v>EPA
Canada
RESEARCH
/INSTITUTE;
United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
EPA/600/R-95/077
June 1995
National Water Research Institute
Burlington, Ontario, L7R 4A6, Canada
0729ASD94
Laboratory Methods for
Soil and Foliar Analysis in
Long-Term Environmental
Monitoring Programs
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EPA/600/R-95/077
June 1995
Laboratory Methods for Soil and Foliar Analysis in
long-Term Environmental Monitoring Programs
by
The Ad Hoc Terrestrial Monitoring Work Group of the Quality Assurance Subgroup
Focusing on Monitoring the Terrestrial Effects of Acidic Deposition
B. A. Schumacher, A. J. Neary, C. J. Palmer, D. G. Maynard,
L Pastorek, I. K. Morrison, and M. Marsh
Prepared for the Quality Assurance Subgroup
John Lawrence, Co-Chairman
Serge Villard, Co-Chairman
Printed on Recycled Paper
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NOTICE
The U.S. Environmental Protection Agency's Office of Research and Development (ORD),
Environment Canada, the Ministry of the Environment - Ontario, and the Canadian Department of
Natural Resources - Great Lakes Forestry Centre, Northern Forestry Centre, and Pacific Forestry
Centre have prepared and funded this methods manual. It has been peer reviewed by the Agency
and approved as an EPA publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
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ACKNOWLEDGEMENTS
This methods manual was prepared as a joint effort between the U.S. Environmental Protection
Agency, Environment Canada, the Ministry of the Environment - Ontario, and the Canadian
Department of Natural Resources - Great Lakes Forestry Centre, Northern Forestry Centre, and
Pacific Forestry Centre. In order to give proper acknowledgement for the efforts of each of the
authors, the following list has been prepared indicating the author, their affiliation, and which
chapters they prepared:
Chapter(s) Prepared
Brian A. Schumacher , Introduction, 6, 9
U.S. Environmental Protection Agency
Environmental Monitoring Systems Laboratory
Analytical Sciences Division \
P.O. Box 93478
Las Vegas, Nevada 89193-3478
USA I
Craig J. Palmer
U.S. Environmental Protection Agency
Environmental Monitoring Systems Laboratory
Monitoring Sciences Division
P.O. Box 93478
Las Vegas, Nevada 89193-3478
USA
Anne J. Neary
Ministry of the Environment - Ontario
125 Resources Road
Etobocoke, Ontario M3P 3V6
CANADA
Douglas G. Maynard
Department of Natural Resources
Northern Forestry Centre
5320 122nd Street
Edmonton, Alberta T6H 3S5
CANADA
Liz Pastorek
Ministry of the Environment - Ontario
125 Resources Road
Etobicoke. Ontario M3P 3V6
CANADA
Introduction, 7, 12, 13
GLP, 14, 15, 16, 19
3, 4, 10, 17, 18, 20
3, 4, 5, 10, 20
HI
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Ian K. Morrison
Department of Natural Resources
Great Lakes Forestry Centre
P.O. Box 490
Sautte Ste. Marie. Ontario P6A 5M7
CANADA
MarRjs Marsh
Ministry of the Environment - Ontario
125 Resources Road
Etobfcoke, Ontario M3P3V6
CANADA
8
11
iv
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Table of Contents
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Table of Contents
Chapter
Page Revision
Notice jj
Acknowledgements iii
1. Introduction 1 of 2 2.1
2. Good Laboratory Practices : i'Of 14 n
3. Total Nitrogen in Plant Tissue •'...' 1 of 6 2.1
4. Total Sulphur in Plant Tissue 1 of 7 2.1
5. P, Mn, Fe, Al, Ca, Mg, and K in Plant Tissue 1 of 7 2.1
6. Particle-Size Analysis 1 of 20 2.1
7- Soil pH i of 6 2.1
8. Organic Carbon , 1 of 8 2.1
9. Total Carbon 1 of 7 1.1
10. Total Nitrogen -j of 5 2.1
11. Extractable Phosphorus 1 of 7 2.1
12. Cation Exchange Capacity : 1 of 12 2.1
13. Exchangeable Cations 1 of 11 2.1
14. Amorphous Iron and Aluminum Oxides 1 of 7 2.1
15. Organic Iron and Aluminum 1 of 10 2.1
16. Iron and Aluminum Oxides i ;.. 1 of 7 2.1
17. Phosphate Extractable Sulphate 1 of 8 2.1
18. Ammonium Chloride Extractable Sulphate 1 of 8 2.1
19. Water Extractable Sulphate 1 of 8 1.1
20. Total Sulphur in Soil 1 of 6 2.1
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Introduction
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Analytics/ Methods for Terrestrial Samples
Introduction
The success of long-term environmental monitoring studies, such as those designed to
measure the effects of the long range transport of atmospheric pollutants, will depend on the type
of data collected and the comparability of the data over the course of the monitoring period. Data
comparability will depend on collection and analysis procedures as well as the natural spatial and
temporal variability of the soil and vegetation. The objective of this document is to present methods
for the collection, preparation, and analysis of soil and plant tissue samples taken as part of a lona-
term study to evaluate the effects of acid rain on terrestrial systems.
The objective of the terrestrial monitoring program is to measure real changes caused by
acidic precipitation. Unfortunately, these changes can be hidden by natural spatial and temporal
variability or variability resulting from errors or changes in the measurement process. Spatial and
temporal variability can be addressed through proper sampling designs. Measurement errors can
be minimized through quality assurance protocols, such as the development of consistent analytical
techniques. It is hoped that this report will assist in selecting and documenting appropriate
analytical methodologies.
Quality Assurance and Quality Control
Quality assurance may be defined as "a system of activities whose purpose is to provide to
the producer or user of a product or service the assurance that it meets defined standards of quality
^ ™tated level of confidence" (Taylor, 1987). The goal of the quality assurance/quality control
(QA/QC) procedures presented in this manual is to ensure that the data collected for a given
parameter is of the highest integrity and that the data quality can be evaluated and documented.
To ensure the integrity and comparability of analyses among various laboratories, several
quality control procedures have been presented in each of the methods. These procedures deal with
the assessment of precision and accuracy and the use of method blanks, quality control preparation
samples (QCPS), and quality control check samples (QCCS). Within each method, acceptance limits
and frequency of use within an analytical batch of these QC samples has been presented. Further,
a suggested run format has been presented showing a potential distribution of the QC samples
within the analytical run.
To assess analytical precision, at least one sample should be analyzed in duplicate with each
run of thirty samples or less. In sample batches that are expected to have large analyte
concentration fluctuations, additional duplicate samples are recommended (recommended rate is
1 duplicate sample per 10 routine samples) to assess precision. To avoid bias associated with
position within the run, non-adjacent duplicate analysis is recommended. For example, if the
suggested run format indicates to run the duplicate sample after the 16th routine sample (i.e., in the
middle of the run), therefore, the associated replicated sample should be selected from the first
(sample numbers 1 to 8) or last (sample number 26 to 30) group of routine samples.
Accuracy can be assessed through the use of standard reference materials (SRMs). For many
of the methods presented in this manual, SRMs are not available from commercial sources. SRMs
are available for total elemental analyses, such as total carbon, in vegetation and soil samples.
Accuracy checks, however, for extractable soil parameters (preparation + analysis) can be created
by using the median values of large Intel-laboratory round-robin studies with soil samples prepared
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and shipped strictly for that purpose. Where available, accuracy standards are recommended to
be run in the middle of the analytical run. .
Method blanks are analyzed to assess if contamination of the sample or sample extracts has
occurred. Contamination can occur from numerous sources, such as glassware, filters and reagents
used during sample preparation or analysis. It is recommended that 3 or more method blanks be
analyzed at the beginning, middle, and end of the run. If contamination is identified within the run
(i.e., the method blank has a concentration greater than the instrument detection limit), two courses
of action can be taken. First, rerun the blank to ensure that the value is above the instrument
detection limit. If the method blank still indicates contamination is present, then all samples
between the last "non-contaminated" blank and the contaminated blank should be rerun until no
contamination is indicated in the new method blank (i.e., the method blank prepared during the rerun
of the routine samples). The second course of action occurs for those methods where significant
preparation is involved in the blanks and thus detectable analyte concentrations will occur in the
method blanks. In this situation, if all the routine sample concentrations are significantly higher
(e.g., 10 times greater) than the blank, analyses should continue with a blank correction occurring
on the final sample results using the mean blank value. In cases where the blank and the routine
samples are close in measured concentrations, then the decision should be made by the laboratory
manager on whether to reanalyze or accept the results.
The QCPS is a matrix matched in-house quality control sample used to monitor accuracy and
long-term between-run precision. This sample should be a soil sample that has been collected and
prepared in bulk and analyzed numerous times through time and across projects/programs. After
each analysis, the resultant analyte concentrations should be incorporated into the database and
a new accuracy window developed for that sample. Over a long period of time, the median value
or long-term mean may approximate the "true" value for the sample. Ideally, more than one QCPS
should be obtained to better characterize the routine analytical range and sample type analyzed (i.e.,
sandy, silty, clayey, and organic soils). Further statistical significance and value of the QCPS, can
be obtained by submitting the sample through interlaboratory round-robin testing. One QCPS is
recommended to be analyzed per run.
The QCCS is a matrix matched standard solution containing the analyte of interest at a known
concentration in the mid-calibration range. The QCCS is analyzed to verify the calibration curve (i.e.,
to monitor and correct for instrumental drift) and should be analyzed at the beginning, after every
ten samples, and after the last sample of each analytical run. This sample should be prepared from
a different stock solution than that used to prepare the initial instrument calibration standards and
should not been taken through any preparation step.
References
Taytor, J.K. 1987. Quality assurance of chemical measurements. Lewis Pub., Chelsea, MI. 328 pp.
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Good Laboratory Practices
Introduction
Providing the client with the best possible data of known and acceptable quality (i.e., with a
specified probability of being correct).must be of foremost concern in the analytical laboratory.
Good laboratory practices may be defined as a group of operations or procedures which comprise
quality assurance within the laboratory, and which are integral to achieving and maintaining the high
quality of output from any laboratory. These operations and procedures should become the code
of behaviour in the laboratory. They apply to workload planning and receiving; data quality objective
setting and sample representativeness; documentation; personnel training; sample storage.
preservation, and preparation; laboratory cleanliness; equipment maintenance; laboratory safety-
instrument calibration; and analytical quality control protocols. >ks part of the commitment to
quality, staff must be aware of their responsibilities, policies and procedures must be documented
and distributed, and the channels of communication between the client and laboratory staff must
remain open. This chapter is meant as a general guide for laboratory managers and supervisors.
It is designed for the laboratory performing routine tests on soil and foliar samples. It does not
provide for unusual samples or complex analytical techniques requiring specialized facilities or
equipment (e.g., work requiring "clean room11 procedures, radioactive or highly toxic substances, etc.).
Workload Planning and Receiving
Workload planning and sample scheduling is necessary to ensure that the client receives
hisyher results in a timely manner and that perishable samples are analyzed without undue delay
(e.g., field moist samples). Any laboratory providing an analytical service must have available space,
staff, necessary facilities, and documented procedures for sample reception, log-in, and
preservation. The latter may include soil and foliar tissue drying, freezing, refrigerating, freeze drying,
and storage. A method of tracking samples as they pass through ihe laboratory is also required.
This is usually done by assigning a unique laboratory number or label to the sample. The protocol
should be documented to guarantee consistency between laboratory personnel and within sample
types. The number or label must be traceable to the original field sample number, client, and date
of receipt at the laboratory. - The chain of custody for each sample must be known so that the
sample's disposition at any time can be determined. Having the analyst initial a form when samples
are removed from and returned to their place of storage and initial tho bench sheet after completion
of an analysis helps to track the progression of samples through the laboratory.
Data Quality Objectives and Sample Representativeness
The data quality required (sensitivity and coefficient of variation at ail levels of detection) must
be established by the client at the outset of the study. This allows the project and laboratory
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managers to work together to achieve these objectives. For the data user, it is critical that the
sample is representative of the environment under observation. It is th'e responsibility of the
sampler and project manager to provide samples which best represent the environment being
measured. For some purposes, a single sample or a homogenized or composite sample prepared
from a series of separate samples may be adequate. More frequently, however, the collection of
replicate samples from the same sampling location is necessary to obtain a measure of variability
in the field. These field replicates must not be confused with laboratory replicates or "sample splits"
which are analyzed routinely in the laboratory to provide a measure of analytical precision.
It is the responsibility of the laboratory analyst to ensure that the method-provides data of
acceptable quality and that this quality is not compromised by the use of inappropriate sampling
containers, preservatives, storage facilities, or handling procedures. Moreover, it is important that
both the laboratory analyst and client know the limitations of the analytical procedures used.
Environmental studies usually require considerable planning. Both the laboratory manager and
project manager or client must have input and assume responsibility for ensuring that the project
data quality objectives are met.
Documentation
The importance of properly documenting procedures in the laboratory cannot be overstated.
Written sample handling, preparation and analytical procedures, maintenance and safety guidelines,
and analytical quality control records should be available. This documentation is required for the
laboratory to establish its credibility; however, it does not, in itself, guarantee that the procedures
are properly followed or that the data produced are of the quality specified. Training and ongoing
evaluation of method performance is also needed.
Staff Training
Laboratory training programs are necessary for all new laboratory personnel and existing staff
whose duties in the lab are changing. The theory and rationale for existing procedures should be
covered in detail.
Sample Preparation
Soil and foliar sample preservation and storage may consist of refrigeration, freezing, freeze
drying, air drying, or oven drying. When the analysis requires field moist samples and the samples
cannot be analyzed immediately, refrigeration at 2 to 4° C or freezing at -20° C is recommended.
Refrigerated samples should be analyzed within one or two days of receipt. The analysis of field-
moist soil samples is often dictated by the project objectives or the tests requested. Irreversible
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°an °CCUr 3S 3 result °f dryFn9- Par^arly in organic soil horizons
T* *"" "* Maynard' 1"1)" M°St methods described j" «•
f°"ar SampISS and« *«•"*>* Notices re,ated to their
Soils
Samples should be dried on non-metallic trays or plastic sheets in an area free from airborne
dust and chemical fumes. Sample drying and grinding/sieving shoulld not be performed in the same
?™\ £* ~mpS °r C'°dS Sh°uld ** broken by hand *° faci«tate drying. Samples should be dried
at 20 to 25° C until a constant mass is attained. Unless specifically requested to do otherwise
samples are disaggregated and sieved. Only the material passing a 2 mm sieve is used for
analysis The > 2 mm material is retained and its weight recorded. The whole sample should be
Some tests, those measuring total amounts of an element or those requiring a very small
sample aliquot, require a homogeneous finely-ground sample. Grinding to < 35 mesh (500 urn) is
recommended for some of the methods outlined in this manual. When grinding a subsample to
pass a specified mesh size, the following steps should be performed:
• The sample should be well mixed before a subsample is removed.
• The subsample should be sieved first to remove all material that is naturally less than the
mesh size used.
• The subsample should be ground using an agate mortar and pestle (to prevent AT
contamination) for short time periods. Frequent re-sieving is necessary until the whole
subsample passes through the sieve. This prevents biasing the sample by discarding any
portion of the sample which is difficult to grind, and it prevents over-grinding of minerals
Over-grinding is not a problem if total amounts of an element are measured, but can be a
problem for the measurement of extractable Fe and Al (Neary and Barnes, 1993).
Foliar Tissue
Foliar samples should be dried in a forced draft oven at between 70 and 80° C. Lower drying
temperatures should be used if volatilization is of concern. Dried samples are ground to pass a 20
mesh (850 /^m) sieve. Large samples may be. first ground through a Wiley Mill using a 10 mesh (2
mm) sieve and then reduced by quartering and put into an Intermediate Wiley Mill or Tecator
Cyclotec (Kalra and Maynard, 1991). These samples may be used for the determination of N, P, K,
Ca, Mg, Na, and S. A non-metallic grinder is recommended if Fe, Mn, Cu and Zn are to be measured.
Samples should be transferred to non-metallic storage containers and tightly sealed for storage.
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Laboratory Cleanliness
Laboratory cleanliness encompasses everything from maintaining an organized workplace
within the laboratory to the quality of the air, water supply, and reagents. A well-designed
laboratory in terms of workstation location helps avoid accidents and reduces the chance of sample
contamination from labware and reagents used for other tests. Shared equipment (balances,
centrifuges, water baths, block digesters, stirrers, vortex mixers, shakers, etc.) should be thoroughly
cleaned after each use to prevent sample contamination and salt build-up from sample extracting
and digesting solutions. Similarly, the sample path in analytical instruments should be thoroughly
rinsedfl lushed with deionized water after each use. This will extend the life of the instrument and
prevent contamination of the next batch of samples analyzed. Burner heads from atomic absorpt.on
spectrophotometers should be cleaned after each use to prevent salt build-up. When equipment
must be shared, it is advisable that all batches of one test be completed before another batch ,s
started.
When cleaning glassware and plasticware consideration must be given to the expected analyte
concentrations in the sample. All new glassware and plasticware should be cleaned with soap and
water. A contaminant-free detergent, such as Decon or Acationox, which rinses well is
recommended. Three short rinses with distilled/deionized water after washing are preferable to one
long rinse. For trace element analysis, labware should be soaked in nitric acid before use. Separate
labware should be used for the analysis of soil and foliar tissue. More rigorous cleaning procedures
and separation of labware is required if sub-mg/L levels are to be measured.
The quality of the air in the laboratory is important for the safety of the staff and to prevent
contamination of the samples. All instruments and equipment should be vented according to the
manufacturer's specifications. Instrument vents and fume hoods should be checked on a routine
basis so that airborne contamination in the form of fumes or dust is prevented. So.l drying sievmg
and grinding should not be performed in the same room used for sample extraction/digestion and
analysis. Even the weighing of dried samples can generate a significant amount of dust and shou d
be done in an area removed from sample analysis. Soil samples and vegetation samples should
be weighed and analyzed in different locations.
A source of purified water is essential to the analytical laboratory. Laboratories having an on-
line supply of purified water should check the conductivity of the water regularly Per,od,c checking
of the analyte of interest can be done by analyzing the purified water as a blank sample ,n_the run
This might be in addition to the matrix-matched method blanks which are part of each batch of
samples analyzed. If water purification (distillation, deionization, etc.) ,s done by the laboratory
staff, regular maintenance schedules and procedures must be documented and foltowedL A
conductivity of below 1 /,S cm'1 for freshly purified water is suitable for most soil and vegetate
^alyses. if the conductivity of the water is above 2/,S cm'1, the water should not be used until the
system is inspected and cleaned. The conductivity of purified water sitting in carboys or ong
Periods will gradually increase to between 1 and 3 /,S cm'1 as ions are leached from the walls of the
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container. If an on-line deionized water tap is present, it may be advisable to discard the first three
litres of water at the beginning of each work day (Robarge and Fernandez, 1986). In cases where
NH+4 or NO"3-free water is required, amine-based deionizing cartridges may be a source of error.
Cleaning of cartridges with formaldehyde has also been found to contribute to contamination
problems.
Reagents must be free of contamination by the analyte. New batches of reagents should be
checked prior to their use. Matrix-matched blank solutions prepared with the new reagent should
be checked against blanks prepared with the old batch of reagent. Fleagents not meeting the purity
requirements should be labelled as such and either discarded or stored separately. Special purity
requirements for reagents or dilution water be must documented in the standard operating
procedure.
Reagent Traceability \
All reagents must be dated upon receipt at the laboratory and on opening to prevent prolonged
storage of reagents beyond their estimated shelf-life (Robarge and Fernandez, 1986). Prepared
reagents should be labelled with the solution name, concentration, date of preparation, and name
of person preparing the reagent. In Canada, WHMIS (Workplace Hazardous Material Information
System) labelling is a requirement for all bought or prepared reagents. WHMIS legislation also sets
strict regulations for the storage of chemicals. The laboratory manager must make sure that all
staff receive WHMIS training.
All powders used for primary standard and reagent preparatiion should be numbered upon
receipt and all stock solutions and reagents prepared using that powder should be numbered and
have a paper trail back to the batch of chemical used. Similarly, all intermediate and working
solutions should be traceable to their source. If certified stock solutions are used instead of neat
chemical powders for standard preparation, all solutions prepared from these stocks should be
traceable back to the original bottle used. When the stock powder or purchased stock solution is
discarded or exhausted, records should be kept and the replacement given a new and unique
number upon receipt. This rigorous record keeping will greatly simplify trouble-shooting for
problems, such as reagent contamination, sensitivity irregularities, calibration errors, etc.
Equipment Maintenance
An equipment maintenance schedule can extend the life of an instrument, reduce repair costs,
and maximize instrument performance. Equipment is often shared by many users in the laboratory.
It is advisable, therefore, to assign one person the responsibility of maintaining a given piece of
equipment. Although all operators should be trained to run daily safety and performance checks
before use and to clean the instrument after use, sharing the routine maintenance of the instrument
among operators does not work well. This is especially true for sophisticated instrumentation such
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as atomic absorption spectrophotometers, ICP emission spectrometers, ion chromatographs,
continuous flow systems, carbon analyzers, and X-ray fluorescence spectrometers. Detailed
equipment maintenance procedures, schedules, and performance checks are usually provided by the
manufacturer. Below are some general guidelines for maintaining and using equipment most
commonly found in the soil/vegetation laboratory.
As much as possible, instruments should be located in areas free from dust and fumes.
Although most analytical instruments are sealed and protected from dust and laboratory chemicals,
a dust cover is advised when the equipment is not in use, especially in particularly corrosive or dusty
environments. Reagents should never be stored on the top of instruments. The level of waste
containers located under instruments should be closely monitored. The pressure on pump lines of
continuous flow systems should be released when not in use. All filters and traps on atomic
absorption spectrophotometers, ICP's, carbon and sulphur analyzers, etc. should be checked,
cleaned, and replaced regularly. Ion chromatograph pumps, and sample chambers and lines on all
instruments should be rinsed well after use, especially if strong salt solutions are used. The
performance of automated shut-down mechanisms should be checked regularly.
Weighing samples and reagents is a daily activity in the soil/vegetation laboratory. Balances
are often used intermittently throughout the day by many lab personnel. The balance should always
be cleaned after each use. Even when sample or chemical spillage does not occur, the weighing
of finely ground soil and vegetation samples will leave a film of dust on the balance. The balance
pan should be removed and the whole balance cleaned after each use with a camel-hair or other
soft-haired brush. Wiping with a moist cloth should be done only after brushing to prevent
scratching and smearing of the balance with moistened sediment.
Balances should be located away from drafts caused by nearby doors, heating and cooling
vents, exhaust fans for instruments, fume hoods, centrifuges etc. A balance table is recommended
even though the newer four-place balances are particularly stable. If the balance must be located
on the laboratory bench, centrifuges, vacuum pumps, stirrers, or any equipment likely to cause
vibration, should not be used on the bench at the same time. Balances should be routinely checked
for accuracy using a certified set of weights. Occasional recalibration may be necessary. Balances
should be turned on in the morning and left on throughout the day.
Centrifuge heads and shakers should be cleaned on a regular basis to avoid salt build-up from
extracting solutions. This build-up may not only contaminate other samples but will eventually cause
pitting of the metal centrifuge head, weakening it, and making it unsafe. Samples must always be
placed in the centrifuge in a manner to ensure that it is balanced and the centrifuge should never
be opened when in use.
Aluminum block digesters are often used for strong acid digestions of soil and vegetation.
These must be used in a fume hood and all non-aluminum hardware (e.g., screws, handles, rivets,
etc.) on either the block itself or side covers should be regularly checked for corrosion by acid
fumes. Metal contamination from such corroded surfaces is possible. Controllers for the block
should be located outside the fume hood.
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Maintenance of pH meters consists primarily of proper electrode care. Combination electrodes
should be kept adequately filled with saturated KCI solution. Loose KCI crystals should be present
at the bottom of the electrode and air bubbles should not be present. The rubber sleeve should
cover the filling hole when the electrode is not in use. Storage buffers and cleaning solutions
recommended by the manufacturer should be used. The electrode should be rinsed between
samples using a stream of deionized water. Wiping of the electrode with laboratory tissue is not
recommended. !
Laboratory Safety
A laboratory safety manual should be available and read by all laboratory personnel. In
Canada, all laboratory personnel are required to undergo WHMIS draining. This training provides
information on storage, labelling and use of chemicals, chemical waste disposal, and transportation.
WHMIS Material Safety Data Sheets (MSDS) must be available for all chemicals used in the
laboratory.
Safe laboratory practices for all the analytical procedures outlined in this manuaj cannot be
covered here; however, some general principles of laboratory safety should be mentioned. Specific
safety precautions during an analysis should be outlined in the (Standard operating procedure.
Instrument operating manuals, provided by the manufacturer, usually outline safety requirements
and should always be kept with the instrument. Emergency shut-down procedures for instruments
should be posted. Safe laboratory practices must never be compromised for the sake of decreasing
sample throughput time. Personnel should develop a positive attitude toward safety.
i
Fire extinguishers, eye wash stations, safety showers, first-aid kits and chemical spill kits
should be present and easily accessible in all laboratory areas. Laboratory evacuation procedures
should be known and practiced by all staff. Fire escape routes must ba marked and kept clear of
carts, equipment, gas cylinders, coat racks, etc. Extreme care must be exercised when handling
flammable or potentially explosive materials (e.g., acetyiides, perchlorates, azides, ozonides, and
peroxides). Flammable materials should be stored separately in specially designed metal storage
cabinets and carried in safety cans. Concentrated acids and bases should be stored in specially
designed vented cabinets and transported in safety bottle carriers. All cylinders must be properly
secured and the tank pressure not allowed to drop below the recommended level. Acids and organic
solutions must be stored separately. Storing powdered reagents in alphabetical order, a practice
commonly seen in the laboratory, should not be done. Oxidizing and reducing agents must not be
put together. Staff should receive training in the cleaning of chemical spills and emergency contact
numbers should be readily available. First aid and cardiopulmonary resuscitation (CPR) training for
staff is also recommended.
The importance of proper analytical technique (e.g., pipetting, preparing solutions with
concentrated acids or bases) and the use of personal safety equipment such as lab coats,
chemical-resistant aprons, gloves, and eye protection must be emphasized by the laboratory
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supervisor. Wearing contact lenses in the laboratory is not advised and open-toed shoes should
not be worn. Food and drink should not be consumed or stored in the laboratory. Smoking is
prohibited by law.
Equipment or procedures emitting chemical fumes should be vented to the atmosphere. This
includes atomic absorption spectrophotometers, ICP emission spectrometers, muffle and
combustion furnaces, and microwave ovens. Fume hoods should be used only to perform
operations for which they were designed. For example, if perchloric acid is used, a specially
designed stainless steel hood without any exposed organic material or sealer and with a proper
washdown facility is required. Fume hoods should be washed down regularly and not modified
without the manufacturer's approval (e.g., removal of baffles). The ventilation efficiency of the hood
should be measured with the sash in its normal position under routine working conditions.
Most laboratory accidents are a result of failing to observe basic safety precautions. Each
worker should ultimately be responsible for his/her own safety and each laboratory supervisor
should try to maintain a safely designed laboratory and ensure than laboratory staff have access
to all necessary safety equipment and information.
Analytical Quality Control
As part of quality assurance within the laboratory, good laboratory practices must include
quality control protocols for ensuring that an analytical method is operating as expected and meets
the predefined data quality objectives. Continual review of these protocols and performance data
will help to identify problems early in the analytical process. Corrective actions can then be taken
with minimal loss of data to the client.
Method Development
Method development is the set of experimental procedures designed to measure a known
amount of a substance in various matrices. Method development ensures and demonstrates that
the extraction of the substance and response of the measurement system to that substance follows
a specific behaviour in a predictable, reliable, and stable fashion. The sample matrix used in the
development process must represent the type of samples which will be analyzed routinely. The
ruggedness and application of a method to the sample matrix for which it is intended must be
determined and the predefined data quality objectives met before the method is adopted. All known
interferences and shortcomings should be listed in the standard operating procedure and made
available to the data user.
A measure of single-operator precision is required before comparison with data obtained by
other analysts and other methods. Inter-laboratory comparisons are useful and should be continued
even after the method is brought "on-line". When changes or improvements to a method are made,
all changes and dates of changes must be documented. An overlap period when the old and new
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methods are run concurrently is necessary until the data collected are representative of all types of
samples received. Differences in sample values and daily performance checks should be monitored
and the methods evaluated. The client/data user must be told how the methods compare so that
historical data may still be useable.
Method Documentation \
The written procedure should include all sample preparation and clean-up steps, detailed
description of the measurement system including instrumental conditions and adjustments,
calibration procedures, performance checks, safety practices, performance characteristics, and data
limitations (Ontario Ministry of the Environment, 1986).
Calibration
Calibration of the measurement system with more than two standards of known concentration
should be performed before the analysis of every batch of samples. It is advisable to prepare
calibration solutions from a primary standard. A primary standard is a substance of high purity,
with the purity known to within very close limits. These standards should be validated against
certified reference standards (e.g., National Institute of Standards and Technology or MIST) and
traceable to the validation.
's
Calibration is performed to establish the relationship between instrument response and analyte
concentration on that particular day. Calibration establishes the linearity of the analytical system.
Sudden changes in the slope and intercept of the calibration curve from day to day should not be
expected and, therefore, the slope and intercept should be monitored with each standardization.
If these changes occur, insufficient instrument warm-up time, instrument degradation, reagent
quality, or error in standard preparation may be the cause.
.i
Working analytical standards are prepared from stock solutions. The concentration of the
stock solution should be high enough that pipetting of less than 1 mL to produce a working
standard is unnecessary. Working standards should be prepared daily if their concentrations are
less than 1 mg/L or if the standards are prepared in a matrix where prolonged storage may cause
changes in the results.
Calibration Performance Checks - Accuracy and Precision
Internal Reference Checks
Quality control check standards (QCCS) should be used to check the accuracy of the
calibration. These standards should be prepared from a different primary standard than was used
in the creation of the calibration standards, or at least a different batch number of the same primary
standard. Using a solution in the low and high end of the calibration range is advisable. Sufficient
volumes of each quality control solution should be prepared to last through several batches of
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calibration standards. The concentrations of these solutions should be read each time the
instrument is calibrated and the measurements should be recorded. This record provides data
which can be used to calculate the between-run precision of the measurement system separately
from sample preparation precision. Limits on this precision may be set and used to identify
calibration problems. In addition to the quality control solutions, a long-term blank comprised of
the dilution matrix used to prepare the quality control solutions will provide a check on the zero
standard or intercept of the calibration curve and the purity of the reagents and distilled/deionized
water used.
External Reference Checks
Externally prepared, certified control solutions, such those obtained from the U.S.
Environmental Protection Agency, may also be analyzed periodically to provide an accuracy check.
Routine participation in an inter-laboratory comparison study gives the analyst an indication of how
well his/her laboratory performs with respect to others.
Interference Checks
An interference in the analysis may occur when a substance other than the analyte is present
in amounts sufficient to affect the results. Interferences are often identified during the method
development stage and steps to prevent these interferences are built into the procedure. During
routine operation, checks may be used to monitor the effectiveness of the methodological
safeguards used to prevent the interference. Interference checks should be close to the threshold
level of the substance found to affect the results. Good laboratory practices require that the analyst
carry out all cross checks available, based on the knowledge of the sample source and matrix, to
provide for the accurate identification and quantification of the parameter being measured.
Sensitivity and Baseline Checks
Changes in the sensitivity of the instrument should be monitored periodically (e.g., every tenth
sample) by analyzing a standard and comparing the peak height to the original calibration. Any
change must be considered when calculating the results. Periodic analysis of a blank, matrix
matched to the standards and samples, may be used to monitor baseline drift.
Method Performance Checks - Accuracy and Precision
Method Blanks
With each batch of samples, three method blanks (reagents only without sample) should be
carried through the entire procedure. These provide a measure of contamination from all possible
sources (reagents, labware, filter paper, handling etc.). The contribution of only the reagents and
instrumental analysis to the overall contamination can be determined from the zero standard and
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long term blank. The mean blank value should be subtracted from the sample result before
reporting the final parameter concentration.
Internal Reference Checks
"Total" elemental analyses are not usually performed on soil samples, with the exception of
total C, N, P, and S. For most purposes, specific fractions or forms of elements are extracted.
These fractions may have been found to relate well to plant growth or soil genesis. As a result, the
accuracy of the method cannot be routinely checked by using spike recoveries. As previously
mentioned, the accuracy of the measurement system can'be controlled. However, the accuracy or
specificity of the extraction/digestion for a particular form of the analyte in the soil must be
determined in the method development stage. For total measurements (especially on foliage),
spiked samples may be used to measure recovery. In this case, the spike should be twice the
endogenous level or ten times the detection limit, whichever is greater. The analyst should note that
the chemical form of the element in the spike may be different than the naturally occurring form in
the soil/foliage. This, in turn, may lead to invalid conclusions about recovery from the sample.
When interferences are indicated by the spike recovery, standard additions may be useful for
accurately determining the amount of analyte present in the sample.
The reproducibility of the method between batches prepared ori different days may be termed
the between-run precision and is measured by replicate control samples. Large amounts of soil or
foliar tissue should be collected, dried, and ground as required for the test and analyzed with each
run of samples. The sample volume should be sufficient for at leasl a year, if not more, of routine
operation. More than one of these between-run control samples should be prepared to represent
the low and high part of the analytical range and different sample matrices (e.g., organic and
mineral soil or different vegetation species). If run over a long period, the standard deviation of the
results of these samples will provide a measure of between-run precision. Control limits may be
set at two or three standard deviations from the long-term mean of the results. Samples failing
outside these limits may indicate problems in batch preparation. If, changes in the analyte occur
due to storage of the control sample over long periods, the change may be detected more easily
by plotting the results on a control chart. A systematic increase or decrease in the values over time
may indicate an unstable sample, or in the case of soil, a sample which may no longer be
homogenous, but which has settled and become sorted. Ensure proper mixing/homogenization prior
to weighing the sample each day.
The reproducibility of a method within the same batch of samples prepared on a specific day
may be called the within-run precision. Some samples within the batch (e.g., every tenth sample)
are prepared in duplicate. The run format should be such that the duplicate samples are not
analyzed side by side within the run. This will eliminate possible bias resulting from position in the
run. Standard deviations of the duplicates can be determined using the following equation:
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where: S = standard deviation of the difference between duplicate pairs,
n - number of duplicate pairs, and
(x, - x-j), = difference of the /th duplicate.
Acceptable duplicate data should conform to limits which are based on historical performance.
External Reference Checks
Certified reference soil and vegetation samples are available from NIST (formerly the National
Bureau of Standards or NBS) and can be run as an accuracy check. Inter-laboratory comparisons
provide a good indication of your performance as it compares to other laboratories. Informal
comparisons can usually easily be arranged by contacting the laboratory supervisor. The Council
on Soil Testing and Plant Analysis based at the University of Georgia provides a soil and plant
laboratory registry which may be used to obtain a list of laboratories doing similar work in Canada
and the United States. In Canada, the Expert Committee on Soil Survey (ECSS), Agriculture Canada,
Land Resource Research Centre, Ottawa Ontario conducts soil round-robins. Similarly, the QA
Subgroup of the Research Monitoring Coordination Committee of the federal Long Range Transport
of Air Pollutants (LRTAP) program conducts a foliage round robin. This is organized through the
Great Lakes Forestry Centre in Sault Ste. Marie, Ontario. LABEX (Laboratories Exchange Program)
is coordinated by the International Soil Reference and Information Centre, Wageningen, the
Netherlands. A foliage sample exchange has also been organized in the past by IUFRO
(International Union of Forestry Research Organizations), and is also based in Wageningen, the
Netherlands.
Method Detection Limits
The method detection limit is usually defined as the smallest amount of analyte which may
be measured under routine operating conditions. It is the minimum amount that can be reliably
discerned as being different from the blank level. There is some degree of imprecision in
measurements at all analyte concentrations and the percent contribution of that imprecision is
generally greater at low levels. Considering this, the reported detection limit must take into account
this imprecision and should not be reported merely as the lowest readable value above the blank
on a particular day. The instrument detection limit may be determined from the standard deviation
of the blank value calculated from a large number of runs. The method detection limit, however, may
be calculated from the mean standard deviation of either a low level standard or preferably replicate
samples at close to blank levels. Generally, three times the standard deviation of the blank or low
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level standard is quoted as the instrument detection limit. A minimum number of replicates required
for the determination of detection limits should be seven.
i
Blind Audit Samples
To avoid special care being given to quality control samples and samples which form part of
an inter-laboratory comparison, it is often valuable to arrange for blind audit samples to be
submitted to the laboratory. These samples may be submitted by the client, laboratory manager,
or external organization. They should be received through the normal channels and should be
unrecognizable from real samples. This allows a better measure of the performance of a method
under routine operating conditions. i
Summary
Ultimately, the integrity of one's data depends on the quality of the operations within the
laboratory. For this reason alone, the importance of good laboratory practices cannot be over-
stated. The above discussion provided only a cursory review of any of the topics. It is advisable
that anyone embarking on terrestrial monitoring work adequately address the area of good
laboratory practices before proceeding. The very nature of long-term environmental monitoring may
mean changes in laboratories and/or laboratory staff over the course of the monitoring period.
Many researchers have found their data sets incomparable or have found uncertainties in historical
data. Frequently, these problems result from improper documentation of laboratory procedures,
quality assurance and control, and measures of accuracy and precision. Addressing these areas
at the outset of a monitoring study will help avoid future disappointment.
References
Kalra, Y.P., and D.G. Maynard. 1991. Methods manual for forest soil and plant analysis. Information
Report NOR-X-319. Forestry Canada, Northwest .Region, Northern Forestry Centre, Edmonton,
Alta.
Maynard, D.G., Y.P. Kalra, and F.G. Radford. 1987. Extraction and determination of sulfur in organic
horizons of forest soils. Soil Sci. Soc. of Am. J. 51:801-806.
Neary, A.J., and S.R. Barnes. 1993. The effect of sample grinding on extractable iron and aluminum
in soil. Can. J. Soil Sci. 73:73-80.
Ontario Ministry of the Environment. 1986. Quality assurance policy and guidelines. Laboratory
Services Branch, Rexdale, Ontario. :
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Peverill, K.I., G.P. Briner, and LA Douglas. 1975. Changes in extractable sulphur and potassium
levels in soils due to oven drying and storage. Aust. J. Soil Res. 13:69-75.
Robarge, W.P., and I. Fernandez. 1986. Quality assurance methods manual for laboratory analytical
techniques. Prepared for the U.S. EPA and USD A Forest Response Program. Corvallis
Environmental Research Laboratory, Corvallis, OR.
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PLANT TISSUE ANALYSIS
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Introduction
Total Nitrogen in Plant Tissue
f °"f -th6- flve major constituents of living matter. As such, its form and
? * °f maj°r in?erest When dealin9 with the 9rowth of terrestrial and aquatic
h na T' n'tr°9en's !ound in a number of forms, all of which are interconvertible given
.hoT'Ca a.nd ™c/°biolog.cal conditions. Soil N accounts for only a small fraction of N
SSth'V^1^1 °n'y **?* Sma" Pr°P°rti0in is directly available to plants
nn , MU °i0*f the N '.8 in,the organic form- Plants obtaiiri most of their N from inorganic
ions, NO3 and NH/( that comprise less than 1% of the total N.
««i- AtmosPhffc deposition of NOX can directly affect the total foliar N concentrations. Further
foliar N content changes may be brought about by indirect effects to the forest ecosystem, such
as a result of acid deposition. Therefore, the measurement of total N in plants is suggested for
long-term terrestrial monitoring programs, such as LRTAP.
Review of Methods
/n '" P'ant tiS8Jie is usually measured by either a wet oxidation (Kjeldahl method) or dry
(Dumas method) procedure (Bremner and Mulvaney, 1982). Considerable modifications
have been proposed for these methods (e.g., Nelson and Sommeirs, 1980). More recently near
infrared reflectance spectroscopy (NIRS) and LECO combustion have been used as alternatives to
* ?°?™ £f ' chemical analysis for total N determination in foliage (Tsay et al., 1982; Wessman
et al., 1988). These are not in widespread use, however, and they involve expensive equipment For
these reasons, chemical wet oxidation techniques are most frequently used.
The principle of the Kjeldahl and Dumas techniques have been 'thoroughly discussed in several
reviews (Bremner, 1965; Nelson and Sommers, 1980; Bremner and MulvaTiey, 1982). The Dumas
22 * *° not q"ant|tatively recover many nitrogenous compounds (e.g., heterocyclic compounds)
ffS™L!U£2S ?K ai?3KZ!!; based on tne Dumas method has not been widely used (Bremner and
Mu vaney, 1982). The Kjeldahl procedure is more commonly used and modifications of the original
Kjeldahl methods have extended the scope of the procedure. The total Kjeldahl N method involves
the digestion of the sample with H2SO4 to convert organic N to NH/-N followed by distillation of the
IRS ^ strong alkali to liberate NH3. Various modifications to the distillation and measurement
°J liS l!beraled^1?1na,v!,.been Proposed. In addition, various methods for the direct measurement
of NH4 in the Kjeldahl digests have also been used. Highly refractory organic N compounds or
compounds containing N-N or N-O linkages are not completely recovered by the Kjeldahl digestion
(Bremner and Mulvaney, 1982). "
Reference Method
The reference method uses the Kjeldahl acid digestion in the
converts organic nitrogen to inorganic ammonium. The measurement
.
distillation to liberate NH3 which is measured by acid titration. The
on a hot plate or in an aluminum digestion block.
presence of a catalyst which
nt of N is done using alkaline
digestion is carried out either
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Summary of Method
Vegetation samples are digested by the regular Kjeldahl technique using a K,,SO4-CuSO4
catalyst in an aluminum block digestor with internal heaters and temperature control Determination
of NH/-N is done by an automated system (Tecator Kjeltec 1030) determining NH3 liberated by
distillation of the digest with 40% NaOH. The NH3 is absorbed in unstandardized H3BO3 and
ammonium borate is formed. The borate is titrated back to H3BO3 using a standard strong acid
(HCI).
Interferences
After digestion, samples must not be allowed to cool in the digestion block as NH3 will be lost
from the (NHJ2SO4 formed by the digestion.
Safety
Protective clothing and safety glasses should be worn when handling strong acids. The
diqestion blocks should be located in the fume hood and, if possible, the temperature controMer
should be located outside the fume hood. The equipment should not be left unattended. The
preparation of the NaOH should also be done in the fume hood. The NaOH pellets should be added
very slowly to the water and in very small portions due to the intense exothermic reaction that
occurs.
Apparatus and Equipment
• digestion block, 20 place, Tecator System 20 1050 or equivalent, with programmable
temperature controller.
• distillation and titration apparatus, Kjeltec Auto 1030 Analyzer or equivalent.
• glass digestion tubes (295 mm x 40 mm), 250 mL to fit block, appropriate to sample and
solution volume used.
• balance, accurate to 0.001 g.
Reagents and Consumable Materials
• sulphuric acid, H2SO4, concentrated, reagent grade (96%).
• water~DI water used in all preparations should conform to ASTM specifications for Type
I reagent grade water (ASTM, 1984).
• catalyst, Kjeltab tablets or equivalent. Each tablet contains 3.5 g Kj,SO4 and 0.4 g GuS04.
• hydrochloric acid, HCI, standard acid 0.01 M.
• boric acid, H3BO3, reagent grade powder.
• ammonium chloride, NH4CI, reagent grade powder.
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• methanol, reagent grade. !
• sodium hydroxide, NaOH, reagent grade pellets.
• sodium hydroxide solution, 40%-Dissolve 10 kg of NaOH in 15 I. DI water.
• bromocresol green.
• bromocresol green solution-In a 100 mL volumetric flask dissolve 0.100 g bromocresol green
in methanol. Dilute to volume with methanol.
• methyl red.
• methyl red solution-In a 100 mL volumetric flask dissolve 0.100 g methyl red in methanol.
Dilute to volume with methanol. j
• receiving solution (Tecator, 1985)-Dissolve 100 g H3BO, in DI water and dilute to 10 L Add
100 mL bromocresol green solution. Add 70 mL methyl red solution. Add 5 mL of 40%
NaOH solution.
• recovery check solution, 5,000 mg-N/L--In a one litre volumetric flask dissolve 19.0927 g
, NH4CI in DI water. Dilute to volume.
Calibration and Standardization
Before analyzing the digested samples, distilled water blanks are run on the Kjeltec 1030
Analyzer until a constant reading of HCI is obtained. A 5 mL aliquot of a recovery check solution
containing 5,000 mg-N/L is analyzed to check the recovery. Recovery should be within ± 10%.
Procedure \
Step 1 - Weigh 0.250 g of plant material (20 mesh) into a digestion tube.
Step 2 - Add 10 mL concentrated H2SO4 to the tube and mix by swirling.
Note: This step should be carried out in a fume hood. \
\
Step 3 - Heat tubes at 200° C in the digestion block until very black (appro* 30 minutes).
Step 4 - Add one catalyst tablet (Kjeltab).
Step 5 - Heat tubes at 200° C for 15-20 minutes until the Kjeltab dissolves.
Step 6 - Increase the block temperature to 300° C and heat for 30 minutes.
Step 7 - Raise the temperature to 425° C and heat tubes until the sample turns a turquoise
green. Digest samples for 20 minutes. \
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Step 8 - Remove the digestion tubes from the block and allow to cool for about 5 minutes.
Note: Do not allow to cool in the heating block as NH3 from the (NI-Q2S04 formed by
digestion will be lost if heated.
Step 9 - Add approximately 30 ml_ DI water and mix well until sample is in solution.
Step 10 - Dilute to approximately 100 mL with DI water.
Step 11 - Follow instructions for the operation of the Kjeltec Auto 1030 Analyzer fTecator, 1985).
Step 12 - Set the alkali pump to deliver 25-30 mL of 40%'NaOH.
Step 13 - Titrate the sample with 0.01 M HCI.
Quality Control
Precision
One sample should be analyzed in duplicate with each run of thirty samples. To eliminate bias
due to position in the run, the routine sample duplicate should be analyzed separately within the
analytical run. Within-run precision is determined from duplicates based on relative percent
difference between the samples at an acceptance limit of a RPD •£ 10%.
Accuracy
Accuracy is determined by analysis of a standard reference material (SRM). Acceptable limits
for accuracy should be ±10% from the known concentration of the standard or within the accuracy
windows supplied by the reference material manufacturer, whichever is larger. It is recommended
that two or more accuracy standards be prepared with each batch of samples. These provide a
check on total between-run precision (digestion and distillation/titration).
Method Blanks
Three method blanks, carried through the extraction procedure, are analyzed with each batch
of samples to measure potential contamination. Method blanks should be run at the beginning,
middle, and end of each analytical run. The concentration of each blank should be less than or
equal to the instrument detection limit.
Quality Control Preparation Sample
A matrix matched in-house quality control preparation sample (QCPS) should be analyzed once
per analytical run. This sample is used to monitor accuracy and long-term between-run precision.
Accuracy of the QCPS should be within ± 10% of the long-term mean. Between-run precision can
be determined by analyzing the QCPS and calculating the cumulative long-term standard deviation.
If values plotted on a control chart deviate from the long-term mean by more than three standard
deviations, the run should be completely reanalyzed, including all digestion and quantification steps.
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Quality Control Check Standard \
A quality control check standard (QCCS) should be analyzed at the beginning/after every ten
samples, and after the last sample of each analytical run. The QCCS should contain all the analytes
of interest with mid-calibration range concentrations. Quantified values of the QCCS should be
within ±10% of the known concentration of the standard.
It is highly recommended that the concentrations of this sample be consistent through time
so that control charts may be plotted to monitor laboratory bias and other potential problems If
analyzed values deviate from the long-term mean by more than three standard deviations the
instrument is re-standardized and re-calibrated prior to any further sample analyses.
Suggested Run Format
QCCS, MB, Samples 1 to 8, QCPS,
QCCS, Samples 9 to 16, MB, DUP,
QCCS, Samples 16 to 25, SRM,
QCCS, Samples 26 to 30, MB, QCCS.
where: QCCS
MB
QCPS
DUP
SRM
quality control check standard
method blank
quality control preparation sample
duplicate sample
standard reference material
Calculations and Reporting
formula:
Report total N as percentage on a dry-weight basis to the nearest 0.01% using the following
N% = (ml sample - mL blank) x N x 1.401
weight (g) of dry soil
where: N = normality of HCI titrant solution.
References
American Society for Testing and Materials. 1984. Annual Book of ASTM Standards, Vol. 11.01,
Standard Specification for Reagent Water, D1193-77 (reapprovsd 1983). ASTM, Philadelphia,
Pennsylvania. ;
Bremner, J.M. 1965. Organic nitrogen in soil. Jn Bartholomew W.V., and F.iE. Clark (ed.) Soil nitrogen
Agronomy 10. Am. Soc. Agron., Madison, WI. p. 93-149.
Bremner, J.M., and C.S. Mulvaney. 1982. Nitrogen-total. In Page, LA. et al. (ed.) Methods of soil
analysis. Agronomy 9. Am. Soc. Agron., Madison WI. p. 595-624.
Nelson, D.W., and L.E. Sommers. 1980. Total nitrogen analysis for soiil and plant tissues. J. Assoc.
Off. Anal. Chem. 63:770-780.
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Tecator. 1985. Kjeltec Auto 1030 Analyzer Manual. Tecator, AB. Hoganas, Sweden. 43p.
Tsay, M.L, D.H. Gjerstad, and G.R. Glover. 1982. Tree leaf reflectance: a promising technique to
rapidly determine nitrogen and chlorophyll content. Can. J. For. Res. 12:788-792.
Wessman, C.A., J.D. Acer, D.L Peterson, and J.M. Melillo. 1988. Foliar analysis using near infrared
reflectance spectroscopy. Can. J. For. Res. 18:6-11.
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Total Sulphur in Plant Tissue
Introduction
Sulphur is an essential element for all biological systems. Historically, its importance as a
plant nutrient and reports of widespread deficiencies, especially in sub-humid areas of intensive
leaching has made it a routine measurement in some laboratories. Most of the sulphur in surface
soils occurs in combination with organic matter (Freney and Williams, 1983). Sulphate is the most
oxidized form of S and most easily taken up by plants and microorganisms (Blair, 1971). Sulphate
(SO*2") is taken up by plants, reduced to S and used to form S-containcng amino acids and other
reduced-S compounds (Stewart et al., 1983). Most plants contain approximately as much S as
phosphorus. '
Concern over the long-range transport and deposition of SO*2" in precipitation has lead to
increased monitoring of the S status of soils. The effects of SO/*" adsorption and desorption on
soil cation leaching has also been the subject of much research. Fundamental to studying the
effects of strong acid precipitation on terrestrial and aquatic systems is an understanding of the
behaviour of S in plant and soil systems. The measurement of total S in plant tissue is, therefore,
suggested for terrestrial monitoring programs, such as LRTAP.
Review of Methods
It has only been within the last 15 years that the difficulty of accurately measuring S in soils
and vegetation has been overcome. Recent advances in analytical techniques have resulted in the
accurate and precise measurement of S in various types of soil and plant materials (Dick and
Tabatabai, 1979; Hogan and Maynard, 1984; Nieto and Frankenberger, Jr., 1985; Maynard et al., 1987).
Several methods are routinely used for the determination of S in environmental samples. These may
be divided into two groups, namely, those involving wet oxidization of the sample and those which
involve direct sample analysis (Hogan and Maynard, 1984). ~
Methods available for the wet oxidation of organic materials are well documented (Beaton et
al., 1968; Tabatabai, 1982; Blanchar, 1986). Acid and alkaline oxidation are the most common
(Blanchar et al., 1965; Tabatabai and Bremner, 1970), as they are dependable, accurate and relatively
rapid (Blanchar, 1986). Full recovery from an acid digestion usually requires the use of perchloric
acid. The danger associated with its use and the special facilities required have meant that, until
recently, acid digestions have been avoided. The recent adaptation of microwave ovens for use in
the laboratory has led to the development of microwave acid oxidation digestion techniques for
foliage which successfully use hydrogen peroxide in place of perchloric acid. Many analysts still,
however, prefer the safer, more rapid, dry combustion techniques.
The wet oxidation technique converts S to SO*2" and produces a solution which can be
analyzed by a variety of methods. Turbidimetry is insensitive, lacks precision and is subject to
numerous interferences (Beaton et al., 1968). Colourimetric methods such as the methylene blue
technique (Technicon, 1972) also have limited application in soils and plant analysis because of
interferences by major nutrient cations (Maynard et al., 1987). The colourimetric method developed
by Johnson and Nishita (1952) was found to be the most sensitive and accurate of the colourimetric
procedures.
Recent publications (Hogan and Maynard, 1984; White and Douthit, 1985; Novozamsky et al.,
1986) have shown that the accurate and precise measurement of S is possible by inductively coupled
plasma-atomic emission spectrometry (ICP-AES) in a range of environmental samples. Precision
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is estimated at ± 2%. Results of analysis of NIST plant material and sediments demonstrated that
the precision and accuracy obtained by ICP-AES are equal to or better than any other technique
currently used. The ICP-AES has several advantages over other methods. It is rapid, flexible, has
a dynamic range, is free from interferences and permits simultaneous multielement analysis. These
factors make it a preferred method of analysis for laboratories possessing ICP-AES capabilities.
Busman et al. (1983) successfully determined total S in plant material using a combination of
ion chromatography following combustion of the foliage in an oxygen flask.
Direct analysis of the sample may be done by combustion of the sample at elevated
temperatures (generally at 1000° C or higher) and measuring the liberated SO, by an infrared
detector. Examples of instruments that combust the sample include the LECO combustion furnace,
Carla-Erba combustion furnace, or the Fisher S analyzer. These methods require little or no sample
preparation for the determination of total S.
Additionally, X-ray fluorescence may be used to quantify S in plant tissues along with many
other elements. This method has been used for the measurement S in a wide variety of plant
materials including lichens (Tomassini et al., 1976). Preparation of vegetation samples for analysis
is done by pressing the dried, ground foliage into a pellet using a wax filler. The method is rapid
but requires between one and two grams of dried sample. This may limit the analysis of low
volume samples. Further, the expense of the instrumentation may make this technique an unfeasible
option for many laboratories.
The LECO S analyzer was originally developed for the determination of S in steel, but because
of its simplicity, speed, and convenience, it has been adapted for use in soil and plant analysis
(Tabatabai, 1982). An initial evaluation of this method by Tabatabai and Bremner (1970) showed
total S results to be unsatisfactory for research that required accurate and precise determinations.
More recent studies have shown that the LECO analyzer equipped with an infrared detector was
capable of providing rapid, accurate analysis of total S in plant material (Hern, 1984; Jackson et al.,
1985). Work at the Ontario Ministry of the Environment has found that the infrared detection system
on the LECO gave unacceptable results for vegetation and organic soil samples. The LECO analyzer
with iodometric titration for the measurement of S was found to give better recovery and precision.
A Fisher S analyzer, using a similar theory as the LECO, was also found to provide rapid, accurate
analysis of total S in plant material (Guthrie and Lowe, 1984).
Reference Method
The reference method for total S in soil samples is dry combustion in a LECO sulphur analyzer
with infrared detection. The analysis of S by LECO-S analyzer has been chosen as a reference
method because it is widely used in North America and has been used successfully by some
laboratories for both soil and foliage samples.
Summary of Method
An air-dried and finely ground sample of soil or a pre-ashed vegetation sample is heated with
an accelerator to 1600 °C in a stream of oxygen. The released sulphur is converted to sulphur
dioxide and is detected by infrared detector (Hern, 1984).
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Interferences and Shortcomings
Large amounts of carbon can prevent proper ignition of the sample. With incomplete
combustion a poor recovery of S may be obtained. This may be overcome by reducing the sample
size and adding LECO Iron Chip Accelerator plus tin. Preashing foliage and highly organic soils at
475 to 500° C for two hours is usually done, but preliminary studies should be done on the type of
samples to be analyzed to determine whether a loss of S occurs during this ashing. Preashed
samples also require accelerator to overcome interference from residual carbon.
j
Moisture deposits on the walls of the delivery tubes or the surface of the dust filter will absorb
SOj. This may be overcome by using magnesium perchlorate between the dust filter and the original
drying tube.
Chlorine in concentrations of less than 1% does not interfere. II1 titration is used to detect S,
the halogens, iodine, chlorine and fluorine may darken the solution iin the titration vessel by the
formation of interhalogen compounds. Poor recoveries may result. A trap inserted in the delivery
tube and filled with 20 mesh antimony metal will prevent these interferences.
Interferences from nitrogen may be overcome by increasing the oxygen flow rate to 1.5
litres/minute.
Safety
Normal safety precautions should be taken when using high-frequency combustion furnaces.
Protective clothing and safety glasses should be worn when handling reagents. Heat resistant
gloves may be needed when placing samples in the furnace. The furnace must be adequately
vented and protected from human contact and combustible materials. Gas cylinders should be
bolted or chained in an upright position.
Fumes of magnesium oxide are toxic. Magnesium perchlorate is a fire and explosion hazard
if it comes in contact with organic materials.
Apparatus and Equipment
• sulphur analyzer with infrared detector, LECO model SC-132. or equivalent.
« balance, accurate to 0.001 g.
• muffle oven, capable of maintaining 500 ± 5° C.
« desiccator and desiccant, P2O5.
• LECO scoop.
• LECO crucibles and covers.
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Reagents and Consumable Materials
• oxygen, high purity.
• compressed air, if needed.
• anhydrous magnesium perchlorate (Mg(C\OJz, 10-20 mesh, or equivalent desiccant specified
by manufacturer for drying gases after combustion and prior to detection.
• magnesium oxide, MgO, reagent grade powder, low in sulphur.
• accelerators, vanadium pentoxide, iron chips, and/or copper metal
Calibration and Standardization
Set up the instrument according to the LEGO operating manual. In general, the instrument
should be calibrated at least once a day or once per batch of samples, whichever is more frequent.
Use either NIST (formerly NBS) reference materials or standards supplied by the manufacturer and
approved by the laboratory or QA manager. The concentration range of the standards must be
representative of the C concentrations expected in the soil samples. A minimum of a two-point
calibration curve should be used. Use of a NIST standard reference material as an initial calibration
check is highly recommended.
Some suggested calibration standards and reference samples include: LEGO brand iron
powder (0.036% S), LEGO coal calibration standard (2.56% S), NIST (NBS) coal reference material
(1.89% S), SU-1A nickel-copper-cobalt ore (9.35% S), and CCU-1 reference material (35.4% S).
Ensure that the anhydrone is dry and the dust filter is clean. Prior to analyzing samples, the
instrument is conditioned by running low level calibration standards until the results are stabilized
to within 5%. Once stable, three blank crucibles (accelerator only) are analyzed followed by three
standards.
Procedure.
Step 1 - Weigh out approximately 3 g of sample into a ceramic crucible. Record sample weight.
Step 2 - Ash sample at 500 °C for 2 hours in the muffle oven. Cool sample in desiccator. Re-
weigh cooled sample and record weight of ashed sample.
Step 3 - Preset power settings on induction furnace according to the manual.
Step 4 - Ignite sample in the crucible for the suggested time period (either given in the operating
manual or as determined through method development work for the particular sample
type being analyzed).
Step 5 - Take sulphur measurement reading.
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Quality Control
Precision
One sample from each batch should be analyzed in duplicate. Within-run precision is
determined from duplicates based on relative percent difference (RPD) between the samples with
an acceptance limit of a RPD £ 10%.
Accuracy i
Accuracy is determined by analysis of a standard reference material (SRM). Acceptable limits
for accuracy should be ±10% from the known concentration of the standard or within the accuracy
windows supplied by the reference material manufacturer, whichever is larger.
Method Blanks \
Two blank crucibles (accelerator only) are analyzed before the run and one blank crucible is
also analyzed in the middle and at the end of each analytical run. The concentration of each blank
should be less than or equal to the instrument detection limit. All results should be blank corrected
using the mean of the acceptable method blank readings.
Quality Control Preparation Sample
A matrix matched in-house quality control preparation sample (QCPS) should be analyzed once
per analytical run. This sample is used to monitor accuracy and long-term between-run precision.
Accuracy of the QCPS should be within ± 10% of the long-term mean. EJetween-run precision can
be determined by analyzing the QCPS and calculating the cumulative long-term standard deviation.
If values plotted on a control chart deviate from the long-term mean by more than three standard
deviations, the run should be completely reanalyzed, including all digestion and quantification steps.
Suggested Run Format
MB, MB, Samples 1 to 8, QCPS,
Samples 9 to 16, MB, DUP,
Samples 16 to 25, SRM,
Samples 26 to 30, MB.
where: MB
QCPS
DUP
SRM
method blank
quality control preparation sample
duplicate sample
standard reference material
Calculations and Reporting
Infra-red system: % S is read directly from the instrument. The sample weight, as. different
from the standard, is taken into account on some instruments.
Results are reported to two significant figures. Results are read to the nearest 0.001%.
Results should be blank corrected.
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References
Beaton, J.D., G.R. Burns and J. Platou. 1968. Determination of sulphur in soils and plant material.
Technical Bull. No. 14, The Sulphur Institute, Washington, D.C.
Blair, G.J. 1971. The sulphur cycle. J. Aust. Inst. Agr. Sci. 37:113-121.
Blanchar, R.W. 1986. Measurement of sulfur in soils and plants. ID MA Tabatabai (ed.). Sulfur in
agriculture. Amer. Soc. Agronomy. Madison, WI. Agronomy 27:457:490.
Blanchar, R.W., G. Rehm, and AC. Caldwell. 1965. Sulfur in plant materials by digestion with nitric
and perchloric acid. Soil Sci. Soc. Am. Proc. 29:71-72.
Busman, L.M., R.P. Dick, and MA Tabatabai. 1983. Determination of total sulfur and
chlorine in plant materials by ion chromatography. Soil Sci. Soc. Am. J. 47:1167-1170.
Dick, WA and MA Tabatabai. 1979. Ion chromatographic determination of sulfate and nitrate in
soils. Soil Sci. Soc. Am. J. 46:847:852.
Freney, J.R. and C.H. Williams. 1983. The sulphur cycle in soil. p. 139-201 In M.B. Ivanov and J.R.
Freney (eds.). The global biogeochemical sulphur cycle. SCOPE Rep. no. 19. John Wiley and
Sons, New York, NY.
Guthrie, T.F., and LE. Lowe. 1984. A comparison of methods for total sulphur analysis of tree
foliage. Can. J. For. Res. 14:470-473.
Hern, J.L 1984. Determination of total sulphur in plant materials using an automated sulphur
analyzer. Comm. Soil Sci. Plant Anal. 15:99-107.
Hogan, G.D. and Maynard, D.G. 1984. Sulphur analysis of environmental materials by vacuum
inductively coupled plasma atomic emission spectrometry (ICP-AES). p. 676-683 Ip Proc.
Sulphur-84, Int. Conf., Calgary, Alberta. June 1984. The Sulphur Development Institute of
Canada, Calgary.
Jackson, LL, E.E. Engleman, and J.L Peard. 1985. Determination of total sulfur in lichens and plants
by combustion-infrared analysis. Environ. Sci. Technol. 19:437-441.
Johnson, C.M. and H. Nishita. 1952. Microestimation of sulphur in plant materials, soils and irrigation
waters. Anal. Chem. 24:736-742.
Maynard, D.G., Y.P. Kalra, and F.G. Radford. 1987. Extraction and determination of sulfur in organic
horizons of forest soils. Soil Sci. Soc. Am. J. £1:801-806.
Nieto, K.F. and W.T. Frankenberger, Jr. 1985. Single column ion chromatography: I. Analysis of
inorganic anions in soils. Soil Sci. Soc. Am. J. 49:587-592.
Novozamsky, I., R. van Eck, J.J. van der Lee, V.J.G. Hauba and E. Temminghoff. 1986. Determination
of total sulphur and extractable sulphate in plant materials by inductively-coupled plasma
atomic emission spectrometry. Comm. Soil Sci. Plant Anal. 17:1147-1157.
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Stewart, J.W.B., C.V. Cole, and D.G. Maynard. .1983. Interactions of biogeochemical cycles in
grassland ecosystems. In B. Bolin and R.B. Cook (eds.). The major biogeochemical cycles and
their interactions. SCOPE Ftep. No. 21. John Wiley and Sons, New York, NY.
Tabatabai, M.A. 1982. Sulfur. ID A.L Page, R.H. Miller, and D.R. Keeney (eds.). Methods of soil
analysis. Part 2. 2nd ed. Agronomy 9:501-538.
Tabatabai, MA and J.M. Bremner. 1970. Comparison of some methods for determination of total
sulfur in soils. Soil Sci. Soc. Am. Proc. 34:417-420.
Technicon. 1972. Sulfate in water and wastewater. Industrial method no. 118-71W. Technicon
Industrial Systems, Tarrytown, NY.
Tomassini, F.D., K.J. Puckett, E. Nieboer, D.H.S. Richardson, and B. Grace. 1976. Determination of
copper, iron, nickel and sulphur by X-ray fluorescence in lichens from the MacKenzie Valley,
Northwest Territories, and the Sudbury District, Ontario. Can. J. Bot. 54:1591-1603.
White, Jr., R.T. and G.E. Douthit. 1985. Use of microwave oven and nitric acid-hydrogen peroxide
digestion to prepare botanical materials for elemental analysis by inductively coupled argon
plasma emission spectroscopy. J. Assoc. Off. Anal. Chem. 68:766-769.
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P, Mn, Fe, Al, Ca, Mg, and K in Plant Tissue
Introduction
Phosphorus
Phosphorus is a naturally occurring element essential to plant growth and microorganism
activity In acid soils, phosphorus is found primarily in combination with iron and aluminum oxides
and oxyhydroxides whereas in alkaline soils, calcium phosphates predominate. Plant available
phosphorus occurs^almost exclusively as orthophosphate in the soil solution.
Phosphorus levels in the environment are increased significantly by man's activities. Acid
deposition may impact on soil and affect the availability of phosphorus to plants. Therefore,
measuring phosphorus levels in plants may provide information on phosphorus availability.
Increased soil acidity may produce increased aluminum availability. In turn, aluminum toxicity is
often manifested in the plant as a phosphorus deficiency.
Manganese
Manganese is essential in plant nutrition and controls the behavior of several other
mtoronutrients. Apparently, the most important function of manganese is related to the plant's
oxidation-reduction processes. Manganese appears to participate in the oxygen-evolving system of
photosynthesis and also plays a basic role in the photosynthetic electron transport system.
Chtoroplasts are the most sensitive of all cell components to manganese deficiencies and react by
showing structural impairment.
Tissue testing is important in determining the plant manganese content since soil analyses
alone are not very reliable in diagnosing the manganese supply to plants. Excesses of manganese
can have a toxic effect on plants which is interrelated with other elements, particularly iron.
The manganese content of plants depends on plant characteristics and the pool of available
manganese in the soil. Generally, the most readily available forms of manganese are found in a
waterlogged acid soil. Manganese toxicity in some field crops might be expected on acid soils with
pH values of 5.5 or toss and high manganese levels. Acid deposition can, therefore, increase
manganese availability.
Iron
Iron is considered the key metal in energy transformations needed for syntheses and other
life processes of cells. Iron deficiencies affect several physiological processes and, therefore, can
retard plant growth. On soils rich in soluble iron, excessive iron uptake by plants can produce toxic
effects. Plant injury due to iron toxicity is most likely to occur in strongly acid soils, acid sulphate
soils, and flooded soils.
Acid soils tend to be higher in soluble inorganic iron forms than either neutral or calcareous
soils. The concentration of iron in soil solutions within common soil pH levels generally ranges from
30 to 550 ug/L, whereas, in very acid soils, it can exceed 2000 fig/L In acid anaerobic soils, Fe may
be toxic to plants whereas in alkalinei well-aerated soils, low concentrations of soluble iron species
may lead to iron deficiencies in the plant. Almost all cases of iron toxjcity and deficiency in plants
are considered to be the result of the soil factors governing iron solubility. These properties, in turn,
are affected by acid deposition which can alter soil pH and, thus, iron availability to the plant.
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Aluminum
Aluminum is a common constituent of all plants although the content varies greatly depending
on soil and plant characteristics. There is some evidence that low levels of aluminum can have a
beneficial effect on plant growth by controlling colloidal properties in the cell. Aluminum injury or
toxicity is often reported for plants grown in acid soils. In soils with pH values below 5.5, aluminum
mobility increases sharply. The mobile aluminum very actively competes with other cations for
exchange sites resulting in impaired nutrient uptake and transport in the plant.
Aluminum toxicity is also frequently associated with increased levels of iron, manganese, and
other heavy metals which are readily available in acid soils. Aluminum excess in plants is known
to reduce internal calcium transport and even induce a calcium deficiency in the plant.
Calcium
Calcium is an essential element for all life forms and enhances biological productivity in plants.
Calcium imparts rigidity to the cell wall and is necessary to growth. It is generally absorbed by
roots by ionic exchange and is thought to affect absorption of other cations and anions. Calcium
is essential for maintenance of selective ion transport in cellular membranes and minimizes root
injury resulting from sodium and hydrogen ions. A deficiency of calcium results in the
disorganization of cellular membrane structure.
In most plants the calcium ion is generally immobile and usually does not move out of older,
lower leaves to younger, upper leaves. Therefore, symptoms of deficiency show at extremities of
the plant in regions of new growth. Calcium uptake and availability depends on the pH of the soil.
Magnesium
Each chlorophyll molecule contains one atom of magnesium, that is, 2.7% of the weight of the
molecule. Magnesium is an activator for many enzymes. The magnesium ton may exist in high
concentrations as magnesium sulphate or chloride since these molecules are highly soluble.
Magnesium can occur in toxic concentrations if soils are low in calcium but this condition is rare.
In contrast to calcium, magnesium is mobile within the plant. ![f deficient, magnesium moves
from older to younger leaves. Therefore, visual symptoms are exhibited by the older leaves having
interveinal yellowing or chlorosis. Magnesium availability decreases in the soil from a pH of 6.5
down to approximately 4.5.
Potassium
Potassium is one of the three most essential nutrients for plants along with nitrogen and
phosphorus. The potassium ion is highly mobile within the plant. Potassium, like magnesium, is
required for the proper functioning of plant enzymes. The presence of potassium in the plant has
also been found to aid in the uptake of other nutrients, namely, anions such as NO,", and in their
movement within the plant.
i
A potassium deficiency can occur in a variety of soils and may be unavailable to plants even
when present in the soil. The most characteristic symptom of deficiency is that of tip and marginal
scorch of the most recently matured leaves. Potassium deficiency has sometimes been associated
with an accumulation of molybdenum in the plant leaves. Potassium availability decreases from pH
6.0 to 4.5.
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Review of Methods
The two most common decomposition procedures are wet oxidation and dry ashing. Wet
oxidation is the destruction of organic matter by high temperature acid digestion. Common acids
used include sulphuric, nitric, hydrochloric, and perchloric acids usually in some combination of two
or three. Sulphuric acid is not recommended for use when digesting tissue high in calcium.
Relatively insoluble calcium sulphate may form and possibly reduce concentrations of other elements
by coprecipitation. Perchloric acid, when hot, is a reactive oxidant and can react with explosive force
when brought into contact with highly oxidizable compounds. Therefore, extreme care is required
when using this acid. Perchloric acid is often used with nitric acid to speed up the digestion
reaction. Further, a combination of perchloric, nitric, and sulphuric acids can be used. Various
procedures with this combination of three acids have been developed with the most effective
combination being a 1:1:3 perchloric:sulphuric:nitric acid mixture. Exact temperature control is
required to avoid the danger of explosion. Temperature control can be maintained through the use
of temperature-controlled digestion blocks. The AOAC Official Methods of Analysis (1984)
recommends a perchloric-nitric digestion for Ca, Cu, Fe, Mg, Mn, K and Zn determinations in plant
tissues. If quantification is to be performed by inductively coupled plasma (ICP) spectroscopy, it
is recommended that the perchloric acid be present in minimal quantities.
Wet oxidation can also be carried out under elevated pressures in a Parr bomb (Sung et al.,
1984) or in sealed ampules placed into an autoclave at 125° C under pressure (Vigler et al., 1980).
Newer methods exist for rapid microwave oven digestions applying the same digestion principles
as those applied under elevated pressure conditions.
A combination of hydrochloric-nitric acid can be used but is not usually strong enough to
completely digest the plant sample. However, if the sample is first ashed to remove carbon, the
hydrochloric-nitric mixture can be employed to dissolve the remaining ash. The ashing temperature
is usually kept below 500° C to prevent volatilization losses. In contrast, the temperature must not
be too low or incomplete organic matter destruction will occur. In some cases, ashing aids
(catalysts or accelerators) may be used to assist in the sample decomposition.
Upon completion of the sample preparation, quantification of the plant nutrients can be
performed by ICP or AAS (atomic absorption spectroscopy). If ICP is selected, a vacuum
spectrometer is required for sulphur analysis. ICP has high speed, multielement capacity, and
computer control capabilities. AAS, while as sensitive as ICP, is more time consuming since
analysis is performed one element at a time instead of simultaneously as in the ICP. Further, AAS
cannot be used to analyze for sulphur or phosphorus.
Reference Method
A dry ashing technique is the reference method due to the difficulties with wet digestions, such
as a toss of calcium with sulphuric acid and the danger of explosion with perchloric acid. This
method is appropriate for all the elements being studied (Al, Fe, Ca, Mg, P, Mn, and K). The shape
and size of ashing vessel can affect ashing efficiency. A high walled, open vesselis recommended.
Silica, pyrex, or well glazed porcelain vessels can be used. Vycor should be used instead of pyrex
if boron analysis is required. The muffle furnace temperature must be raised slowly to prevent
flaming of the sample. Highly carbonaceous tissue may require an ashing aid such as magnesium
nitrate or nitric acid. The ash, after complete organic destruction has occurred, should be white and
free of black carbon particles. The ash is combined with a nitric-hydrochloric acid mixture and
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heated to dissolve the ash. This will help to ensure release of aluminum and iron. During the acid
digestion, hydrogen peroxide (H-PJ is added to ensure that manganese oxides are solubilized.
Final elemental quantification should be performed by ICP-AES using a vacuum spectrometer.
Summary of Method i
,!•
A weighed sample is ashed at 500° C for three hours. The ash is heated in a nitric-
hydrochlorec acid digestate for four hours. Hydrogen peroxide is added and the volume reduced to
1 mL The solution is made up to volume and analyzed by ICP.
Interferences and Shortcomings \ . .
Volatilization losses can occur on ashing leading to low results. This can be minimized by
sequential temperature ashing.
Sequential ICP is less prone to interferences than simultaneous ICP. Spectral overlap can
occur but can be compensated for by an interelement correction factors after monitoring and
measuring the interfering element. Scattered light, emission from the torch, and several other
factors can produce background emission leading to erroneously high results. ICP has the
capability of measuring the background intensity at a selected distance adjacent to the analytical
line and correcting the reading by background subtraction.
Safety
i
Normal laboratory safety practices should be observed. Protective clothing and safety glasses
should be worn especially when handling HCI, HNO3, and H2O2.
Apparatus and Equipment \
• crucibles, porcelain. 1
• watchglasses. i •
I
• polystyrene, centrifuge tubes, 15 mL
• polyethylene dropping bottles. \
i
• muffle furnace, capable of heating to 500° C.
• hot plate, capable of heating to 100° C, or equivalent. !
• top-loading balance capable of weighing to 0.01 g.
• balance calibration weights, 3-5 weights covering expected range.
• inductively coupled plasma atomic emission vacuum spectrometer with computer and
auto-sampler.
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Reagents and Consumable Materials
• nitric acid, concentrated (specific gravity 1.41)--Ultrapure grade, Baker Instra-Analyzed or
equivalent.
• hydrochloric acid, concentrated (12 M HCI, specific gravity 1.19)-Ultrapure grade, Baker
Instra-Analyzed or equivalent.
• hydrogen peroxide, 50%.
• water-DI water used in all preparations should conform to ASTM specifications for
Type I reagent grade water (ASTM, 1984).
• stock solutions for: potassium, calcium, magnesium, phosphorus, iron, aluminum, and
manganese.
• argon, oxygen-free.
Calibration and Standardization
To correctly calibrate and standardize the ICP, refer to operating manual for the instrument.
Calibrate by analyzing a calibration blank (0 mg/L standard) and a series of at least three additional
standards within the linear range of the instrument. Calibration standards should be prepared in
the extraction solution. If an ICP is used, a multi-element standard may be prepared and analyzed.
The concentration of standards should bracket the expected sample concentration; however, the
linear range of the instrument should not be exceeded.
Procedure
Step 1 - Weigh 0.50 g of vegetation into a crucible using a top-loading balance.
Step 2 - In a muffle furnace dry and ash the sample at 150° C for 15 minutes, at 250° C for 60
minutes and at 500° C for 3 hours.
Step 3 - Wet the sample ash after cooling with DI water.
Step 4- Add 1 mL of concentrated HNO3 and 3 mL of concentrated HCI.
Step 5 - Place on a hot plate at 35° C (or low setting) for 10 minutes, then raise temperature to
just below boiling.
Step 6 - Cover the crucible with watchglass and digest for four hours. Remove crucible and
wash watchglass with DI water adding washings to crucible.
Step 7 - Add 2 drops HjO2 and return to hot plate at 90-95° C.
Step 8 - Reduce volume to 1 mL by evaporation.
Step 9 - Transfer to 15 mL centrifuge tube washing crucible with DI water. Dilute to 10 mL
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Step 10 - Mix sample well.
Step 11 - Analyze samples by ICP.
Quality Control I
Precision
One sample should be analyzed in duplicate with each run of thirty samples. To eliminate bias
due to position in the run, the routine sample duplicate should be analyzed separately within the
analytical run. Within-run precision is determined from duplicates based oa relative percent
difference between the samples at ah acceptance limit of a RPD s 10%.
Accuracy
Accuracy is determined by analysis of a standard reference material (SRM). Acceptable limits
for accuracy should be ±10% from the known concentration of the standard or within the accuracy
windows supplied by the reference material manufacturer, whichever is larger.
Method Blanks
Three method blanks are taken through the entire digestion procedure to measure potential
contamination. Method blanks should be run at the beginning, middle, and end of each analytical
run. Blank concentrations should be negligible and less than the instrument detection limit. Results
are blank corrected using the mean of the acceptable method blank; readings.
Quality Control Preparation Sample \ .
A matrix matched in-house quality control preparation sample (QCPS) should be analyzed once
per analytical run. This sample is used to monitor accuracy and long-term between-run precision.
Accuracy of the QCPS should be within ± 10% of the long-term meam. Between-run precision can
be determined by analyzing the QCPS and calculating the cumulative! long-term standard deviation.
If values plotted on a control chart deviate from the long-term mean by more than three standard
deviations, the run should be completely reanalyzed, including all digestion and quantification steps.
Quality Control Check Standard i
i
A quality control check standard (QCCS) should be analyzed alt th« beginning, after every ten
samples, and after the last sample of each analytical run. The QCCS should contain all the analytes
of interest with mid-calibration range concentrations. Quantified values of the QCCS should be
within ±10% of the known concentration of the standard.
It is highly recommended that the concentrations of this sample be consistent through time
so that control charts may be plotted to monitor laboratory bias and other potential problems. If
analyzed values deviate from the long-term mean by more than three standard deviations, the
instrument is re-standardized and re-calibrated prior to any further sample analyses.
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Suggested Run Format
QCCS, MB, Samples 1 to 8, QCPS,
QCCS, Samples 9 to 16, MB, DUP,
QCCS, Samples 16 to 25, SRM,
QCCS, Samples 26 to 30, MB, QCCS.
where: QCCS
MB
QCPS
DUP
SRM
quality control check standard
method blank
quality control preparation sample
duplicate sample
standard reference material
Calculations and Reporting
The calculations for all the metals are as follows:
Metal Gug/g) - metal fug/mL) in solution x 10 mL
0.5 g
All sample results are method blank subtracted and should account for any dilution factors.
References
American Society for Testing and Materials. 1984. Annual Book of ASTM Standards.Vol. 11.01,
Standard Specification for Reagent Water, D1193-77 (reapproved 1983). ASTM, Philadelphia,
Pennsylvania.
AOAC. 1984. Official Methods of Analysis. Section 3.014.
Sung, J.F.C., A.E. Nevissi. and F.B. Dervalle. 1984. Simple sample digestion of sewage and sludge
for multi-element analysis. J. Envfr. Sci. Health A19:959-972.
Vigler, M.S., A.W. Vames, and HA Strecker. 1980. Sample preparation techniques for AA and ICP
spectroscopy. Am. Lab. 12:31-34.
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Particle-Size Analysis
Introduction
Particle-size analysis (PSA) is a measurement of the size distribution of the individual mineral
particles in a soil sample (Gee and Bauder, 1986). Particle-size analysis; provides fundamental data
that can be used for many purposes by many different scientific disciplines. Soil scientists use PSA
data in soil classification, evaluation of field texture, quantification of clay movement in soil horizons,
determination of the relationship of parent material to the solum, chemical adsorption properties,
base exchange capacity, water retention, unsaturated hydraulic conductivity, permeability, aeration,
and soil plasticity. Engineers can evaluate tillage properties, potential rates of siltation in
waterways, and determine the suitability of soil materials for foundations, landfills, roadways,
sewage disposal, etc. using PSA data. Geologists and fluvial geomprphologists use PSA in the
evaluation of sedimentation and alluvial properties.
Particle-size analysis characterizes particles with sizes ranging from boulders (>60 cm
diameter) to clays (<0.002 mm diameter). A breakdown of the particles by Canadian Soil Survey
Committee (CSSC) and the United States Department of Agriculture (UJ5DA) nomenclatures and the
defined size limits of their mean diameters are as follows (McKeague, 1978):
CSSC Nomenclature
clay, fine
clay, coarse
silt, fine
silt, medium
silt, coarse
sand, very fine
sand, fine
sand, medium
sand, coarse
sand, very coarse
gravel
cobble
stone
boulder
USDA Nomenclature
clay
silt
sand, very fine
sand, fine
sand, medium
sand, coarse
sand, very coarse
gravel, fine
gravel, coarse
cobble
stone
boulder
Mean diameter size range
0.0002 mm or less
0.002 to 0.0002 mm
0.002 mm or less
0.005 to 0.002 mm
0.02 to 0.005 mm
0.05 to 0.02 mm
0.05 to 0.002 mm
0.1 to 0.05 mm
0.25 to 0.1 mm
0.5 to 0.25 mm
1.0 to 0.5 mm
2.0 to 1.0 mm
10.0 to 2.0 mm
76.0 to 10.0 mm
76.0 to 2.0 mm
250.0 to 76.0 mm
600.0 to 250.0 mm
600.0 mm or greater
In general, particles with mean diameters greater than 2 mm (gravel or larger) are determined in the
field by visual volume estimation and the sand, silt, and clay fractiiohs are determined in the
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laboratory. More accurate quantification of the gravel and cobble fractions can be obtained through
sieving and weighing of the sample before and after the removal of these individual size fractions.
Since soil scientists are predominantly concerned with the sand, silt and clay fractions, the
remainder of this discussion focuses on particles less than 2 mm in diameter.
Review of Methods
Particle-size analysis can be divided into three different phases: (1) sample pretreatment, (2)
sample dispersion, and (3) weight contribution of «ach size fraction to the totart sample weight.
Each phase is comprised of several different processes which are discussed separately.
Sample Pretreatment
Numerous pretreatments have been developed to achieve complete aggregate dispersion in
samples. The pretreatments are primarily for the removal of cementing and binding agents such
as organic matter, iron oxides, carbonates, and soluble salts. A brief discussion of each
pretreatment follows with a more complete discussion presented in Gee and Bauder (1986).
The effect of organic matter on sample dispersion varies greatly with different soil types.
Organic matter acts as a binding agent among particles giving the soil the appearance of having
a coarser texture than if just the mineral components were analyzed. Organic matter is most
commonly removed using hydrogen peroxide (H2O2). Hydrogen peroxide is added to the soil until
the organic matter is decomposed as generally indicated by a lack of effervescence from the
sample. Other oxidants that have been used include sodium hypochlorite (NaOCI), sodium
hypobromite (NaOBr), and potassium permanganate (KMnO^ (Gee and Bauder, 1986).
Iron oxides, such as hematite and goethite, can form strong binding agents among soil
particles as either discrete crystals or coatings on particle surfaces (Gee and Bauder, 1986). Iron
oxide removal usually involves the reduction and solubilization of iron using Mehra and Jackson's
(1960) sodium dithionite-sodium citrate-sodium bicarbonate (DCB) method. This procedure consists
of multiple washings with the DCB solution until the soil is gray (gleyed), and subsequent washings
with sodium citrate and/or sodium chloride to remove all iron from the system, saturate the
exchange sites with sodium, and flocculate the sample. Iron oxides are an intricate part of the
mineralogical composition, their removal can change the particle-size distribution and lead to
erroneous interpretations of other soil chemical properties that are commonly related to PSA
contents (El-Swaify, 1980). The procedure should, therefore, be used with caution.
Carbonates are commonly removed from the soil by washings with dilute 0.2 N HCI, 1 N HCI,
or an acidified sodium acetate (1 M NaOAc, pH 5). Sodium acetate is recommended because it is
not as harsh as HCI and saturates the exchange sites with sodium. Once again caution should be
exercised. Limestone and dolomite particles can be removed resulting in a change in particle-size
distribution and textural classification of the soil (Kilmer and Alexander, 1949).
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In alkaline soils, soluble salts of calcium, magnesium, and sodium may be present in
concentrations high enough to cause particle flocculation. The addition of sodium-based chemical
dispersants (to be discussed) further hinders aggregate dispersion by increasing the salt content.
Therefore, the salts must be removed prior to sample dispersion. Removal of excess salts can be
accomplished by multiple washings with deionized water. Gee and Bauder (1986) suggest that the
washings should be continued until the leachate salt concentration drops below 10 mM.
Sample Dispersion \
• - ' 'l
Almost all of the analytical methods for the determination of particlei-size distribution require
complete sample dispersion prior to quantifying the individual size fractions. Without complete
dispersion, PSA results are biased to the coarser fractions since various sized aggregates are
measured and not individual soil particles. Sample dispersion can be performed by mechanical
(including electrical), chemical methods, or a combination of both.
Common mechanical methods for soil dispersion include: (1) tho use of a "milkshake11 mixer,
(2) shaking overnight on a reciprocating shaker, or (3) ultrasonic disioersion of the sample. The
"milkshake" mixer and overnight shaking processes rely on shearing action and turbulent mixing of
the sample to disaggregate the soil while the ultrasonic dispersion separates particles through
cavitatipn (Gee and Bauder, 1986). Particle fragmentation is a concern with these methods due to
their inherently violent nature. Mechanical dispersion techniques are often combined with chemical
dispersing agents to ensure complete sample dispersion.
Chemical dispersion agents work on the principle of particle repulsion. The addition of the
dispersing agent elevates the zeta potential by saturating the exchange sites with a monovalent
cation, such as Na* or NH4+ (Gee and Bauder, 1986). Numerous dispersing chemicals have been
used, including ammonium hydroxide (NH4OH), sodium hydroxide (NaOH), sodium carbonate
(Na2COg), sodium silicate (Na^SiOJ, sodium oxalate (Na^OJ, and several sodium phosphate
compounds such as sodium hexametaphosphate, sodium pollyphosphate, and sodium
metaphosphate. Certain types of chemical dispersion, however, may cause mineral destruction and
dissolution. \
Quantification Methods
Sand Fractions \
|
Sands are separated from the silts and clays by wet sieving after sample dispersion. The wet
sands are oven-dried, passed through a nest of separatory sieves, and weighed to determine the
individual sand fraction contents. The success and simplicity of the sieve method has meant the
development of very few other methods for the measurement of the sand fraction. Six additional
methods to quantify sand fractions mentioned in the literature include the use of the optical
microscope, computer-assisted image analysis, visual accumulation tube, sedimentation balance,
elutriation, and hydrometer.
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Optical microscopy allows for direct observation of the sample with sand fraction percentages
determined by grain counting techniques. Additional information about the sand grains, such as
colour, shape, and surface morphology, is also obtained and can be preserved via photographs.
The major drawbacks of this method are operator fatigue and the extensive time requirements to
perform sufficient grain counts to satisfy counting statistics (Yamate and Stockham, 1979).
Another method using the grain counting principle is computer-assisted image analysis (Graf,
1979a). Complete dispersion of the sample is a key factor in this analysis since counting is
performed by contrast of the individual grains and against the background. If the sample is not
completely dispersed, the computer combines the overlapping grains and count them into a larger
size fraction. Since the system is computer operated, the elimination of operator fatigue is a major
advantage of the method. This method is still experimental, yet it may have potential for future PSA
application.
The remaining four methods for measurement of the sand fraction are based on the settling
of grains in a liquid medium. The rate at which different particles settle is directly related to their
size (radius). Falling particles follow Stokes1 law in which the terminal fall velocity of the particle
is defined as follows:
v =
where v = velocity (cm/sec),
g = gravitational acceleration (cm/sec2),
q, = particle density (g/cm3),
q, = liquid density (g/cm3),
d = particle diameter (cm), and
tl = liquid viscosity (g/cm" sec).
Stokes' law assumes that the particles are smooth, spherical, and noninteractive with each other,
that terminal velocity is reached immediately at the start of the settling process, and that the
viscosity of the liquid controls the rate of settling. Separation of the various particle sizes can be
achieved by homogenizatfon of the soil suspension and decanting all that remains above the plane
z = -h (in cm) after a given time / (in sec) as follows (Gee and Bauder, 1986):
18 T^
Settling times for silt and clay fractions are provided in Tables 1 and 2.
-------
Table 1. Time
Temperature
(°C)
20.0
20.5
21.0
21.5
22.0
22.5
23.0
23.5
24.0
24.5
25.0
25.5
26.0
26.5
27.0
27.5
28.0
28.5
29.0
29.5
30.0
required for particles to settle to
0.02 mm
hr:min:sec
0:04:48
0:04:34
0:04:31
0:04:28
0:04:25
0:04:22
0:04:19
0:04:15
0:04:13
0:04:10
0:04:07
0:04:04
0:04:01
0:03:59
0:03:56
0:03:53
0:03:51
0:03:48
0:03:46
0:03:43
0:03:41
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a depth of 10 cm**.
0.005 mm
hr:min:sec
:14:13
:13:19
:12:26 (
:11:34
:10:43
:09:53
1:09:04
1:08:15
1:07:28
1:06:41
1:05:56
1:05:11
1:04:27
1:03:43
1:03:01
1:02:19
1:01:38
1:00:58
1:00:18
0:59:39 !
0:59:01
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0.002 mm
hrmin
8:00
7:54
7:49
7:43
7:38
7:32
7:27
7:22
7:17
7:12
7:07
7:02
6:57
6:52
6:48
6:44
6:39
6:35
6:31
6:26
6:22
a = assuming q. - 2.65 g/cm3, adjusted for q, and T| for temperature variations.
b = from USDA/SCS, 1992.
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Table 2. Sampling depths in cm for the clay fraction (<0.002 mm) for given temperature and settling
times". . .
Temperature
(°C)
20.0
20.5
21.0
21.5
22.0
22.5
23.0
23.5
24.0
24.5
25.0
25.5
26.0
26.5
27.0
27.5
28.0
28.5
29.0
29.5
30.0
4.5
hours
5.82
5.89
5.96
6.04
6.11 .
6.18
6.25
6.33
6.40
6.48
6.55
6.63
6.70
6.78
6.85
6.93
7.01
7.09
7.16
7.24
7.32
a = assuming q. - 2.65 g/cm3, adjusted
b - from USDA/SCS,
1992.
5.0
hours
6.47
6.55
6.63
6.71
6.79
6.87
6.95
7.03
7.11
7.20
7.28
7.36
7.45
7.53
7.62
7.70
7.79
7.87
7.96
8.05
8.13
for q, and i
5.5
hours
7.11
7.20
7.29
7.38
7.47 ,
7.55
7.64
7.73
7.83
7.92
8.01
8,10
8.19
8.28
8.38
8.47
8.57
8.66
8.75
8.85
8.95
1 for temperature
6.0
hours
7.76
7.86
7.95
8.05
8.14
8.24
8.34
8.44
8.54
8.64
8.74
8.84
8.94
9.04
9.14
9.24
9.34
9.45
9.55
9.65
9.76
variations.
6.5
hours
8.41
8.51
8.61
8.72
8.82
8.93
9.03
9.14
9.25
9.36
9.46
9.57
9.68
9.79
9.90
10.01
10.07
10.23
10.35
10.46
10.57
The visual accumulation tube is a convenient and sjmple method to use but is restricted to
sand fraction quantification (Schiebe et al., 1981). The researcher simply pours the sand into a long
water-filled column and at specified times measures the height of the accumulated sands at the
bottom of the tube. The individual fraction heights are converted into percentages by dividing by the
total sand column height. The major difficulty with this method is determining the exact height of
the column since a flat surface rarely exists in the settled sands.
Sedimentation balances work by measuring the weight of the settled particles from the soil
suspension by placement of the weighing pan within the settling column. Siebert (1979) reported
excellent correlations between the sedimentation balance and the optical microscope using glass
beads, the hydrometer (to be discussed) on geologic samples, and the pipette (to be discussed) on
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marine sediments. Complications may exist if the weighing pan is suspended into the suspension
from above due to particle settling on balance parts other than the weighing pan.
Elutriation methods, i.e. constant flow systems, depend upon rising currents of water with
different velocities to separate particles (Kilmer and Alexander, 1949; Boavers and Jones, 1966). With
decreasing current velocity, finer particles are carried to the top of one collection container and
transferred to another container (with a different flow velocity) while the coarser particles settle in
the initial collection container. Once the particles are separated, particle contents are determined
by drying and weighing of the separated fractions. Failures in this system result from the excessive
time required (up to 48 hours) to separate clay fractions, clogging in transfer tubes by larger
particles, difficulty in maintaining a constant flow velocity causing {sedimentation. Beavers and
Jones (1966) found that if properly maintained, the elutriator produced1 clay contents within ± 2% of
the results obtained by the pipette method.
i
The hydrometer, although designed primarily to determine silt and clay fractions, can also be
used to determine sand contents. The hydrometer and a similar measurement instrument, the
plummet, are usually used to determine total sand content and not individual sand fractions. Both
instruments work on the principle that density differences occur as a result of particle settling.
Density differences change the buoyancy of the suspension resulting in different hydrometer or
plummet readings, that are directly related to the various particle sizes, at different times. Problems
with this method include difficulty in reading the hydrometer, sample disturbance during instrument
insertion, particle settling on the hydrometer surface, and particles settling below the hydrometer
leaving an area of decreased density. The increased speed of analysis, however, may override these
relatively small errors depending upon operator need and final data usage (Kilmer and Alexander,
1949; Gee and Bauder, 1986). Liu et al. (1966), compared the hydrometer with the pipette method
and found good correlations (r = 0.899 to 0.980) in clay contents between the two methods in a
wide variety of soils from eleven states with the hydrometer method yielding slightly greater clay
contents than the pipette method.
Silt and Clay Fractions
Most PSA method development has been devoted to quantifying the silt and clay fractions.
New methods were developed to decrease the extensive time required to obtain the results using
earlier methods. Some of these earlier methods included: optical microscopy, decantation,
centrifugation, elutriation, the use of floats and the manometer, pijaetting, and the hydrometer
method. The majority of the earlier methods, excluding grain counting by optical microscopy, were
based on particle settling following Stokes' Law.
One of the earliest methods used to quantify silt and clay fractions was through repeated
decantation, i.e., the Atterburg method (Stein, 1985). This method involves shaking a dispersed soil
into suspension, and the removal of the supernatant containing particles of the desired size and
finer until a clear supernatant was obtained. Decantation can also t>e used in the separation of
sands from the silt and clay fractions. Although this method is effective for the separation of silt
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and clay fractions, its major drawback is the extensive time required for the analyses. Kilmer and
Alexander (1949) reported that about 7 days were required to separate eight to ten samples.
A variation on the decantation method was the use of a centrifuge to shorten the settling time.
Centrifugation markedly increased the gravitational acceleration (fl) as defined in Stokes' Law
thereby shortening the time required for particle settling. After centrifuging, the supernatant was
decanted and the soil resuspended into solution. The process was repeated until a clear
supernatant was obtained.
The manometer and float techniques measure particle-size distribution based on changes in
suspension density. The manometer measures resultant pressure head changes within the
suspension due to the settling of particles below the measurement arm. Various floats of different
densities were placed in the suspension and the time was recorded for a given density float to
reach a certain depth in the suspension. Alternatively, the depth to which the float sank after a
specified settling time was recorded. The time or depth measurement was converted to particle-size
percentages using variations of Stokes' Law. The manometer and floats both had the inherent
problem of remaining in suspension throughout the measurement period which allowed for particle
sedimentation on instrument surfaces, thereby affecting the final results. Furthermore, the
manometer was difficult to read due to small pressure changes and floats of good quality were
difficult to obtain (Rose, 1954).
The most common technique for PSA of silt and clay fractions is the pipette method. This
method is perhaps the •standard" method to which most other PSA techniques have been compared.
The pipette method consists of bringing the dispersed sample into suspension, allowing an
appropriate settling time, and sampling to a specified depth using a pipette of known volume. The
extracted aliquot is dried, weighed, corrected for the weight contribution of the dispersion agent, and
converted into weight percent silt or clay. A variation on the pipette method, the Andreason pipette,
employs the same technique except that the pipette is permanently mounted in the sedimentation
cylinder (Siebert, 1979). Problems arise from the use of the Andreason pipette due to settling of the
sample on the pipette and a disruption of the particle settling pathway.
With advances in electronic and X-ray technologies and improvements in various sensing
devices, several new methods have been developed to enhance the speed of PSA. These methods
work on the principles of: (1) photoextinction of white light, (2) low-angle forward scattering
(Fraunhofer diffraction) of laser light, (3) X-ray absorption, and (4) electrical conductivity. Methods
employing photoextinction and X-ray absorption techniques are based on particle settling following
Stokes' Law.
The hydrophotometer and the turbidity meter are two instruments that determine PSA by
photoextinction of white light. PSA is performed by passing a light beam through a suspension of
a dispersed sample and measuring the amount of light transmitted through the sample. Singer et
al. (1988) reported that five samples could be analyzed in 1 hr, 40 min using the hydrophotometer.
These authors, in a comparison experiment of the Malvern Laser Sizar (to be discussed), Electrozone
Particle Counter (to be discussed), the Sedigraph (to be discussed), and the hydrophotometer using
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sorted and glacial silt standards, reported that all four methods worked well on the sorted silt
standards. However, they reported that the hydrophotometer displayed a slight shift toward the
coarser particle sizes in the mean and mode of the particle-size distribution when the glacial silt
standards were used. Moreover, they found the hydrophotometer to have the poorest precision
among the four instruments for polymodal samples. Samples containing silt-clay mixtures posed
the greatest analytical problems due to light dispersion, increased fluid! viscosity, and particle-
particle interactions. Initial turbulence preventing the settling of the silt and clay particles during
sample suspension is also a problem with the hydrophotometer.
. Two instruments that use a laser light source for PSA are the Leeds and Northrup Microtrac
II1 and the Malvern Laser Sizer2. These instruments operate by passing the las'er beam through
a continuous flow sample cell and quantifying the emergent beam. As particle size increases, the
amount of scattered light increases, but the angle of scattering decreases. A collector lens focuses
the scattered light to a solid state photodiode detector array which translates the signal to a
proportional volumetric measurement. The range in measured particle sizes is from 700 to 0.7
microns for the Microtrac II Standard Range Analyzer and from 60 to 0.12 microns for the Microtrac
II Small Particle Analyzer. The Malvern Laser Sizer employs three different collector lenses, with
differing focal lengths, giving a measurable particle size range of 564 to 1,2 microns (Singer et at.,
1988). Measurement times can be set from 1 to 999 seconds with a sample size requirement of
approximately 0.05 grams to 2 grams for the Microtrac II Small Particle Analyzer and 0.5 to 20
grams for the Microtrac II Standard Range Analyzer while the Malvern Laser Sizer requires about
10 minutes per sample per lens. One notable advantage of these systems is that they are not
dependent on particle settling and are thus free from the secondary effects of particle shape,
density, optical properties, and settling fluid viscosity. In a comparison study conducted by Singer
et al. (1988), the Malvern Laser Sizer displayed a slight shift in the mean and mode of the particle-
size distribution toward the coarser fractions and had the poorest resolution when sorted silts and
glacial silt standards were tested. An additional problem with the laser system was that different
collector lenses do not yield the same particle-size distribution where overlapping measurable
particle sizes occurred. The occurrence of this type of problem in the Microtrac II systems is
unknown by the author.
'i
The Micromeritics Sedigraph 51003 combines X-ray absorption and particle settling for the
quantification of silt and clay particles. A finely collimated beam of low energy X-rays is passed
through a vertically moving sample cell. The sample cell is moved vertically at a controlled rate (to
increase the effective settling time) and the emergent X-ray beam is detected and converted into
particle-size data. The Sedigraph 5100 has a measurement range of 300 to 0.1 microns equivalent
spherical diameter and uses a 50 mL dispersed sample whose precise concentration is not required.
The time required to process one sample with particle sizes ranging from 100 to 1 micron is
1 Leeds and Northrup, Sumneytown Pike, North Wales, PA 19454.
2Malyem Instruments Inc., 10 Southville Road, Southborough, MA
3 Micromeritics Instrument Corporation, One Micromeritics Drive,
01772
Norcross, GA 30093-1877
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generally around 30 minutes with 100 minutes required for analysis to an endpoint of 0.2 microns
using the Sedigraph 5000 (Welch et al., 1979; Berezin and Voronin, 1981). Multiple scans are required
for this wide range in particle sizes. In comparison studies with the pipette method, the Sedigraph
indicated a finer particle-size distribution and was more precise than the pipette (Welch et al., 1979;
Berezin and Voronin, 1981). Several concerns with the Sedigraph system are that Instrument down
time may be a factor (Schiebe et al., 1981), organic matter sensitivity (Berezin and Voronin, 1981),
availability of qualified operators (Schiebe et al., 1981). Although the precise sample concentration
is not required, a given concentration range is required for optimal instrument performance. At
suspension concentrations of less than 2% by volume, the Sedigraph was found to be both accurate
and precise, yet above that point, a shift toward the finer particle sizes was noted (Singer et al.,
1988). Stein (1985) reported that soils high in montmorillonite caused problems with the Sedigraph
due to the thixotropic properties of montmorillonite.
The Coulter Counter TAII4 and the Elzone 1805 (also cited in the literature as the Electrozone
System) determine PSA by measuring changes in the electrical conductivity as particles pass
through a small orifice. As the particles pass through the orifice, the electrolyte is displaced, and
a resistance between the electrodes (on both sides of the orifice) is created. This resistance
increase is measured by impulse .height and discriminantly counted in a multichannel analyzer. A
dilute sediment concentration and continual mixing of the sample are required to prevent large
particle settling and to eliminate particle coincidence (i.e. particles moving through the orifice at the
same time resulting in the counting of a coarser particle size). These instruments are capable of
measuring particle sizes from about 1200 to 0.4 microns and require between 10 seconds and 10
minutes per sample. Similar to the laser techniques, the Coulter Counter TAII and Elzone 180 are
true sizing instruments that measure particles regardless of their densities, shapes, and the settling
liquid viscosity, which are important factors in methods employing particle settling. Several major
disadvantages of these systems are (1) multiple orifice sizes are required to cover the range in
particle sizes for soils, (2) electrolyte selection, (3) orifice clogging, (4) particle coincidence, the
necessity to remove the previously measured coarser particles prior to analysis with each
sequentially smaller orifice (Graf. 1979b), and (5) the inability of the system to measure the complete
clay fraction. Pennington and Lewis (1979) compared the Coulter Counter TAII with the pipette
method using a variety of Idaho soils and found good correlation between the two methods for the
silt fractions (r2 - 0.80). For the clay fractions, however, the Coulter Counter results were between
one-third to one-half lower than the pipette when,clay contents ranged between 29 and 33%.
Reference Method
The reference method for PSA, even in light of the more sophisticated technology now
available, is still the separation of sand fractions and the pipette method to quantify silt and clay
fractions. Organic matter removal using hydrogen peroxide and sample dispersion using sodium
4 Coulter Electronics, Inc., 650 W. 20th Street, Hialeah, FL 33010
s Particle Data, Inc., P.O. Box 265, Elmhurst, IL 60126
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hexametaphosphate are recommended. The use of the hydrometer is a viable alternative due to its
increased speed in comparison with the pipette and can be used if time is a critical factor. Other
previously discussed methods ha\e limitations, such as limited particle size ranges either on the
coarse (i.e., can not determine particles up to 2 mm in diameter) or fine (i.e., generally limited to
around 0.1 micron and coarser fractions) ends, instrument cost, multiple reading requirements to
capture entire particle-size distribution, excessive time requirements for analysis ^or sample
preparation, increased technician skill requirements, and do not have widely accepted and
documented standard operating procedures, which decreases their usefulness for PSA. However,
with continual improvements in the electronics and X-ray technologies, fast, reliable particle-size
determination of the silt and clay fractions may soon be possible.
NOTE: This reference method is for mineral horizon PSA analysis only.
Summary of Method
Organic matter is removed from the sample. The sand fractions are separated from the silt
and clay fractions by wet sieving. The silt and clay separates are suspended in water. Aliquots are
taken from the suspension under the specified conditions, dried, and then weighed. The resulting
gravimetric data allows calculation of the percentages of each particle-size fraction.
Interferences and Shortcomings '<
. i .
The sedimentation cylinder should not be disturbed nor may the temperature vary while the
soil in suspension is settling. The use of forceps, finger cots, cotton or vinyl gloves are required to
avoid adding weight from moisture, body salts, or body oils when handling weighing bottles, pans,
etc. Other sources of error include the deviation of the specific gravity of the soil particles from the
assumed 2.65 g/cm3, rising air bubbles after mixing interfering with particle settling, changes in
settling velocity due to friction on the sides of the cylinder, and the fact that the particles, especially
the clays, are not spherical.
Safety
Use forceps or heat resistant gloves to remove weighing containers from hot ovens. Use
waterproof gloves and safety goggles while handling H2O2. Follow standard laboratory safety
practices when handling reagents and equipment.
Apparatus and Equipment
• Erlenmeyer flask, beaker, or other suitable container, 250 ml_ or equivalent.
• Beakers, 125 ml_ or equivalent.
• Reciprocating shaker, 120 oscillations per minute.
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• Sedimentation cylinders, (1 litre graduated cylinders, optional).
• Stirrer, motor-driven.
• Stirrer, hand. Fasten a circular piece of perforated plastic to one end of a rod.
• Shaw pipette rack, or equivalent.
• Pipettes, 25 mL automatic (Lowry with overflow bulb, or equivalent).
• Polyurethane foam pipe-insulation; constant temperature batch (± 1° C); or temperature
controlled room (±1° C).
• Steve shaker, 1.25 cm vertical and lateral movement, 500 oscillations per minute, or
equivalent. Unit must accommodate a nest of sieves.
• Glass weighing bottles, 90 mL, wide-mouth with screw caps, or equivalent, capable of
withstanding intermittent heatings to 110° C.
• Top loading balance (0.01 g sensitivity).
• Electronic analytical balance (0.1 mg sensitivity).
• Set of sieves, square-mesh, woven phosphor-bronze or stainless steel wire cloth; U.S. Series
and Tyler Screen Scale equivalent designation as follows:
Nominal U.S. Tyler
Opening (mm) No. Mesh Size
10 18 16
0.5 35 32
0.25 60 60
0.105 MO 15°
0.053 270 270
• Receiving pan, used with sieves.
• Cover glasses, watch glasses, or equivalent
• Thermometer, range 10 to 120° C.
• Desiccator and desiccant, P2O5.
• Hot plate (block digester, optional).
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Reagents and Consumable Materials
• Hydrogen peroxide (H2O.j), 30 to 35 percent.
• Sodium carbonate (Na2COg).
• Sodium hexametaphosphate
• water-Water used in all preparations should conform to AS1FM specifications for Type I
reagent grade water (ASTM, 1984).
• Dispersing agent - Dissolve 35.7 grams (NaPOg), and 7.94 grams of NaCO3 per litre of
detonized (DI) water.
• Aluminum weighing dishes, or equivalent.
Calibration and Standardization
Calibrate thermometers periodically to ensure that they are measuring temperature accurately.
Temperature of the suspensions should not vary more than ± 1° C.
Procedure
The reference method is divided into four procedures. The first procedure is the removal of
organic matter which may be excluded for samples with low organic matter contents at the
discretion of the analyst. The remaining three procedures, namely, sample dispersion, sand fraction
quantification, and silt and clay quantification, are essential for the determination of PSA.
NOTE: The procedures described are for mineral horizons only.
Procedure One - Removal of Organic Matter \
I
Step 1 - Weigh 10 ± 0.01 g air-dried soil into a tared Erlenmeyer flaslk, beaker, or equivalent. For
soils with low clay contents, it may be necessary to double the amount of soil in order
to have a sufficient measurable quantity of clay. Doubling the amount of soil does not
require any adjustments to the remaining steps in this procedure.
Step 2 - Add 50 ml_ of DI water, followed by 5 ml_ of H2O2. Cover the flask with a watch glass.
If a violent reaction occurs, repeat the H2O2 treatment periodically until no more frothing
occurs.
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Step 3 -
Heat the flask to approximately 90° C on an electric hot plate. Add H2O2 in 5 mL
amounts at 10-15 minute intervals until the organic matter is destroyed as determined
visually by the lack of effervescence. Continue heating for approximately 30 minutes to
remove excess H2O2.
NOTE:
Some long-chain organics tend to produce a stable foam which continues to build upon
heating. The foam can be controlled by periodic streams of water or methanol from a
wash bottle. -
Do not let samples on hot plate boil dry or certain clays may collapse and may not be
successfully dispersed.
Dry the sample overnight in an oven at 105° C, cool the sample in a desiccator, and
weigh the H2O2-treated sample plus flask to the nearest 0.01 g. Record the weight of
the soil + flask. The oven-dry soil weight can then be calculated by subtracting the
flask weight from the soil + flask weight. Use the weight of the oven-dry, H2O2-treated
sample as the soil weight (Treated Sample Weight) for calculating percentages of the
particle-size fractions..
Procedure Two - Sample Dispersion and Separation of Sand from Silt and Clay
NOTE:
Step 4 -
Step 1 -
Step 2 -
Add 10 mL of dispersing agent to the flask containing the oven-dry treated sample.
Bring the volume to approximately 200 mL using DI water. Stopper the flask and shake
overnight on a horizontal reciprocating shaker at 120 oscillations per minute.
Place a 270-mesh sieve on top of the sedimentation cylinder. Wash the dispersed
sample onto the sieve with DI water. Avoid using jets of water because they may break
the fine mesh of the sieve. Silt, clay, and some very fine sand will pass through the
sieve into the cylinder. The sand fractions will remain in the sieve. It is important to
wash all particles of less than 0.05 mm diameter through the sieve. Gently tapping the
sieve clamp with the side of the hand may facilitate sieving.
NOTE: A clamp and ring stand may be used to hold the sieve in place.
Step 3 - Continue washing the sand until suspension volume in the cylinder is approximately 900
mL
NOTE: For soils high in silt, wet sieving may be inadequate to separate aH the silt from the
sand fraction. In this situation, it is recommended that the sample remaining on the
sieve be air-dried overnight on the sieve resting on a watch glass to catch any silt
passing through during drying. Dry sieve the sample the next day and add any portion
passing through the sieve to the sedimentation column prior to proceeding to step 4.
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Step 4 - Remove the sieve from the cylinder. Wash the sand into a tared evaporating dish or
original flask with DI water. Dry the sand overnight at 105° C.
Step 5 - Dilute the silt and clay suspension in the cylinder to 1 litre with DI water. Cover the
cylinder with a watch glass.
Procedure Three - Sand Fraction Quantification.
Step 1 - Wash the sands quantitatively from the sieve (Procedure Two, Step 4) into a beaker or
equivalent drying container with DI water and dry the sample overnight at 105° C.
Step 2 - Remove the sands from the oven and allow to cool to room temperature in a desiccator.
Weigh the total dry sand to the nearest 0.01 g and record the weight.
Step 3 - Stack sieves from the largest mesh opening (1.0 mm) on top to the smallest mesh
opening on the bottom. Below the smallest mesh sieve, place the receiving pan.
Step 4 - Quantitatively transfer the dried sand to the top sieve. i
Step 5 - Cover and secure the stack of sieves in the shaker. Shake the nest of sieves for 3
minutes. ;
Step 6 - Quantitatively transfer the sand fractions to tared aluminum weighing dishes (tared to
the nearest 0.01 g). Weigh each sand fraction to the nearest 0.01 g and record the
weight. The sand fractions contained on top of the sieves are the very coarse, coarse,
medium, fine, and very fine listed from top to bottom sievw, respectively.
Procedure Four - Silt and Clay Quantification by Pipetting
\
NOTE: All pipetting should be performed in a location free from drafts and temperature
fluctuations. A temperature-controlled room, constant-tem|3erature water bath, or foam
insulation may be used.
f -
NOTE: A blank sample should be prepared with each run to determine the weight contribution
of the dispersing agent to the weight of the pipetted aliquot. The blank sample should
contain 10 ml_ of the dispersing agent only, diluted to 1 litre. The blank sample should
proceed through all the steps in the following procedure. The net weight of this blank
will enter into the final fraction quantification as the RBLANK in the formulae.
Step 1 - Allow 12 to 24 hours for the suspension temperature to equilibrate to room temperature
if temperature control is being maintained by use of either foam insulation, or a constant
temperature room, or to equilibrate in the constant temperature water bath.
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Step 2 - Refill sedimentation cylinder to the 1 litre mark with DI water at the same temperature
as the analysis to be performed.
Step 3 - Stir the material in the sedimentation cylinder for 6 minutes with the motor-driven stirrer.
Stir 8 minutes if the suspension has been standing for more then 16 hours. If stoppers
of adequate size are available, it is preferable to stopper the cylinder and invert. Care
must be taken to ensure that the stopper is held tightly in the cylinder. After the cylinder
has been inspected to ensure that the fine particles are not adhering to the glass walls
or bottom of the cylinder, repeat the inversion procedure at least 6 additional times.
Step 4 - Remover the stirrer and either (1) cover the cylinder with a length of polyurethane foam
pipe-insulation, (2) immerse the cylinder in a constant-temperature water bath, or (3)
place the cylinder in a temperature-controlled room.
Step 5 - Record the temperature of the solution in the cylinder by gently lowering a thermometer,
5 cm into the suspension. Support the thermometer with a clamp to reduce disturbance
of the suspension.
NOTE: A separate cylinder of DI water may be used to monitor the temperature.
Step 6 - Stir the suspension for 30 seconds with a hand stirrer using an up-and-down motion.
Record the time when the stirring is complete. Do not move, stir, or otherwise disturb
the cylinder from this point until all pipetting has been completed.
Step 7 - Determine the appropriate settling time required for the 0.02- to 0.005 mm fraction
(medium silt) using Table 1. The resultant pipetting for this fraction will be referred to
as Pipette #1 in the calculations.
Step 8 - About 30 seconds before the sedimentation time has elapsed, slowly lower the Lowry
automatic pipet 10 cm into the suspension. (A 25-mL volumetric pipet premarked for a
10-cm depth and clamped firmly in place on a stand may be used.)
Step 9 - At the appropriate time, slowly fill the pipet (allow for about 12 seconds). Carefully
remove the pipet from the suspension.
Step 10 - Wipe the outside of the pipet clean and empty the contents into a drying container, such
as a wide mouth glass weighing bottle or aluminum weighing tins, tared to the nearest
0.1 mg. Rinse the pipette once with DI water and add the rinse water to the contents
of the bottle.
Step 11 - Dry the bottle or tin and contents in an oven overnight at 105° C. Cool in a desiccator
over phosphorus pentoxide (P2Og). Weigh and record net weight to nearest 0.1 mg.
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Step 12 - Repeat steps 7 through 11 for the 0.005 to 0.002 mm (PipBtte #2) and the <0 002 mm
(Pipette #3) clay fraction. The <0.002 mm fraction may bte pipetted at a time ranging
from 5 to 8 hours depending on the temperature and the depth of sampling as listed in
Tables 1 and 2. The use of Table 1 is strongly recommended.
Step 13 - Determination of the fine clay fraction (<0.0002 mm mean diameter) must be performed
by centnfuging due to the excessively long time required for sedimentation (up to 39
days) and the influence of Brownian motion on the settling of Fine clay particles (Kilmer
and Alexander, 1949). Required centrifuging times may be calculated by incorporation
of the appropriate gravitational acceleration ($) into Stokes' Law. A more complete
presentation of this procedure is presented by Sheldrick (1984).
Quality Control
Precision
One sample should be analyzed in duplicate with each run of thirty samples. The standard
deviation among similar size fraction should be within 3.0 weight % for the sand and silt fractions
and within 2.0 weight percent for clay fractions.
Accuracy
Accuracy is determined by analysis of a standard reference material (SRM). Acceptable limits
for accuracy should be ±10% from the known fraction concentration of the standard or within the
accuracy windows supplied by the reference material manufacturer, whichever is larger.
Method Blanks
One method blank should be analyzed with each batch of thirty samples. A method blank
consists of a 25 mL aliquot of the diluted dispersion solution (10 mL of the hexametaphosphate
dispersion solution diluted to 1 L), dried, and weighed. The method blank weight is used in the final
calculation of the silt and clay contents.
Quality Control Preparation Sample
A well-characterized soil having a minimum of 5 percent sand, silt, and clay should be analyzed
once per analytical run. This sample is used to monitor accuracy and long-term between-run
preasion. Quantified values of the QCCS should be within ±10% of the known quantities within the
sample with checking only being performed on the total sand, silt, and clay fractions Between-run
p;ec*10" can *? determined bv analyzing the QCPS and calculating the cumulative long-term
standard deviation If values plotted on a control chart deviate from the long-term mean by more
than three standard deviations, a new QCPS should be obtained.
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Suggested Run Format
MB, Samples 1 to 8, QCPS,
Samples 9 to 16, DUP,
Samples 16 to 25, SRM,
Samples 26 to 30.
where: MB = method blank
QCPS - quality control preparation sample
DUP = duplicate sample
SRM = standard reference material
Calculations
NOTE: All quantities of individual size fractions are calculated in weight percent.
SAND =
I VERY COARSE MEDIUM + FINE + VERY }
_ [COARSE SAND * SAND * SAND SAND FINE SAND]
Treated Sample Weight
X100
SILT = 100% - (SAND + CLAY)
= (Oven-dry Net Weight of Pipette #3 - RBLANK) x 1000 x 1(X)
Treated Sample Weight 25
VERY _ Oven -dry net weight of very coarse sand x 1QO
COARSE SAND Treated Sample Weight
COARSE SAND
MEDIUM
(Oven-dry net weight of coarse sand) x
Treated Sample Weight
°f
* ino
Treated Sample Weight
FINE SAND = (Oven-dry net weight of fine sand) x 10Q
Treated Sample Weight
VERY = (Oven -dry net weight of very fine sand) x
FINE SAND Treated Sample Weight
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COARSE SILT = SILT - (MEDIUM SILT + FINE SILT)
MEDIUM SILT =
of Pipette #1 - ODNW of Pipette #2) y 1000 v
Treated Sample Weight
25
FINE SILT = (OPNW of Pipette #2 - ODNW of Pipette #3) x 1000 x
Treated Sample Weight 25 •
where: ODNW = oven-dry net weight.
References ;
i
American Society for Testing and Materials. 1984. Annual Book of A8TM Standards, Vol. 11.01,
Standard Specification for Reagent Water, D-1193-77 (reapproved 1983). ASTM, Philadelphia.
PA. ;
j
Beavers, A.H., and R.L. Jones. 1966. Elutriator for fractionating silt. Soil Sci. Soc. Am. Proc.
30:126-128.
Berezin, P.N., and A.D. Voronin. 1981. Use of a Sedigraph for the particle-size analysis of soils. Sov.
Soil Sci. 13:101-109.
i
El-Swaify, S.A. 1980. Physical and mechanical properties of oxisols. In BXG. Theng (ed.) Soils of
triable charge. New Zealand Soc. Soil Sci., Lower Hutt, New 2baland. pp. 303-324.
Gee, G.W., and J.W. Bauder. 1986. Particle-size analysis. In A. Klute (ed.), Methods of Soil Analysis,
Part 1. Agronomy 9:383-411.
Graf, J. 1979a. Sizing with modern image analyzers. In Stockham, J.D., and E.G. Fochtman (ed.)
Particle size analysis. Ann Arbor Sci. Pub., Inc. Ann Arbor, MI. pp. 35-44.
Graf, J. 1979b. The "Electrozone" counter: Applications to nonaqueouss particle-fluid systems. In
Stockham, J.D., and E.G. Fochtman (ed.) Particle size analysis. Ann Arbor Sci. Pub., Inc. Ann
Arbor, MI. pp. 65-76. \
Kilmer, V.J., and LT. Alexander. 1949. Methods of making mechanical;analyses of soils. Soil Sci.
68:15-24. ;
Liu, T.K., R.T. Odell, W.C. Etter, and T.H. Thornburn. 1966. A comparison of clay contents determined
by hydrometer and pipette methods using reduced major axis analysis. Soil Sci. Soc. Am. Proc.
30:665-669.
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McKeague, JA (ed.) 1978. Manual on soil sampling and methods of analysis. Canadian Soc. Soil
Sci., Ottawa, Canada.
Mehra, O.P., and M.L Jackson. 1960. Iron oxide removal from soils and clays by a dithionite-citrate
system buffered with sodium bicarbonate. In Clays and Clay Min. Proc. 7th Conf. Natl. Acad.
Sci. Natl. Res. Council Pub., Washington, DC. pp. 237-317.
Pennington, K.L, and G.C. Lewis. 1979. A comparison of electronic and pipet methods for mechanical
analysis of soils. Soil Sci. 128:280-284.
Rose, H.E. 1954. The measurement of particle size in very fine powders. Constable and Comp. Ltd.
London, England.
Schiebe, F.R., N.H. Welch, and LR. Cooper. 1981. Measurement of fine silt and clay size distributions.
Trans. Am. Soc. Agric. Eng. 491-496.
Sheldrick, B.H. (ed.) 1984. Analytical methods manual 1984. Land Resource Res. Inst. Contribution
84-30. Land Resource Res. Inst. Ottawa, Ontario.
Siebert, P.C. 1979. Simple sedimentation methods including the Andreason pipette and the Cahn
sedimentation balance. In Stockham, J.D., and E.G. Fochtman (ed.) Particle Size Analysis. Ann
Arbor Sci. Pub., Inc. Ann Arbor, MI. pp. 45-56.
Singer, J.K., J.B. Anderson, M.T. Ledbetter, I.N. McCave, K.P.N. Jones, R. Wright. 1988. An
assessment of analytical techniques for the size analysis of fine-grained sediments. J. Sed.
Pet. 58:534-543.
Stein, R. 1985. Rapid grain-size analyses of clay and silt fraction by Sedigraph 5000D: Comparison
with Coulter Counter and Atterberg methods. J. Sed. Pet. 55:590-615.
United States Department of Agriculture/Soil Conservation Service 1992. Soil survey laboratory
methods manual. Soil Survey Investigations Report No. 42. U.S. Government Printing Office,
Washington, DC.
Welch, N.H., P.B. Allen, and D.J. Galindo. 1979. Particle-size analysis by pipette and Sedigraph. J.
Environ. Qual. 8:543-546.
Yamate, G., and J.D. Stockham. 1979. Sizing particles using the microscope. In Stockham, J.D., and
E.G. Fochtman (ed.) Particle size analysis. Ann Arbor Sci. Pub., Inc. Ann Arbor, ML pp. 23-33.
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SoilpH
Introduction
Soil pH is one of the most indicative chemical measurements in soil. Soil pH is a measure of
the hydrogen ion activity in the soil solution and can, therefore, be considered as the intensity factor
for soil acidity. The importance of soil pH is that it is integral to many other soil properties such
as the solubility of compounds, the availability of plant nutrients, thei relative bonding of ions to
exchange sites, and the activity of various soil microorganisms. It is inteqral in the sense that soil
pH is both a symptom and a cause of the many reactions that occur within soil.
Soil pH is recommended as a parameter for terrestrial monitoring programs for several
reasons. One of the effects of acid deposition on soil is soil acidification. Decreases in soil pH
resulting from soil acidification will reflect the overall decline in base saturation and an increase in
exchangeable acidity (Bache, 1980). Soil pH can be measured with relatively high precision by
present analytical methods. The spatial variability of soil pH is also lower than a majority of other
soil properties (Arp and Krause, 1984). The temporal variability in Hhis parameter can also be
minimized through the selection of appropriate methods. i
Review of Methods
The value obtained for pH in a soil is very dependent upon the method used for its
measurement. Several factors that influence the measured pH value include the soil/solution ratio
e ectrolyte concentration, CO2 content of the soil/solution, temperature, nature and placement of the
electrodes, and treatment of a soil prior to measurement. Common soil/solution ratios on a weight
basis include 1:1,1:2,1:4,1:5, and 1:10, and saturated pastes (McLean, 1982). Common electrolytes
5iS™ "?r. measurement °f soil pH include deionized water, 0.01 M CaCI2, 1 N KCI and 0002 M
CaClj, (Cappo et al., 1987). The higher the electrolyte concentration, the larger the amount of
exchangeable acidity displaced from soil exchange sites with the subsequent lowering of the pH of
the soil solution. The displacement of hydrogen ions by the catioms of the salts can also be
accompanied by the displacement of exchangeable aluminum, which upon hydrolysis, increases the
H ions in the soil solution. The CO2 content of soil influences the measured pH through the
formation of carbonic acid as it dissolves in water. One reason for stirring and allowing a soil to
stand for a period of time prior to measurement is to allow the soil! CO, content to come into
equilibrium with CO2 in the air. Commonly used times are 30 minutes or 60 minutes.
The temperature at the time of measurement affects not only the activity of the H+ ion but
the calibration of the instrument. The placement of the electrodes in relationship to the sediment
or the supernatant can have a marked effect. This effect has been attributed to the liquid junction
potential at the calomel electrode and to differences in H+ concentration with distance from soil
particles (McLean, 1985). The prior treatment of a soil can affect the pH measurement. The method
of drying and storage conditions of the soil can be important. The pH of air-dried soils are generally
lower than field moist samples. !
Reference Method
The reference solutions for mixing with soil for the soil pH measurement are 0.01 M CaCI, and
deionized water. Measurement in these two solutions has been recommended by the Canadian
Society of Soil Science (McKeague, 1976), the American Society of Soil Science (McLean, 1985) and
the U.S. Environmental Protection Agency (Cappo et al. 1986), and are widely used by soil testing
laboratories. Measurement of soil pH in 1 N KCI is not recommended since the solution is not
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representative of electrolyte concentrations found in most natural soils. The high electrolyte
concentration of the KCI solution will result in excessive amounts of exchangeable acidity being
displaced by this electrolyte. Measurement of soil pH in 0.01 M CaCI2 has the advantage of
minimizing temporal variability in soil pH due to seasonal salt fluctuations, is independent of
soil/solution ratios, and it minimizes potential problems with liquid junction potentials by flocculating
soil suspensions. Another standard solution for measuring soil pH is deionized water for which
numerous amounts of data are available for comparison. These two measures of soil pH are highly
correlated. If both pH values are measured, inaccurate data points can be identified as not being
internally consistent.
The reference soihsolution (by weight) ratios are 1:1 for mineral soils and 1:5 for organic soils.
The saturated paste technique for measuring soil pH is not recommended due tq poorer precision
of measurement and potential problems with liquid junction potentials. Laboratory methods are
recommended over field methods due to the difficulty of obtaining reproducible results in the field.
Improved control over temperature, sample mixing, and subsampling are possible in the laboratory
whereas this control is lost during field pH measurements.
A standard KCI combination electrode with a sleeve is preferred for measurement over the
double electrode system. The combination electrodes are simpler to handle and maintain, can be
used on smaller samples, have a constant distance between the junction and the glass electrodes,
and are less prone to potential stoppage of flow of electrolyte from the calomel electrode.
A period of one hour is suggested for equilibration time with regular stirrings at 15 minute
intervals to allow the soil to come to equilibrium with the CO2 in the air in the laboratory. The
method presented is an adaptation of the method presented by Cappo et al. (1986).
Summary of Method
Two suspensions of each soil sample are prepared, one in DI water, and one in 0.01 M CaCI2.
The pH of each suspension is measured with a pH meter and a combination electrode.
Interferences and Shortcomings
Soils high in salts, especially sodium (Na+) salts, may interfere with the pH reading and the
electrode response time.
Clays may clog the KCI junction and may slow the electrode response time. Thorough cleaning
of the electrode between samples can help avoid this problem.
Wiping the electrode dry with cloth, laboratory tissue, or similar materials or removing the
electrode from solution when the meter is not on standby may cause electrode polarization and,
therefore, should be avoided.
The initial pH of a non-alkaline soil will usually be as much as 0.5 pH unit greater than the pH
taken after the sample has set for 30 minutes or longer.
The pH will vary as much as 1.0 pH unit between the supernatant and soil sediment. Always
place the electrode junction at the same distance above the surface of the soil to maintain
uniformity in pH readings.
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Safety
No specific hazards are associated with this procedure or with the required reagents Normal
laboratory safety practices are to be observed. Protective clothing and safety glasses should be
worn, especially when handling concentrated HCI and dry Ca(OH)2 to prepare reagents.
Apparatus and Equipment
• digital pH/mV meter, capable of measuring pH to ± 0.01 pH unil! and potential to ± 1 mVand
temperature to ± 0.5° C. The meter must also have automatic temperature compensation
capability. i
• pH and reference electrodes, high quality, low-sodium glass. Gel-Type reference electrodes
must not be used. A combination electrode is strongly recommended, and the procedure
is written assuming that one is used. The Orion Ross combination pH electrode, or
equivalent, with a retractable sleeve junction is preferred. At least two electrodes, one a
backup, should be available to the analytical laboratory.
' 'i
• beakers, plastic or paper containers, 50 mL
• glass stirring rods or disposable stirrers, one per sample.
i
Reagents and Consumable Materials
• pH buffers of pH = 4, pH = 7, and pH = 10, for electrode calibration.
• buffer of pH 4.0 as a quality control check standard (QCCS). The QCCS can be purchased,
or it can be prepared from 0.05 M potassium hydrogen phthalate (KHC8H4O4 or KHP). This
buffer must be from a different container or lot than the standards used for electrode
calibration.
Dry KHP for 2 hours at 110° C, cool to room temperature in a desiccator. Weigh 10.21 g of
KHP, dissolve it in DI water, and dilute the solution to 1.000 L To preserve the KHP
solution, add 1.0 mL of chloroform or one crystal (about 10 mm in diameter) of thymol per
litre of the buffer solution. This solution has the following pH values at the temperatures
given:
TfCl
15
20
25
30
pH
3.999
4.002
4.008
4.015
water-Water used in all preparations should conform to ASTM specifications for Type I
reagent grade water (ASTM, 1984).
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• stock calcium chloride solution (CaCI2, 1.0 M)--Dissolve 55.493 g of anhydrous CaCI2 in DI
water and dilute to 500 mL
• calcium chloride 0.01 M CaCI2-Dilute 20 mL of stock 1.0 M CaCI2 to 2.000 L with DI water.
If the pH of this solution is not between 5 and 6.5, adjust the pH by addition of dilute
Ca(OH)2 or HCI, as needed. Verify the concentration of the CaCI2 solution by measuring the
electrical conductivity. The specific conductivity should be 2.32 ± 0.08 mmho/cm at 25° C.
If it is not, prepare fresh solution.
• calcium hydroxide (Ca^HJJ-Dissolve 0.185 g Ca(OH)2 in 1 L of DI water.
• hydrochloric acid (HCI)-Dilute 1 mL concentrated HCI to 1 L with DI water.
• potassium chloride (KCI, 3 M)-Dissolve 224 g of KCI in DI water and dilute to 1 L.
• potassium chloride (KCI, 0.1 M)-Dissolve 74.5 g of KCI in DI water and dilute to 1 L
Calibration and Standardization
For storage and readings, the electrode need only be immersed to cover the liquid junction of
the reference electrode (typically about 2.5 cm).
Rinse electrode with DI water between each sample and each buffer to prevent solution carry-
over. Do not rub or blot electrode dry because this may produce a static electric charge and,
thereby, polarize the electrode.
To prepare the pH electrode for use, move the band covering the fill hole and fill the reference
reservoir to the hole with 3 M KCI filling solution. Allow 5 minutes for the ceramic frit to become wet
with filling solution before immersing the electrode in sample or buffer. The retractable sleeve
junction allows easy cleaning of clay particles and other insoluble compounds that clog the junction
and, thereby, produce drift and slow response.
Each analyst must be thoroughly acquainted with the procedure and familiar with all
instrument functions. Read and follow all operating and start-up procedures for the pH meter.
Leave the instrument on standby and verify that the combination electrode is connected and that
the level of reference filling solution is at least 3 cm above the sample surface. Check the
temperature calibration by measuring room temperature of a solution with the electrode and meter
and with a thermometer.
Calibrate the electrode at a minimum of two points that bracket the expected pH and that are
three pH units or more apart. Use standardized buffers of pH 4, 7, and 10 for samples in the
expected pH range.
Stir pH 4.0 buffer solution for 30 seconds, read the pH after equilibration, and adjust the meter,
if necessary. Perform the step again, using the pH 7.0 buffer. Repeat measurements and
adjustments until readings for both buffer solutions are within 0.1 pH units of the respective true
buffer values. Repeat the process substituting a pH 10.0 buffer in place of the pH 4.0 buffer for
soils of pH greater than 7.0.
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Procedure
Step 1 - Prepare two suspensions of each soil sample, one in DI water and one in 0.01 M CaCI2,
using soil-to-solution ratios of 1:1 for mineral horizons and 1:5 For organic horizons. For
mineral horizons, add 20 ml of the appropriate solution to 20.00 g soil. For organic
horizons, add 25 ml_ solution to 5.00 g soil.
Step 2 - Aljow soil to absorb solution without stirring, then thoroughly stir the soil-solution
mixture for 10 seconds with a glass stirring rod or disposable stirrer. Stir again for 10
seconds after 15, 30, 45, and 60 minutes.
Step 3 - After the final stirring, allow the suspension to settle for 1 minute. Place the pH
electrode in the supernatant of the soil suspension.
For mineral soils, the electrode junction should be below the solution surface and above
the soil-solution interface.
Some organic soils swell, so there is no free water available. As long as the electrode
junction is below the surface of the organic material, an acceptable, repeatable reading
is generally attained. If the reading is not stable, add enough solution to cover the
electrode junction. When the reading is stable, record pH to the nearest 0.01 pH unit.
Step 4 - Report the pH of the soil:DI water suspension and the soil:O.OI M CaCI2 suspension for
each sample.
Steps- After measurements are completed, store the electrode in 0.1 M KCI manufactured
storage solution. Do not let the sensing element and reference junction dry out. The
level of the storage solution should be 1 inch below the filling solution level to prevent
influx of the storage solution. Periodically check that the electrode reservoir is full of
filling solution. <
Quality Control
i
Precision
One sample should be analyzed in duplicate with each run of thirty samples for each of the
following solutions: DI water and 0.01 M CaCI2. To eliminate bias due to position in the run, the
routine sample duplicate should be analyzed separately within the analytical run. Within-run
precision is determined from duplicates based on absolute difference between the samples at an
acceptance limit for the difference of £ 0.1 pH unit. \
Quality Control Preparation Sample
\
A matrix matched in-house quality control preparation sample (QCPS) should be analyzed once
per analytical run. This sample is used to monitor accuracy and long-term between-run precision.
Accuracy of the QCPS should be within ± 0.1 pH unit of the long-term mean. Between-run precision
can be determined by analyzing the QCPS and calculating the cumulative long-term standard
deviation. If values plotted on a control chart deviate from the long-term mean by more than three
standard deviations, the run should be completely reanalyzed.
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Quality Control Check Standard
A quality control check standard (QCCS), a pH 4.0 standard that is'either from a different
purchased lot or created at a different time, should be analyzed at the beginning, after every ten
samples, and after the last sample of each analytical run. Values for the QCCS should be 4.00 ±
0.05 pH units.
It is highly recommended that the concentrations of this sample be consistent through time
so that control charts may be plotted to monitor laboratory bias and other potential problems. If
analyzed values deviate from the long-term mean by more than three standard deviations, the
instrument is re-standardized and re-calibrated prior to any further sample analyses.
Suggested Run Format
QCCS, Samples 1 to 10,
QCCS, Samples 11 to 20,
QCCS, Samples 20 to 30, QCPS, DUP, QCCS.
where: QCCS
QCPS
DUP
quality control check standard
quality control preparation sample
duplicate sample
NOTE: The run format presented is per solution (i.e., DI water or 0.01 M
Calculations and Reporting
No calculations are required to obtain pH values in pH units.
References
American Society for Testing and Materials. 1984. Annual Book of _ --- -
Standard Specification for Reagent Water, D1193-77 (reapproved 1983). ASTM,
Pennsylvania.
Arp, PA and H.H. Krause. 1984. The forest floor: lateral variability as revealed by systematic
sampling. Can. Soc. Soil Sci. 64:421-437.
Bache B W. 1980. The acidification of soils. ID Hutchinson, T.a, and M. Havas (eds.). Effects of acid
precipitation on terrestrial ecosystems. NATO Conference Series, Volume 4. Plenum Press, New
York. pp. 183-202.
Cappo KA LJ. Blume, GA Raab, J.K. Bartz, and J.L Engels. 1987. Analytical methods manual for
the Direct/Delayed Response Project soil survey. U.S. E^^e^l Prot^wn ^en°y'
Environmental Monitoring Systems Laboratory, Las Vegas, NV. EPA 600/8-87/020.
McKeague, JA (ed.). 1978. Manual on soil sampling and methods of analysis. Can. Soc. of Soil Sci.
212 pp.
McLean, E.O. 1982. Soil pH and lime requirement. In AL Page (ed.). Methods of soil analysis.
Part 2. 2nd ed. Agronomy 9:199-224.
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Organic Carbon
Introduction
In soil, carbon (C) occurs in mineral and organic forms. In acid soils, where carbonate
minerals are generally lacking, most C occurs in organic matter. Organic matter consists of plant,
animal, and microbial remains in various stages of decomposition plus their highly-altered
derivatives. The latter are collectively referred to as humic matter or humus. Humus maintains the
physical condition, improves moisture holding capacity, serves as an energy source for soil
organisms, and increases the nutrient holding capacity (particularly of nitrogen and phosphorus) of
the soil. Further, quantification of carbon, in conjunction with total N and S, provides insight about
the potential for uptake or release of N and/or S by the soil organic matter due to microbial activity
(Blume et al., 1990). Therefore, the measurement of organic carbon iin the soil is suggested for
terrestrial monitoring programs, such as LRTAP.
In addition to humus (and considered "organic" because of their origin) are several highly-
condensed, nearly-elemental forms of C, such as charcoal and graphite. These may form a
significant portion of the total organic C present in soils with a history olF frequent fires. In contrast
to humus, these highly-condensed forms are relatively inert. Thus, it is desirable to discriminate not
only between mineral and organic C» but between the readily oxidizable and inert organic C forms.
The mineral and inert carbon forms are indistinguishable from the organic carbon forms using
the total carbon procedure presented in Chapter 9. Thus, this method is presented to address the
determination of organic C in soils where carbonates or noticeable quantities of charcoal are
present. In most acid soils, however, the determination of carbon cam be made using either this
method or the total carbon method presented in Chapter 9. j
Review of Methods
There are a number of approaches to the determination of organic C in soil: (1) as the
difference between total (i.e. all forms) C and the mineral (chiefly carbonate) forms present, (2) by
determining total C after removing inorganic C by acid treatment, (3) by dry combustion (in a muffle
furnace at moderate temperature) with the organic content expressed ais weight "loss-on-ignition",
and (4) by organic matter reduction of Cr2O72" and the subsequent titrirnetric determination of the
unreacted Cr2O7^ by ferrous sulphate. The first three methods estimate total organic C, without
discrimination between humus and elementary C. Further, the first two methods require specialized
equipment. Loss-on-ignition, while straightforward and acceptable for descriptive purposes, may
provide over-estimate of reactive organic materials in soils of high charcoal content, or in certain
clay soils where weight loss may be associated with loss of water or hydroxyl groups.
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Schollenberger (1927) introduced a rapid titrimetric method whereby soil organic matter is
oxidized by a saturated solution of potassium dichromate in concentrated sulphuric acid with
application of heat. The unreduced chromic acid is then back-titrated with ferrous ammonium
sulphate solution. Walkley and Black (1934) and later Walkley (1947) modified this by omitting the
heating step, utilizing only the heat of dilution of H2SO4 with water. The procedure is about 75%
effective in recovering organic C with the exact figure varying somewhat with the soil. Walkley and
Black (1934) found a mean recovery of 76% for a range of British and foreign soils, necessitating
a multiplying factor of 1.32 to yield a result equivalent to organic C by combustion methods. Allison
(1960) and others, however, suggested a much broader range varying with soil group. In the
absence of a specific value, however, the original Walkley-Black (1934) value (76%) is frequently
used. The original Wajkley-Black procedure has been modified by a number of authors. Readily
available manuals, all giving variants of the "Walkley-Black" method include: Metson (1956), Jackson
(1958), Allison (1965), McKeague (1978), Nelson and Sommers (1982), Heffernan (1985), and Kalra and
Maynard (1991).
Reference Method
The following procedure is that presented by Walkley (1947) with minor wording changes only.
Ferrous ammonium sulphate [FetNHJ^SO^'eHjX)) may be substituted for ferrous sulphate; ortho-
phenanthroline ferrous complex ("Ferroin"), instead of diphenylamine, may be used as the indicator.
Summary of Method
A sample is oxidized with 1 N_ potassium dichromate and concentrated sulphuric acid. After
20 to 30 minutes, the reaction is halted by dilution with water. Phosphoric acid and diphenylamine
indicator are added to the solution. The excess dichromate is potentiometrically back-titrated with
ferrous sulphate. A blank is carried throughout the procedure to standardize the ferrous sulfate
solution. Percent organic C is reported on an oven-dry soil basis.
Interferences and Shortcomings
Chloride (Cr), if present in substantial quantity, interferes with the Walkley-Black procedure
through the formation of chromyl chloride (CrOjCy. Where problematic, the soil can be leached free
of Cr before analysis (Schollenberger, 1945). Alternatively, the CI" can be precipitated as AgCI by
the use of H^O4 containing 25 g Ag2SO4 per L in the digestion. A correction factor equal to one-
twelfth of the Cr content may also be used in soils with a CI:C ratio up to 5:1 (Walkley 1935,1947;
Jackson, 1958; Nelson and Sommers, 1982).
Nitrate (NO3') interferes only if the ratio of C to NO3' is toss than 20:1 (Heffernan, 1985; Kalra
and Maynard, 1991).
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While higher oxides of manganese (principally MnO2, but also Mn2O3 and Mn3OJ present a
potential interference, it would appear that only freshly-precipitated MnOa is capable of competing
with Cr2O7 for oxidizable substances. Thus, MnO2 interference can be generally discounted in most
soils (Walkley, 1947; Allison, 1965; Nelson and Sommers, 1982). If, however, large quantities of
reactive Mn oxides were to be encountered their effect can be nullified by pretreatment with FeSO
(Walkley, 1947; Jackson, 1958).
Ferrous iron (Fez+), if present, is oxidized to Fe3* by Cr2O7* leading to a high result. However,
thorough air-drying of even highly-reduced soils before analysis is sufficient to convert the Fe2* to
Fe3+ and negate the effect (Walkley, 1947; Jackson, 1958; Nelson and Stammers, 1982). The amount
of Fe2+ present in well-aerated soils is so small that its effect can be neglected. Iron or steel
equipment should be avoided because of the possibility of introducing reducing material in the form
of metallic Fe (Jackson, 1958; Allison, 1965; Nelson and Sommers, 1982; Heffernan, 1985).
Safety
All operations should be carried out in well-ventilated conditions. Protective clothing including
eye protection should be worn at all times, and especially when handling concentrated acids.
Special care must be taken when adding water to concentrated H2SO4. Oiphenylamine has
carcinogenic properties and should be handled with extreme caution. Gloves and a respirator should
be worn when preparing the indicator solution.
Apparatus and Equipment
• flasks, Erlenmeyer, 500 mL
• dispenser, capable of accurately dispensing 25 mL
• shaker, reciprocating, Eberbach or equivalent.
• burette, 100 mL
• balance, accurate to 0.001 g.
Reagents and Consumable Materials
• sulphuric acid, H2SO4, concentrated, reagent grade (not less tfian 96%).
• phosphoric acid, H3PO4, concentrated, reagent grade.
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• water-DI water used in all preparations should conform to ASTM specifications for Type
I reagent grade water (ASTM, 1984).
• diphenylamine indicator-To 0.5 g of indicator powder, add 20 ml_ water and 100 ml cone.
potassium dichromate solution, KgCr^, 1.0 {*-In a one litre volumetric flask, dissolve
49.04 g Ks,Cr2O7 in DI water. Dilute to volume with DI wateir.
ferrous sulphate solution, 1 N~In a one litre volumetric flask, dissolve 278.0-g of FeSO4»7H2O
in DI water. Add 15 mL cone. H2SO4. Dilute to volume with DI water.
Calibration and Standardization
Standardization of the FeSO4«7H2O is required for the accurate determination of organic
carbon contents. Standardization is performed using the method blank. Normality of the
standardized ferrous sulphate solution is calculated using the formula presented in the 'Calculations
and Reporting* section. Calibration standards should be prepared in the extraction solution.
Procedure
Step 1 - Grind sufficient soil for convenient sampling to pass a 0.5-mm screen, avoiding mortars
of steel or iron. Transfer a weighed quantity, not exceeding 10 g and containing about
10 to 25 mg of organic carbon, to a 500 ml Erlenmeyer flask.
Step 2 - Add 10 mL of 1.0 N Kj,Cr2O7 solution and swirl flask gently to disperse soil in the solution.
Step 3 - Rapidly add 20 mL of concentrated H2SO4.
Step 4 - Immediately shake the flask once or twice and allow it to stand for 20 to 30 minutes.
Step 5 - Add 200 to 300 mL DI water, 10 mL of concentrated H3PO4, and 1 mL diphenylamine
indicator solution.
Step 6 - Run in 1.0 N FeSO4«7H2O solution from a burette until the solution is purple or blue.
Step 7 - Continue adding 1.0 N FeSO4«7H2O solution in portions of about 0.5 mL until the colour
flashes to green, which it does with little or no warning.
Step 8 - Add 0.5 mL 1.0 N K^O, solution and complete the titration by adding 1.0 £ FeSO4»7H2O
solution dropwise until the last trace of blue disappears. Record volume of titrant used.
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Quality Control
Precision
One sample should be analyzed in duplicate with each run of thirty samples. To eliminate bias
due to position in the run. the routine sample duplicate should be analyzed separately within the
analytical run. Within-run precision is determined from duplicates based on relative percent
difference between the samples at an acceptance limit of a RPD £ 10%.
Accuracy
Accuracy is determined by analysis of a standard reference material (SRM). Acceptable limits
for accuracy should be ±10% from the known concentration of the standard or within the accuracy
windows supplied by the reference material manufacturer, whichever is larger.
Method Blanks
One method blank, carried through the extraction procedure, are analyzed with each batch of
thirty samples to determine the exact normality of the FeSO4»7H2O solution.
Quality Control Preparation Sample
A matrix matched in-house quality control preparation sample (QCPS) should be analyzed once
per analytical run. This sample is used to monitor accuracy and long-term between-run precision.
Accuracy of the QCPS should be within ± 10% of the long-term mean. Between-run precision can
be determined by analyzing the QCPS and calculating the cumulative long-term standard deviation.
If values plotted on a control chart deviate from the long-term mean by more than three standard
deviations, the run should be completely reanalyzed, including all digestion and quantification steps.
Quality Control Check Standard
I
A quality control check standard (QCCS) should be analyzed at the beginning, after every ten
samples, and after the last sample of each analytical run. The QCCS should contain all the analytes
of interest with mid-calibration range concentrations. Quantified values of the QCCS should be
within ±10% of the known concentration of the standard.
It is highly recommended that the concentrations of this sample be consistent through time
so that control charts may be plotted to monitor laboratory bias and other potential problems. If
analyzed values deviate from the long-term mean by more than three standard deviations, the
instrument is re-standardized and re-calibrated prior to any further sample analyses.
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Suggested Run Format
QCCS, MB, Samples 1 to 8, QCPS,
QCCS. Samples 9 to 16, DUP,
QCCS, Samples 16 to 25, SRM,
QCCS, Samples 26 to 30, QCCS.
where: QCCS = quality control check standard
MB - method blank
QCPS = quality control preparation sample
DUP = duplicate sample
SRM = standard reference material
Calculations and Reporting
The following calculations assume that a total of 10.5 mL K.,Cr2O7 solution is used and that
the recovery is greater than 75%. If a soil-specific recovery other than 75% is identified, then that
factor should be used. If the results are to be expressed in terms of "readily oxidizable organic C",
the recovery factor is omitted (Jackson 1958).
1 mL 1.0 N KgC^Oy is equivalent to 0.003 g C.
(1) Determine exact normality of FeSO4«7H2O solution from the method blank as follows:
Normality of FeSO4-7H2O = _ 10.5 __
mL FeSO4«7H2O used
(2) Determine volume of K^Cv reduced as follows:
mL KjCr^ Reduced = mL 1 N 1^Cr2O7 used - (mL FeSO4«7H2O x N FeSO4«7H2O)
(3) Calculate "total organic C" (%) as follows (the calculation assumes that the oxidation is 76%
efficient)
Total Organic C % = (mL tC.Cr.py reduced) x 0.395
weight (g)
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(4) Alternatively, assuming the soil organic matterorganic C ratio to be 1.724, the results may be
expressed as "total organic matter* (OM)
Total OM% m (ml K.Cr.OJ reduced x Q.eifll
weight (g)
(5) Alternatively, the results may be expressed as "readily oxidizable organic C" (the calculation
omits recovery factor) !
Readily Oxidizable Organic C % = fmL tCCr.O, reduced! x 0.3
weight (g)
(6) Alternatively, the results may be expressed as "readily oxidizabla organic matter".
Readily Oxidizable OM % = fmL ICCr,OTl reduced x 0.517
weight (g)
References
i
Allison, LE. 1960. Wet combustion apparatus and procedure for organic and inorganic carbon in soil
Soil Sci. Soc. Am. Proc. 24:36-40.
Allison, LE. 1965. Organic carbon, pp. 1367-1378 In C.A. Black (ed), Methods of soil analysis. Part
2. Chemical and microbiological properties. Am. Soc. Agron., Agron. No. 9.
American Society for Testing and Materials. 1984. Annual Book of ASTM Standards, Vol. 11.01,
Standard Specification for Reagent Water, D1193-77 (reapproved 1983). ASTM, Philadelphia,
Pennsylvania.
Heffernan, B. 1985. A handbook of methods of inorganic chemical analysis for forest soils, foliage
and water. CSIRO, Canberra, 281 pp.
Jackson, M.L 1958. Soil chemical analysis. Prentice-Hall Inc., Englewood Cliffs, N.J. pp. 498.
Kalra, Y.P., and D.G. Maynard. 1991. Methods for forest soil and plant analysis. Forest. Can. Inf Rep
Nor-x-319. pp. 116.
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McKeague, JA (ed.) 1978. Manual on soil sampling and methods of analysis, 2nd ed. Can. Soc. Soil
Sci. Ottawa, pp. 212. .
Metson, A.J. 1956. Methods of chemical analysis for soil survey. N.Z Dep. Sci. Ind. Res. Soil Bur.
Bull. 12. pp. 208.
Nelson, D.W., and LE. Sommers. 1982. Total carbon, organic carbon, and organic matter. In A.L
Page, R.H. Miller, and D.R. Keeney (eds.). Methods of soil analysis. Part 2, 2nd ed. Agronomy
9:539-580.
Schollenberger, C.J. 1927. A rapid approximate method for determining soil organic matter. Soil Sci.
24:65-68.
Schollenberger, C.J. 1945. Determination of soil organic matter. Soil Sci. 59:53-56.
Walkley, A. 1935. An examination of methods or determining organic carbon and nitrogen in soils.
J. Agrte. Sci. 25:598-609.
Walkley, A, 1947. A critical examination of a rapid method for determining organic carbon in soils -
effect of variations in digestion conditions and of inorganic soil constituents. Soil Sci.
63:251-263.
Walkley, A., and LA. Black. 1934. An examination of the Degtjareff method for determining soil
organic matter, and a proposed modification of the chromic acid titration method. Soil Sci.
37:29-38.
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Total Carbon
Introduction
In soil, carbon occurs in both mineral and organic forms. In soils affected by acidic
deposition, carbonate minerals (limestone and dolomite) are generally lacking and thus, most C
occurs in organic matter. As a result of this lack of inorganic carbon forms, total carbon analyses
in eastern forest soils affected by acidic deposition are often referred to as total organic carbon
(TOC) measurements.
Organic carbon forms consist of plant, animal, and microbial remains in various stages of
decomposition plus their highly-altered derivatives. The latter are collectively referred to as humic
matter or humus. Humus maintains the physical condition, improves moisture holding capacity,
serves as an energy source for soil organisms, and increases the nutrient holding capacity
(particularly of nitrogen and phosphorus) of the soil. In addition to humius (and considered "organic"
because of their origin) are several highly-condensed, nearly-elemental forms of C, such as charcoal
and graphite. These nearly-elemental forms may compose a significant portion of the total organic
C present in soils with a history of frequent fires. In contrast to humus,, these highly-condensed
forms are relatively inert in the soil.
Total carbon is the sum of all inorganic and organic forms of carbon in the soil (Nelson and
Sommers. 1982). Quantification of total C, in conjunction with total N aind S, provides insight about
the potential for uptake or release of N and/or S by the soil organic matter due to microbial activity
(Blume et al, 1990). The carbon content of a soil affects sulphate adsorption and cation exchange
properties of the soil, and TC content is sometimes used for certain soil taxonomic classifications
such as the determination of a mollic (and thus, Mollisols) and histic epipadons (Soil Survey Staff,
1975). Therefore, the measurement of total carbon in the soil is suggested for terrestrial monitoring
programs, such as LRTAP.
Review of Methods
Total carbon determinations for soil involve the conversion of all forms of C to CO2 by either
dry combustion or wet digestion techniques. The evolved CO2 is then quantified by gravimetric,
titrimetric, volumetric, manometric, spectrophotometric, or gas chromatographic techniques (Nelson
and Sommers, 1982).
!
Direct analysis of the sample may be done by combustion of the sample at elevated
temperatures (generally at 1000° C or higher; 1300° C is optimal for the completion destruction of
calcium carbonate) and measuring the liberated CO2 by an infrared or thermal conductivity
spectroscopy. Combustion is performed in a resistance furnace or an induction furnace in the
presence of a stream of oxygen or CO2-free air. Various catalyst or accelerators may be used to
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ensure complete combustion of all carbon forms. Dry combustion procedures using either high-
temperature (approximately 1500° C) or induction furnaces are most commonly found in
commercially-available automated total C analyzers (Nelson and Sommers, 1982). Examples of
Instruments that combust the sample include the LEGO CHN analyzers and Carla-Erba C and N
analyzers. These methods require little or no sample preparation for the determination of totafC.
The wet digestion analysis of soils for total C by chromic acid digestion has long been a
standard method giving good agreement with the dry combustion techniques (Nelson and Sommers,
1982). Schollenberger (1927) introduced a rapid titrimetric method whereby soil organic matter is
oxidized by a saturated solution of potassium dichromate in concentrated sulphuric acid with
application of heat. The unreduced chromic acid is then back-titrated with ferrous ammonium
sulphate solution. Walkley and Black (1934) and later Walkley (1947) modified this by omitting the
heating step, utilizing only the heat of dilution of H2SO4 with water. The procedure is about three-
quarters effective in recovering organic C wjth the exact figure varying somewhat with the soil.
Walkley and Black (1934) found a mean recovery of 76% for a range of British and foreign soils,
necessitating a multiplying factor of 1.32 to yield a result equivalent to organic C by combustion
methods. Allison (1960) and others, however, suggested a much broader range varying with soil
group. Alternately, wet combustion may be carried out in a Van-Slyke-Neil apparatus and evolved
CO2 estimated by manometric procedures (Bremner, 1949; Nelson and Sommers, 1982). However,
it should be noted that these methods are only comparable to the dry combustion techniques in
soils in which no carbonates or inert forms of carbon (e.g., charcoal or graphite) are present.
Reference Method
The reference method for total C in soils is dry combustion in a carbon analyzer with either
infrared or thermal conductivity detection. The analysis of total C employing this technique has been
chosen as a reference method because it is widely used in North America and has been used
successfully by some laboratories for both soil and foliage samples.
Summary of Method
A soil sample is oxidized at temperatures greater than 1000° C (1300°C preferred) with
catalysts as specified by the instrument manufacturer. The evolved CO2 is then quantified by either
infrared or thermal conductivity detection. Percent total C is reported on an oven-dry soil basis.
Depending on the type of instrument present in the laboratory, total N and H can also be
simultaneously quantified from the same sample aliquot using this technique.
Interferences and Shortcomings
Although moisture can interfere with certain carbon-hydrogen-nitrogen (CHN) analyses by
producing large responses for total H, this interference can be eliminated in elemental analyses with
moisture traps. Drying can cause losses of volatile organic materials containing C and N, and
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decomposition and loss of certain carbonates and ammonium salts. Samples may be freeze-dried
to minimize these losses if approved by the quality assurance (QA) manager.
Ambient N2 and CO2 not associated with the sample present possible gaseous interferences.
Care must be taken with the blank to hold N2 and CO2 below the required instrument detection limit.
The use of high purity carrier gas or helium traps helps reduce CO2 contamination.
Soil residue can accumulate at the top of the combustion column. The column should be
cleaned if sufficient residue accumulates that analytical results are affected.
Safety
Normal safety precautions should be taken when using high-temperature combustion furnaces.
Protective clothing and safety glasses should be worn when handling reagents. Heat resistant
gloves may be needed when placing samples in the furnace. The Furnace must be adequately
vented and protected from human contact and combustible materials. Gas cylinders should be
bolted or chained in an upright position.
Apparatus and Equipment
• carbon analyzer with infrared detector, Carlo-Erba model 1500, or equivalent.
i
• balance, accurate to 0.001 g.
• balance calibration weights, 3-5 weights covering expected range.
Reagents and Consumable Materials
• oxygen, high purity. i
• compressed air, if needed,
• catalysts and combustion accelerators, vanadium pentoxidle or as recommended by
instrument manufacturer.
• absorbents, as needed.
• vials, crucibles, boats/or tin sample capsules, as required by instrument.
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Calibration and Standardization
Set up the instrument according to the instrument manufacturer's instructions regarding
calibration and standardization. In general, the instrument should be calibrated at least once a day
or once per batch of samples, whichever is more frequent. Use either NIST (formerly NBS) reference
materials or standards supplied by the manufacturer and approved by the laboratory or QA
manager. The concentration range of the standards must be representative of the C concentrations
expected in the soil samples. A minimum of a two-point calibration curve should be used. Use of
a NIST standard reference material as an initial calibration check Is highly recommended.
Procedure
Step 1 - Weigh out 100 mg of air-dried mineral soil or 20 mg of organic soils into appropriate
sample holder. Record sample weight.
Step 2 - Perform sample analysis following manufacturer's instructions.
Step 3- Take carbon measurement reading (in counts).
Quality Control
Precision
One sample should be analyzed in duplicate with each run of thirty samples. To eliminate bias
due to position in the run, the routine sample duplicate should be analyzed separately within the
analytical run. Within-run precision is determined from duplicates based on relative percent
difference between the'samples at an acceptance limit of a RPD s; 10%.
Accuracy
Accuracy is determined by analysis of a standard reference material (SRM). Acceptable limits
for accuracy should be ±10% from the known concentration of the standard or within the accuracy
windows supplied by the reference material manufacturer, whichever is larger.
Method Blanks
Three method blanks, containing all accelerators or catalysts used in routine sample analysis
In their appropriate proportions, should be carried through the combustion procedure with each
batch of samples to measure potential contamination. Method blanks should be run at the
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beginning, middle, and end of each analytical run. The concentration of «ach blank should be less
than or equal to the instrument detection limit. Ail results should be blank corrected using the mean
of the acceptable method blank readings.
Quality Control Preparation Sample j
A matrix matched in-house quality control preparation sample (QCPS) should be analyzed once
per analytical run. This sample is used to monitor accuracy and long-term between-run precision.
Accuracy of the QCPS should be within ± 10% of the long-term mean. Between-run precision can
be determined by analyzing the QCPS and calculating the cumulative long-term standard deviation.
If values plotted on a control chart deviate from the long-term mean by more than three standard
deviations, the run should be completely reanalyzed, including all digestion and quantification steps.
Quality Control Check Standard
A quality control check standard (QCCS) should be analyzed at the beginning, after every ten
samples, and after the last sample of each analytical run. The QCCS should contain all the analytes
of interest with mid-calibration range concentrations. Quantified values of the QCCS should be
within ±10% of the known concentration of the standard. I
It is highly recommended that the concentrations of this sample be consistent through time
so that control charts may be plotted to monitor laboratory bias and other potential problems. If
analyzed values deviate from the long-term mean by more than three standard deviations, the
instrument is re-standardized and re-calibrated prior to any further sample analyses.
Suggested Run Format
QCCS, MB, Samples 1 to 8, QCPS, i
QCCS, Samples 9 to 16, DUP, ;
QCCS, Samples 16 to 25, SRM, '!
QCCS, Samples 26 to 30, QCCS.
" " ,i
where: QCCS = quality control check standard
MB = method blank
QCPS = quality control preparation sample i
OUP = duplicate sample '
SRM = standard reference material
Calculations and Reporting
Each laboratory should consult the manufacturer's instruction manual for the determination
of the appropriate formulae to be used in the calculation of total C.
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For some total C analyzers, % C can be read directly from the instrument. The sample weight,
as different from the standard, is taken into account on some instruments.
Results are reported to two significant figures. Results are read to the nearest 0.01%. Results
should be blank and moisture corrected.
In general, the total organic carbon (assuming the lack of or negligible quantities inorganic
carbonates in the soil) may be calculated as follows:
C (wt %) = (instrument reading - blank) x instrument factor .
(sample weight (g) x moisture correction)
NOTE: The moisture correction factor is as follows:
[(1 - moisture content in %) + (100 + moisture content in %)]
References
Allison, LE. 1960. Wet combustion apparatus and procedure for organic and inorganic carbon in soil.
Soil Sci. Soc. Am. Proc. 24:36-40.
Blume, LJ., BA Schumacher, P.W. Shaffer, K.A. Cappo, M.L Papp, R.D. van Remortel, D.S. Coffey,
M.G. Johnson, and D.J. Chaloud. 1990. Handbook of methods for acid deposition studies:
Laboratory analyses for soil chemistry. EPA/600/4-90/023. U.S. Environmental Protection
Agency, Environmental Monitoring Systems Laboratory, Las Vegas, NV.
Nelson, D.W., and LE. Sommers. 1982. Total carbon, organic carbon, and organic matter. In A.L
Page, R.H. Miller, and D.R. Keeney (eds.). Methods of soil analysis. Part 2. 2nd ed Agronomy
9:539-580.
Scholtenberger, C.J. 1927. A rapid approximate method for determining soil organic matter. Soil Sci.
24:65-68.
Soil Survey Staff. 1975. Soil taxonomy: A basic system of soil classification for making and
interpreting soil surveys. USDA-SCS Agric. Handb. 436. U.S. Gov. Print. Office, Washington,
DC.
Walkley, A. 1947. A critical examination of a rapid method for determining organic carbon in soils -
effect of variations in digestion conditions and of inorganic soil constituents. Soil Sci.
63:251-263.
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Walkley, A., and IA Black. 1934. An examination of the Degtjareff method for determining soil
organic matter, and a proposed modification of the chromic acid titration method. Soil Sci.
37:29-38.
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Total Nitrogen
Introduction
Nitrogen is one of the five major constituents of living matter. As such, its form and
concentration are of major interest when dealing with the growth of terrestrial and aquatic
organisms. In nature, nitrogen is found in a number of forms, all of which are interconvertible given
the correct chemical and microbiological conditions. Soil N accounts for only a small fraction of N
in the lithosphere, and of this fraction, only a very small proportion is directly available to plants
since greater than 90% of the N is in the organic form. Plants obtain most of their N from inorganic
ions, NOa and NH4+, that comprise less than 1% of the total N.
Atmospheric deposition of NOX can directly affect the total soil N concentrations. Further, soil
N content changes may be brought about by indirect effects to the forest ecosystem, such as a
result of acid deposition. Therefore, the measurement of total soil N is suggested for long-term
terrestrial monitoring programs, such as LRTAP. Nitrate and ammonium levels in soils are usually
negligible and the spatial and temporal variability associated with these parameters is much greater
than for total N.
Review of Methods
Total N in soil is usually measured by either a wet oxidation (Kjeldahl method) or dry oxidation
(Dumas method) procedure (Bremner and Mulvaney, 1982). Considerable modifications have been
proposed for these methods (e.g., Nelson and Sommers, 1980). More recently, near infrared
reflectance spectroscopy (NIRS) and LECO combustion have been used as alternatives to the
conventional chemical analysis for total N determination. However, these are not in widespread use
and involve expensive equipment. Thus, the chemical wet oxidation techniques remain the most
frequently used.
The principle of the Kjeldahl and Dumas techniques have been thoroughly discussed in several
reviews (Bremner, 1965; Nelson and Sommers, 1980; Bremner and Mulvaney, 1982). The Dumas
methods do not quantitatively recover many nitrogenous compounds (e.g., heterocyclic compounds)
and the automated N analyzer based on the Dumas method has not been widely used (Bremner and
Mulvaney, 1982). The Kjeldahl procedure is more commonly used and modifications of the original
Kjeldahl methods have extended the scope of the procedure. The total Kjeldahl N method involves
the digestion of the sample with H2S04 to convert organic N to NH/-N followed by distillation of the
digest with strong alkali to liberate NH3. Various modifications to the distillation and measurement
of the liberated NH, have been proposed. In addition, various methods for the direct measurement
of NH/ in the Kjeldahl digests have also been used. Highly refractory organic N compounds or
compounds containing N-N or N-O linkages are not completely recovered by the Kjeldahl digestion
(Bremner and Mulvaney, 1982). However, in most soils, little of the available N is in the refractory
form. Similarly, NO,-N and NO2-N are not adequately recovered but are not usually present in
significant amounts in undisturbed forest soils.
Reference Method
The reference method uses the Kjeldahl acid digestion in the presence of a catalyst which
converts organic nitrogen to inorganic ammonium. The measurement of N is done using alkaline
distillation to liberate NH3 which is measured by acid titration. The digestion is carried out either
on a hot plate or in an aluminum digestion block.
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Summary of Method
Soil samples are digested by the regular Kjeldahl technique using a K^O4-CuSO4 catalyst in
an aluminum block digestor with internal heaters and temperature conltrol. Determination of NH/-N
is done by an automated system (Tecator Kjeltec 1030) determining MH, liberated by distillation of
the digest with 40% NaOH. The NH3 is absorbed in unstandardized H3BO3 and ammonium borate
is formed. The borate is titrated back to H3BO3 using a standard strong acid (HCI).
Interferences
i
After digestion, samples must not be allowed to cool in the digestion block as NH3 will be lost
from the (NH4)2SO4 formed by the digestion. !
Safety
Protective clothing and safety glasses should be worn when handling strong acids. The
digestion blocks should be located in the fume hood and, if possible, the temperature controller
should be located outside the fume hood. The equipment should not be left unattended. The
preparation of the NaOH should also be done in the fume hood. The NaOH pellets should be added
very slowly to the water and in very small portions due to the intense exothermic reaction that
occurs.
Apparatus and Equipment
• digestion block, 20 place, Tecator System 20 1050 or equivalent, with programmable
temperature controller.
l
• distillation and titration apparatus, Kjeltec Auto 1030 Analyzer or equivalent.
* glass digestion tubes (295 mm x 40 mm), 250 mL to fit block, apiaropriate to sample and
solution volume used.
• balance, accurate to 0.001 g.
Reagents and Consumable Materials \
• sulphuric acid, HjSO^ concentrated, reagent grade (96%).
• water-DI water used in all preparations should conform to A8TWI specifications for Type
I reagent grade water (ASTM, 1984). ,
!
• catalyst, Kjeltab tablets or equivalent. Each tablet contains 35 g JC,SO4 and 0.4 g CuSO4.
• hydrochloric acid, HCI, standard acid 0.01 M. |
• boric acid, H3BO3, reagent grade powder. \
\
• ammonium chloride, NH4CI, reagent grade powder.
• methanol, reagent grade.
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• sodium hydroxide, NaOH, reagent grade pellets.
• sodium hydroxide solution, 40%~Dissolve 10 kg of NaOH in 15 L DI water.
• bromocresol green.
• bromocresol green solution-In a 100 ml_ volumetric flask dissolve 0.100 g bromocresol green
in methanol. Dilute to volume with methanol.
• methyl red.
• methyl red solution-In a 100 ml_ volumetric flask dissolve 0.100 g methyl -red in methanol.
Dilute to volume with methanol.
• receiving solution (Tecator, 1985)-Dissolve 100 g H3BO, in DI water and dilute to 10 L. Add
100 mL bromocresol green solution. Add 70 ml_ methyl red solution. Add 5 ml_ of 40%
NaOH solution.
• recovery check solution, 5,000 mg-N/L-In a one litre volumetric flask dissolve 19.0927 g
NH4CI in DI water. Dilute to volume.
Calibration and Standardization
Before analyzing the digested samples, distilled water blanks are run on the Kjeltec 1030
Analyzer until a constant reading of HCI is obtained. A 5 mL aliquot of a recovery check solution
containing 5,000 mg-N/L is analyzed to check the recovery. Recovery should be within ± 10%.
Procedure
Step 1 - Weigh 0.250 g of organic horizons, 0.500 g of Ah horizons (60 mesh), or 1 to 2.00 g of
mineral soil (60 mesh) into a digestion tube.
Step 2 - Add 10 mL concentrated H2SO4 to the tube and mix by swirling.
Note: This step should be carried out in a fume hood.
Step 3 - Heat tubes at 200° C in the digestion block until very black (approx. 30 minutes).
Step 4 - Add one catalyst tablet (Kjeltab).
Step 5 - Heat tubes at 200° C for 15-20 minutes until the Kjeltab dissolves.
Step 6 - Increase the block temperature to 300° C and heat for 30 minutes.
Step 7 - Raise the temperature to 425° C and heat tubes until the sample turns a turquoise
green. Digest samples for 20 minutes.
Step 8 - Remove the digestion tubes from the block and allow to cool for about 5 minutes.
Note: Do not allow to cool in the heating block as NH3 from the (NHJj.SO* formed by
digestion will be lost if heated.
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Step 9 - Add approximately 30 mL DI water and mix well until sample is in solution.
Step 10 - Dilute to approximately 100 mL with DI water.
Step 11 - Follow instructions for the operation of the Kjeltec Auto 1030 Analyzer (locator, 1985).
Step 12- Set the alkali pump to deliver 25-30 mL of 40% NaOH.
Step 13 - Titrate the sample with 0.01 M HCI. I
i
Quality Control
Precision
StloUrld ^ ana|y*!d in duplicate with each run of thirty samples. To eliminate bias
« £ run< the r?u ine sample duP|icat«» should be analyzed separately within the
EL*' Wlltin'run Photon is determined from duplicates based on relative percent
difference between the samples at an acceptance limit of a RPD x 10%. ""-»» percent
Accuracy
Accuracy is determined by analysis of a standard reference material (SRM) Acceptable limits
11!*10? fr°m the kn°Wn concentrati°n °f the standard or witSe TaS;uScy
* th8 refe/en€e matuerial manufacturer, whichever is larger. It is recommended
™. more accuracy standards be prepared with each batch of samples. These provide a
check on total between-run precision (digestion and distillation/titration). I»OVK» a
Method Blanks
1313 *-kS> ?arr'8d thr°U9h the extraction Procedure, are analyzed with each batch
M f to measure potential contamination. Method blanks should be run at
h mid?e> u "^ end of eacn ana|ytica' r"n- The concentration of each blank should be
less than or equal to the instrument detection limit. ««uiu o«
Quality Control Preparation Sample
il!!?tri? matc!2?d ir»-nouse quality control preparation sample (QCP8) should be analyzed once
* 'orS8 2an)ft is used to monitor accuracy and long-term between-run pTeciston
lQCPSt S-h°Uld. ^ Within * 10% of the Ion9-term mean. EJetween-run precision can
5Y ana|yan9 the QCPS and calculating the cumulative long-term standard deviation
hd °nKa ^"l01 ^ de,viate from the "ortfl-tarm mean by more than three standard
, the run should be completely reanalyzed, including all digestion and quantification steps
Quality Control Check Standard
q 2* ^"V? ?*?k standard (QCCS) sh°"ld be analyzed at the beginning, after every ten
*l"riS' 'rS !ampl8 °f each ana|ytical run- The QCCS should contain all the analytes
?*umidlcallbratlon range concentrations. Quantified values of the QCCS should be
±10% of the known concentration of the standard.
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It is highly recommended that the concentrations of this sample be consistent through time
so that control charts may be plotted to monitor laboratory bias and other potential problems. If
analyzed values deviate from the long-term mean by more than three standard deviations, the
instrument is re-standardized and re-calibrated prior to any further sample analyses.
Suggested Run Format
QCCS, MB, Samples 1 to 8, QCPS,
QCCS, Samples 9 to 16, MB, DUP,
QCCS, Samples 16 to 25, SRM,
QCCS, Samples 26 to 30, MB, QCCS.
where: QCCS
MB
QCPS
DUP
SRM
quality control check standard
method blank
quality control preparation sample
duplicate sample
standard reference material
Calculations and Reporting
formula:
Report total N as percentage on a dry-weight basis to the nearest 0.01% using the following
N % = (mL sample - mL blank) x N x 1.401
weight (g) of dry soil
where: N= normality of HCI titrant solution.
References
American Society for Testing and Materials. 1984. Annual Book of ASTM Standards, Vol. 11.01,
Standard Specification for Reagent Water, D1193-77 (reapproved 1983). ASTM, Philadelphia,
Pennsylvania.
Bremner, J.M. 1965. Organic nitrogen in soil. In Bartholomew W.V., and F.E. Clark (ed.) Soil nitrogen.
Agronomy 10. Am. Soc. Agron., Madison, WI. p. 93-149.
Bremner, J.M., and C.S. Mulvaney. 1982. Nitrogen-total. In Page, LA et al. (ed.) Methods of soil
analysis. Agronomy 9. Am. Soc. Agron., Madison WI. p. 595-624.
Nelson, D.W., and LE. Sommers. 1980. Total nitrogen analysis for soil and plant tissues. J. Assoc.
Off. Anal. Chem. 63:770-780.
Tecator. 1985. Kjeltec Auto 1030 Analyzer Manual. Tecator, AB. Hoganas, Sweden. 43p.
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Extract able Phosphorus
Introduction
Phosphorus (P) is an essential nutrient for plant growth and is required in relatively large
quantities by plants. It is essential for energy transfer reactions within all cells and is a constituent
of many proteins, nucleic acids, and coenzymes. Information on the phosphorus status of soils has
long been regarded as being very important to the proper management of soils for agricultural
purposes. More recently, it has become recognized as important in forest management as well.
Trie availability of P to plants is influenced by soil pH. Phosphorus is most available to plants at
a soil pH of 6 to 7. At pH lower than 6, P becomes fixed in iron and aluminum compounds, and, at
pH above 7, calcium phosphates precipitate to an extent which limits P availability.
i *
From the above discussion, it can be seen that soil acidification resulting from atmospheric
deposition can influence the availability of P to plant species. Since P is an important macronutrient
for plants upon which growth rates and general health depend, it should be monitored as a
parameter for terrestrial monitoring programs. ,
Review of Methods
Extractants i
Phosphorus is of concern largely in relation to its role in plant growth and, therefore, methods
for extracting soil P should best reflect the plant's ability to extract P from the soil. Standardizing
methods is difficult because the effectiveness of an extractant depends on soil type.
Many extractants have been used to extract plant available P. The most common, and perhaps
the ones which best correlate with plant P uptake, are NaHCOJNaOH (Olsen) and NH4F/HCI (Bray
methods). The Olsen method is widely used on soils with high base status. The extractant
decreases the amount of Ca in solution by causing precipitation of CaCO, resulting in the
dissolution of calcium phosphates. It also may remove some of the less strongly bound P from Al
compounds. Since the method does not remove strongly bound P from Al and Fe oxides, the P
levels obtained from acidic soils can be very low, often below detectable limits. The Bray method
is more appropriate on acidic soils. The combination of HCI and NH4F removes readily acid soluble
P forms, including Ca-phosphates. The NH4F dissolves Fe- and Al-phosphates which are prevalent
in acid soils. In calcareous soils, CaCO3 rapidly neutralizes the acid (Randall and Grava, 1971), and
low estimates of available P result. Also, insoluble compounds may form as a result of reactions
of CaFa with P (Smillie and Syers, 1972).
The objective of most extractions is to approximate the relative amount of P that may be
available for plant growth, therefore, it is logical to use extractants which are similar to those within
the rhizosphere, such as root exudates. Organic ions, such as oxalate, malate, citrate, and acetate,
would be appropriate for such use. These extractants have not been extensively used for soil
testing, but have been used in research and were found to be effective extractants (Lopez-
Hernandez et al., 1986; Ball and Williams, 1968). Holford and Callis (1985b) found a combination of
ammonium lactate-acetic acid to be a superior method for gauging plaint response to soil P levels
than other methods, including Bray 1, Bray 2, and NaHCO3, on moderately acid and alkaline soils in
Australia. On acidic soils neutral fluoride extractants and 0.01 M sulphuric acid were better
indicators of response than ammonium lactate-acetic acid.
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Extraction Methods
When choosing an extraction method, it is necessary to consider the purpose of the
measurements and the type of soils being monitored. Phosphorus availability to plants, particularly
forest species, is of primary concern when studying the terrestrial effects of acidic atmospheric
deposition. Detrimental effects would be seen first on soils which are both sensitive to acid
deposition (low acid buffering, capacity) and which are already low in P. Moderately acidic and
acidic brunisols and podzols meet these criteria and, therefore, the extractant chosen should be a
good indicator of plant-available P for these soils. There must also be evidence available linking
extractable P levels to growth of tree species. For these reasons, the Bray methods have been
chosen as means of determining extractable P for the terrestrial effects related to LRTAP studies.
Thomas and Peaslee (1973) describe NH4F-HCI (the Bray extractant) methods as being appropriate
for soils with low to medium CEC that have been moderately to highly weathered, and Olsen and
Sommers (1982) state that the Bray method is most successful on acid soils.
Numerous modifications to the originally described Bray method have been made to suit local
conditions. These have resulted in considerable intralaboratory variation in shaking times,
soihextractant ratios, and extractant concentrations. In general, the amount of P extracted
increases with increased shaking time and speed as well as with decreased soihsolution ratios.
The Bray 2 method increases the concentration of HCI from 0.025 N to 0.10 N in order to
dissolve more Ca-phosphates. In acid and moderately acid soils, this does not appreciably improve
the correlation to plant uptake, and, for these soils the Bray 1 method is appropriate and is widely
used. Due to the common use of the Bray 1 method, and its appropriateness for acid and
moderated acid soils it is the method suggested for use in terrestrial effects studies.
Olsen and Sommers (1982) suggest a 1:7 soihsolution ratio. This ratio would appear to be
appropriate for acid and moderately acid soils, although lower ratios (i.e., 1:50) are more suited to
less acidic soils. McKeague (1978) recommends a soihsolution ratio of 1:10 (2.5 g soil to 25 mL
solutfon). We also recommend the 1:10 ratio as it will result in the extraction of slightly greater
amounts of P, is more convenient, and will still correlate well with plant uptake of P.
Shaking times vary from 1 minute (Olsen and Sommers, 1982) to 30 minutes (Halford and
Cullis, 1985a). Although more P is extracted with increased shaking times, Olsen and Sommers
(1982) and McKeague (1978) use a standard one minute shaking time.
Analysis of Extract
Phosphorus in the extracting solution has most often been measured by colorimetric methods.
Molybdate blue methods are the most sensitive and common. A phosphomolybdate complex is
reduced to give a blue colour. The reducing agent chosen depends upon the solution P
concentration, the concentration of interfering substances, such as arsenates and silicates, and the
extracting solution. Olsen and Sommers (1982) and Sheldrick (1984) recommend the use of SnC^
as a reducing agent for analysis of Bray extracts. However, the coloured solution produced with
SnCI, is only stable for about 2 hours and must be mixed immediately before each analysis.
Furthermore, at a fixed concentration of the reducing agent, the absorbance degrades with
increasing P concentration. Additionally, the solution can produce precipitates and coat the flow cell.
The latter problem can be minimized by periodically cleaning the apparatus with acid (Smith and
Scott, 1983).
Ascorbic acid as a reducing agent has for the most part replaced SnCI2 in measurements and
was recommended by the Canada Soil Survey Committee (McKeague, 1978). The reagent is stable
for 24 hours and problems with precipitates are not encountered, however, it is not as sensitive as
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SnCI2 because colour development is weaker. For soil monitoring programs, the sensitivity obtained
with ascorbic acid is more than adequate. The ascorbic acid reduction method using ammonium
molybdate to* develop colour is, therefore, preferred for use in LRTAP monitoring programs.
Reference Method
The reference method for the determination of extractable P from soil samples for terrestrial
effects of acid deposition is the use of 0.03 N NH4F + 0.025 N HCI as an extract solution (Bray 1)
followed by a cotorimetric analysis using ammonium molybdate with ascorbic acid as a reducing
agent. Automated segmented flow cplourimetric systems are commonly used for the measurement
of P and curve calibration programs used for determining the standard calibration curve. The
procedure described here, however, describes a manual method, keeping equipment costs to a
minimum.
Summary
A 2.5 g soil sample is shaken with 25 mL of extracting solution for 1 min. The solution is then
filtered. Approximately 5 mL aliquot is transferred to a 25 mL volumetric; flask and the pH is reduced
to 5 using HLSO4. Ammonium molybdate reagent is added and the content of P is determined
colorimetricaily. The percent transmittance of the solution is read using a colorimeter. The Bray 1
P is reported in mg/kg.
Interferences and Shortcomings
•i
Some labs add carbon black to assure a clear filtrate with less interference for colorimetric
determination of P. Olsen and Sommers (1982) do not suggest the use of carbon black and suggest
extra filtration steps be used.
For calcareous soils, a 1:50 soihextracting solution should be used. Incomplete dissolution
of Ca-phosphates may still be observed such that a different method is required.
Boric acid is sometimes used to eliminate possible interference firom fluorides. However, the
routine use of boric acid in most soils has not been established.
Safety
All operations should be carried out in well-ventilated conditions. Protective clothing including
eye protection should be worn at all times, and especially when handling concentrated acids.
Special care must be taken when adding water to concentrated H2SO4. Use sodium bicarbonate and
water to neutralize and dilute spilled acids.
Apparatus and Equipment
• flasks, Erlenmeyer, 50 mL. ;
• dispenser, capable of accurately dispensing 25 mL.
• shaker, reciprocating, Eberbach or equivalent.
• filter paper, Whatman #42. . :
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• colorimeter.
• colorimeter tubes, glass, 1 cm light path.
• balance, accurate to 0.001 g.
Reagents and Consumable Materials
• sulphuric acid (H2SOJ, concentrated, reagent grade.
• hydrochloric acid (HCI), concentrated, reagent grade.
• water—DI water used in all preparations should conform to ASTM specifications for Type
I reagent grade water (ASTM, 1984).
• hydrochloric acid (HCI), 0.5 N: Dilute 20.2 ml_ of concentrated HCI to a volume of 500 mL with
DI water.
• ammonium fluoride (NH4F), 1 N-In a one litre volumetric flask, dissolve 37 g of HN4F in
DI water. Dilute to volume with DI water. Store in a polyethylene bottle.
• extracting solution-Add 15 ml of 1.0 N NH4F and 25 ml of 0.5 N HCI to 460 mL of DI water
to give a solution of 0.03 N HN4F and 0.025 N HCI.
• ammonium molybdate-Dissolve 20.0 g of (NHJ6MO7024«4H20 in 225 mL of concentrated
H.,SO4. Dilute to 2 litres with DI water.
• ascorbic acid—Dissolve 1.76 g of ascorbic acid in 200 mL of DI water.
NOTE: This solution must be made fresh each day before use.
• P stock standard-In a 1 litre volumetric flask, dissolve 0.876 g of KH2PO4 (which has been
dried for 24 hours at 80° C) in the extracting solution. Dilute to volume with extracting
solution. This gives a 200 mg/L (w/v) P standard.
Calibration and Standardization
Calibration standards should be prepared in the extraction solution. Prepare a standard curve
by plotting the transmittances of at least 5 standards (up to 10 standards are commonly used)
against P concentration on semilogarthmic graph paper. Construct the calibration curve by finding
the "best" fit line of the plotted standard concentrations. P is reported in mg/L.
Procedure
Step 1 - Place 2.5 g of soil (air dried and sieved through 2 mm sieve) into a 50 mL Erlenmeyer
flask.
Step 2 - Add 25 mL of extracting solution. Shake for 1 minute
Step?- Filter through Whatman #42 paper.
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Step 4 - Pipet an aliquot of the extract containing 1 to 20 /^g of P (usually 5 mL) into a 25 mL
Steps- Add 0.5 mL of 5 N H2SO4 to reduce pH to 5.
Step 6 - Add DI water to bring the volume to about 20 mL, then add 4 mL of ammonium
molybdate reagent. Make to volume and mix
Step 7 - Read absorbance at 880 /im after 10 min. The colour is s tabla for 24 hours.
Quality Control
Precision i
One sample should be analyzed in duplicate with each run of thirty samples. To eliminate bias
due to position in the run, the routine sample duplicate should be analwied separately within !he
analytical run. Within-run precision is determined from duplicates based on relative percent
difference between the samples at an acceptance limit of a RPD *• 10%.
Accuracy
Accuracy is determined by analysis of a standard reference material (SRM). Acceptable limits
for accuracy should be ±10% from the known concentration of the standaird or within the accuracy
windows supplied by the reference material manufacturer, whichever iis larger.
Method Blanks
are
analyzed with each batch
' blanks should be run at
concentration of each blank should be
lae blank corrected using the
Three method blanks, carried through the extraction procedure,
of samples for each cation to measure potential contamination. Method
the beginning, middle, and end of each analytical run. The concentra
less than or equal to the instrument detection limit. All results should
mean of the acceptable method blank readings.
Quality Control Preparation Sample
A matrix matched in-house quality control preparation sample (QCPS) should be analyzed once
per analytical run. This sample is used to monitor accuracy and long-term between-run precision
Accuracy of the QCPS should be within ± 10% of the long-term mean. Between-run precision can
be determined by analyzing the QCPS and calculating the cumulative long-term standard deviation.
If values plotted on a control chart deviate from the long-term mean by more than three standard
deviations, the run should be completely reanalyzed, including all digestion and quantification steps.
Quality Control Check Standard
A quality control check standard (QCCS) should be analyzed at the beginning, after every ten
samples, and after the last sample of each analytical run. The QCCS should contain all the analytes
of interest with mid-calibration range concentrations. Quantified values of the QCCS should be
within ±10% of the known concentration of the standard.
i
It is highly recommended that the concentrations of this sample be (consistent through time
so that control charts may be plotted to monitor laboratory bias and other potential problems. If
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analyzed values deviate from the long-term mean by more than three standard deviations, the
instrument is re-standardized and re-calibrated prior to any further sample analyses.
Suggested Run Format
QCCS, MB, Samples 1 to 8, QCPS,
QCCS, Samples 9 to 16, MB, DUP,
QCCS, Samples 16 to 25, SRM,
QCCS, Samples 26 to 30, MB, QCCS.
where: QCCS = quality control check standard
MB = method blank
QCPS = quality control preparation sample
DUP = duplicate sample
SRM = standard reference material
Calculations and Reporting
To calculate the concentration of extractable P as follows:
mg Pykg soil = mg P/mL solution x 10
This calculation assumes an aliquot of 5 mL of extracting solution was used.
References
American Society for Testing and Materials. 1984. Annual Book of ASTM Standards, Vol. 11.01,
Standard Specification for Reagent Water, D1193-77 (reapproved 1983). ASTM, Philadelphia,
Pennsylvania.
Holford, I.C.R., and B.R. Cullis. 1985a. Effects of phosphate buffer capacity on yield response
curvature and fertilizer requirements of wheat in relation to soil phosphate tests. Aust. I. Soil
Res. 23:647-653
Holford, I.C.R., and B.R. Cullis. 1985b. An evaluation of eight soil phosphate extractants on acid
wheat growing soil.
McKeague, J.A. (ed). 1978. Manual on soil sampling and methods of analysis. Can. Soc. Soil Sci.
Ottawa, Ont.
Olsen, S.R., and LE. Sommers. 1982. Phosphorus. In Page, A.L. (ed.) Methods of soil analysis. Part
2. Chemical and microbiological properties. 2nd ed. Agronomy 9. Am. Soc. Agron., Madison, WI,
U.S.A.
Randall, G.W., and J. Grava. 1971. Effect of soil: Bray no 1. ratios on the amount of phosphorus
extracted from calcareous Minnesota soils. Soil Sci. Soc. Am. Proc. 35:112-114.
Sheldrick, B.H. (ed). 1984. Analytical methods manual. Land Resource Research Institute. Agriculture
Canada, Ottawa. LRRI Contribution No. 84-30.
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Smillie, G.W., and J.K Syers. 1972, Calcium fluoride formation during eixtraction of calcareous soils
with fluoride: II. Implications to the Bray P-1 test. Soil Sci. Soc. Am. Proc. 36:25-30.
Smith, K.A., and A. Scott. 1983. Continuous-flow and discrete analysis;. In Smith, K.A. (ed) Soil
analysis. Marcel Dekker. New York.
Thomas, G.W., and D.E. Peaslee. 1973. Testing of soils for phosphorus. In Walsh, L.M., and J D
Beaton (ed.) Soil testing and plant analysis. Soil Sci. Soc. Inc. Madison, WI.
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Cation Exchange Capacity
Introduction
Cation exchange capacity (CEC), usually expressed in milliequivalents (meq) per 100 g of soil,
is a measure of the quantity of readily exchangeable cations neutralizing negative charge in the soil
(Rhoades, 1982). Negative charge in the soil can be derived from several sources which fall into two
categories: permanent and pH-dependent (or variable) charge. Permanent charge sites are the
result of isomorphic substitution within the crystal structure of layer silicate minerals which are
commonly referred to as clay minerals. Permanent charge CEC is independent of pH, electrolyte
concentrations, ion valences and the dielectric constant of the medium. The pH dependent CEC is
derived from broken bonds at mineral edges, dissociation of acidic functional groups in organic
matter and sesquioxides present in the soil, and preferential adsorption of certain ions on the
charged particle surfaces. In general, as the pH, electrolyte concentration, dielectric constant of the
medium, and the ion valences increase, the net contribution of pH-dependent charge to the overall
CEC increases.
A close approximation of the CEC can be made by the summation of the exchangeable base
cations of Ca2+, Mg2*, Na+, and K+ and the exchangeable acidity as determined by BaCI2-TEA
extraction or the addition of exchangeable Al extracted by KCI (USDA/SCS, 1972).
Review of Methods
Most CEC methods begin with the displacement of existing cations with a saturating salt to
provide one index cation on the exchange complexes. Many different replacing cations have been
used to study the exchange characteristics of soils. Some of these include NH4 (Grove et al., 1982;
Mrozet al., 1985; Richter, 1986; Johnson et al., 1991; Soil Survey Laboratory Staff, 1992), K* (Gillman
and Uehara, 1980), Ba2+ (Kalisz and Stone, 1980; Rhoades, 1982; Hendershot and Duquette, 1986;
Ross and Bartlett, 1992), and Sr2* (Matsue and Wada, 1985). After removing or accounting for
excess saturating salt, the index cation is replaced with a different saturating salt and subsequently
measured to provide an estimate of the CEC of soil.
Although the choice of the replacing cation is considered arbitrary (Thomas, 1982), the
selection of accompanying anions can be important. If the final replacing solution contains SO4 ,
specific adsorption of this anion can affect the measured CEC in SO^2" adsorbing soils (Matsue and
Wada, 1985; Hendershot and Duquette, 1986). Although some concern has been expressed
regarding the selection of CI" as the replacement anion (due to ion pairing with divalent cations),
Rhue and Reve (1990) did not see any differences in measured CEC when compared to CIO/. Large
differences have been found between soil CEC measured in buffered (pH 7) acetate salts when
compared to neutral salts (Kalisz and Stone, 1980; Grove et al., 1982). In acid forest soils, the
recommendation has been made to measure CEC with unbuffered salts (Rhoades, 1982; Hendershot
et al., 1993). The concentration of the unbuffered salts has also been shown to influence the
measured CEC (Wada and Okamura, 1980).
Two saturating. solutions containing NH4+ are commonly used for CEC determination.
Ammonium acetate (1.0 N NH4OAc) buffered at pH 7.0 yields a CEC which is close to the total cation
exchange capacity for a specific soil. This saturating solution is commonly used for soil
comparisons because it has the advantage of extracting all samples at the same pH (USDA-SCS,
1972). In acid soils, this estimate results in a high CEC value relative to the CEC found under field
conditions because of adsorption of NH4+ ions to the pH-dependent exchange sites that exist above
the soil's natural pH level (Grove et al., 1982). The overestimation will not occur when an unbuffered
neutral salt solution such as ammonium chloride (1.0 N NH4CI) is used. During the extraction, the
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solution assumes the pH of the soil. The NH4CI CEC has been termed "effective" CEC because it
provides more unambiguous information about the behaviour of the soil under field conditions.
Reference Method
The reference approach to long-term monitoring methods for cation exchange capacity is to
measure the effective CEC with 1 N NH4CI. Buffered salt solutions will not be used, as they are not
considered representative of the field forest soil condition. A 1 N NH4CI salt solution was chosen
over other commonly used solutions (NaCI, KCI, and BaCy due to its predominant use for extracting
exchangeable cations in studies of forested ecosystems in North America.
Summary of Method
The soil sample is saturated with NH4+ from a solution of NH4CI. Excess NH4*
ethanol rinses. The NH4+ is displaced by Na+ and is measured by one of three methods: automated
is removed by
distillation-titration, manual distillation-automated titration, or ammonium disiplacement-f low injection
analysis. These methods are based on Doxsee (1985), Rhoades (1982), and USDA/SCS (1984).
The NH4CI saturating solution should be retained for the exchangeable cation determinations
(Chapter 13). \
NOTE: This method has been written assuming the use of a mechaniical extractor.
Interferences and Shortcomings
Inconsistency in the NH4* saturating and rinsing steps is the greatest source of error. Soils
containing an abundance of minerals, such as biotite, muscovite, illite, and vermiculite, may retain
interlattice NH4+ and produce artificially high results. The use of a mechanical extractor minimizes
inconsistency. :
Safety
Wear protective clothing (laboratory coat and gloves) and safety glasses when preparing
reagents, especially when concentrated acids and bases are used. The use of concentrated acids
and hydroxide solutions should be restricted to a fume hood.
Apparatus and Equipment
Apparatus for Saturation Procedure
• mechanical extractor, 24-place (Figure 1).
• reciprocating shaker.
• balance, capable of weighing to 0.01 g.
• balance calibration weights, 3-5 weights covering expected range.
• wash bottle.
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Figure 1. Mechanical Extractor.
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Apparatus for Analysis
The apparatus and equipment needed are specific to the selected analytical method. It is not
necessary to have equipment for all three analytical methods.
Ammonium Displacement-Flow Injection
• flow injection analyzer (FIA), Lachat or equivalent, modified for ammonium chemistry with
630 11 m interference filter and consisting of:
a. sampler.
I
b. analytical manifold with 200 jiL sample loop.
c. in-line heater.
d. colorimeter equipped with a 10 mm flow cell.
e. printer.
• balance, capable of weighing to 0.001 g. \
• balance calibration weights, 3-5 weights covering expected range.
Automated Dlstlllatlon-Titratlon \
• steam distillation-titration apparatus, Kjeltec Auto 1030 Analyser, or equivalent.
• printer, Alphacom 40, or equivalent. |
• digestion tubes, 250 ml, straight neck.
• balance, capable of weighing to 0.1 g.
• balance calibration weights, 3-5 weights covering expected range.
• policeman, rubber. <
Manual Distillation/Automated Utratlon
• automatic titrator with autosampler, Metrohm or equivalent.
« Kjelclahl flasks. 800 mL
• balance, capable of weighing to 0.1 g.
• balance calibration weights, 3-5 weights covering expected range.
• policeman, rubber.
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Reagents and Consumable Materials for Saturation Procedure
• washed analytical filter pulp, Schleicher and Schuell, No. 289 (see procedure section for
washing procedure).
• glacial acetic acid (HC^OJ, concentrated, reagent grade.
• ammonium hydroxide (NH4OH), concentrated, reagent grade.
• ammonium chloride (NH4CI), reagent grade, 1 N - Dissolve 535 g in DI water and dilute to
10 L
• ethanol (CjHjOH), 95 percent, U.S.P.
• Nessler's reagent-
Add 4.56 g potassium iodide (KI) to 30 mL DI water in a beaker. Add 5.68 g mercuric iodide
(Hgl;,). Stir until dissolved.
Dissolve 10 g NaOH in 200 mL DI water.
Transfer NaOH solution to 250 mL volumetric flask. Add Hg solution slowly. Dilute to
volume and mix thoroughly. Solution should not contain a precipitate. It can be used
immediately.
• water—Water used in all preparations should conform to ASTM specifications for Type I
reagent grade water (ASTM, 1984).
• syringes, disposable, 60 mL polypropylene (use one sample tube, one reservoir tube, and
one tared extraction syringe for each sample).
• rubber tubing, 1/8 x 1/4 inch (for connecting syringe barrels).
• bottles, linear polyethylene (LPE), 25 mL
• tubes, glass, centrifuge or culture, with caps, 25 mL
• weighing pans, disposable.
Reagents and Consumable Materials for Analysis
The reagents and consumable materials needed are specific to the selected analytical
procedure. It is not necessary to have the reagents and materials for all three analytical
procedures.
Ammonium Displacement-Flow Injection Analysis
The reagents and consumable materials used depend on recommendations of the
manufacturer of the FIA and may vary by make and model.
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• hydrochloric acid, 0.1 N-Purehased or prepared by the following procedure: Add 150 mL
concentrated HCI to approximately 15 L DI water, dilute to 18 L Standardize 0.1 N HCI by
titration against dried primary standard grade sodium carbonate to the methyl orange end-
point.
• nitroferricyanide reagent-Dissolve 40 g potassium sodium tart rate (KNaC4H4O6) and 30 g
sodium citrate (Na3C8H5O7«2H2O) in 500 ml DI water. Add 10 g sodium hydroxide pellets
(NaOH). Add 1.5 g sodium nitroferricyanide (Na2Fe(CN)5NO»2l-l2O)a dilute to 1.00 L, and mix
well. Store in a dark bottle. Prepare fresh solution monthly.
• sodium hypochlorite reagent-Dissolve 20 g sodium hydroxide and 20 g boric acid in 150 mL
of DI water. Add 800 mL 5 percent solution NaOCI. Dilute to 1.00 L with DI water. Store
in a dark bottle. Prepare fresh solution monthly.
• sodium phenate reagent-Dissolve 95 ml of 88 percent liquified phenol in 600 ml DI water.
While stirring, slowly add 120 g NaOH. Cool. Add 100 ml ethanol and dilute to 1.00 L
Store in a dark bottle.
• nitrogen standard solution, 1,000 mg NH4-N/L~Dissolve 3.819 g ammonium chloride (NH4CI),
dried at 105* C, in DI water and dilute to 1.00 L
• working standards-Pipet 15.0,10.0,6.0, and 2.0 mL of the nitrogen standard solution, above,
into 100 mL volumetric flasks. Bring to volume with 0.1 N HCI. This will yield 150, 100, 60,
and 20 mg NH4-N/L working standards. Pipet 5 mL of the 100 mg NH4-N/L working standard
into a 100-mL volumetric flask and dilute to volume with 0.1 N HCI. This provides a 5 mg
NH4-N/L working standard. Prepare fresh working standards weekly.
• water-Water used in all preparations should conform to ASTM specifications for Type I
reagent grade water (ASTM, 1984).
• weighing pans, disposable.
Automated Dlstlllatlon-Tltnition
• sodium chloride (NaCI).
• antifoam, silicons spray bottle.
• hydrochloric acid (HCI), 0.10 N, standardized.
• boric acid (H3BOg), 4 percent (w/v) aqueous solution-Add 720 13 boric acid to about 4 L DI
water in a large stainless steel beaker. Heat to near boiling and stir until crystals dissolve.
Add to a 5 gallon Pyrex bottle about 12 L DI water. Transfer hot solution through a large
polyethylene funnel into the bottle. Dilute to 18 L with DI wateir and mix well.
• water—Water used in all preparations should conform to ASTM specifications for Type I
reagent grade water (ASTM, 1984).
• compressed air.
• weighing pans, disposable. '
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Manual Dlatillatlon-Aiitomatlc Tltratlon
• sodium chloride (NaCl).
• antifoam mixture-Mix equal parts of mineral oil and octanol.
• boric acid (H-jBO-j), 4 percent.
• hydrochloric acid (HCI), 0.1 N.
• sodium hydroxide (NaOH), 1 N-Add 500 mL 50 percent NaOH so|ution to 8 L of DI water in
a 9.5 L Pyrex solution bottle. Dilute to 9 L with DI water and mix well.
• zinc, granular.
• water-Water used in all preparations should conform to ASTM specifications for Type I
reagent grade water (ASTM, 1984).
• compressed air.
• beakers, plastic disposable, 250 mL
• weighing pans, disposable.
Calibration and Standardization
Calibration standards should be prepared in the extraction solution.
Flow Injection Analysis Calibration
For the FIA, use standards containing 0, 5, 20, 60, 100, and 150 mg NH4-N/L to develop a
calibration curve. A regression of the standard curve should have an intercept close to zero. Air
bubbles can produce sharp sudden peaks which destroy the calibration curve. In the event of air
bubbles, the calibration curve and all samples analyzed since the last quality control check sample
(QCCS) must be reanalyzed. Standard values should not vary by more than 5 percent relative
standard deviation (%RSD).
Tltratlon Calibration
Titrants used in the automated titrations are calibrated prior to analysis to establish the
normality. The normality is checked weekly. Should the check value differ from the normality by
more than 5 percent, two additional checks are run and the mean of the three check values is used
as the normality. The same standard titrant should be used for all samples within a batch.
Procedure
Before proceeding with the analytical procedure, the analyst should be certain that all QC
procedures have been implemented, all labware has been cleaned properly, and valid instrumental
detection limits (IDLs) have been obtained.
NOTE: This method has been written assuming the use of a mechanical extractor.
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Pulp Washing
i
NOTE: Commercial filter pulps often are contaminated and will have to be washed.
Step 1 - Prepare sample tubes by tightly compressing a 0.5 g ball of filter pulp into bottom of
syringe barrel with a modified plunger. Modify the plunger by removing the rubber
portion of the plunger, and cut off the plastic protrusion.
Step 2 - Add 50 mL 0.1 N HCI to the syringe containing the pulp and extract rapidly.
Step 3 - Add 50 mL DI water to the syringe containing the pulp and extract rapidly.
Step 4 - Repeat Step 3 again.
Step 5 - Remove any excess solution from the washed pulp by applying gentle suction to the
syringe tip. Proceed immediately with an extraction.
Extraction \
*
Step 1 - For mineral soils, weigh 5.00 g air-dry soil, place in sample itube, and record exact
weight. Place sample tube in upper disc of extractor and connect to inverted, tared
extraction syringe, the plunger of which is inserted in the slot of the stationary disc of
the extractor. Fill the syringe to the 25 mL mark with NH4CI. Stir sample and NH4CI with
glass stirring rod for 15 seconds, rinse rod with NH4CI, and fill syringe to the 30 mL
mark. Let stand for 20 minutes.
For organic soils, weigh 1.25 g of air-dried soil into a small glass tube and record exact
weight. Add 2 mL ethanol as a wetting agent. (If the organic soil wets easily, it is not
necessary to add the ethanol.) When the soil is moistened, add 15 mL NH4CI, cap, and
shake for one hour on a reciprocating shaker. Place sample tube in upper disc of
extractor and connect to inverted, tared extraction syringe, the plunger of which is
inserted in the slot of the stationary disc of the extractor. Then quantitatively transfer
the sample and NH4CI to the sample tube and fill to the 25 mL mark with NH4CI. Let
stand for 20 minutes.
NOTE: Up to 35 mL may be used for transfer; see Step 2.
Step 2 - Put reservoir tube on top of sample tube; extract rapidly until NH4CI is at a depth of 0.5
to 1.0 cm above sample. Turn off extractor. Add about 45 rnL NH4CI to reservoir tube,
turn on extractor, and extract overnight or for approximately 16 hours. Do not allow the
soil to dry between the time the extractor is turned off and back on.
NOTE: If 35 mL are used in Step 1, reduce 45 mL to 35 mLL Total NH4CI used during
extraction should be approximately 70 mL j
Step 3 - The next morning, switch off extractor and pull plungers down as far as extractor will
allow. Disconnect syringes from sample tubes, leaving rublber connectors on sample
tubes. Weigh each syringe containing the NH4CI extract to the nearest 0.01 g. The final
weight and tare weight are used to calculate the volume of ammonium acetate extract
(Section 12.0), according to the formula in the calculation and reporting section.
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Step 4 -
Step 5 -
Step 6 -
Mix the extract in each syringe by shaking ma™^y- *'^^^
polyethylene bottle twice with small volumes ofthe extract solutionjheniilljhe^bottle
with extract solution and discard the excess. This solution is reserved for analysis of
exchangeable cations as described in Chapter 13.
Return uooer 2-disc unit to starting position. Attach the syringes to the sample tubes,
and rTnsePS?e fides of sample tubes with ethanol from a wash bottle. Fill sample tubes
to the 20 mL mark stir, and let stand for 15 to 20 minutes. Place reservoir tube in
lamole tube" Extract rapidly until the level of ethanol is 0.5 to 1.0 cm above sample.
Tumoff ex?acto?and add enough ethanol to the reservoir to ensure an excess over the
capacity of the syringe. Extract for 45 minutes.
After the extractor has stopped, turn off the switch, pull the plungers_ down^ remove
svrinaes and discard the ethanol wash. Return the upper unit of the extractor to
sfficfoptionreattach syringes to the sample tube, fill reservoir tubes with about 45
^SS^S^SMBimd time for approximately 45 minutes. When extractor
&rt5Stf£»a« syringes and discard ethanol. After the s ^SSnff!^
collect a few drops of ethanol extract on a spot plate. Test for residual NH, in each
SmSe by adding four drops of Nessler's reagent to one drop of solution It the> test
isT positive fre orange eiidpoint) repeat another ethanol extraction of the affected
sar^te^ndle'st bousing Nessli's reagent until a negative test is obtained.
Analytical Procedure Using FIA
Step 1 •
Add 50 mL of 0.1 N HCI and extract at a setting of 10 (approximately one hour) and
record volume.
Step 2- Disconnect syringes and save the HCI extract for FIA analysis.
Step 3 - Operate the FIA according to manufacturer's instructions.
Step 4 - Read mg NH4-N/L; if concentrations exceed calibration standards, dilute in the instrument
calibration range and reanalyze.
Analytical Procedure using Automated Distillation-Titration
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Analytical Procedure using Manual Distillation-Automated Titration
Step 1 - Transfer sample to an 800 mL Kjeldahl flask.
Step 2 - Add
40
2 9 flranular ** and
Step 3 - Set upKjeldahl distillation apparatus and distill until 175-180 mL of distillate is collected
in a 250 mL plastic beaker containing 50 mL of 4 percent boric acid solution.
St6P *" ESStoX** beakef t0 distillation apparatus and operate according to manufacturer's
Step 5 - Read mL titration and record with the normality of titrant.
Quality Control
Precision
due to
due to
nmnh "aly^d in delicate w,!th each run of thirty samples. To eliminate bias
in the run, the routine sample duplicate should be analyzed separately within the
E5- Wi!tin-run P,fecision is determined from duplicates baled I on ^3T Seem
difference between the samples at an acceptance limit of a RPD «s 10%. '«««• percent
Accuracy
Accuracy is determined by analysis of a standard reference material (SRM). Acceptable limits
^nH^°UraCy "Wl*,.*10^ fr°m the known concentration of the standard or witWrfthe accuracy
windows supplied by the reference material manufacturer, whichever is larger. accuracy
Method Blanks
method. blan.ks- carr'ed through the extraction procedure, are analyzed with each batch
J? rm-HCJ? Cat^,n t°,measur,f P°ten«a' contamination. Method blanks should be run art
9> d e< and end of each ana|yt'cal run. The concentration of each blank should be
than or equal to the instrument detection limit.
Quality Control Preparation Sample
A matrix matched iivhouse quality control preparation sample (QCPS) should be analyzed once
"8 f a"l5't ls used to monitor accuracy and long-term between-run precision.
°US ^lhin i 10% of the tons-t6^ "i^n. Eletween-run precision can
the,?CPS and calculating the cumulative long-term standard deviation.
Ho- 0rVa ^"u01 chart deviate from tne ton9-term mean by more than three standard
deviations, the run should be completely reanalyzed, including all digestion and quantification steps.
Quality Control Check Standard
A quality control check standard (QCCS) should be analyzed at the beginning, after every ten
samples, and after the last sample of each analytical run. This standard should have
concentrations at about mid-calibration range. Quantified values of the QCCS should be within ±10%
of the known concentration of the standard.
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It is highly recommended that the concentrations of this sample be consistent through time
so that control charts may be plotted to monitor laboratory bias and other potential problems. If
analyzed values deviate from the long-term mean by more than three standard deviations, the
instrument is re-standardized and re-calibrated prior to any further sample analyses.
Suggested Run Format
QCCS, MB, Samples 1 to 8, QCPS,
QCCS, Samples 9 to 16, MB, DUP,
QCCS, Samples 16 to 25, SRM,
QCCS, Samples 26 to 30, MB, QCCS.
where: QCCS
MB
QCPS
DUP
SRM
quality control check standard
method blank
quality control preparation sample
duplicate sample
standard reference material
Calculations and Reporting
Flow Injection Analysis
CEC (meq/100 g) = {([Final Sol. Vol.] x [Inst. Reading]) x ([Total Diluted Vol.] + [Aliquot
l q aj vol.]) x 1 L} + {[1,000 mL x 1 meq + 14 mg x 1] -t- (sample wt. x (1-
[MOIST]) + (100 + [MOIST])}
Titration
[CEC (meq/100 g)]
{[Titrant Volume] x [Normality] x 1} + {[sample wt.] x (1 - [MOIST])
+ (100 + [MOIST]) x-100}
NOTE: moisture corrections [MOIST] are applicable if sample extracted \snoi oven-
dried, thereby, removing all free water. The moisture correction factor is as follows:
[(1 - moisture content in %) + (100 + moisture content in %)]
References
American Society for Testing and Materials. 1984. Annual Book of ASTM Standard^VolL11.01,
Standard Speciffcatfon for Reagent Water, D-1193-77 (reapproved 1983). ASTM, Philadelphia,
PA.
Doxsee, K. 1985. Cation Exchange Capacity in Nursery Soils Using FIA (Flow Injection Analysis).
Am. No. 1503-15. Weyerhaeuser Technology Center, Research Division, Tacoma, WA.
Gillman G P and G. Uehara. 1980. Charge characteristics of soils with variable charge minerals:
II. Experimental. Soil Sci. Soc. Am. J. 44:252-255.
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Grove, J.H., C.S. Fowler, and M.E. Surnner. 1982. Determination of the charge character of selected
acid soils. Soil Sci. Soc. Am. J. 46:32-38.
Hendershpt, W.H. and M. Duquette. 1986. A simple barium chloride method for determining cation
exchange capacity and exchangeable cations. Soil Sci. Soc. Am. J. 50:605-608.
Hendershot W.H., H. Lalande, and M. Duquette. 1993. Ion exchange and exchangeable cations
]Q M.R. Carter, (ed.). Soil sampling and methods of analysis. Can. Soc. Soil Sci Lewis
Publishers, Ann Arbor, MI. p. 167-176
Johnson, C.E., A.H. Johnson, and T.G. Siccama. 1991. Whole-tree clear-cutting effects on
exchangeable cations and soil acidity. Soil Sci. Soc. Am. J. 55:502-508.
Kalisz, P.J. and E.L Stone. 1980. Cation exchange capacity of acid forest humus layers Soil Sci
Soc. Am. J. 44:407-413.
Matsue, N., and K. Wada. 1985. A new equilibrium method for cations exchange capacitv
measurement. Soil Sci. Soc. Am. J. 49:574-578.
Mroz, G.D., M.F. Jurgensen, and D.J. Frederick. 1985. Soil nutrient changes following whole tree
harvesting on three northern hardwood sites. Soil Sci. Soc. Am. J. 49:1552-1557.
Rhoades J.D 1982 Cation exchange capacity. In A.L Page (ed.). Methods of soil analysis, Part 2
2nd edition. Agronomy 9:149-158.
Rhue, R.D., and W.H. Reve. 1990. Exchange capacity and adsorbed-cation charge as affected by
chloride and perchlorate. Soil. Sci. Soc. Am. J. 54:705-708.
Rtehter, D.D. 1986. Sources of acidity in some forested Udults. Soil Sci. J3oc,, Am. J. 50:1584-1589.
Ross, D.S. and R.J Bartlett. 1992. Ionic strength effects on acidity and cations leached from forest
floor cores. Soil. Sci. Soc. Am. J. 56:1796-1799.
Thomas G.W. 1982. Exchangeable cations. In A.L Page (ed.). Methods of soil analysis, Part 2
2nd edition. Agronomy 9:159-165.
U.S. Department of Agriculture/Soil Conservation Service. 1972. Procedures for collecting soil samples
and methods of analysis for soil surveys. Soil Survey Investigations Rep No 1 US
Department of Agriculture. U.S. Government Printing Office, Washington, D.C. v '
U.S. Department of Agriculture/Soil Conservation Service. 1984. Soil survey laboratory methods and
procedures for collecting soil samples. Soil Survey Investigations Rep. No. 1, U.S. Department
of Agriculture. U.S. Government Printing Office, Washington, D.C.
Wada, K, andI Y. Okamura. 1980. Electric charge characteristics of Ando A, and buried A, horizons
soils. J. Soil Sci. 31:307-314.
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Exchangeable Cations
Introduction
Exchangeable cations are those cations (positively charged tons) in soil that can be «xchanged
with cations of an added salt solution. They are comprised of exchangeable bases and
exchanteabfe acidity. The quantity of the exchangeable cations required to neutralize the negative
chaVgein soil is defined as the cation exchange capacity (CEC). The negative charge ,rrso, arises
from factors such as isomorphous substitution within layer silicate minerals, broken chemical bonds
on the edges and external surfaces of soil minerals, and the dissociation of acidic functional groups
in soil organic compounds.
Exchanqeable bases commonly found in soils, presented in order of relative abundance, include
cateiun^agnesium, potassium, and sodium. Exchangeable acidity is primarjy composed of
excnangeabli aluminum and hydrogen. The exchange complex of most so.ls fate ;somewhere
beuveen beina saturated with basic or acidic cations. The fraction of the CEC that is comprised
ot e^hSngeable baS is drihed as the base saturation and is extremely important in determining
whether a soil is relatively acidic or basic. Soil acidification occurs when base cations are replaced
with acidic cations in the soil.
The exchangeable cations are essential nutrients for plant growth. Acidic inputs to soil result
in a replacement of the base cations with acidic cations. A concern for forest health is that aodic
deposSion could result in a diminishing supply of essential nutrients and a simultaneous^crease
In Aluminum due to increased Al solubility that can be toxic to plant roots. Andersson (1986)
JonchSedThat^cdicdeposition has contributed to an accelerated decline in the base saturation
ofseveral soils in Sweden over the period of 1943-1984. Nutrient def ,c,enc,es^ magne s «" • «*
calcium have been correlated to key symptoms of forest decline in Europe (Krause et al., 1986).
As exchangeable cations are leached from the soil, the soil loses its ability to buffer further
acidic incuts In the absence of other buffering mechanisms such as sulphate adsorption soil
wat£ tSs to tocome more acidic and increase in its concentration of aluminum and heav?mto^
The decline in base saturation in soils therefore has important implications to the quality of streams
and lakes in forested ecosystems (Reuss and Johnson, 1986)
Review of Methods
By definition, any added cation should be able to replace an ^han.9aenab^^^^
It is for this reason that the choice of the replacement cat,on has oftenlbeen constdere d as
somewhat arbitrary. The consequence is that a tremendous variety of replacement solutions have
been used by soil scientists for the characterization of exchangeable cat,ons ,n soil Thei ony
cations that might be excluded from the selection process are those one would wish to.measure
such as 5*+ Mo2* K* Na+, and Al**. The only exception to this approach is that Na* is used in
certain soHregiofs wtere its'cTntribution to the exchangeable+bases is minimal. The most common
replacement cations are NH»*. Ba2+, and S^ although U+, K+, and Cs+ have all been used.
The anion in the replacement solution cannot be considerecI as an arbitrary'Choice. Antons
can be selected to provide a neutral salt solution such as with Cf or SO4 salts or a pH buffered
^alt solution such as 1 N NKOAc (pH 7). The quantity of acidic cations, such as aluminum, in soils
%w dependent on the pHofihe soil. Any anion that alters the soil pH will alter the amount of
exchangeable acidity measured as well as the CEC.
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J??H ^ f. °" ? the rep]f cement solutton can nave an impact on measured soil properties
h C(f °; ^y ,f act,°rf affect the measured CEC of a soil including the pH of the extract ng
r8 elec!.ro|yte 'eve • the va'ence (charge) of the counter ion, and the dielectric constant of
the extracting and wash solutions. The magnitude of a change in measured CEC for any of these
tte sotl ependS °n the S01' Or9anic matter content and tne Predominant clay minerals present in
The measurement of exchangsable acidity and CEC can be approached in two different ways.
One approach is to try and estimate the "effective" acidity or CEC or that measurement which
SSS^^Sff^f* tht f ct?a.' oon?tloz in the fiekj- ™s ls fi|enera"y SSSSEdSSh
neutral unbuffered salts. A recent technique has been to use an unbuffered BaCL salt to saturate
the soj and then measure the CEC of the soil at soil solution concentration similar to a field soil
£™9 hAcomPuls«ye ^placement of Ba with the addition of a dilute solution of MgSO, (Rhoades
A' £2 alternative approach is to measure the "total potential" acidity or CEC by buffering the soil
TO pM o.o. T
A wide variety of soihsolution ratios have been used in the measurement of exchangeable
cations. The mam constraint is that an excess of cations be added to effectively replace the
exchangeable cations in the soil. The equilibration time with the soil also varies considerably from
a few minutes to overnight extractions. Filtering or centrifuging are both used to obtain a clean
extract for chemical analysis after equilibration with the soil. Filtering tends to provide a cleaner
solution as organic materials may float in solutions which are centrifuged. A disadvantage of filter
materials is that they may be found to contaminate the extract with low levels of base cations and
w»ho,rf^m«aSU*ra«ble CEC.:, For thls reason. » is important to run reagent blanks (extractions
without soil) to test for possible contamination in the extraction process.
The majority of CEC methods require a displacement of the saturating ion by another salt
solution. The displacement is often accomplished after the soil has been washed free of excess
saturating solution. The solutions used to wash the soil vary from cleionized water to alcohol
solutions (methyl, ethyl, or isopropyl alcohols).
Reference Method
The reference approach to long-term monitoring methods for exchangeable bases is to
measure the exchangeable bases, effective acidity, and effective CEC with 1 N NKCI. Buffered salt
f?,UMun™ Wl1 not,b? used as *** are not considered representative of the forest soil condition. A
1 N NH4CI salt solution was chosen due to its predominant use for extracting exchangeable cations
in studies of forested ecosystems in North America. The NH4+ ton will not interfere with the
measurement of any of the exchangeable cations and has been shown (Robarge, 1988) to be an
essentially equivalent extractant of exchangeable Al as 1 N KCI (the most common exchangeable Al
extractant). The use of just one extracting solution also reduces the time and analytical
requirements for measurements. A compulsive replacement method is mot being recommended as
it is felt that it would be too time consuming and operator dependent for long-term monitoring
purposes.
=««/ ef,fecj'Y8 CEC is measured by replacing the NH4+ ton with Na+ after washing the soil with
50% ethyl alcohol to remove excess soil solution NH4+ ion and testing for art absence of cr in the
wash with a AgNO3.
Two choices for extraction procedures are provided in the methods section. In addition to a
common equilibration procedure that does not require any specialized equipment, an automated
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equilibration procedure is also included as many of the soil laboratories in Canada are presently
using this equipment.
Summary of Method
Previously prepared extracts from the CEC procedure a^ analyzed for aluminum calcium,
"—
is a measure of the concentration of that cation in the extract.
Dhotocurrents from the photomultiplier tubes are processed by a computer system. ™e signa is
pyoportional to £e analyte concentration and is calibrated by analyzing a senes of standards (U.S.
EPA, 1983; Fassel, 1982)
Fmi^inn snectroscoov (ES) can be used to measure potassium and sodium. The sample is
the element.
Interferences and Shortcomings
effects.
background effects, or using a narrower slit width.
phosphate in the extractant.
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The ,mos-t c0"1"100 Physical interference in the analysis of soils exchange solutions is salt
fSF fh>Hbum-r«or n?bulizer- Chough dilution will reduce this problem Twif also
change the matrix and any effect it may have on the instrument read-out.
Sandard i
u*ual|y compensated for by analyzing samples and all calibration standards,
JSX F01*01 standards in the same matrix. Matrix effects may be tested by
5ddltlons- and comparison with an alternative method of analysis. When the
8 * ^"^ * readily C°rr*Cted' the analyses must *
prot®?ive closing (laboratory coat and gloves) and safety glasses' when preparing
0 Whe" hco?clntrated adds and bases are used. The UM of concentratedac 31
Apparatus and Equipment
Determination by Atomic Absorption
• burner, as recommended by the spectrophotometer manufactureir. When nitrous oxide is
used as the oxidant, a nitrous oxide burner is required.
* Si^H3?"0^-'31!!08' Sirgle element 'amps preferred; multi-elerriemt lamps may be used
Electrodeless discharge lamps may be used where available, j
• balance, capable of weighing to 0.1 g. \
• balance calibration weights, 3-5 weights covering expected range.
Determination by Inductively Coupled PA
'asma ,
inductively coupled plasma-atomic emission spectrometer. >
• balance, capable of weighing to 0.01 g.
i'
• balance calibration weights, 3-5 weights covering expected range.
Determination by Emission Spectroscopy '
• flame photometer, direct-reading or internal-standard type; or an atomic absorption
spectrometer operated in the flame emission mode. aosorpuon
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balance, capable of weighing to 0.001 g.
balance calibration weights, 3-5 weights covering expected range.
Reagents and Consumable Materials
Acids used in the preparation of standards and for sample processing must be ofutow*igh
purity grade (e.g., Baker Ultrex grade or SeaStar Ultrapure grade). To min.mize «noerrtMrt»nerf
cations in standard solutions by evaporation, store solutions in linear or high density polyethylene
Stttes uSsma I containers to reduce the amount of dry element that may be picked up from the
tettte walls" wten tffSHS is poured. Shake each container thoroughly before use to redissolve
any accumulated salts from the walls.
Detonized (DI) water used for preparing or diluting rea9ents^tandards and samples must
meet purity specifications for Type I reagent water as given in ASTM D 1193 (ASTM, 1984).
Determination by Atomic Absorption
• hydrochloric acid, concentrated (12 M HCI)-Ultrapure grade, Baker Instra-Analyzed or
equivalent.
• HCI (1 percent v/v)~Add 5 mL concentrated HCI to 495 ml. DI water.
• nitric acid, concentrated-Ultrapure grade, Baker Instra-Analyzed or equivalent.
• HN03 (0.5 percent v/v HNCg-Add 0.50 ml HNO3 to 50 mL DI water and dilute to 100 mL
• primary standard solutions-Prepare frcm ultra-high purity grade chemicals as directed in
the individual procedures. Commercially available stock standard solutions may also be
used.
• dilute calibration standards-Prepare a series of standards of
appropriate stock metal solution in the specific matrix to cover the concentration range
desired. Prepare all calibration standards in concentration units of mg/L.
• fuel-Commercial grade acetylene with in-line filter is generally acceptable.
• oxidant-Air may be supplied from a compressed-air line, a laboratory compressor, or from
a cySer oppressed air. Nitrous oxide is supplied from a cylinder of compressed gas.
• lanthanum chloride (LaCy matrix modifier solution-Dissolve 29 g La2O3. s'°w|y and in sma"
portions, in 250 mL of concentrated HCI.
Caution: Reaction is violent. Dilute to 500 mL with DI water.
• water-Water used in all preparations should conform to ASTM specifications for Type I
reagent grade water (ASTM, 1984).
• weighing pans, disposable.
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Baker
80D ""- —"*•'«' HO to <00 mL n water and d»u,e ,o 1.00
f8^1"'0 8'avity 1.41)-Ultrapu,. grade. Baker tostra-Analyzed or
£JLtoS.
HNO, (SO percent vAO-Add 500 mL concentrated HNO, to <00 mL DI water and dilute to
• argon, oxygen-free.
* «™ ««-«»• for Type I
• weighing pans, disposable.
Determination by Emission Spectroscppy
NOTE: Uthium is used to suppress fonization of K+ and Na+.
•**
lithlum so'utk!n
• acetylene (commercial grade or better).
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water-Water used in all preparations should conform to ASTM specifications for Type I
reagent grade water (ASTM, 1984).
weighing pans, disposable.
Calibration and Standardization
Calibration standards should J^-P^
Within each class of instruments (^ JCP- « ^SirS^Sa^s of at toast three additional
Calibrate by analyzing a calibration blank (0 1 mg £ s*andar JJ jCp jssusedl a multi-element standard
Procedure
detection limits (IDLs) have been obtained.
General procedures for AA, ICP, and ES are given in following sections.
Procedure for Determinations by Atomic Absorption Spectrometry
Differences among AA sp«ct^^
applicable to every instrument. ^^^^oS toltowraSS lamp for the analysis, allow the
instrument. In general, after ^^f^^SSlSK^ Instrument is operated in a double-beam
lamp to warm up for a minimum of 15m nutes ^"'f s*h®t n^ monochromator at the correct
mode. During this period, align the •nstrument ^tion we ^^ cgthode current
wavelength, select the Pr°Pf Vm0"?^L°manuf actur^ ^^Subsequently, light the flame and regulate
according to the recommendation irt •*• ^"jJfJJS 5£Sr flow rate for maximum percent
Calcium and Magnesium
Step 1 -
Step 2 - Calibrate the instrument.
Step 3 - Analyze the samples.
solution) as the dilullon agent.
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Step 4- Dilute and __
as outlined in
i any samples for which the concentration exceeds the linear range,
Step 5 - Record results as mg/L in the soil extract.
Potassium and Sodium
Step 1 - Calibrate the instrument.
Step 2- Analyze the samples.
*•
Step 4- Ftecord results as mgyt in the soil extract.
Procedure for Determinations by Inductively Coupled Plasma
Step 1 -
Step2-
81613 3 '
8yStem With the "*"hod blank
W water
Step 5 - Record results for Caa+; Mga+; Na+; and AI3+. Analyze K+ by AAS or ES.
Procedure for Determinations by Emission Spectroscopy
Step 1 - Calibrate the instrument.
Step 2 - Analyze the samples.
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by ICP.
Step 3 -
Step 4 - Record results for K+ and Na+. Analyze Ca- and Mg~ by ICP or AAS and
Quality Control
Precision
difference between the samples at an acceptance limit of a RPD 5: 10%.
Accuracy
Method Blanks
toss than or equal to the instrument detection limit.
Quality Control Preparation Sample
•n i..__ _I^**A^I r\n a rvintrnl nnart deviate TlOm intJ luiiy taiiii iii»««ii "j _i:_— ^tf,r\f>
i and quantification steps.
Quality Control Check Standard
- control check standard (QCCS) should be analyzed at
o, ,nraresi ^^s^^^^£^^^-^^:^ -
within ±10% of the known concentration of the stanaaro.
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Suggested Run Format
QCCS, MB, Samples 1 to 8 QCPS
QCCS, Samples 9 to 16, MB, DUP,'
2S2' £amples 1S to 25* SRM,
QCCS, Samples 26 to 30, MB, QCCS
where: QCCS
MB
QCPS
DUP
SRM
quality control check standard
method blank
quality control preparation sample
duplicate sample
standard reference material
Calculations and Reporting
The appropriate reporting units for the exchangeable cations are rneq/100 g of soil.
Final Extract Volume = [Final wt. (g) + Tare wt. (g)] + 1.0105 !
SSTt&Tfi^
Exchangeable Cation (meq/100 g) =
Mll'+^r^^^
correction, if applicable)] |x 100 fl)) t(samPle «"*• m mfl) x (moisture
sr-s ^^ •— *
[(1 - moisture content in %) + (100 + moisture content in %)]
References
Soii.'!?^1'0 dep°Sition and its ef(Mts °" •" •*•«» of Nordto Europe. Water, Air
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Reuss J O and D.W. Johnson. 1986. Acid deposition and the acidification of soils and waters.
Ecotogicalstudies; v.59. Springer-Verlag, New York.
Rhoades, J.D. 1982. Cation exchange capacity. In A.L Page (ed.). Methods of soil analysis, Part 2.
2nd edition. Agronomy 9:149-158.
U.S. Envfronmental Protection Asency.
method 200.0. atomic absorption ™°.?'™ ™ entanalysls of Water and wastes.
l Ohio.
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Introduction
Amorphous Iron and Aluminum Oxides
to
^^
responsible for most of the SO4 adsoratfon SSSt a^SmS 10™*'^' amorphous phase is
Johnson and Todd (1983) found howewr that nniJ Iho ??,*' 1I78; lRajan- 1978- Sin9h, 1980).
with S04 adsorption. Part oflis dTs^epaSS may b? 2S.±l^0'*Si ^P* Si9nlfi^ntly
extractant used is not specific for the remo'SfoT a^p&Fe SdAlSS S°" C°nditi°nS' the
Review of Methods
^thod for amorphous Fe and A. oxides
Sfi^^ 4 as described by
0 In order to isolate the amorphous
Schwertmann
inorganic fraction
..
removal from the sample prior NH Ox treatment ^r?d i?hntn * ^aker (1<)83) suggests magnetite
should also be exercised l/rt^2d St al" (198|1) Su99est that
JohnsonandTodd (1983) report
Washington. The mineral ha^t^SSS^1L9aa^1rOm -^ HamPs^e Sd
Ministry of the Environment, unpublished datS McKeaaul anri n 8S22£8imIlar trends
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samples, NH^xtractable Fe
these could be explained by the PfeseJ^fa°* ^"^ than NH I Ox, in some cases up to five times
every sample (n=27), »*d«»£an«ne«^ extraction can be used as an
* spectes- rt ls felt that this reaflent
investigation before it is used routinely.
^
Fe and A.
monitoring programs. On a smaller
rn°n.i^g ^es»arcn programs, the
the reference procedure is outlined
Reference Method
The above evidence suggests that. NH4Ox
amorphous Fe and Al species from <*rta. "
purpose. We. therefore, do not «W« t
fractions on soil samples connected with
by Agriculture Canada (1984).
Th. NH.OX exKacUon -jf^^'SjSlirt^
extraction, it is suggested that in order to Mobtan an spre se n«^ sduepto' rinding. It is advisable
less than 0.5 mm be used This will i ^JMJJJJJ Cftrst and then grind the remaining sample to
SSS^JKE!^^ ^ -ided- For
refer to Neary and Barnes (1993).
Summary of Method
Soil samples are extracted for four
reduces Fe and Al to tower va to no? «JJ» • ^SS^Sm dissolution of crystalline
be performed in the dark as ultraviolet light I tes bee n to un a ro p ^^^ rf 1964). samples are
Interferences and Shortcomings
necessan--
NOTE: If dBuflco wi* delonized (DI) water is used, matrix-matched oalibrato, s«andards and
QC solutions must also be diluted.
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Safety
Apparatus and Equipment
• tubes, polystyrene, disposable, 17 X 100 mm, with caps, 15 mL
• dispenser, Oxford or equivalent, capable of dispensing 10 mL liquid.
• glazed weighing paper.
• flasks, volumetric, 1 L
• filter paper, Whatman #1.
• shaker, reciprocating, Eberbach or equivalent.
• centrifuge, capable of holding 17 X 100 mm, 15 mL tubes, and reaching 2500 rpm.
• pH meter, and electrode.
• balance, accurate to 0.001 g.
• tubes to fit AAS or ICP autosampler.
Reagents and Consumable Materials
• ammonium oxalate, (NHJaC2O4.H8O, crystals, reagent grade.
• oxalic acid, H2C204-HZ0, crystals, reagent grade.
oonform to
Calibration and Standardization
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the high and one in the tow part of the operating range.
wav^S^^^
Al and an air/acetylene flame is used for Fe.
Procedure
Step 1 -
Onto tared, glazed weighing paper, weigh 0.250 g of air-dried soil, ground to pass
through a 0.5 mm (35 mesh) sieve.
Step 2 -
Step 3 -
Step 4 -
Step 5 -
Step 6.-
helps to prevent enhanced Al results.
Transfer soil to a labelled, 15 ml disposable tube.
Using the dispenser, add 10 mL acid ammonium oxalate solution to each tube.
Cap tubes and shake at low speed on a reciprocating shaker for four hours in the dark.
Remove tubes from shaker and centrifuge at 2500 rpm until clear.
Decant supernatant into clean, labelled disposable tubes. Filter samples if sediment is
present.
Step 7 - Analyze samples by AAS or ICP.
Quality Control
Precision
One sample shou,d be anaiyzed ,„
Accurscy
Method Blanks
Three n,e,hod blanks, carried
the
* each blank snou* be
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Quality Control Preparation Sample
* resuits shouid be
*•
nAa!y\t^
Accuracy of the QCPS should be within i : 10% of the^nn t«™ J«9 D"! between-run precision.
be determined by analyzing the QCPS and cataiStinn 5KS^i ?• ^ Between-"« Precision can
Quality Control Check Standard
Suggested Hun Format
i
QCCS, MB, Samples 1 to 8, QCPS
QCCS, Samples 9 to 16, MB, DUP,
QCCS, Samples 16 to 25, SRM,
QCCS, Samples 26 to 30, MB, QCCS
where: QCCS
MB
QCPS
DUP
SRM
quality control check standard
method blank
quality control preparation sample
duplicate sample
standard reference material
Calculations and Reporting
Fe
n in solution x -in mi
(250 mg soil x 1000 jug/mg)
Al
in solution
(250 mg soil x 1000 //g/mg)
ml x 100
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References
Aflrteulture Canada. 1984. Analytical Methods
Research Institute. Ottawa, Ontario. LRRI
Alexander E.B. 1974. Extractable iron in relation to soil age on terraces along the Truckee River,
Nevada. Soil Sci. Soc. Am. Proc. 38:121-124.
Pennsylvania.
Baril R and G. Brttton. 1967. Anomalous values of free iron in some Quebec soite containing
' magnetite. Can. J. Soil Sci. 47:261.
^
Johnson, D.W., and D.E. Todd. 19.83 Relationships among iron, aluminum, carbon, and sulphate ,n
a variety of forest soils. Soil Sci. Soc. Am. J. 47.792-800.
McKeague, J.A., and J.H. Day. 1966. Dithionite and oxalate-ext ractabte Fe and AJ as aids in
differentiating various classes of soils. Can. J. Soil Sci. 46.13-22.
Neary A.J and S.R. Barnes. 1993. The effect of sample grinding on extractable iron and aluminum
in soils. Can. J. Soil Sci. 73:73-80.
Parfitt R.L, and R.S.C. Smart. 1978. The mechanism of sulf ate, adsorption on iron oxides. Soil
Sci. Soc. Am. J. 42:48-50.
Raian S S S 1978. Sulf ate adsorbed on hydrous alumina, ligands displaced, and changes in surface
charge. Soil Sci. Soc. Am. J. 42:39-44.
Soc. Am. J. 45:645-649.
84:194-204.
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Schwertmann, U. 1973. Use of oxalate for Fe extraction from soils. Can. J. Soil Sci 53-244-246
so^in some acw f°re
ite °" OXalat6- and djtni«nite-extractable iron. Soil Sci.
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Organic Iron and Aluminum
Introduction
a negative influence on SO/" adsorption (Johnson and Todd, 198,3).
" 8* sus«pS ,o fcWeation, the measurement of this fraction is necessary.
Since this is expected to be a relatively su-ble parameter, this measurement need oply be done
initially to help characterize the soil.
Review of Methods
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the possible modification of the extracted humic substances, although some evidence has been
presented to refute this argument. Smith and Lorimer (1964) found that the fulvic acid extracted by
, ^*H3d. I81!"!13' comP°sition to that extracted by water. Similarly, Schnitzer and Skinner (1968)
found that the f ulvic acid removed by 0.5 N NaOH and 0.1 N HCI had similar elemental compositions
Hayes (1985), on the other hand, found that strong alkali and associated chemical oxidation
destroyed organic matter.
Neutral salts of mineral or organic acids have also been used, however, their success depends
upon the ability of their anions to interact with the cations in combination with humic material in the
soil. Bremner and Lees (1949) tested the sodium salts of eight inorganic acids, ten organic acids
as well as NaOH and NaCO3. They concluded that Na-pyrophosphate (Na4P2O7)" was the best in
providing a mild but reasonably efficient extractant. They recommend a concentration of at least
0.1 M. Below this minimum concentration, yield varies with concentration and results in decreased
6 ^nC£^hnitZ8r 6t a'" (1958) alS° fOUnd that Na«P*°7
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were commonly slightly higher. Schuppli et al., (1983) experimentedon Canadian •""repml
using high speed centrifugation, Superfloc + tow speed centrifugat.cn, and both of the above with
ultrafiltration. In the tropical soils, Superfloc + low speed centrifuging removed all the Al silicates
but concentrated goethite in the remaining suspended material. Similar results were obtained with
high speed centrifugation but with less goethite. .This effect was not nearly as pronounced in the
Canadian soils. They concluded that high speed centrifugation or tow speed cen r.fugation +
Superfloc alone (without ultrafiltration) was adequate for most temperate region so.ls. Ultraf iltration
seems most suited to laboratories not equipped with a high speed centrifuge. To date, the form
of Fe and Al remaining in ultracentrifuged or ultrafiltered samples has not been adequately
characterized.
The pH of the extracting solution has been of some concern to analysts. If maintaining the
integrity of the humic material is necessary for the identification of the^organics, an alkaline solution
such as unbuffered Na4P2O, may not be suitable. Bremner and Lees (1949) speculated that,.order
to decrease the potential alteration of proteins, the pH of the extractant should be 7 Data compiled
from a series of studies shows the pH 7 method to be superior for minimizing the alteration of
humic substances (Hayes, 1985). Kononova (1966) also recognized the need to adjust the phTto 7
but recommended that because the efficiency of the extractant, for humic acids espeaally, is better
at a higher pH, a pH of 9 should.be used. Certainly, if one's primary interest is the measurement
of organic Fe and Al, rather than the characterization of the organics, a higher pH ,s probably
suitable. A comparison performed by the Ontario Ministry of the Environment founcI that the Fe and
Al extracted at pH 10 with 0.1 M Na4P2O7 correlates better with organic C than 0.1 M Na4P2O7 at pH
7 or 0.01 M Na4P2O7 at pH 7.
Adopted standard methods in Canada, the U.K. and the U.S. involve the use of 0.1 M Na4P2O7
at pH 9.6 or 10 with either high speed centrifugation or the addition of Superftoc + tow speed
centrifugation. Of these methods, Loveland and Digby (1984) report that Na4P2O7 at pH 10 with high
speed centrifugation (20,000 rpm) is a more consistent treatment, particularly for Al.
• The Canadian and U.K. methods use samples ground to less than 0.5 mm whereas the U.S.
EPA methods (Blume et al., 1990) use a larger sample aliquot and the less than 2 mm sample. In
order to obtain a representative sample, it is suggested that if less than one gram of sample is
chosen for extraction, the sample should be ground to less than 0.5 mm prior to •**£"•
Overwinding must be avoided. The extracts may be analyzed by erther atomic absorption
spectrophotometry (AAS) or by inductively coupled plasma optical emission spectroscopy (ICP-OES).
Reference Method
The reference extraction method for the removal of organically-bound Fe and Al from soil is
treatment with 0.1M Na4P2O7 unbuffered or at pH 10. Centrifuging at 20,000 rpm ^6™m™"d*°>
although Superftoc + low speed centrifugation (with ultrafiltration optional) maybe usedL as rt is
recognized that not all laboratories are equipped with a high speed centrifuge. This method was
chosen for the following reasons:
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• the efficiency of the alkaline extraction for humic material,
• identification of the humic substances is not required,
• the results of previous work indicating its usefulness for the approximate differentiation
solls (McKea9ue e< +• ** a"d «^**^«
Adoption of this method by the Canadian, USDA, and British soil classification systems.
that
^increase.
(Sh3king)
iS Su99ested since Bremner and Lees (1949) found
materia' 6XtraCted (as ™asured b* <*'«"* N) does
Summary of Method
lfJmpl8 °f au~dried S0ll> 9round to less tnan °'5 mm- is ^ighed into a plastic centrifuge
d Pyr0ph°SPhate solution <°'1 M at PH 10) is added, the tube is capped
rf - - , is cemrifge
speed for 15 rmnutes. Alternatively, 0.5 mL of 0.1% Superfloc s added and the tub? is
hrou^ T? ** 15 ^ minUt6S " """ SO'Uti°nS " *-' Turbid "rnptes should
ICP 9 ^ membrane filter Ir°n 3nd a'Uminum are dete^™d in the supernatant
Interferences and Shortcomings
mavc n9 an reSU8 lJild-up on the *»"* head
may occur. This may be alleviated somewhat by the aspiration of large amounts of deionized water
(D II between samples. Alternatively, samples may be diluted prior to analysis (Fe and Al tevete
^^ (aVaiIabte °" S°me inst— ^ « d^ted with HNO3
mat-matChed caption standards and QC
of Surno H " S3mpleS are turbld High "P"*1 <*ntrifuging or the addition
of Superfloc and low speed centnfuging is required to obtain extracts which are free of suspended
nrn* t '* is ^^ that a Iar96 amount «* ** F« and AJ extracte(i by this method is in the
organic form, it is possible that other forms of iron and aluminum may be extracted by this method
The results can, therefore, only be used for the approximate differentiation of organic Fe and Al
n
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Safety
No specific hazards are associated with this procedure or with the required reagents. Normal
laboratory safety practices are to be observed. Protective clothing and safety glasses should be
worn when handling concentrated HNO3 or H^PO, or NaOH. Standard safety precautions should be
observed when analyzing samples using a nitrous oxide/acetylene flame.
Apparatus and Equipment
• centrifuge tubes, plastic 50 mL, Oak Ridge type, screw closure.
• screw caps to fit centrifuge tubes.
• tube rack to hold centrifuge tubes.
• tubes to fit AAS or ICP autosampler.
• spatula.
• glazed weighing paper.
• dispenser, capable of accurately dispensing 30 mL of liquid.
• reciprocating shaker, Eberbach, end-over-end shaker, or equivalent.
• centrifuge with head to hold 50 mL tubes (preferably capable of attaining a speed of 20,000
rpm or 510 X G. This is not mandatory, as a speed of 2000 rpm may be used with the
addition of Superftoc).
• balance, accurate to 0.001 g.
• pH meter.
• 0.1 pm membrane filters and filter apparatus (optional).
Reagents and Consumable Materials
• sodium pyrophosphate (Na4P2O7), anhydrous reagent grade powder or Na4P2O7"
• nitric acid (HNOg), concentrated, reagent grade.
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• Superfloc (N-100) only required if a high speed centrifuge is not available. Two known
sources for this product are: Cyanamid of Canada, Montreal, Quebec or from the American
Cyanamid Co. of Wayne, New Jersey, U.S.A.
• water-DI water used in all preparations should conform to ASTM specifications for Type
I reagent grade water (ASTM, 1984).
• sodium hydroxide (NaOH), 0.1 M solution or phosphoric acid (H3POJ as required to adjust
the pH of the pyrophosphate to 10.
• sodium pyrophosphate solution, 0.1 M-Into a one litre volumetric fllask, weigh out 26.59 g
anhydrous Na4P2O7, oven dried and cooled in a desiccator (44.61 g if using Na4P2O,»10H O)
Dissolve in DI water and dilute to one litre. Adjust to pH 10 with NaOH or H PO . * "
Calibration and Standardization
Within each class of instruments (AAS and ICP), the calibration procedure varies slightly
Calibrate by analyzing a calibration blank (0 mg/L standard) and a series of at least three additional
standards within the linear range of the instrument. If an ICP is used, a multi-element standard
may be prepared and analyzed. For AAS determinations, the instrument must be calibrated for each
anaiyte by using a separate standard. The concentration of standards should bracket the expected
sample concentration; however, the linear range of the instrument should not be exceeded
i
Prepare calibration standards so that the final concentration of N:a4P2O7 in the standards is
equal to that of the sample extracts. It is advisable to have two quality control solutions one in
the high and one in the tow part of the operating range.
To correctly set-up the AAS, refer to the operating manual for the instrument. Optimal
wavelengths are: 309.3 rim for Al and 248.3 r,m for Fe. A nitrous oxide/acetylene flame is used for
Al and an air/acetylene flame is used for Fe.
Procedure
Step 1 - Onto tared, glazed weighing paper, weigh 0.300 g of air-dried soil, ground to pass
through a 0.5 mm (35 mesh) sieve.
NOTE: It is advisable to sieve out the naturally less than 0.5 mm fraction first and then
grind the remaining sample aliquot to pass through the 0.5 mm sieve. This eliminates
undue grinding of the naturally less than 0.5 sample and helps prevent enhanced Al
results (Neary and Barnes, 1993). \
Step 2 - Transfer into a labelled 50 mL plastic centrifuge tube.
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Step 3 -
Step 4 -
Step 5 -
Step 6 -
Using the dispenser, add 30 mL of 0.1 M Na4P2O7 extracting solution to each tube.
Cap tubes and shake overnight on a reciprocating shaker at tow speed or on an end-
over-end shaker at 40 to 50 rpm.
Remove tubes from shaker. Uncap tubes and centrifuge at 20,000 rpm for 15 minutes.
Alternatively, add 0.5 mL of 0.1% Superfloc solution to the sample and centrifuge at 1500
for 15 to 30 minutes or until clear. Filter any turbid" samples through 0.1 pirn membrane
filters.
Carefully decant supernatant into labelled 15 mL disposable tubes. Acidify extracts with
3 drops of concentrated nitric acid and let samples sit overnight allowing any suspended
matter to settle.
Step 7 - Analyze for Fe and Al by AAS or ICP.
Quality Control
Precision
One sample should be analyzed in duplicate with each run of thirty samples. To eliminate bias
due to position in the run, the routine sample duplicate should be analyzed separately within the
analytical run. Within-run precision is determined from duplicates based on relative percent
difference between the samples at an acceptance limit of a RPD * 10%.
Between laboratory precision can be determined by analyzing the Agriculture Canada ECSS
round-robin samples.
Accuracy
Accuracy is determined by analysis of a standard reference material (SRM). Acceptable limits
for accuracy should be ±10% from the known concentration of the standard or within the accuracy
windows supplied by the reference material manufacturer, whichever is larger.
Method Blanks
Three method blanks, carried through the extraction procedure, are analyzed with each batch
of samples for each cation to measure potential contamination. Method blanks should be run at
the beginning, middle, and end of each analytical run. The concentration of each blank should be
less than or equal to the instrument detection limit. All results should be blank corrected us.ng the
mean of the acceptable method blank readings.
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Quality Control Preparation Sample
A matrix matched in-house quality control preparation sample (QCF'S) should be analyzed once
per analytical run. This sample is used to monitor accuracy and long-term between-run precision
Accuracy of the QCPS should be within t 10% of the long-term mean. Between-run preclstor,can
tfvJurJTtiri* ^ **"? ^KQCP? 3nd calculatin9 t^ cumulative long-term standard deviation.
If values plotted on a control chart deviate from the long-term mean by more than three standard
dev.at.ons, the run should be completely reanalyzed, including all digestion and quantification steps.
Quality Control Check Standard
A quality control check standard (QCCS) should be analyzed at the beginning, after every ten
samples, and after the last sample of each analytical run. The QCCS should contain all the analytes
of interest with mid-calibration range concentrations. Quantified values of the QCCS should be
within ±10% of the known concentration of the standard.
It is highly recommended that the concentrations of this sample t*. consistent through time
so that control charts may be plotted to monitor laboratory bias and other potential problems If
analyzed values deviate from the long-term mean by more than three standard deviations 'the
instrument is re-standardized and re-calibrated prior to any further sample analyses.
Suggested Run Format
QCCS, MB, Samples 1 to 8, QCPS,
QCCS, Samples 9 to 16, MB, DUP,
QCCS, Samples 16 to 25, SRM,
QCCS, Samples 26 to 30, MB, QCCS.
where: QCCS
MB
QCPS
DUP
SRM
quality control check standard
method blank
quality control preparation sample
duplicate sample
standard reference material
Calculations and Reporting
If a chart recorder is used, peak heights are measured in mm from a baseline drawn between
peaks. Calculate Fe and Al concentrations, accommodating for sensitivity changes throughout the
run, blank values and dilution factors. Results are reported as % Fe and ;M in the soil. Results are
reported to two significant figures.
(%) = Fe (uo/mL) in solution x 3Q ml x 100 ''
(300 mg soil x 1000
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(%) = Al ffjg/mU in solution x 30 mL x 100
(300 mg soil x 1000
References
Pennsylvania.
^^
Soil Sci. 60:141-143.
Bascomb, C.L 1968. Distribution of pyrophosphate extractable iron and organic C in soils of various
groups. J. Soil Sci. 19:251-268.
Monitoring Systems Laboratory, Las Vegas, NV. EPA/600/4-90/023.
• ":
a
ssat
and Sons, New York. 692p.
Hu^te. A.a, ami J.G. McCoH. 1984. Soil cation ,ea=Mn9 by -acHd rain- w«h varying nr,ra,e:su,fa,e
ratios. J. Envir. Qual. 13:366-371.
...
exlraols of some soil horizons. Geoderma 26:95-105.
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Kononova. M.M. 1966. Soi. Organic Matter. Perma8on Press. Inc..
New York. 544p.
of forms
and a,umlnum
°n«arto (Ores,
"9ands dlsplaced'
. SC«
peat Can. J.
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Iron and Aluminum Oxides
Introduction
be done once to help character,* the so.l.
Review of Methods
is the optimum pH for extraction.
method were comparable to the ^'^e^yn^i^d^rniBht shaking with citrate-
™ Canada adopted the USDWCS me,hod as
their standard procedure. t
identified.
Samp«e extracts may be analyzed by either atornta ^bsorptton
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Reference Method
The sample extracts may be analyzed for Fe and Al by either ICP or MS.
Summary of Method
Interferences and Shortcomings
iS USed' '"atrix-matched ca.ibration standards and QC
°f Fe and MJro^ silicates fay the citrate-dithionite extraction is generally
W^r< rt should.be noted that ^is extractant also removes organic Fe
, ,xides 9°ntent is desired, the pyrophosphale-actractable Fe and /S
rSu« a I- **, ! "l^J and subtracted from the results of this analysis. In this casethe
results are also subject to the inherent limitations of the pyrophosphate tachnique
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Safety
No specific hazards are associated with this procedure or with the required reagents Normal
analyzing samples using a nitrous oxide/acetylene flame.
Apparatus and Equipment
• plastic centrifuge tubes, 50 mL, with screw caps.
• dispenser, capable of accurately dispensing 25 mL
• reciprocating shaker (Eberbach or equivalent) or end-over-end shaker (40-50 rpmj.
• spatula.
• glazed weighing paper.
• tubes to fit AAS or ICP autosampler,
• tube racks to hold 50 mL centrifuge tubes and AAS autosampler tubes.
• balance, accurate to 0.001 g.
Reagents and Consumable Materials
• sodium dithionite (hydrosulphite) (Na2SaOJ, reagent grade.
• sodium citrate (Na3C6H5O7-2H2O), reagent grade.
• nitric acid (HNCg, concentrated, reagent grade.
Cyanamid Co. of Wayne, New Jersey, U.S.A.
• water-DI water used in all preparations should conform to ASTM specifications for Type
I reagent grade water (ASTM, 1984).
dissolve 200 grams of
- . sodium citrate solution (0.68 M)-In a one litre
NaaCeH5O7-2H20 in DI water. Dilute to volume with DI water.
Calibration and Standardization -
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Procedure
Step 2 -
Step 3 -
Step 4-
11 ls 'dy1?3*118 t° sie» out the naturally less than 0.5 mm fraction first and then
Transfer weighed sample to a labelled 50 mL plastic centrifuge tube.
Using the dispenser, add 25 mL of 0.68 M sodium citrate solution.
Using a calibrated scoop, add 0.4 g of sodium dithionite.
— « at taw speed, or on an end-
Step 6 -
Step 7 -
Remove caps and centrifuge tubes for 15 minutes at 20,000 rpm Alternatively add 0 5
mL of 0.1% Superf loc solution to the sample and centrifuge J w5%£^*j^^
the suPernatar|t into tubes to fit autosampler of the AAS Acidifv
t
Step 9 - Analyze samples by AAS or ICP.
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Quality Control
Precision
difference between the samples at an acceptance limit of a RPD & 10%.
Accuracy
windows supplied by the reference material manufacturer, whichever is larger.
Method Blanks
mean of the acceptable method blank readings.
Quality Control Preparation Sample
Quality Control Check Standard
within ±10% of the known concentration of the standard.
Suggested Run Format
QCCS, MB, Samples 1 to 8, QCPS,
QCCS, Samples 9 to 16, MB, DUP,
QCCS, Samples 16 to 25, SRM,
QCCS, Samples 26 to 30, MB, QCCS.
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where: QCCS
MB
QCPS
DUP
SRM
quality control check standard
method blank
quality control preparation sample
duplicate sample
standard reference material
Calculations and Reporting
Deakslf 3Thea?«reSI.deAr,i^Sed'tpefkllei9hts are measured *" mm from a baseline drawn between
Fe (%) = Fe fua/mU in solution x 9s ml x 5 x 100
(500 mg soil x 1000 Aig/mg)
(%) • Al toa/mLl in solution x 9$ ml. x 5 x 100
(500 mg soil x 1000 pg/mg)
References
Agriculture Canada 1984. Analytical Methods Manual 1984. B.H. Sheldrick (eel) Land Resource
Research Institute, Ottawa, Ontario. LRRI Contribution NoTwSl ^source
Jacksoa 1953- Iron oxide removal from soils ancl
for JestVn9 and Materials. 1984. Annual Book of ASTM Standards, Vol. 11 01
°' ^^^ Wat6r< D1193'77 fr^P"^ 1983)- ASTM, ?niladelPhia,
r ' and S>C; Fang' 1964' Iron or al"minum coatings in relation to suifate
adsorption characteristics of soils. Soil Sci. Soc. Am. Proc. 28:632-<}35.
h^ I--W. Zelazny, and DC. Martens. 1981. Effect of photolytte oxalate treatment on soil
hydroxy-mterlayered vermiculites. Clays and Clay Min. 29:429-434.
Jackson M.L 1956. Soil Chemical Analysis-Advanced Course published by the author, Dept. of
Soils, University of Wisconsin, Madison, Wisconsin. 991 p.
• i
aluminum
su"ate in
Lazerte, B. 1989. Aluminum speciation and organic carbon in waters of Central Ontario In
Chefsea Ml p^QsSoT"1*1 Chemistry and Tojdcoto9y °* Aluminum, Chapt. 1. Lewis Pub.,
Mehra, O.P. and M.L Jackson. 1960. Iron oxide removal from soils and clays by a dithionite-citrate
32T151!? fe M*ith ,S°dJium bicarbonate- Proceedings of the 7th National ConfertSS of
Clays and Clay Minerals. Permagon Press, New York. pp. 317-327.
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Neary, A.J., and S.R. Barnes. 1993. The effect of sample grinding on extractable iron and aluminum
in soils. Can. J. Soil Sci. 73:73-80.
Neary AJ E. Mistry, and L Vanderstar. 1987. Sulphate relationships in some central Ontario forest
soils. Can. J. Soil Sci. 67:341-352.
Raad, AT., R. Protz, and R.L Thomas. 1969. Determination of Na-dithionite and NH4-oxalate
extractable Fe, Al and Mn in soils by atomic absorption spectroscopy. Can. J. Soil Sci.
49: 89-94.
Rajan, S.S.S. 1978. Sulfate adsorbed on hydrous alumina, ligands displaced, and changes in surface
charge. Soil Sci. Soc. Am. J. 42:39-44.
*
Schwertmann, V. 1964. Differenzierung der Eisenoxide des Bodens durch extraktion mit
ammoniumoxalat-Losung. Zeischrift fur Pflanzenernah rung Dunfung Bodenkunde 105:194-202.
Sheldrfck, B.H., and J.A. McKeague. 1975. A comparison of extractable Fe and Al data using methods
followed in the U.S.A. and Canada. Can. J. Soil Sci. 55:77-78.
Singh, B.a 1984. Sulfate sorption by acid forest soils: 2. Sulfate adsorption isotherms with and
without organic matter and oxides of aluminum and iron. Soil Sci. 138:294-297.
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Phosphate Extractable Sulphate
Introduction
Sulphur, in the form of sulphate (SO/), is the principal anion in acidic: deposition in eastern
North America. Concern over the long-range transport and deposition of SO/ in precipitation has
lead to increased monitoring of the sulphur status of soils. In most mineral soils, the majority of
extractable S is in the SO/ form. In contrast, in organic horizons up to 50% of the total extractable
S may be organically-bound (Maynard et al., 1987). The ability of soils to adsorb sulphate is one
of the principal factors affecting the rate and extent of soil and watershed response to acidic
deposition. Quantification of existing pools of adsorbed sulphate on a soil, concurrent with
measurements of sulphate adsorption capacity of that soil, provide useful information for
understanding the status and for predicting the future response of the soil to acidic deposition. The
measurement of extractable sulphate is, therefore, suggested for terrestrial monitoring programs
such as LRTAP. '
Review of Methods
The preparation and storage of soils is important in the determination of extractable SO/ in
soils. Several studies have shown that drying significantly altered the SO/ content of the soil,
particularly in organic horizons (Peverill et al., 1975; David et al., 1982; Ssarle and Sparling, 1987).
Moreover, storage of air-dried samples at room temperature (20 to 25° ID) for between 12 and 78
weeks resulted in significant increases in SO/ concentrations (Maynard et al., 1987; Searle and
Sparling, 1987). The increased sulphate concentrations observed were not consistent among soils.
For extractable SO/ measurements in organic horizons to be meaningful, the test should be
performed on field-moist samples. A representative sample can be obtained by mixingyhomogenizing
the moist organic horizon prior to analysis. The changes in SO/ concentration in mineral soils
caused by drying and storage were much less than organic soils. For this reason and because of
the difficulty in obtaining a representative sample from moist samples, air-dried soils are
recommended for the analysis of mineral soils.
Numerous extractants have been proposed for the removal of SO/ from soils (Beaton et al.,
1968; Tabatabai, 1982). Most of the experimental work has involved using mineral agricultural soils
in an attempt to correlate extractable SO/ with plant growth and S uptake. Many of the techniques
used on agricultural soils have been adapted for use on forest soils.
^ Sulphate extraction methods in soils can be broken into two groups based on the fraction of
SO/ removed. The two groups include those extractants that remove readily soluble SO/ and
those extractants that remove readily soluble SO/ plus adsorbed SO/. The extractants used to
remove readily soluble SO/ (H2O and weak salt solutions, such as CaClj, or NH4CI), are preferred
for organic and mineral soils containing no appreciable amounts of adsorbed SO/. In contrast,
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phosphate extractants, generally employing a Ca(H2POJ2 solution (500 mg P/L), are recommended
for soils containing sesquioxides, allophane, or kaolinitic clays which have the potential to adsorb
^. The latter class of soils generally includes the mineral horizons of most forest soils.
Eight extractants (including H2O, several weak salts, and Cafl^POJJ were evaluated for
extractable SO,* on five forest organic soil horizons (Maynard et at., 1987). The most consistent
extractant for SO4* was 0.01 M NH4CI (1:10 soil to solution ratio), although all the weak salt
extractants remold similar amounts of SO.-. Water as an extractant gave the most ^variabe
results. The phosphate extractant did not remove any more SO,2" in the orgamc soils and gave more
variable results than the NH4CI extractant. The strong phosphate solution was also noted to reduce
the life of the ion chromatography column.
A phosphate extractant is preferred for mineral soils to ensure that strongly adsorbed SO,*
is extracted. Calcium dihydrogen phosphate (Ca^POJJ solution, containing 500 mg | P/L is
preferred over Na or K phosphates since Ca enhances particle f locculation in clayey soils and makes
filtering more convenient.
Over the last decade, the development of ion chromatography (1C) and inductively coupled
plasma atomic emission spectrometry (ICP-AES) have made the quantifi teat ion of S^and SO rap,d
and more accurate. The determination of total S in a soil extract can be done by ICP-AES, but this
technique is not specific for SO/". Ion chromatography is, however, specific to the SO4 ion (Dick
and Tabatabai, 1979; Nieto-and Frankenberger, Jr., 1985; Maynard et al., 1987) and ,s, therefore,
preferred for sulphate quantification in soil extracts.
Reference Method
The reference method for the extraction of the readily soluble SO4* plus strongly adsorbed
SO * phases from mineral soils employs a CafhLPOJ, extraction solution containing 500 mg P/L
Sulphate in the extract is then quantified by ion chromatography. This method is primarily for use
in the determination of extractable sulphate contents in mineral soil horizons but may be used for
the analysis of organic horizons.
Summary of Method
Air-dried mineral horizons which have been disaggregated and passed through a 2-mm sieve
are extracted with Ca(H2POJ2 solution containing 500 mg P/L (2:20 air-dried soil to solution ratio)
For oS«s, 'the but sample is homogenized in the field-moist state, pri or to£—
of an aliquot for extraction with a Ca^POJ, solution conta,n.ng 500 mg P/L. Sulphate in the
extracts is determined by suppressor or single column ton chromatography.
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Interferences and Shortcomings
The high levels of phosphate in the Ca(H2POJ2 extracts usually require adjustments to. the
eluent strength and instrument settings in order to provide a good separation between sulphate and
phosphate. These adjustments may cause increased elution time for SO,*. The column-life may
also be shortened by the use of strong P solutions.
Colloidal material and certain organic compounds may interfere with the SO^ peak on the
chromatogram. Membrane filtering will help remove colloidal material and extend the life of the
guard cartridges.
As with other soluble salts in soils, the amount of SO,2" extracted varies with the
soihextractant ratio. This ratio should, therefore, remain constant if samples are to be compared.
Safety
Wear protective clothing (laboratory coat and gloves) and safely glasses when preparing
reagents. Follow the safety precautions of the manufacturer when operating instruments.
Apparatus and Equipment
• ion chromatograph, pump and conductivity detector. Either suppressed ion chromatography
(SIC) such as the Dionex models, or single column ion chromatography (SCIC) such as the
Waters systems.
• anion separator column and appropriate guard column. i
• automated sampler and injection system and sample vials or tubes to fit sampler.
• data recording system, integrator or strip chart recorder.
• balance, accurate to 0.001 g.
• reciprocal shaker.
• vacuum filtration apparatus and funnels.
• vacuum membrane filter apparatus (optional).
• volumetric flask, 2 L
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• volumetric flask, 1 L
• Nalgene bottles, 60 ml_
Reagents and Consumable Materials
• calcium dihydrogen phosphate (Ca^OJ^O), reagent grade crystals.
• sodium sulphate (Na2SOJ, reagent grade crystals.
. water-DI water used in all preparations should conform to ASTM specifications for Type
I reagent grade water (ASTM, 1984).
• Cad^CX), extracting solution (500 mg P/L)~Weigh out 4.0(39 g C.O-tfOJ.-H.p into a 2 L
volumetric flask. Dissolve in DI water and dilute to 2 L volume.
• sulphate standard stock solution (1,000 mg SOJU-Wetfi 1-4790 g Na2SO4 (oven
dried at 105' C and cooled in a desiccator) and transfer to a 1 L volumetric flask
Dissolve in Ca(H2POJ2 extracting solution and dilute to one litre. Store refrigerated
at 4° C.
• working sulphate standards prepared from the stock solution to cover expected range of
S? concentrations in sample extracts. Use CafltfOJ, extracting solution during d.lufon
of the standard stock solution.
• appropriate eluent for the 1C system and anion separator column used, (see manufacturer's
recommendations).
• filter paper, Whatman #42.
• membrane filters, 0.45 pm (optional).
Calibration and Standardization
Follow the set-up procedures outlined in the manufacturer's operating manual for the specif to
Mn^^T-M through the system and stabilize the baseline. Adjust the recorder unt.l
Sote approximately 10% and the high standard is approximately 90% of the chart.
Use a minimum of three standards plus a zero standard to calibrate the system. The
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Sh°Uld n0t ta exceeded- T* calibratfon
fresh daiiy-
should
Procedure
Stepl- Weigh 2.00 g of air-dried soil into a 60-mL Nalgene bottle.
Step 2 - Add 20 mL of Ca(H2POJ2 extracting solution to each bottle.
Step3- Shake for 1 h on a reciprocal shaker at 1 to 2 cycles per second.
SUSpenslon usi"9 Whatman #™ filter paper in a Buchner
Step 5 -
Step6-
NOTE: Alternately, samples may be centrifuged until the supernatant is clear.
Vacuum filter the samples through 0.45 /urn membrane filters.!
Analyze samples by ion chromatography within 24 hours.
° ** transferred into vials or tubes )iDeciffc to the type of
NOTE: Samples should be stored at 4° C prior to sample analysis.
Quality Control
Precision
*" analyZ6d in duplicate with each run of thirty samples. To eliminate
r°Utine Sample duP|icate should
feren we duplicates
difference between the samples at an acceptance limit of a RPD n 10%.
Accuracy
Accuracy is determined by analysis of a standard reference material (SRM) Acceotable
hrmts for accuracy shouW be ±10% from the known concentration of the standard ' or^SS the
accuracy wndows supplied by the reference material manufacturer, whichever is larger
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Method Blanks
Three method blanks, carried through the extraction proceduire, are analyzed with each batch
of samples for each cation to measure potential contamination. Method blanks should be run at
the beginning, middle, and end of each analytical run. The concentration of each blank should be
toss than or equal to the instrument detection limit. All results should be blank corrected using the
mean of the acceptable method blank readings.
Quality Control Preparation Sample
A matrix matched in-house quality control preparation sample (QCPS) should be analyzed
once per analytical run. This sample is used to monitor accuracy and long-term between-run
precision Accuracy of the QCPS should be within t 10% of the long-term mean. Between-run
precision can be determined by analyzing the QCPS and calculating the cumulative long-term
standard deviation. If values plotted on a control chart deviate from the long-term mean by more
than three standard deviations, the run should be completely reanalyzed, including all digestion and
quantification steps. ,
Quality Control Check Standard
A quality control check standard (QCCS) should be analyzed at the beginning, after every ten
samples, and after the last sample of each analytical run. The QCCS should contain all the analytes
of interest with mid-calibration range concentrations. Quantified values of the QCCS should be
within ±10% of the known concentration of the standard.
It is highly recommended that the concentrations of this sample be consistent through time
so that control charts may be plotted to monitor laboratory bias and other potential problems. If
analyzed values deviate from the long-term mean by more than three standard deviations, the
instrument is re-standardized and re-calibrated prior to any further sample analyses.
Suggested Run Format
»
QCCS. MB. Samples 1 to 8, QCPS,
QCCS, Samples 9 to 16, MB, DUP,
QCCS, Samples 16 to 25, SRM,
QCCS. Samples 26 to 30, MB, QCCS.
where: QCCS
MB
QCPS
DUP
SRM
quality control check standard
method blank
quality control preparation sample
duplicate sample
standard reference material
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Calculations and Reporting
!0" ?f S°4* iS calculated bV read'"9 the peak area (or height) of the sample
n CUrVe" ^ cateu'*ion program used shoukT take Into
the run as detected by the ln-run
For mineral soils, calculate extractable sulphate concentrations as follows:
Extractable SO,* (mg S/kg) = SO.* (molL) in solution x extract volume fmL)
dry soil weight (g)
samDte , mUSt * based on % molsture in the field-moist
sample. Therefore, the following formula is applicable:
Extractable SO,* (mg S/kg) - SO,* fmo/Ll in solution x a*, act volume fmLl
(initial sample weight (g) x moisture correction)
NOTE: The moisture correction factor is as follows with the moisture content presented as
a fraction of the whole (e.g., 0.75):
[(1 - moisture content in %) + (100 + moisture content in %)]
i
References
American Society for Testing and Materials. 1984. Annual Book of ASTM Standards Vol 11 01
Standard Specification for Reagent Water, D1193-77 (reapproved 1983). ASTM, Philadelphia
Pennsylvania.
Beaton, J D., G.R. Burns, and J. Platou. 1968. Determination of sulphur in soils and plant material
Technical Bull. No. 14. The Sulphur Institute, Washington, D.C.
David. MB., MJ. Mitchell, and J.P. Narkus. 1982. Organic and inorganic sulfur constituents of a forest
soil and their relationship to microbial activity. Soil Sci. Soc. Am. J. 46:529-536.
Dick, W.A.. and MA Tabatabai. 1979. Ion chromatographic determination of sulfate and
nitrate in soils. Soil Sci. Soc. Am. J. 46:847:852.
Maynard, D.G., Y.P. Kalra, and F.G. Radford. 1987. Extraction and determination of sulfur
in organic horizons of forest soils. Soil Sci. Soc. Am. J. 51:801-806.
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Nteto, K.F., and W.T. Frankenberger, Jr. 1985. Single column ton chromatography: I. Analysis of
inorganic antons in soils. Soil Sci. Soc. Am. J. 49:587-592.
Peverill, K.I., G.P. Briner, and LA, Douglas. 1975. Changes in extractable sulphur and potassium
levels in soil due to oven drying and storage. Aust. J. Soil Res. 13:69-75.
Searte, P.L, and G.P. Sparling. 1987. The effect of air-drying and storage conditions on the amounts
of sulphate and phosphate extracted from a range of New Zealand topsoils. Cornm. Soil Sci.
Plant Anal. 18:725-734.
Tabatabaf, MA 1982. Sulfur. In A.L Page (ed.). Methods of soil analysis, Part 2. 2nd ed. Amer. Soc.
Agronomy, Madison, WI. Agronomy 9:501-538.
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Ammonium Chloride Extractable Sulphate
Introduction
North °f SU'Phate (S°^' iS the ***** anfon in 51cidic deposition in eastern
North Amenca. Concern over the long-range transport and deposition of SO.2"
eactabsin
extractable S is in the SO4 form. In contrast, in organic horizons up to i50% of the total extractabte
S may be organically-bound (Maynard et al., 1987). The ability of soils to adsorb sutohale is one
dl±-tSriPft S0'8 affeCtin9 ^ rate and 6Xtent °f S0il and waters'*d resiTe3 o ^S
deposition. Quant,f,cat,on of existing pools of adsorbed sulphate on a soil concurrent with
measurements of sulphate adsorption capacity of that soil, provide useful wSSi S
' '9 the f UtUre reSP°nse of ** «* to acidt CSn iS
'"S> theref°re' SU996Sted f°r terre«trial ^onitorin^p oghams!
Review of Methods
soils uH K °f u°ilS JS imP°rtant in the dete^'nation of extractable SO* in
soHs. Several stud,es have shown that drying significantly altered the SO,* content of the soil
part,cularly ,n organic horizons (Peverill et al, 1975; David et al, 1982; Searto and SparHnfl 1987)'
Moreover, storage of air-dried samples at room temperature (20 to &° C) or bTtweS r3 f and 78
' "
causd
C0ncentrations observed ^re not consistent among soils.
'" °r9an''C h°riZ°nS tO *» "«*««, the test shou.d be
mpleS> ArePresenta«ve sample can be obtained by mixing/homogenizing
" "" tO ana'SiS'
emed or th
recommended for the analysis of mineral soils.
mhe
than °rganic S0ils' For this raason a"d because of
samPte ^om moist samples, air-dried soils are
"u™erous exfactants have been proposed for the removal of SO*1 from soils (Beaton et al
; Tabatabai, 1982). Most of the experimental work has involved using mSS«£^^oi
in an attempt to correlate extractable SO4* with plant growth and S uptake. Many of thetcnniques
used on agncultural soils have been adapted for use on forest soils • , . tecnmques
SO - emd t " br°ken int° tw° 9rouPs based on *e fraction of
S04 removed. The two groups include those extractants that remove readily soluble SO * and
those extractants that remove readily soluble SO,* plus adsorbed SO*. The extractants used "o
remove readily soluble SO4* (H2O and weak salt solutions, such as CaCL or NH CO a"e prefefreS
for orgamc and mineral soils containing no appreciable amounts of adsc r£d SO* n contrast
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phosphate extractants, generally employing a Ca(H2POJ2 solution (500 mg P/L), are recommended
for soils containing sesquioxides, allophane, or kaolinitic clays which have the potential to adsorb
SO,2". The latter class of soils generally include the mineral horizons of most forest soils.
Eight extractants (including H2O, several weak salts, and Ca(H2PO,y were evaluated for
extractable SO,2" on five forest organic soil horizons (Maynard et al., 1987). The most consistent
extractant for SO,2- was 0.01 M NH4CI (1:10 soil to solution ratio), although all the weak salt
extractants removed similar amounts of SO,2". Water as an extractant gave the most variable
results The phosphate extractant did not remove any more SO,2" in the organic soils and gave more
variable results than the NH4CI extractant. The strong phosphate solution was also noted to reduce
the life of the ton chromatography column.
A phosphate extractant is preferred for mineral soils to ensure that strongly adsorbed SOf
is extracted. Calcium dihydrogen phosphate (Ca^PCgj solution, containing 500 mg/L P, is
preferred over Na or K phosphates since Ca enhances particle ftocculation in clayey soils and makes
filtering more convenient.
Over the last decade, the development of ion chromatography (1C) and inductively coupled
plasma atomic emission spectrometry (ICP-AES) have made the quantification of S and SO4 rapid
and more accurate. The determination of total S in a soil extract can be done by ICP-AES, but this
technique is not specific for SO,2-. Ion chromatography is, however, specific to the SO4 ion (Dick
and Tabatabai, 1979; Nieto and Frankenberger, Jr., 1985; Maynard et al., 1987) and is, therefore,
preferred for sulphate quantification in soil extracts.
Reference Method
The reference method for the extraction of readily soluble SO,* from organic soils employs a
001 M NH,CI extraction solution. Sulphate in the extract is then quantified by ion chromatography.
This method is primarily for use in the determination of extractable sulphate contents in organic so.l
horizons.
Summary of Method
Field-moist organic horizons are homogenized in a Waring blender and extracted with 0.01 M
NH4CI (approx. 2:20 air-dried soihsolution). Sulphate in the extracts is determined by suppressor or
single column ton chromatography.
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Interferences and Shortcomings
Colloidal material and certain organic compounds may interfere with the SO/ peak on the
chromatogram. Membrane filtering will help remove colloidal material and extend the life of the
guard cartridges.
As with other soluble salts in soils, the amount of SO/ extracted varies with the
soihextractant ratio. This ratio should, therefore, remain constant if samples are to be compared.
Safety
Wear protective clothing (laboratory coat and gloves) and safety glasses when preparing
reagents. Follow the safety precautions of the manufacturer when opBrating instruments.
Apparatus and Equipment
• ton chromatograph, pump and conductivity detector. Either suppressed ion chromatography
(SIC) such as the Dionex models, or single column ion chromatography (SCIC) such as the
Waters systems. •
• anion separator column and appropriate guard column.
• automated sampler and injection system and sample vials or tubes to fit sampler.
• data recording system, integrator or strip chart recorder.
• balance, accurate to 0.001 g.
• reciprocal shaker. \
• vacuum filtration apparatus and funnels. :
• vacuum membrane filter apparatus (optional).
• volumetric flask, 1 L
i
• Nalgene bottles, 60 mL
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Reagents and Consumable Materials
• ammonium chloride (NH4CI), reagent grade powder.
• sodium sulphate (Na^O^, reagent grade crystals.
• water-DI water used in all preparations should conform to ASTM specifications for Type
I reagent grade water (ASTM, 1984).
• NH4Cl extracting solution, 0.01 M-Weigh out 0.5350 g NH4CI and transfer to a 1 L volumetric
flask. Dissolve in DI and dilute to one litre.
• sulphate standard stock solution (1,000 mg/L SOJ-Weigh 1.4790 g Na2SO4 (oven
dried at 105° C and cooled in a desiccator) and transfer to a 1 L volumetric flask.
Dissolve in NH4CI extracting solution and dilute to one litre. Store refrigerated at 4°
C.
• working sulphate standards prepared from the stock solution to cover expected range of
SO^ concentrations in sample extracts. Use NH4CI extracting solution during dilution of the
standard-stock solution.
• appropriate eluent for the 1C system and anion separator column used, (see manufacturer's
recommendations).
• filter paper, Whatman #42.
• membrane filters, 0.45 pirn (optional).
Calibration and Standardization
Follow the set-up procedures outlined in the manufacturer's operating manual for the specific
instrument. Pump eluent through the system and stabilize the baseline. Adjust the recorder until
zero is approximately 10% and the high standard is approximately 90% of the chart.
Use a minimum of three standards plus a zero standard to calibrate the
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Procedure
Step 1 - Thaw sample if previously frozen.
Step 2 - Homogenize field moist sample in a Waring blender until visually homogeneous.
Step 3 - Weigh an aliquot of the f ield-moist soil that would approximate 2 g on a dry weight
basis into a 60-mL Nalgene bottle. ^ 9
a^it;°"!l^lic'uot should *» P^ared for drying and determination of the %
moisture so that SO,* can be calculated on a dry weight basis.
Step 4 - Add 20 mL of 0.01 M NH4CI extracting solution to each bottle.
Step 5 - Shake for 1 h on a reciprocal shaker at 1 to 2 cycles per second.
SUSpenslon usi"9 Whatman #42 filter paper in a Buchner
Step 7 -
Steps-
NOTE: Alternately, samples may be centrifuged until the supernatant is clear.
Vacuum filter the samples through 0.45 /«n membrane filters.
Analyze samples by ton chromatography within 24 hours.
O * transferred into vials or tubes specific to the type of
NOTE: Samples should be stored at 4° C prior to sample analysis.
Quality Control
Precision
te Sh°Uld "• ana|Vz0d in duplicate with each run of thinty samples To eliminate
° h8 '""• *e r°Utlne S3mle dup"'Cate should ** analyzed
we * fr°m duplicates
difference between the samples at an acceptance limit of a RPD * 10%.
relative
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Accuracy
Accuracy is determined by analysis of a standard reference material (SRM). Acceptable
limits for accuracy should be ±10% from the known concentration of the standard or within the
accuracy windows supplied by the reference material manufacturer, whichever is larger.
Method Blanks
Three method blanks, carried through the extraction procedure, are analyzed with each batch
of samples for each cation to measure potential contamination. Method blanks should be run at
the beginning, middle, and end of each analytical run. The concentration of each blank should be
IMS than orequal to ihe instrument detection limit. All results should be blank corrected usmg the
mean of the acceptable 'method blank readings.
Quality Control Preparation Sample
A matrix matched in-house quality control preparation sample (QCPS) should be analyzed
once per analytical run. This sample is used to monitor accuracy and long-term between-run
precision. Accuracy of the QCPS should be within ± 10% of the long-term mean. Between-run
precision can be determined by analyzing the QCPS and calculating the cumulate long-term
standard deviation. If values plotted on a control chart deviate from the long-term mean by more
than three standard deviations, the run should be completely reanalyzed, including all digestion and
quantification steps.
Quality Control Check Standard
A quality control check standard (QCCS) should be analyzed at the beginning, after every ten
samples, and after the last sample of each analytical run. The QCCS should f"^^""^
of interest with mid-calibration range concentrations. Quantified values of the QCCS should be
within ±10% of the known concentration of the standard.
It is highly recommended that the concentrations of this sample be consistent through time
so that control charts may be plotted to monitor laboratory bias and other potential problems If
analyzed values deviate from the long-term mean by more than three standard dev.at.ons, the
instrument is re-standardized and re-calibrated prior to any further sample analyses.
Suggested Run Format
QCCS, MB, Samples 1 to 8, QCPS,
QCCS, Samples 9 to 16, MB, DUP,
QCCS, Samples 16 to 25, SRM,
QCCS, Samples 26 to 30, MB, QCCS.
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where: QCCS
MB
QCPS
DUP
SRM
quality control check standard
method blank
quality control preparation sample
duplicate sample
standard reference material
Calculations and Reporting
Extractable SO,2" (mg S/kg) = SO.2- frn0/n in solution x avtra,r*
(initial sample weight (g) x moisture correction)
N01E:
foltows wiih the
[(1 - moisture content in %) + (100 + moisture content in
%)]
References
American Society for Testing and Materials. 1984. Annual Book of ASTM Standards Vol
Beaton , do G.R. Burns, and J. Platou. 1968. Determination of sulphur in soils and plant material
Technical Bull. No. 14. The Sulphur Institute, Washington, D.C. maieriai.
nH u'P- NarkUS< 1982' Organic and Inor9anic sulfur constituents of a forest
soil and their relationship to microbial activity. Soil Sci. Soc. Am. J. 46:529-536.
Dick, W A, and M.A, Tabatabai. 1979. Ion chromatographic determination of £ ulfate and
nitrate in soils. Soil Sci. Soc. Am. J. 46:847:852.
Maynard, D.G., Y.P. Kalra, and F.G. Radford. 1987. Extraction and determination of sulfur
in organic horizons of forest soils. Soil Sci. Soc. Am. J. 51:801-806.
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Nfeto, K.F., and W.T. Frankenberger, Jr. 1985. Single column ion chromatography: I. Analysis of
inorganic antons in soils. Soil Sci. Soc. Am. J. 49:587-592.
Peverill, K.I., GP. Briner, and LA. Douglas. 1975. Changes in extractable sulphur and potassium
levels in soil due to oven drying and storage. Aust. J. Soil Res. 13:69-75.
Searle P L and G P Sparling. 1987. The effect of air-drying and storage conditions on the amounts
J^S^^o^ extracted from a range of New Zealand topsoi.s. Comm, So,. Sa.
Plant Anal. 18:725-734.
Tabatabai, MA 1982. Sulfur. In A.L Page (ed.). Methods of soil analysis, Part 2. 2nd ed. Amer. Soc.
Agronomy, Madison, WI. Agronomy 9:501-538.
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Water Extractable Sulphate
Introduction
Review of Methods
The preparation and storage of soils is important in the determination of extractable SO * in
££ , , StUdl6S ^ Sh°Wn that dfyin9 Si9"^ant.y altered the SO ? ^3^ the so"
part,cularly ,n orgamc horizons (Peverill et al., 1975; David et al., 1982; Sear te a~d SparHna 1987?'
Moreover, storage of air-dried samples at room temperature (20 to 25° Q f I tetwee ^12 aid 78
from moist
^
those extractants that remove readily soluble SO/ plus adsorbed SO/ The Lractants used to
remove read.ly soluble SO/ (HaO and weak salt solutions, such as S(^ « (JSS^ pSS£
for organ,c and mineral soils containing no appreciable amounts of adsorbe SO * n contrast
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phosphate extractants, generally employing a OOtfOJ. solution (500 mg
foTsoils containing sesquioxides, allophane, or kaolinitic clays wh,ch have the potonbaT to adsorb
. The latter class of soils generally include the mineral horizons of most forest so.ls.
Eight extractants (including H2O, several weak salts, and Ca^POJJ were evaluated for
extractabte SO * on five forest organic soil horizons (Maynard et al., 1987). The most cons.stent
eSracant for So" wls 0.01 M NH4CI (1:10 soil to solution ratio), although all the weak salt
SSSl^Sid^nltar amounts of W Water as an extractant gave the <™*™*»
Results. The phosphate extractant did not remove any more SO.- in the orgamc so.ls ^ gave more
viable results than the NH4CI extractant. The strong phosphate solufon was also noted to reduce
the life of the ion chromatography column.
A phosphate extractant is preferred for mineral soils to ensure that strongly adsorbed SO£
is extracted. Calcium dihydrogen phosphate (Ca(H2POM solution, conta,n,ng 500 mg/L P ,s
^rred over Na or K phosphates since Ca enhances particle f locculation ,n clayey so.ls and makes
filtering more convenient.
Over the last decade, the development of ion chromatography (1C) and i
plasma atomic emission spectrometry (ICP-AES) have made »a quaflMntion. of
and more accurate. The determination of total S in a soil extract can be done by
technique is not specific for SO4*. Ion chromatography is, however, specrf ,c to the
and Tabatabai, 1979; Nieto and Frankenberger, Jr., 1985; Maynard et al, 1987) and ,s, therefore,
preferred for sulphate quantification in soil extracts.
Reference Method
ssr
^
for organic horizons bearing in mind the method limitations ,nd,cated .n the "Review of
Methods" section.
Summary of Method
Air-dried mineral horizon samples, which have been disaggregated and passed through a 2-mm
sieve arf exacted with deionized water (1:20 air-dried soihwater). Sulphate in the extracts »
determined by suppressor or single column ion chromatography.
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Interferences and Shortcomings \
Colloidal material and certain organic compounds may interfere with the SO,* peak on the
chromatogram. Membrane filtering will help remove colloidal material and extend the life of the
guard cartridges.
As with other soluble salts in soils, the amount of SO^ extracted varies with the
soil:extractant ratio. This ratio should, therefore, remain constant if samples are to be compared.
Safety
Wear protective clothing (laboratory coat and gloves) and safety glasses when preparing
reagents. Follow the safety precautions of the manufacturer when operating instruments.
Apparatus and Equipment
• ion chromatograph, pump and conductivity detector. Either suppressed ion chromatography
(SIC) such as the Dionex models, or single column ion chromatography (SCIC) such as the
Waters systems.
• anion separator column and appropriate guard column. •
• automated sampler and injection system and sample vials or tubes- to fit sampler.
• data recording system, integrator or strip chart recorder.
• balance, accurate to 0.001 g.
• reciprocal shaker.
• vacuum filtration apparatus and funnels.
• vacuum membrane filter apparatus (optional).
• volumetric flask, 1 L
• Nalgene bottles, 100 ml_.
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Reagents and Consumable Materials
• sodium sulphate (Na2SOJ, reagent grade crystals.
• water-DI water used in all preparations should conform to ASTM specifications for Type
I reagent grade water (ASTM, 1984).
• sulphate standard stock solution (1,000 mg/L SOJ-Weigh 1.4790 g Na2SO4 (oven
dried at 105° C and cooled in a desiccator) and transfer to a 1 L volumetric flask.
Dissolve in DI water and dilute to one litre. Store refrigerated at 4° C. •
• working sulphate standards prepared from the stock solution to cover expected range of
SO,,2" concentrations in sample extracts.
• appropriate eluent for the 1C system and anion separator column used, (see manufacturer's
recommendations).
• filter paper, Whatman #42.
• membrane filters, 0.45 pm (optional).
Calibration and Standardization
Follow the set-up procedures outlined in the manufacturer's operating manual for the specific
instrument. Pump eluent through the system and stabilize the baseline. Adjust the recorder until
zero is approximately 10% and the high standard is approximately 90% of the chart.
Use a minimum of three standards plus a zero standard to calibrate the system. The
standards are analyzed and a calibration curve produced by plotting peak area or height against
concentration. The concentration of standards should bracket the expected sample concentration;
however, the linear range of the instrument should not be exceeded. The calibration curve should
be close to linear. Calibration standards should be prepared fresh daily. Calibration standards
should be prepared in the extraction solution, in this case DI water.
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Procedure
;i
Step 1 - Weigh 4.00 g of air-dried mineral soil into a 100-mL Nalgene bottle.
NOTE: If this method is to be used for an organic soil horizon, homogenize the sample
in a Waring blender until visually homogeneous and weigh a 2.00 gram sample into the
100-mL Nalgene bottle.
NOTE: An additional aliquot should be prepared for drying and determination of the %
moisture so that SO*2" can be calculated on a dry weight basis. '
Step 2 - Add 80 mL of DI water to each bottle.
Step 3- Shake for 1 h on a reciprocal shaker at 1 to 2 cycles per second.
Step 4 - Vacuum filter the resulting suspension using Whatman #42 filter paper in a Buchner
funnel.
NOTE: Alternately, samples may be centrifuged until the su|Dernatant is clear.
Step 5 - Vacuum filter the samples through 0.45 /urn membrane fliters.
Step 6 - Analyze samples by ion chromatography within 24 hours.
NOTE: Samples may have to be transferred into vials or tubes specific to the type of
sampler used.
NOTE: Samples should be stored at 4° C prior to sample analysis.
Quality Control
Precision
One sample should be analyzed in duplicate with each run of thirty samples. To eliminate
bias due to position in the run, the routine sample duplicate should be analyzed separately within
the analytical run. Within-run precision is determined from duplicates based on relative percent
difference between the samples at an acceptance limit of a RPD ** 10%
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Accuracy
Accuracy is determined by analysis of a standard reference material (SRM). Acceptable
limits for accuracy should be ±10% from the known concentration of the standard or within the
accuracy windows supplied by the reference material manufacturer, whichever is larger.
Method Blanks
Three method blanks, carried through the extraction procedure, are analyzed with each batch
of samples for each cation to measure potential contamination. Method blanks 'should be run at
the beginning, middle, and end of each analytical run. The concentration of each blank should be
tess than or equal to the instrument detection limit. All results should be blank corrected using the
mean of the acceptable method blank readings.
Quality Control Preparation Sample
A matrix matched in-house quality control preparation sample (QCPS) should be analyzed
once per analytical run. This sample is used to monitor accuracy and long-term between-run
precision. Accuracy of the QCPS should be within ± 10% of the long-term mean. Between-run
precision can be determined by analyzing the QCPS and calculating the cumulative long-term
standard deviation. If values plotted on a control chart deviate from the long-term mean by more
than three standard deviations, the run should be completely reanalyzed, including all digestion and
quantification steps.
Quality Control Check Standard
A quality control check standard (QCCS) should be analyzed at the beginning, after every ten
samples, and after the last sample of each analytical run. The QCCS should contain all the analytes
of interest with mid-calibration range concentrations. Quantified values of the QCCS should be
within ±10% of the known concentration of the standard.
It is highly recommended that the concentrations of this sample be consistent through time
so that control charts may be plotted to monitor laboratory bias and other potential problems. If
analyzed values deviate from the long-term mean by more than three standard deviations, the
instrument is re-standardized and re-calibrated prior to any further sample analyses.
Suggested Run Format
QCCS, MB, Samples 1 to 8, QCPSt
QCCS. Samples 9 to 16, MB, DUP,
QCCS, Samples 16 to 25, SRM,
QCCS, Samples 26 to 30, MB, QCCS.
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where: QCCS
MB
QCPS
DUP
SRM
quality control check standard
method blank
quality control preparation sample
duplicate sample
standard reference material
Calculations and Reporting
The concentration of SO/*" is calculated by reading the peak area (or height) of the samples
against the standard calibration curve. The calculation program used should take into account
sensitivity changes throughout the run as detected by the in-run standard. All sample results are
method blank subtracted.
For mineral soils, calculate extractable sulphate concentrations as follows:
Extractable SO^ (mg S/kg) = SQf (mg/L) in solution x extract volume (mL)
dry soil weight (g)
For organic samples, the sample weight must be based on % moisture in the field-moist
sample. Therefore, the following formula is applicable:
Extractable SO4*" (mg Sykg) = SO,2" (mg/L) in solution x extract volume (mL)
(initial sample weight (g) x moisture correction)
NOTE: The moisture correction factor is as follows with the moisture content presented as
a fraction of the whole (e.g., 0.75): '•
[(1 - moisture content in %) •*• (100 + moisture content in %)]
References
American Society for Testing and Materials. 1984. Annual Book of ASTM Standards, Vol. 11.01,
Standard Specification for Reagent Water, D1193-77 (reapproved 1983). ASTM, Philadelphia,
Pennsylvania.
Beaton, J.D., G.R. Burns, and J. Platou. 1968. Determination of sulphur iin soils and plant material.
Technical Bull. No. 14. The Sulphur Institute, Washington, D.C.
David, M.B., M.J. Mitchell, and J.P. Narkus. 1982. Organic and inorganic sulfur constituents of a forest
soil and their relationship to microbial activity. Soil Sci. Soc. Am. J. 4(3:529-536.
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Dick, W.A., and MA Tabatabai. 1979. Ion chromatographic determination of sulfate and
nitrate in soils. Soil Sci. Soc. Am. J. 46:847:852.
Maynard, D.G., Y.P. Kalra, and F.G. Radford. 1987. Extraction and determination of sulfur
in organic horizons of forest soils. Soil Sci. Soc. Am, J. 51:801-806.
Nieto, K.F., and W.T. Frankenberger, Jr. 1985. Single column ion chromatography: I. Analysis of
inorganic antons in soils. Soil Sci. Soc. Am. J. 49:587-592.
Peyerill, K.I., G.P. Briner, and LA. Douglas. 1975. Changes in extractable sulphur and potassium
levels in soil due to oven drying and storage. Aust. J. Soil Res. 13:69-75.
Saarte, P.L, and G.P. Sparling. 1987. The effect of air-drying and storage conditions on the amounts
of sulphate and phosphate extracted from a range of New Zealand topsoils. Comm. Soil Sci.
Plant Anal. 18:725-734.
Tabatabai, MA 1982. Sulfur. In AL Page (ed.). Methods of soil analysis, Part 2.2nd ed. Amer. Soc.
Agronomy, Madison, WI. Agronomy 9:501-538.
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Total Sulphur
Introduction
Sulphur is an essential element for all biological systems. Historically, its importance as a
plant nutrient and reports of widespread deficiencies, especially in sub-humid areas of intensive
leaching, has made it a routine measurement in some laboratories. Most of the sulphur in surface
soils occurs in combination with organic matter (Freney and Williams, 1983). Inorganic SO^-S,
although it comprises a small fraction of the total S in most soils, is important to the cycling of S
in the soil.
Concern over the long-range transport and deposition of SO^ in precipitation has lead to
increased monitoring of the sulphur status of soils. The determination of total sulphur is useful for
characterizing relationships between inputs of sulphur from acidic deposition and soil sulphur pools.
Fundamental to studying the effects of strong acid precipitation on terrestrial and aquatic systems
is an understanding of the behaviour of sulphur in these systems. The measurement of total S is,
therefore, suggested for terrestrial monitoring programs.
i
Review of Methods
It has only been within the last 15 years that the difficulty of accurately measuring S in soils
has been overcome. Recent advances in analytical techniques have resulted in the accurate and
precise measurement of S in various types of soils (Dick and Tabatabai, 1979; Hogan and Maynard,
1984; Nieto and Frankenberger, Jr., 1985; Maynard et al., 1987). Several methods are routinely used
for the determination of S in environmental samples. These may be divided into two groups, namely,
those involving wet oxidization of the sample and those which involve direct sample analysis (Hogan
and Maynard, 1984).
Methods available for the wet oxidation of organic materials are well documented (Beaton et
al., 1968; Tabatabai, 1982; Blanchar, 1986). Acid and alkaline oxidation are the most common
(Blanchar et al., 1965; Tabatabai and Bremner, 1970), as they are dependable, accurate and relatively
rapid (Blanchar, 1986). Full recovery from an acid digestion usually requires the use of perchloric
acid. The danger associated with its use and the special facilities required have meant that, until
recently, acid digestions have been avoided. The recent adaptation of microwave ovens for use in
the laboratory has led to the development of microwave acid oxidation digestion techniques for
foliage and soils which successfully use hydrogen peroxide in place of perchloric acid. Acid
digestions for the measurement of total S in soils requires the use of hydrofluoric acid to destroy
the silicate matrix and ensure complete recovery of S. A HNO3-HCIO4-HF mixture is commonly used.
For this reason, many analysts have preferred the safer, more rapid, dry combustion techniques.
The wet oxidation technique converts S to SO/1" and produces a solution which can be
analyzed by a variety of methods. Turbidimetry is insensitive, lacks precision and is subject to
numerous interferences (Beaton et al., 1968). Colourimetric methods such as the methylene blue
technique (Technicon, 1972) also have limited application in soils and plant analysis because of
interferences by major nutrient cations (Maynard et al., 1987). The colourimetric method developed
by Johnson and Nishita (1952) was found to be the most sensitive and accurate of the colourimetric
procedures. Ion chromatography has not been used extensively for the measurement of total S, but
has been shown to be an excellent method for the determination of SO42" and its companion anions,
as well as for cation determinations, in waters and soil extracts.
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Recent publications (Hogan and Maynard, 1984; White and Douthit, 1985; Novozamsky et al.f
1986) have shown that the accurate and precise measurement of S is possible by ICP-AES in a
range of environmental samples. Precision is estimated at ± 2%. Results of analysis of NIST plant
material and sediments demonstrated that the precision and accuracy obtained by ICP-AES are
equal to or better than any other technique currently used. The ICP-AES has several advantages
over other methods. It is rapid, flexible, has a dynamic range, is free from interferences and permits
simultaneous multielement analysis. These factors make it a preferred method of analysis for
laboratories possessing ICP-AES capabilities.
Direct analysis of the sample may be done by combustion of the sample at elevated
temperatures (generally at 1000° C or higher) and measuring the liberated SO, by an infrared
detector. Examples of instruments that combust the sample include the LECO combustion furnace,
Carla-Erba combustion furnace, or the Fisher S analyzer. These methods require little or no sample
preparation for the determination of total S.
Additionally, X-ray fluorescence (XRF) may be used to quantify soil S along with many other
elements. The method has been used to a lesser extent on soils than the aforementioned methods
(tabatabai and Bremner, 1975). The use of XRF technology is, however, a tedious, time consuming
process. Sample preparation may include sample fusion into a borate glass disk or pressing the
sample into a pellet under high pressure. Unfortunately, without fusion of the sample into a glass,
the natural variability in the sample matrix causes difficulty in obtaining representative standards
and can result in interelement interferences. Finally, the expense of the instrumentation makes this
method an unfeasible option for many laboratories.
The LECO S analyzer was originally developed for the determination of S in steel, but because
of its simplicity, speed, and convenience, it has been adapted for use in soil and plant analysis
(Tabatabai, 1982). An initial evaluation of this method by Tabatabai and Bremner (1970) showed
total S results to be unsatisfactory for research that required accurate and precise determinations.
The Ontario Ministry of the Environment has found that the infrared detection system on the LECO
gave unacceptable results for highly organic soil samples.
Reference Method
The reference method for total S in soil samples is dry combustion in a LECO sulphur analyzer
with infrared detection. The analysis of S by LECO-S analyzer has been chosen as a reference
method because it is widely used in North America and has been used successfully by some
laboratories for both soil and foliage samples. However, the use of other manufacturer's
instruments for total sulphur determinations is acceptable.
Summary of Method
An air-dried and finely ground soil sample is heated with an accelerator to 1600° C in a stream
of high purity oxygen. The released sulphur is converted to sulphur dioxide and is detected by
infrared detector (Hem, 1984).
Interferences and Shortcomings
Large amounts of carbon can prevent proper ignition of the sample. With incomplete
combustion, a poor recovery of S may be obtained. This may be overcome by reducing the sample
size and adding LECO Iron Chip Accelerator plus tin. Preashing of highly organic soils at 475 to
500° C for two hours may be performed, but preliminary studies should be. done on the type of
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samples to be analyzed to determine whether a toss of S occurs during this ashing. Preashed
samples also require accelerator to overcome interference from residual carbon.
Moisture deposits on the walls of the delivery tubes or the surface of the dust filter will absorb
SOj. Tnismay be overcome by using magnesium perchlorate between the dust filter and the original
Interferences from nitrogen may be overcome by increasing tlie oxygen flow rate to 1.5
litres/minute.
Safety
Normal safety precautions should be taken when using high-tempeirature combustion furnaces.
Protective clothing and safety glasses should be worn when handling reagents. Heat resistant
gloves may be needed when placing samples in the furnace. The furnace must be adequately
vented and protected from human contact and combustible materials. Gias cylinders should be
bolted or chained in an upright position.
Fumes of magnesium oxide are toxic. Magnesium perchlorate is a fire and explosion hazard
if it comes in contact with organic materials.
Apparatus and Equipment
• Sulphur analyzer with infrared detector, LEGO model SC-132, or equivalent.
• Balance, accurate to 0.001 g.
• LEGO scoop.
j . :.
Reagents and Consumable Materials
• Oxygen, high purity.
t
• Compressed air, jf needed.
• Anhydrous magnesium perchlorate (Mg(CIOJ2, 10-20 mesh, or equivalent desiccant specified
by manufacturer for drying gases after combustion and prior to detection.
• Magnesium oxide, MgO, reagent grade powder, tow in sulphur.
• LEGO combustion boats, sulphur-free and appropriate for use with the equipment used.
• Accelerators, vanadium pentoxide, iron chips, and/or copper metal
Calibration and Standardization t
Set up the instrument according to the LECO operating manual. In general, the instrument
should be calibrated at least once a day or once per batch of samples, whichever is more frequent.
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Use either NIST (formerly NBS) reference materials or standards supplied by the manufacturer and
approved by the laboratory or QA manager. The concentration range of the standards must be
representative of the C concentrations expected in the soil samples. A minimum of a two-point
calibration curve should be used. Use of a NIST standard reference material as an initial calibration
check is highly recommended.
Some suggested calibration standards and reference samples include: LEGO brand iron
powder (0.036% S), LEGO coal calibration standard (2.56% S), NIST (NBS) coal reference material
(1.89% S), SU-1A nickel-copper-cobalt ore (9.35% S), and CCU-1 reference material (35.4% S).
Ensure that the anhydrone is dry and the dust filter is clean. Prior to analyzing samples, the
Instrument is conditioned by running low level calibration standards until the results are stabilized
to within 5%. Once stable, three blank crucibles (accelerator only) are analyzed followed by three
standards.
Procedure - Infra-red Detection
Step 1 - Weigh out 0.250 g air dried and finely ground soil into a LEGO combustion boat. Record
sample weight.
Step 2 - Preset power settings on induction furnace according to the manual.
Step 3 - Ignite the sample in the crucible for the suggested time period. Seven minutes has been
found to work well for soils but this should be determined for the range of soil types
specific to the laboratory.
Step 4 - Take a sulphur measurement reading.
QualityControl
Precfsfon
One sample from each batch should be analyzed in duplicate. Within-run precision is
determined from duplicates based on relative percent difference (RPD) between the samples with
an acceptance limit of a RPD £ 10%.
Accuracy
Accuracy is determined by analysis of a standard reference material (SRM). Acceptable limits
for accuracy should be ±10% from the known concentration of the standard or within the accuracy
windows supplied by the reference material manufacturer, whichever is larger.
Method Blanks
Two blank crucibles (accelerator only) are analyzed before the run and one blank crucible is
also analyzed in the middle and at the end of each analytical run. The concentration of each blank
should be less than or equal to the instrument detection limit. All results should be blank corrected
using the mean of the acceptable method blank readings.
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Quality Control Preparation Sample
A matrix matched in-house quality control preparation sample (QCPS) should be analyzed once
per analytical run. This sample is used to monitor accuracy and tang-term between-run precision.
Accuracy of the QCPS should be within ± 10% of the tang-term mean. Between-run precision can
be determined by analyzing the QCPS and calculating the cumulative long-term standard deviation.
If values plotted on a control chart deviate from the tang-term mean by more than three standard
deviations, the run should be completely reanalyzed, including all digestion and quantification steps.
Suggested Run Format
MB, MB, Samples 1 to 8, QCPS,
Samples 9 to 16, MB, DUP,
Samples 16 to 25, SRM,
Samples 26 to 30, MB.
where: MB
QCPS
DUP
SRM
method blank
quality control preparation sample
duplicate sample
standard reference material
Calculations and Reporting
Infra-red system: % S is read directly from the instrument. The sample weight, as different
from the standard, is taken into account on some instruments.
Results are reported to two significant figures. Results are read to the nearest 0.001%.
Results should be blank corrected.
References \
Beaton, J.D., G.R. Burns and J. Platou. 1968. Determination of sulphur in soils and plant material.
Technical Bull. No. 14, The Sulphur Institute, Washington, D.C.
Blanchar, R.W. 1986. Measurement of sulfur in soils and plants. In MA Tabatabai (ed.). Sulfur in
agriculture. Amer. Soc. Agronomy. Madison, WI. Agronomy 27:457:490.
Dick, W.A. and M.A. Tabatabai. 1979. Ion chromatographic determination of sulfate and nitrate in
soils. Soil Sci. Soc. Am. J. 46:847:852.
Freney, J.R. and C.H. Williams. 1983. The sulphur cycle in soil. p. 139-201. In M.B. Ivanovand J.R.
Freney (eds.). The global biogeochemical sulphur cycle. SCOPE Rep. no. 19. John Wiley and
Sons, New York, NY.
Hern, J.L 1984. Determination of total sulfur in plant materials using an automated sulphur analyzer.
Comm. Soil Sci. Plant Anal. 15:99-107.
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Hogan, G.D. and Maynard, D.G. 1984. Sulphur analysis of environmental materials by vacuum
inductively coupled plasma atomic emission spectrometry (][CP-AES). p. 676-683 Ip Proc.
Sulphur-84, Int. Conf., Calgary, AJberta. June 1984. The Sulphur Development Institute of
Canada, Calgary.
Johnson, C.M. and H. Nishita. 1952. Microestimation of sulphur in plant materials, soils and irrigation
waters. Anal. Chem. 24:736-742.
Maynard, D.G., Y.P. Kalra and F.G. Radford. 1987. Extraction and determination of sulfur in organic
horizons of forest soils. Soil Sci. Soc. Am. J. 51:801-806.
Nieto, K.F. and W.T. Frankenberger, Jr. 1985. Single column ion chromatography: I. Analysis of
inorganic anions in soils. Soil Sci. Soc. Am. J. 49:587-592.
Novozamsky, I., R. van Eck, JJ. van der Lee, V.J.G. Hauba and E. Temminghoff. 1986. Determination
of total sulphur and extractable sulphate in plant materials by inductively-coupled plasma
atomic emission spectrometry. Comm. Soil Sci. Plant Anal. 17:1147-1157.
Tabatabai, MA 1982. Sulfur. In A.L Page (ed.). Methods of soil analysis. Part 2. 2nd ed.
Agronomy 9:501-538.
Tabatabai, M.A. and J.M. Bremner. 1970. Comparison of some methods for determination of total
sulfur in soils. Soil Sci. Soc. Am. Proc. 34:417-420.
Tabatabai, M.A. and J.M. Bremner. 1975. An alkaline oxidation method for the determination of total
sulfur HI soils. Soil Sci. Soc. Am. Proc. 34:62-65.
Technfcon. 1972. Sulfate in water and wastewater. Industrial method no. 118-71W. Technicon
Industrial Systems, Tarrytown, NY.
White, Jr., R.T. and G.E Douthit. 1985. Use of microwave oven and nitric acid-hydrogen peroxide
digestion to prepare botanical materials for elemental analysis by inductively coupled argon
plasma emission spectroscopy. J. Assoc. Off. Anal. Chem. 68:766-769.
* IT.S. GOVERNMENT PRINTING OFFICE: 1995 - 650-006/2205&
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