
is of considerable interest because of the toxicity of selenium and its reported presence in almost all
conceivable types of paper. A 2,3-diaminonaphthalene (DAN) analytical procedure is preceded by
isolation of selenium with toluene 3, 4-dithiol and includes the use of EDTA, sodium fluoride, and
sodium oxalate masking agents. The reactions in the procedure are thought to involve the formation
of a piazselenol as follows:
Sample+
So” + i:;XX)
p azseIenoI
The piazselenol emits at 570 mji when excited at 317 msz.
The use of the DAN fluorometric procedure affords the sensitivity necessary for trace analysis
and at the same time retains a precision of measurement comparable to competitive methods. The
isolation step and masking agents are used to prevent background fluorescence and foreign ion effect.
APPARATUS
The apparatus required for the analyses is:
1. Farrand Mark I Spectrofluorometer with 10mm slits and RCA IP 121 phototube
2. Wiley Mill fitted with a 10 mesh screen or a Williams patent hammermill and double-cone
homogenizer.
3. Parr bomb calorimeter
4. Beckman zeromatic pH meter
5. Sorval SS-l centrifuge
REAGENTS
The analysis requires the following reagent grade chemicals:
1. Hydrochloric acid
2. Sodium hydroxide
3. Nitric acid
4. 72 percent perchioric acid
2
-------
Selenium
5. Ethylene chloride
6. Carbon tetrachioride
7. Cyclohexane
8. 2,3-diaminonaphthalene (DAN) (Aldrich Chemical Co.)
9. Toluene-3,4-dithiol zinc salt (Eastman Organic Chemicals)
10. Sodium fluoride
11. Sodium oxalate (Na 2 C 2 04)
12. Sodium selenite (Na 2 Se0 3 )
13. Ethylenediaminetetraacetic acid disodium salt (EDTA)
14. Sulfuric acid
15. Quinine
Prepare solutions of the above reagents as follows:
1. Prepare 0.1 N HC I by diluting 8.3 ml of concentrated HC1 to 1,000 ml with distilled water.
2. Prepare 0.1 N NaOH by dissolving 4.0 g of NaOH in 1,000 ml of distilled water.
3. Dissolve 480 g of NaOH in 1,000 ml of distilled water to produce an approximately 12 N
solution.
4. Prepare 0.1 percent DAN fresh daily by dissolving 0.1 g of DAN in 100 ml of 0.1 N HC1.
5. Prepare a suitable dithiol solution fresh daily by adding 1 g of toluene-3,4-dithiol zinc salt to
several milliliters of ethanol. Add several drops of concentrated HC1 and heat the mixture under
the tap to produce a clear solution. Finally, dilute the solution to 100 ml with ethanol.
6. Prepare 0.1 M sodium fluoride by dissolving 4.2 g in 1,000 ml of distilled water.
7. Prepare 0.1 M sodium oxalate by dissolving 13.4 g in 1,000 ml of distilled water.
8. Prepare 0.1 M EDTA by dissolving 37.2 g in 1,000 ml of distilled water.
9. Prepare a standard solution of selenium by dissolving 2.1881 g of Na 2 Se0 3 in 1,000 ml of
distilled water. The concentration of this solution is 1,000 .ig Se per milliliter.
10. Dilute 2.8 ml of concentrated H 2 SO 4 to 1,000 ml, thus producing a 0.1 N solution.
11. Prepare a stock standard of quinine sulfate by dissolving 0.0100 g of quinine in 1,000 ml of
0.1 N H 2 SO 4 thus producing a concentration of 10 ig per milliliter. Prepare a working standard
by diluting 1 ml of stock standard to 10 ml with 0.1 N H 2 SO 4 . These solutions should remain
in the dark when not in use.
SAFETY PRECAUTIONS
Adequately ventilate the spectrofluorometer lamp housing (with a hood if possible) because
ozone is produced when the instrument is in use.
Take extreme caution during the nitric-perchloric acid digestions. These should never be taken to
dryness because salts of perchloric acid are explosive.
Follow the manufacturer’s instructions when using the Parr bomb, and follow general laboratory
safety practices at all times.
3
-------
METHODS OF SOLID WASTE TESTING
SAMPLE PREPARATION
Grind, mix, and dry refuse and compost as described in an earlier chapter. Pelletize l-g samples
and ash m a Parr bomb using 10 to 15 ml of water as an absorbing solution under 30 atm. of oxygen
pressure. Tap cool the bomb for 10 mm and release the excess oxygen over a 2-mm period. Filter
the bomb contents and washings through Whatman #12 filter paper and adjust the volume to 25 ml.
Place glass fiber millipore filters containing particulates collected by isokinetic stack sampling
in a 100 ml beaker and add 15 ml of a l0 I HNO 3 and HC1O 4 acid mixture. Heat the mixture cau-
tiously to remove oxides of nitrogen. After adding 25 ml of water, bring the solution to a boil, allow
to cool, and pass through a sintered glass filter. Adjust the volume to 50 ml. Extract samples (1 g) of
incinerator residue in the same manner.
PROCEDURE
1. Transfer 25 ml or an aliquot containing
not less than 0.01 pg of Se into a 125-mi
separatory funnel, adjust the volume to
35 ml, and add 50 ml of concentrated HC1.
2. Add 4 ml of freshly prepared 1 percent
zinc dithiol suspension in ethanol. Mix and
let stand 1 5 mm.
3. Extract consecutively with a 10 ml and 5
ml mixture of ethylene chloride and carbon
tetrachlonde (1: 1) and combine the organic
phases in a stoppered test tube.
4. Add 1 ml of 72 percent HC1O 4 and 10
drops of concentrated HNO 3 and boil off
organic solvent in boiling water bath.
5. Heat cautiously until fumes of HCIO 4 are
in evidence. Add 1 ml of H 2 0, heat until
fumes of HC1O 4 are apparent again, and
add 10 ml of 1-120.
6. Adjust the pH to 2 with NaOH and add
0.5 ml each of 0.1 M aqueous solutions of
EDTA, NaF, and Na 2 C 2 04.
7. Readjust the pH to 2 with HCI and add 4
ml of freshly prepared 0.1 percent DAN
in 0.1 N HC1.
8. Place the tubes in a 50 C H 2 O bath for
20 mm and tap cool.
9. Transfer contents of tubes to 1 2 5-mI sep-
aratory funnels containing 10 ml of cyclo-
hexane. Extract and collect organic phase
in a centrifuge tube.
10. Centrifuge at 2,000 rpm for 2 mm and
read on spectrofluorometer exciting at
370 mp and emitting 517 mp
STANDARDIZATION AND CALIBRATION
Prepare a calibration curve by analyzing standard solutions ranging from 0.005 to 1.0 pg Se. A
linear relationship should be obtained by plotting the concentration of selenium versus the fluorescent
intensity. A distilled water blank should give negligible readings at the analytical wave lengths.
Bracket each sample reading by readings of 1 pg per milliliter quinine sulfate solution to account
for fluctuations in lamp intensity.
4
-------
Selenium
CALCULATIONS
The formula needed to calculate the concentration of selemum in samples is:
/ MM X TQC\/lO
C = (MM x T 8 ) (S)( II—
\MMX TQS/\VS
where
C = concentration of selenium in 10 ml cyclohexane
MM = meter multiplier setting
T 9 = percent transmittance of the sample
S = slope of the calibration curve
TQC = percent transmittance of quinine for the calibration curve
TQ 9 = percent transmittance of quinine for the sample run
V 8 = volume of sample analyzed
METHOD EVALUATION
Eleven samples of standard solution containing 1 pg of selenium per 10 ml of test solution were
analyzed over a 2-day period. The results from these runs gave a standard deviation of 0.078 and a
relative error of 2.57 percent. The standard deviation indicates ± 0.03 pg of selenium for the
analyses of 1 pg standard solutions.
BIBLIOGRAPHY
1. Watkinson, J.H. Fluorometric determination of selenium in biological material with 2,3-
diaminonaphthalene. Analytical Chemistry, 38:92-97, Jan. 1966.
2. Lott, Peter F., Peter Cukar, George Moriber, and Joseph Solga. 2,3-Diaminonaphthalene as a
reagent for the determination of milligram to submicrogram amounts of selenium, Analytical
Chemistry, 35. 1159-1163, Aug. 1963.
3. Clark, R.E.D. o-Dithiols in analysis, Analyst 82:182-185, Mar. 1957.
4. Dye, W.B., E. Bretthauer, H.J. Seim, and C. Blincoe. Fluorometric determination of selenium in
plants and animals with 3,3’—diaminobenzidine, Analytical Chemistry, 35:1687-1693,
Oct. 1963.
5. Johnson, H. Determination of selenium in solid waste, Environmental Science and Technology,
4:850-853, Oct. 1970.
5
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PART III
MICROBIOLOGICAL METHODS
-------
METHODS FOR BACTERIOLOGICAL EXAMINATION
OF SOLID WASTE AND WASTE EFFLUENTS
Mirdza L. Peterson
INTRODUCTION AND GENERAL LABORATORY PROCEDURES 2
Introduction 2
General laboratory procedures 2
COLLECTION AND PREPARATION OF SAMPLES 5
Method for collection of solid waste or semi-solid waste samples 5
Method for collection of liquid samples—quench and industrial waters or
leachate 5
Method for collection of mcinerator stack effluents 6
Method for collection of dust samples 6
Method for preparation of solid and semi-solid samples for analyses 8
BACTERIOLOGICAL EXAMINATION OF WASTE AND RELATED MATERIALS 8
Method for preparation of decimal dilutions of a solid, semi-solid, or
liquid waste matenal 8
Methods for total viable bactenal cell number 8
Methods for presence of members of coliform group 10
M ethod to determine the presence of viable heat-resistant spore number 12
Methods to detect enteric pathogemc bacteria 14
Method for examination of stack effluents 18
Method for examination of dust 18
REFERENCES 18
*Semor Research Microbiologist, Solid Waste Research Laboratory, National Environ-
mental Research Center, Cincinnati.
-------
METHODS OF SOLID WASTE TESTING
INTRODUCTION AND GENERAL LABORATORY PROCEDURES
Introduction
Developing methods to detect and enumerate bacterial pathogens in solid waste and waste
effluents, particularly those found in fecal and food waste, is an important microbiological research
goal of this Solid Waste Research Laboratory (1). Attempts to isolate such organisms from solid
waste on a routine basis have not been fruitful because of low initial numbers and/or relatively
short periods of survival. Pathogenic microorganisms in waste are constantly subjected to such
debilitating environmental factors as chemical additives, drying, freezing, heat, and pH extremes.
These factors often affect cultivation of these organisms in media originally designed for diagnostic
purposes. For these reasons, attention was directed primarily toward the development of methods
for the detection and enumeration of a group of organisms that are of significance in the fields
of public health and sanitation. Three procedural lines of investigation were undertaken:
1. To develop suitable methods for indicating the sanitary quality of solid waste before and after
processing or disposal.
2. To develop suitable methods for determining the efficacy of operational procedures for
removing or destroying the microorganisms.
3. To develop suitable methods for indicating the health hazard of solid waste in which pathogenic
agents may be present in small numbers.
An investigation was made to evaluate presently employed bacteriological methods applicable to
solid waste and related matenals. This evaluation led to the establishment of reliable methods that
are well-suited to routinely measuring, under practical conditions, the bacteriological quality of solid
waste, incinerator residue, industnal and quench waters, leachate, stack emissions, and dust in and
around waste processing areas. The methods described here will determine:
1. Total number of viable bacterial cells
2. Total coliforms
3. Fecal coliforms
4. Heat-resistant spores
5. Enteric pathogens, especially Salmonella sp.
It should be noted that minor changes in technical procedure may result in marked changes in the
validity of the data.
General Laboratory Procedures
Glassware washing.
All glassware known to contain infectious material must be sterilized by autoclaving before
washing. All glassware that is to be used in microbiological tests must be thoroughly washed before
sterilization, using a suitable detergent and hot water, and followed by hot water and distilled water
rinses. Six to 12 rinses may be required to remove all traces of inhibitory residues from the glass
surface.
2
-------
Bacteriological Examination
Sterilization.
Dry heat is used for the sterilization of glass sampling bottles, foil-covered flasks, beakers,
graduates, pipettes packed tightly in sealed cans, or articles that are corrosively attacked be steam.
Recommended time-temperature ratio for dry heat sterilization is 170 C for 2 hr.
Saturated steam under pressure (or autoclaving) is the most frequently used sterilization method.
Media, dilution water, and materials (rubber, paper, cotton, cork, heat-stable plastic tubes, and
closures, for example) are sterilized by autoclaving at 121 C. Sterilization time for media and dilution
water (for volumes up to 500 ml) is 15 miii; 1,000-ml quantities are held for 20 mm, instruments
for 15 mm, gloves for 20 mm, and packs for 30 mm (measured from the time the autoclave temper-
ature reaches 121 C).
Membrane filters are sterilized for 10 mm at 121 C with fast steam exhaust at the end of the
sterilization process.
Heat-sensitive carbohydrates and other compounds are sterilized by passage through a cellulose
esther membrane or another bacteria-retaining filter.
Culture media
The use of dehydrated media is recommended whenever possible, since these products offer the
advantages of good consistency from lot to lot, require less labor in preparation, and are more
economical. Each lot should be tested for performance before use.
Measurement of the final pH of a prepared culture medium should be accomplished colon-
metrically after autoclaving and cooling. Acceptable pH range is 7.0 ± 0.1.
Media should be stored in a cool, dry, and dark place to avoid dehydration, deterioration, and
adverse light effects. Storage in the refrigerator usually prolongs the shelf-life of most media. Media
should not be subjected to long periods of storage, because certain chemical reactions may occur in
a medium even at refrigerator temperatures.
Many of the media referred to below can be obtained from commercial sources in a dehydrated
form with complete information on their preparation. These media will therefore be listed but not
described in this section. Described in this section are those media that are formulated from
ingredients or from dehydrated materials. Culture media (Difco or BBL products) are listed as
follows:
Bacto-agar
Bismuth sulfite agar
Blood agar
Brain heart infusion broth
Brilliant green agar
Brilliant green lactose bile, 2 percent
Coagulase mannitol agar
Dextrose
E. C. broth
Eosin methylene blue agar, Levine
Fluid thioglycollate medium
Gelatin
H-broth
Indole nitrite medium
KCN medium
3
-------
METHODS OF SOLID WASTE TESTING
Lactose
Lactose tryptose broth
Lauryl tryptose broth
Lysine decarboxylase medium
M-Endo broth
M-FC broth
MacConkey’s agar
Malonate broth, Ewing modified
Maltose
Mannitol
Mannitol salt agar
Methyl red-Voges Proskauer medium
Nitrate broth
Nutrient agar
Phenol red broth base
Phosphate buffer, APHA, pH 7.2
Sabouraud’s dextrose agar
Salmonella-Shigella agar
SBG enrichment broth
Selenite-F enrichment broth
SIM medium
Simmons citrate agar
Sucrose
Triple sugar iron agar
Trypticase soy agar
Tryptone glucose extract agar
Urea agar base concentrate (sterile)
XLD agar
Culture media requiring preparation.
1. Blood Agar: Suspend 40 g of trypticase soy agar in a liter of distilled water. Mix thoroughly.
Heat with agitation and boil for 1 mm. After solution is accomplished, sterilize by autoclaving for
15 mm at 121 C. Cool agar to 45 to 50 C, and add 5 to 7 percent sterile, defibrinated sheep blood,
mixing evenly throughout the medium. Pour into sterile Petn dishes. After solidification, invert
dishes and incubate overmte.
2. Phenol Red Broth Base: Dissolve 15 g in a liter of distilled water. Add 5 to 10 g of desired carbo-
hydrate. Use Durham fermentation tubes for detection of gas formation. Arrange tubes loosely in
suitable containers and sterilize at 116 to 118 C for 15 mm.
3. Phosphate Buffer Solution: To prepare stock phosphate buffer ;olution, dissolve 34.0 g
potassium dihydrogen phosphate, KH 2 P0 4 , m 500 ml distilled water, adjust to pH 7.2 with iN NaOH,
and dilute to 1 liter with distilled water. Add 1.25 ml stock phosphate buffer solution to 1 liter
distilled water. Dispense in amounts that will provide 99 ± 2.0 ml or 9 ± 0.2 ml after autoclaving
at 121 C for 15 mm.
4
-------
Bacteriological Examination
COLLECTION AND PREPARATION OF SAMPLES
Method for Collection of Solid Waste or Semi-Solid Waste Samples
Equipment and materials.
Necessary items are as follows:
1. Sample containers, specimen cups, sterile, 200-mi size (Falcon Plastics, Los Angeles)
2. Sampling tongs, stenle (stainless steel, angled tips, 18 in. long)
3. Shipping container, insulated, refrigerated, 6 by 12 in. I.D.
4. Disposable gloves
Procedure.
1. Using sterile tongs, collect 20 to 40 random 100- to 200-g samples and place in sterile sampling
containers. When collecting samples from contaminated sources, wear disposable gloves and avoid
contaminating the outside of the container.
2. Identify samples on tag and indicate time and date of sampling. If incinerator residue samples are
taken, record operating temperatures of incmerator.
3. Deliver samples to laboratory. It is recommended that the examination be started preferably
within 1 hr after collection,* the time elapsing between collection and examination should in no
case exceed 8 hr.
Method for Collection of Liquid Samples-Quench and Industrial Waters or Leachate
Equipment and materials.
Necessary items include a screw-capped, 250-ml, sterile sample bottle or a l6-oz, sterile plastic
bag.
Procedure
Collect sample in bottle or plastic bag, leaving an air space in the container to facilitate mixing of
the sample before examination. When collecting samples from contaminated sources, wear disposable
gloves and avoid contammating the outside of the container.
Identify and deliver samples to laboratory. When shipping samples to laboratory, protect con-
tainers from crushing and maintain temperature below IOC dunng a maximum transport time
of 6 hr. Examine within 2 hr. If water sample contains residual chlorine, a dechlorination agent
such as sodium thiosulfate is added to collection bottles to neutralize any residual chlorine and to
prevent a continuation of the bactericidal action of chlorine during the time the sample is in
transit to the laboratory. Enough sodium thiosulfate is added to the clean sample bottle before
sterilization to provide an approximate concentration of 100 mg per liter in the sample.
Sif sample is shipped to a laboratory for analysis and examination cannot begm within 1 hr of collection, the
container must be insulated and sample maintained below 10 C dunng the maximum transport of 6 hr. Such samples
should be refrigerated upon receipt in the laboratory and processed within 2 hr.
5
-------
METHODS OF SOLID WASTE TESTING
Method for Collection of Inc inerator Stack Effluents
Equipment and materials.
Necessary items include an Armstrong portable sampler (2), equipped with sampling assembly
(Figure 1). The sampler is mounted on a steel plate (6 by 12 in.) and can be enclosed by a metal
cover with a handle attached. On one side of the base is a vacuum pump with a 6-ft cord and
switch. The pump is capable of drawing up to 1 cu ft per mm of air (vacuum of 5.6 in. [ 14.3 cm] of
water). On the other side of the base, a 700-ml, wide-mouth, Pyrex bottle contains 300 ml of 0.067
M phosphate buffer solution (pH 7.2) prepared by standard methods (3). The two-hole rubber
stopper has a 1-in. (2.54 cm) piece of cotton-plugged glass tubing in one of the two holes. The
stopper, glass tube, and contents of the bottle are maintained sterile. The bottle is held to the
base plate by three removable spring clips, which are attached at the base and at a wire triangle
slipped over the top of the bottle. The sampling probe is made of stainless steel tubing of appropriate
diameter (e.g., 0.25-rn. ID. [ 0.64 cm]). The probe end has a right-angle bend so that the opening
faces the stack-gas current. The tubing must be long enough to reach all parts of the stack. The tubing
is coiled to permit additional cooling of the gases and is straight for 1 or 2 ft (30.48 or 60.96 cm) at
a right angle to the other straight length. Before use, the sampling probe is sterilized by dry heat
sterilization. It is important to keep the inside of the probe dry to minimize adsorption of micro-
organisms on the walls of the tubing. When sampling, the probe is inserted into the stack at locations
that will yield a representative sample. The other end of the sterile probe is inserted through the
sterile rubber stopper to approximately 0.5 in. (1.27 cm) above the buffered water. This is done to
reduce the frothing that would occur if the probe were mserted below the surface; enough froth
results in capturing the microorganisms.
Procedure.
1. Draw stack effluent through the sterile stainless steel tube by a 1.0 cfm vacuum pump; cool
the tube with a water jacket.
2. Obtain a 10-cu-ft sample by drawing the stack effluent for 10 mm.
3. Identify sample on tag and examine within 4 hr. The Armstrong portable sampler provides
a method for qualitative, nomsokinetic sampling and is adjustable to isokinetic conditions.
Method for Collection of Dust Samples
Equipment and materials.
Necessary items include the following:
I. Andersen sampler (4)
2. Trypticase soy agar containing 5 percent sheep blood (6 plates per sample)
3. Eosin methylene blue agar
Procedure.
I. Draw air through the sterile, assembled sampler at 1.0 cfm with a vacuum of 15 in. of mercury.
2. Remove agar plates from the sampler, cover, and incubate at 35 ±0.5 C. Use aseptic technique
throughout the procedure.
6
-------
II
11
CARRYING CASE
0
PHOSPHATE BUFFER
SAMPLING PROBE
FLOWMETER
6”
VACUUM
PUMP
12”
I — I
0
I
Figure 1. Portable sampler for microorganisms in mcinerator stack emission.
-------
METHODS OF SOLID WASTE TESTING
Method for Preparation of Solid and Semi-Solid Samples for Analyses
Equipment and materials.
Necessary items are as follows:
1. Cold phosphate buffer, 0.067 M, pH 7.2, sterile (3)
2. Blender, Waring (Model 1088), sterile
3. Balance, with weights, 500-g capacity
4. Tongs, sterile
5. Beakers, two, 5,000 ml and 1,000-ml sizes, sterile, covered with aluminum foil before stenli-
zation.
Procedure.
1. Using aseptic technique, composite all random samples into a 5,000-ml beaker. Mix well.
2. Weigh 200 g of the subsample into a 1,000-ml beaker.
3. Transfer the weighed sample to a sterile blender.
4. Add 1,800 ml of sterile, phosphate buffered solution to the blender.
5. Homogenize for 15 sec at 17,000 rpm (5).
6. Prepare a series of decimal dilutions as described below in “Methods for Preparation of Decimal
Dilutions of a Solid, Semi-Solid, or Liquid Waste Material.”
Solid waste and residue samples for enteric pathogenic bacteria are examined directly without homog-
enization.
BACTERIOLOGICAL EXAMINATION OF WASTE AND RELATED MATERIALS
Method for Preparation of Decimal Dilutions of a Solid, Semi-Solid, or Liquid Waste Material
Immediately after homogenization of any sample (see procedure under Method for Preparation of
Solid and Semi-Solid Samples for Analyses) transfer a I -ml portion of the homogenate (10-1 d ii)
to a dilution bottle containing 99 ml of phosphate buffered solution. Stopper and shake the bottle
25 times.
Prepare dilutions as indicated in Figure 2. Again shake each dilution vigorously 25 times after
adding an aliquot of sample.
These dilutions are used to inoculate a series of selected culture media for the detection of various
groups of microorganisms as described in the following sections of this paper
Methods for Total Viable Bacterial Cell Number
The chief cultural method for determining total viable bacterial densities has been the agar plate
method (3, 6, 7). Experience indicates that an enumeration of total number of viable bacteria
multiplying at a temperature of 35 C may yield useful information concerning the sanitary quality
of the waste entering a processing or a disposal site and provide useful information in judging the
efficiency of procedures used in solid waste processing and/or disposal operations. The viable
microbial count also provides valuable information concerning the microbiological quality of
environmental aerosols existing in or around a waste processing plant or a disposal site.
8
-------
\0
I
Solid or semi- solid
waste sample
200 gms
1
ml
e
Figure 2. Preparation of decimal dilutions.
-------
METHODS OF SOLID WASTE TESTING
Equipment, materials, and culture media.
1. Pipettes, 1.1 ml with 0.1 ml and 1 ml graduations
2. Dilution blanks, phosphate buffered solution, 99 ml ± 1 ml (cold)
3. Culture dishes (100 x 15 mm), plastic, sterile
4. Water bath for tempering agar, 45 ± 1 C
5. Incubator 35 ± 0.5 C
6. Colony counter, Quebeck
7. Sterile glass spreader, bent rod
8. Trypticase soy agar with 7 percent defibrinated sheep blood (TSA + blood)
9. Tryptone glucose extract agar (TGE)
Prepare TGE agar as indicated on label and hold in a melted condition in the water bath (45 C).
Dissolve ingredients of TSA and heat to boiling. Stenhze by autoclaving at 121 C for 15 mm.
Cool to 45 C and add sheep blood. Dispense in Petri plates and allow to solidify. Invert plates and
place them in incubator overnight to dry.
Procedure for bacterial count by pour plate.
1. Pipette 1 ml, 0.1 ml, or other suitable volume of the sample into each of appropriately marked,
duplicate culture plates, being sure to shake each dilution bottle vigorously 25 times to resuspend
material that may have settled out.
2. Add 10 to 12 ml of melted TGE agar to the sample in the Petri plate.
3. Mix dilution and the agar medium by rotating or tilting the plate.
4. Allow plates to solidify as rapidly as possible after pouring.
5. Invert plates and incubate them at 35 C ± 0.5 C for 24 ± 2 hr.
6. Count all colomes using Quebeck colony counter, the objective being to count plates with
30 to 300 colorues.
7. Compute the colony count per gram of waste (wet weight) or related solid material, and per
100 ml of water. The number of bacteria should not include more than two significant figures.
Procedure for bacteria! count by streak plate.
1. Dispense 0.1 ml samples of the serially diluted homogenate (or liquid) on the surface of each
of appropnately marked, duplicate TSA + blood agar plates.
2. Using a sterile glass spreader and starting with the highest dilution plates, spread the inoculum
evenly over the agar surface.
3. Invert plates and incubate them at 35 C for 24 hr ± 2 hr.
4. Count the number of colonies on plates with 30 to 300 colonies.
5. Select and mark colonies for further testing.
Methods for Presence of Members of Coliform Group
The coliform bacteria have long been used in the United States as indicators of fecal pollution in
sanitary bacteriology. Some members of the coliform-group organisms are found in the feces of
warm-blooded animals, in the guts of cold-blooded animals, in soils, and on many plants. Studies
have shown that warm-blooded animal feces from humans, animals, or birds may at any time contain
disease-producing microorganisms (8). It was pointed out that cold-blooded animal feces are
quantitatively insignificant as a source of pollution, but the coliform bacteria from plants or soils
10
-------
Bacteriological Examination
that have been recently exposed to fecal pollution have the same significance as those from feces.
On the other hand, the coliform bacteria deriving from soils or plants that have not been exposed to
recent fecal contamination has less public health significance.
Adequate treatment of waste before disposal and proper operational design of a waste processing
plant should remove all coliform organisms. Treated or processed waste containing coliform bacteria
demonstrates an inadequate treatment and should be considered of sanitary significance. The
contamination of waste by fecal matter may be one avenue of transmission of pathogenic micro-
organisms to the environment and man.
The presence of fecal matter in waste and related materials is determined by the standard tests
for the coliforrn group described in Standard Methods for the Examination of Water and Waste
Water (3). The completed Most Probable Number (MPN) procedure is employed. The testing method
includes the elevated temperature test (44.5 C) that indicates the fecal or nonfecal origin of
coliform bacteria. Comparative laboratory studies conducted showed that the MPN estimate is the
most suitable method for achieving a representative enumeration of the coliform organisms in solid
waste and waste effluents (9).
Equipment and materials.
1. Pipettes, sterile—deliveries to 10 ml, 1 ml (1.1 ml), and 0. 1 ml
2. Media prepared in fermentation tubes:
Lauryl tryptose broth
Brilliant green lactose bile broth, 2 percent
Lactose tryptose broth
E.C. broth
3. Media for plating.
Eosin methylene blue agar plates
Nutrient agar slants
4. Dilution blanks, phosphate buffer solution, sterile, 99-ml or 90-ml amounts
5. Incubator, adjusted to 35 C ± 0.5 C
6. Water bath, adjusted to 44.5 C ± 0.2 C
Procedure for total coliform group.
Presumptive Test.
1. Inoculate a predetermined volume of sample into each of 5 lauryl tryptose broth tubes. The por-
tions of the sample used for inoculation should be decimal multiples and submultiples of I ml.
2. Incubate the fermentation tubes at 35 ± 0.5 C for 24 ± 2 hr.
3. Examine for the presence of gas. If no gas is formed, incubate up to 48 ± 3 hr. Record the
presence or absence of gas formation at each examination of the tubes, regardless of the amount.
Confirmed Test.
1. Submit all presumptive test tubes showing any amount of gas at the end of 24- and 48-hr
incubation to the confirmed test. Using a sterile platinum loop 3 mm in diameter, transfer one loop-
ful of medium from.. the presumptive test fermentation tube to a fermentation tube containing
brilliant green lactose bile broth.
2. Incubate the inoculated brilliant green lactose bile broth tube for 48 ±3 hr at 35 ± 0.5 C.
The presence of gas in any amount m the fermentation tube of the brilliant green lactose bile
broth within 48 ± 3 hr indicates a positive confirmed test.
11
-------
METHODS OF SOLID WASTE TESTING
Completed Test.
1. Submit all confirmed test tubes showing any amount of gas to the completed test. Streak an
eosin methylene blue agar plate from each brilliant green bile broth tube as soon as possible after
the appearance of gas.
2. Incubate the plates at 35 ± 0.5 C for 24 ± 2 hr.
3. Fish one or more typical or atypical colonies from plating medium to lactose tryptose broth
fermentation tubes and nutrient agar slants.
4. Incubate the broth tubes and the agar slants at 35 ± 0.5 C for 24 ± 2 hr or 48 ± 3 hr if gas is
not produced in 24 hr.
5. Prepare gram stained smears from the nutrient agar slants if gas is produced in any amount
from lactose broth.
6. Examine smears under oil immersion. If typical coliform staining and morphology are found
on the slant, the test may be considered completed and the presence of coliform organisms demon-
strated.
Procedure for fecal coliform group (E. C. broth).
1. Submit all gas positive tubes from the Standard Methods presumptive test (lauryl tryptose
broth) to the fecal coliform test. Inoculate an E. C. broth fermentation tube with a 3-mm ioop of
broth from a positive presumptive tube.
2. Incubate the broth tube m a water bath at 44.5 ± 0.2 C for 24 hr. All E. C. tubes must be
placed in the water bath within 30 mm after planting.
3. Gas production in the E. C. broth fermentation tubes within 24 hr ± 2 hr is considered a posi-
tive reaction indicating fecal origin.
Computing and recording most probable number (MPN).
The calculated estimate and the 95 percent confidence limits of the MPN described in the
13th edition of Standards Methods for Examination of Water and Waste Water (3) are presented in
Table I. This table is based on five 10-mi, five 1 .0-ml, and five 0. 1-ml sample portions. When the series
of decimal dilutions such as 1.0, 0.1, and 0.01 ml are planted, record 10 times the value in .the table,
if a combination of portions of 0.1, 0.01, and 0.001 ml are planted, record 100 times the value in
the table. MPN values for solid samples are calculated per g of wet weight; MPN for liquid samples
are recorded per 100 ml.
Method to Determine the Presence of Viable Heat-Resistant Spore Number
It is important to enumerate those heat-resistant, spore-forming microorganisms in waste, incin-
erator residue, and quench or mdustnal waters that survive a temperature of 80 C for as long as 30
mm. With respect to mere survival of heat, most microorganisms in an actively growing (vegetative)
state are readily killed by exposures to temperatures of about 70 C for 1 to 5 mm (10). Cells inside
solid material such as discarded meat products may escape heat longer because the heat does not
penetrate immediately to the center of solid masses. Large masses of nonfluid solid matter require
a long time (1 V 2 to 2 hr), even m the autoclave (121 C), to be heated thoroughly enough for the
center to reach a sporocidal temperature. Other reports point out (11) that although internal air
temperatures of municipal incinerators usually range from 1,200 to 1,700 F (650 to 925 C) in
continuous operation, intermittent use and overcharging of the incinerator and moisture content of
the waste may interfere with sterilization of the residue.
12
-------
Bacteriological Examination
TABLE 1.
MPN INDEX AND 95 PERCENT CONFIDENCE LIMITS FOR
VARIOUS COMBINATIONS OF POSITIVE AND NEGATIVE RESULTS
WHEN FIVE l0-ML PORTIONS, FIVE 1-ML PORTIONS, AND FIVE
0.1-ML PORTIONS ARE USED.*
No. of Tubes Giving 95% Con- No. of Tubes Giving 95% Con-
Positive Reaction out of MPN fidence Limits Positive Reaction out of MPN fidence Limits
________________ _______ Index _____________ _______________________ Index _____________
per per
5 of 10 5 of 1 5 of 0.1 100 ml Lower Upper 5 of 10 5 of 1 5 of 0.1 100 ml Lower Upper
I Each ml Each ml Each
ml Each ml Each ml Each m
0 0 0 <2
o 0 1 2 <0.5 7 4 2 1 26 9 78
0 1 0 2 <0.5 7 4 3 0 27 9 80
0 2 0 4 <0.5 11 4 3 1 33 11 93
4 4 0 34 12 93
1 0 0 2 <0.5 7
1 0 1 4
-------
METHODS OF SOLID WASTE TESTING
A test of this type reveals operational problems of a waste processing plant and identifies the
unsatisfactory quality of waste effluents of a municipal incinerator.
Equipment and materials.
1. Test tubes, sterile, screw capped, 20 x 150 mm
2. Pipettes, sterile, graduated, 10-mi
3. Water bath, electrically heated, thermostatically controlled at 80 ± 0.5 C, equipped with
thermometer (range 0 to 110 C), NBS certified. Volume of water should be sufficient to absorb
cooling effect of rack of tubes without drop in temperature greater than 0.5 C.
4. Test tube support for holding tubes
Procedure.
1. Transfer 10 ml from each original sample and from each successive dilution thereof to screw-
capped test tubes, being careful to avoid contaminating the lip and upper portion of tube with
sample.
2. Place tubes in a rack.
3. Place rack of tubes in water bath at 80 C for 30 mm. Tubes should be immersed so that the
water line is approximately 1 ‘/2 in. above the level of samples in the tubes.
4. At the end of the 30-mm holding period, remove the rack of tubes from the water bath and
place in cold water for 5 mm to cool.
5. Determine viable heat-resistant spore count by agar pour-plate method (see, Procedure for
Bacterial Count by Pour Plate under Methods for Total Viable Bactenal Cell Number).
6. Report results as “viable heat-resistant spore count per gram.”
Methods to Detect Enteric Pathogenic Bacteria
Fecal pollution in the environment because of untreated and improperly disposed of waste may
add enteric pathogenic bacteria to a body of water or a water supply. The most common type of
pathogen that may be found in untreated waste is Salmonella. The wide distribution of the many
types of salmonellae in many species of ammals with which man has contact or may use as food
makes it difficult to prevent transmission to man (12). Infections may occur through food, milk, or
water contaminated with infected feces or urine, or by the actual ingestion of the infected animal
tissues (13). Salmonella has been found in many water supplies (14), polluted waters (15-17), raw
municipal refuse, and in incinerator residue (18, 19).
The detection of enteric pathogenic bacteria such as salmonellae and shigellae in municipal solid
waste before and after a treatment and/or disposal determines the microbiological quality of the
material and serves as a procedure to determine the efficacy of a waste treatment process in removing
or destroying the waste-borne pathogens. Results obtained in the testing may also be used for the
design of epidemiological studies in other programs.
The method described below has been tested in the field and has been described by Peterson and
Klee(18) and Spino(19), using mcubation temperatures of 39.5 C and 41.5 C.
Equipment, materials and media.
1. Incubator, 37 C
2. Water baths, constant temperature, 39.5 C and 41.5 C
14
-------
Bacteriological Examination
3. Flasks, wide-mouth, 500-mi
4. Membrane filter holder
5. Flasks, vacuum, 2,000-mi
6. Balance, with weights, 1 00-g capacity
7. Needle, inoculating
8. Media and reagents:
Selenite brilliant green/sulfa enrichment broth
Selenite F ennchment broth
Eosin methylene blue (EMB) agar
Saimoneila-Shigella (SS) agar
Bismuth sulfite (BS) agar
McConkey’s agar
Brilliant green (BG) agar
Triple sugar iron (TSI) agar
Urea medium
XLD agar
Salmonella antiserums
Shigella antiserums
Biochemical media (15)
9. Diatomaceous earth (Johns-Manvile, Celite 505), stenie
Procedure to detect pathogens in solid waste and incinerator residue.
1. Add a previously weighed, 30-g sample to each of two flasks containing 270 ml Selenite F
enrichment broth, and also to each of two flasks containing 270 ml Selenite brilliant green/sulfa
(SBG) enrichment broth. Shake to mix.
2. Incubate one Selenite F and one SBG flask at 39.5 C and the other two at 41.5 C for 16 to
18 hr.
3. After incubation, streak one loopful from each enrichment medium on each of four plates of
Salmonella-Shigella and other selective enteric media.
4. Incubate the plates at 37 C for 24 to 48 hr and pick suspicious colonies to triple sugar iron
agar slants.
5. Incubate the slants at 37 C for 24 hr and complete identification by appropriate methods as
descnbed by Edwards and Ewing (20). Isolation, preliminary identification, and biochemical testing
are described in Figure 3 and in Table 2.
Procedure to detect pathogens in quench or industrial waters and in leachate.
1. Place enough sterile diatomaceous earth on the screen of a stainless steel membrane filter
holder to form a I-in, layer.
2. Filter 800-mi sample through the earth layer.
3. Remove one-half the diatomaceous earth layer with a sterile spatula and place into 90 ml of
Selenite F ennchment broth; place other half of the earth layer into 90 ml of Selenite brilliant
green/sulfa enrichment broth. Shake both flasks to mix.
4. Incubate both flasks in a water bath at 39.5 C for 16 to 18 hr.
5. Proceed as directed in steps 3 through 5 of Procedure to Select Pathogens in Solid Waste and
Incinerator Residue.
15
-------
METHODS OF SOLID WASTE TESTING
L
Selenite F
2 7 0ml
I Solid was rresidue’
Green/SulIa 270 ml
1
Incubate at 39.5 C and 41.5 C
I
I
I
I
I
EMS
SS
BS
BG
MacConkey’s
A
ar
liii
Agar
1
Agar
4 Plates each
Agar
Agar
I
Incubate at 37 C
I
Triple Sugar
Iron agar
J
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Urea
medium
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I
[ 2- to 4-hour reading of urea medium
+
Proteus
group
(Reincubate negat 1
urea medium) I
I H S+J
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I
Salmonella polyvalent
antiserum 5
Shigella polyvalent antiserums
Salmonella polyvalent antiserums
+
—
Identify
serolog—
Do biochemical
ically; confirm
biochemically.
tests.
I
+
Identify serologically;
confirm biochemically.
Do biochemical
tests.
If not readily identifiable,
proceed to biochemical tests.
Figure 3. Isolation and preliminary identification.
16
-------
TABLE 2. DIFFERENTIATION OF ENTEROBACTERIACEAE BY BIOCHEMICAL TESTS.
(I) Cost. blotypus 01£ Ikn..n prodac. g s a S on..’ colt.,.. I . , . . . ’ tact... ..d on. olO°’ly s.d d.carbooytot. .oelthto.
(2) £ (ppM, S ch.trn. awe S efl..Utdta btogo, Posatypbt A s.d Pull .... s.d t i .e co h n . otdlntIIy do o c t (cant dolc Itol po oly S cdak...-.,os do.. got lca.ot .nbt.on
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— cc ..)odty ..gstlo. • nobly po IUo. ,..cUn
Source identification of Enterobactersaceae by P R. Edwards and W H. Ewing Third edition, 1972, p 24 Reproduced by permission,
Burgess Publishing Company, Minneapolis, Minnesota
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I
-------
METHODS OF SOLID WASTE TESTING
Method for Examination of Stack Effluents
As described in Methods for Collection of Incinerator Stack Effluents (using the Armstrong
sampler), the microorganisms are impinged into a 300-mi phosphate buffer solution.
1. Filter 100 ml of the “inoculated” phosphate buffer solution through a 0.45 HA membrane
filter (3).
2. Transfer membrane filter with sterile forceps to a culture plate containing trypticase soy agar.
3. Incubate culture plate under constant saturated humidity for 20 hr (± 2 Kr) at 35 C.
4. After incubation, remove cover from culture plate and determine colony count with the aid of
a low-power (10-15 magnifications) binocular, wide-field microscope. Characterize colonies using
specific isolation media.
5. Remove a l0-ml portion of the “inoculated” phosphate buffer solution and examine for viable
heat-resistant spores as directed in steps 1 through 6 of the procedure under Method to Determine
the Presence of Viable Heat-Resistant Spore Numbers.
Microbial counts are reported as organisms per cubic foot of air. If the sample is not taken
under isokinetic conditions, the results are qualitative. If the stack velocity is known and remains
relatively constant, however, the flow rate of the sampler can be adjusted to isokinetic conditions
to yield quantitative results.
Method for Examination of Dust
As described in Methods for Collection of Dust Samples, the Andersen sampler is used with two
types of media—trypticase soy agar (TSA-BBL product) containing 5 percent sheep blood, and eosin
methylene blue agar (EMB-Difco product). The TSA/blood agar is used to isolate a wider range of
fastidious organisms such as ‘Staphylococci, Streptococci, and Diplococci. The EMB agar is used to
isolate gram-negative bacteria. The plates are incubated aerobically at 37 C for 24 hr. (Preliminary
studies showed that few organisms in the dust would grow under anaerobic conditions.) Enumeration
of colonies is made with a Quebec colony counter. Microbial count is reported as organisms per
cubic foot of air. At times, when microbial counts are high, the sampling time is 0.25 mm, thus yield-
ing 0.25 cu ft air.
REFERENCES
1. Hanks, T.G. Solid waste/disease relationships. U. S. Dept. of Health, Education, and Welfare,
Public Health Service Pub!. No. 999-UIH-6, Cincinnati, National Center for Urban and Indus-
trial Health, 1967.
2. Armstrong, D.H. Portable sampler for microorganisms in incinerator stack emissions. Applied
Microbiology, 19 (l):204-205, 1970.
3. American Public Health Association. Standard methods for the examination of water and
waste water. New York, American Public Health Association, 1971.
4. Andersen, A.A. New sampler for the collection, sizing and enumeration of viable airborne
particles. Journal of Bacteriology, 76:471-484, 1958.
5. Peterson, ML. and F.J. Stutzenberger. Microbiological evaluation of incinerator operations.
Applied Microbiology. 18(1): 8-13, 1969.
18
-------
Bacteriological Examination
6. American Public Health Association, Inc. Standard methods for the examination of dairy
products microbiological and chemical. New York, American Public Health Association, mc,
1960.
7. Harris, A.H., and M.B. Coleman. Diagnostic procedures and reagents. New York, American
Public Health Association, Inc. 1963.
8. Clark, H.F., and P.W. Kabler. Revaluation of the significance of the coliform bacteria. Journal
of American Water Works Association, 56:931-936, 1964.
9. Smith, L., and M.A. Madison. A brief evaluation of two methods for total and fecal coliforms
in municipal solid waste and related materials. Cincinnati, U. S. Environmental Protection
Agency, National Environmental Research Center. Unpublished data, 1972.
10. Frobisher, M. Fundamentals of microbiology, 6th ed. Philadelphia, W. B. Saunders Co., 1957.
p. 151-152.
11. Barbeito, M. S. and G.G. Gremillion. Microbiological safety evaluation of an industrial refuse
incinerator. Applied Microbiology, 16:291-295, 1968.
12. Dauer, Carl C. 1960 Summary of disease outbreaks and a 10-year resume. Public Health Report,
76,no. l0,Oct. l96l.p 915.
13. Dubos, Rene. Bacterial and mycotic infections of man. Philadelphia, J. B. Lippincott, 1958.
14. Weibel, S. R., FR. Dixon, R.B. Weidner, and L.J. McCabe. Waterborne-disease outbreaks
1946-1960. Journal of the American Water Works Association, 56:947-958, Aug., 1964.
15. Spino, D.F. Elevated-temperature techniques for the isolation of Salmonella from streams.
Applied Microbiology, 14:591, 1966.
16. Scarce, L.E. and M.L. Peterson. Pathogens in streams tributary to the Great Lakes. In:
Proceedings; Ninth Conference on Great Lakes Research, Chicago, March 28-30, 1966.
Public No. 15. Ann Arbor, Univ. of Mich., 1966. p. 147.
17. Peterson, M.L. The occurrence of Salmonella in streams draining Lake Erie Basin. In: Proceed-
ings; Tenth Conference on Great Lakes Research, Toronto, Apr. 10-12, 1967, Ann Arbor, Univ.
of Mich., 1967. p. 79.
18. Peterson, M.L. and A.J. Klee. Studies on the detection of salmonellae in municipal solid
waste and incinerator residue. International Journal of Environmental Studies, a: 125-132, 1971.
19. Spino, D. Bacteriological study of the New Orleans East\ Incinerator. Cincinnati, U.S. Environ-
mental Protection Agency, National Environmental Research Center, 1971.
20. Edwards, P.R. and W.H. Ewing. Identification of Enterobacteriaceae. Minneapolis, Burgess
Publishing Co., 1972.
19 *
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