-------
0.700
FIGURE 10
0.600
0.500
0.400
0.300
0.200
,u_i
0.100
U_i.
0.000
50.0
45.0
40.0
35.0
25.0
30.0
15.0
20.0
10.0
0.0
5.0
Page No. ?-15
-------
Page No. 2-16
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
FIGURE
1
i
1-
/
>
1
-
1
1 1 1
1 ~
§'
1
, m?(
-
• t 1
1
i
MM
|
1 .
0.0 5.0 10.0 T5.0 20.0 25.0 30.0 35.0 40.0 45.0 50 *
-------
EFFLUENT MONITORING PROCEDURE: Preparation of Calibration Graphs
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Graph Paper
1. General comments
2. Labeling the
graph paper
1. Remove the page containing
figure 1.
2. Lay it on a desk or any
other place where it will
be convenient for you to
write on it.
1. Draw two lines on figure 1
so that it looks like
figure 2.
2. Label figure 1 so that it
looks 1 ike figure 3.
2a. For the remainder of this procedure, you will
actually use figure 1 and some example absorbance
and concentration values to prepare a calibration
graph. Additional figures are also included to
demonstrate the instructions.
2b. You will have to furnish your own piece of graph
paper when you want to prepare other calibration
graphs.
la. Use a pencil, since you may have to do some
erasing during the preparation of the calibration
graph.
2a. mg/1 stands for milligrams per liter. It is
an expression of concentration. If the amount of
chemical constituent present in the sample is
extremely small, the label yg/1 (micrograms per
liter) might be used. A stands for absorbance.
2b. The mg/1 line is a horizontal line. It is called
the X axis, or abscissa. The A line is called the
Y axi s, or ordi nate.
Page No. 2-17
-------
EFFLUENT MONITORING PROCEDURF: Preparation of Calibration Graphs Pa9e No. 2-18
OPERATING PROCEDURES
A. Graph Paper
(continued)
STEP SEQUENCE
3. Examine the example absorb-
ance and concentration
values in the column at
the right.
4. Note that the lowest mg/1
value is 0.0 and the high-
est is 50.0.
5. Mark the mg/1 axis on
figure 1 so that it looks
like figure 4.
INFORMATION/OPERATING GOALS/SPECIFICATIONS
3a.
mg/1
A
0.0
0.000
5.0
0.060
10.0
0.120
20.0
0.250
30.0
0.340
40.0
0.470
50.0
0.590
A of sample = 0.180
3b. It is data for a series of standards.
3c. Each pair of values (e.g. 5.0 and 0.060)
represents a point on the graph.
3d. Later, you will complete the calibration graph by
drawing a straight line through the seven points.
TRAINING
GUIDE NOTES
5a. Note that the entire length of the mg/1 axis was
used. Always use as much of this line as is
convenient. Do not, for example, use only one-
half of the mg/1 axis to mark off the values.
5b. Also note that each of the large squares is marked
as a whole number of mg/1.
5c. Two of the smaller squares equal 1 mg/1.
-------
EFFLUENT MONITORING PROCEDURE: Preparation of Calibration Graphs
OPERATING PROCEDURES
STEP SEQUENCE
——1——————————1
INFORMATION/OPERATING GOALS/SPECIFICATIONS
I RAINING
GUIDE NOTES
A. Graph Paper
(continued)
6. Note that the lowest A
value is 0.000 and the
highest is 0.590.
7. Mark the A axis on figure
1 so that it looks like
figure 5.
6a. It is generally not considered good practice to
have A values greater than 0.6 or 0.7.
7a. Note that the entire length of the A axis was used-
Always use as much of this line as convenient.
Do not, for example, use only one-half of the A
axis to mark off the values.
7b. Also note that each of the large squares is marked
as a whole number of A units.
7c. One of the smaller squares equals 0.01 A units.
7d. If transmittance measurements were being made,
the Y axis or ordinate, would be marked T. T axes
are always marked from 0 (bottom of axis) to
100 (top of axis).
3. Drawing the
calibration graph
1. On figure 1 draw a vertical
line from the 50.0 mg/1
point of the mg/1 axis to
the top of the graph.
2. On figure 1 draw a horizon-
tal line from the 0.590
point of the A axis to the
right side of the graph.
la. Figure 1 should now look like figure 6.
2a. Figure 1 should now look like figure 7.
2b. The intersection of these two lines is the point
represented by a concentration of 50.0 mg/1
and an absorbance of 0.590.
Page No. 2-19
-------
^EFFLUENT MONITORING PROCEDURE: Preparation of Calibration Graphs
Page No. 2-20
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Graph Paper
(conti nued)
3. Using the same technique as
in 1 and 2 above, locate
the next five points on
figure 1.
4. Lay your ruler on figure 1.
5. Look along the edge of
the ruler.
6. Draw a line between the
0.0 - 0.000 and the 50.0 -
0.590 points.
3a. The point located at 0.0 and 0.000 is at the
intersection of the mg/1 and A axes.
3b. Your graph should now look like figure 8. Some
analyses may require more than five points.
4a. So one end of it lies at the 0.0 - 0.000 point,
and at the 50.0 - 0.590 point.
5a. The other five points (represented by the inter-
sections of the horizontal and vertical lines do
not all lie along the edge of the ruler.
6a. Note that some of the points lie slightly above
the line, some lie slightly below the line, and
some lie on the line. If one point is consider-
ably off the line, some error in preparing the
particular standard was probably made.
6b. This is the line of best fit for the seven points.
Always draw the line of best fit when preparing
calibration graphs.
6c. The calibration graph is now complete.
6d. Figure 1 should now look like figure 9.
6e. After you have prepared a few calibration graphs,
you will find that you won't have to draw the
horizontal and vertical lines to locate the points.
You'll be able to move your pencil along the graph
paper and put dots at the appropriate points.
You'll then draw the line of best fit through them
to the 0.0 - 0.000 point.
-------
,E^nLU^NTBiiiMU^^1^0RJ_NG__PiR0C>EDyiR^: preparation of Calibration Graphs
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
Determining the
Concentration of the
Chemical Constituent
in the Sample.
C. Sample Dilution
1. Locate 0.180 on the A axis.
2. Draw a horizontal line to
the right side of the paper
3. Locate the intersection of
this horizontal line and
the sloping calibration
graph.
4. From this intersection,
draw a vertical line down
to the bottom of the paper.
5. Note that the vertical line
crosses the mg/1 axis at
15.3.
If it was necessary to
dilute the sample, the
value read from the mg/1
axis must be multiplied by
a dilution factor.
la. This was the absorbance of the sample.
2a. It should now look like figure 10.
4a. It should now look like figure 11.
5a. Recall that on the mg/1 axis, 2 of the small
squares equal 1 mg/1.
5b 15,3 mg/1 is therefore the concentration of the
chemical constituent being measured in the sample.
la. The dilution may have been necessary so that the
A value for the sample would not be greater than
the A value obtained for the highest concentration
standard; 0.590 in this set of example data.
lb. The dilution factor is the ml of sample taken
for dilution, divided into the ml to which it was
diluted; e.g., if 10.0 ml of the original sample
were diluted to 1000 ml (as in a volumetric flask)
the dilution factor would be 1000/10, or 100.
lc. In some determinations, you may prepare more than
one dilution of the sample. Look at the mq/1
axis of figure 1 and assume that three dilutions
of the sample gave values of 2.2, 24.0, and 48.0
mg/1 , before correcting for the dilution factor.
It is common practice to use the 24.0 value,
since it lies nearest the middle of the calibra-
tion graph.
Page No. 2-21
-------
A PROTOTYPE FOR DEVELOPMENT OF
ROUTINE OPERATIONAL PROCEDURES
for the
DETERMINATION OF TOTAL PHOSPHORUS (as P)
OR OF ORTHOPHOSPHATE (as P), SINGLE REAGENT METHOD
as applied in
WASTEWATER TREATMENT FACILITIES
and in the
MONITORING OF EFFLUENT WASTEWATERS
National Training Center
Municipal Operations and Training Division
Office of Water Program Operations
U.S. Environmental Protection Agency
CH.PHOS.EMP.la.3.76
Page No. 3-1
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
This Operational Procedure was developed by:
NAME Timothy R. Counts
ADDRESS Water and Wastewater Technical School, Box 370,
Neosho, Missouri 64850
POSITION Chemist-Instructor
EDUCATION AND TECHNICAL BACKGROUND
B.S. Chemistry, Missouri Southern State College
Missouri Secondary Level Teacher's Certificate, Chemistry
2 years Industrial Laboratory Technician
1 year Water and Wastewater Technical School,
Wastewater Laboratory Analyst
Page No.
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
1. Objective:
To determine orthophosphate, mg P/liter or total phosphorus, mg P/liter.
2. Description of Analysis:
Orthophosphate* in dilute solution will react with ammonium molybdate and
antimony potassium tartrate to form a heteropoly acid. This acid is re-
duced to an intensely blue-colored complex, molybdenum blue, by ascorbic
acid with the amount of blue produced being proportional to the amount
of orthophosphate present.
In the procedure this is accomplished by the addition of a combined reagent
to a 50 ml sample and a set of orthophosphate standards, followed by a wait
for color development. A photometer or spectrophotometer is used to measure
the absorbance of the samples and standards. The orthophosphate concentra-
tions of samples are read directly from a graph prepared by plotting the
absorbance values of the standards against their concentration.
This analytical procedure utilizes reactions that are specific for the
orthophosphate ion. In order to obtain the total phosphorus concentrations
of samples, all non-orthophosphate phosphorus forms must be converted to
the orthophosphate ion. In the procedure this is accomplished by digesting
samples with ammonium persulfate and sulfuric acid. This step does not
affect the original orthophosphate content of the sample, but ensures con-
version of all other forms of phosphorus to orthophosphate. Direct
orthophosphate colorimetry may then be performed on the sample as described
in the preceding paragraph, and the results obtained reported as total
phosphorus, mg P/liter.
*The orthophosphate ion, (P0^)E ion, is the smallest and simplest of the
phosphorus-oxygen radicals. It consists of four oxygen atoms tetrahedrally
arranged around and bonded to a central phosphorus atom. The more complex
and commercially important phosphates, the poly or multiphosphates
(P2°7* P3°l0* etc')' are typically formed by linking orthophosphate units.
The term "phosphate" is a general one and may apply to any one of hundreds
of compounds. The {P0^)= ion is distinguished by the prefix "ortho" and
is correctly called the orthophosphate ion.
3. Applicability of this Procedure:
a. Range of Concentration:
0.01 to 1.00 mg P/liter
(The range may be extended for samples by dilution.)
Page No. 3-4
-------
AFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
b. Pretreatment of Samples:
This procedure includes the persulfate digestion for the total Phosphorus
determination as specified in the Federal Register Guidelines. These
Guidelines do not specify any pretreatment for the orthophosphate
determination.
c. Treatment of Interferences in Samples:
This procedure includes directions for removal of turbidity or suspended
solids from samples for the orthophosphate determination. It also includes
the modification to prevent adsorption of phosphorus on metal precipitates
in samples for the total phosphorus determination as publicized in the
"Changes and Errata. . ." for the Source of Procedure*. For either de-
termination it includes the treatment for samples which have been preserved
with mercury chloride. Arsenate is the one additional interference listed
in the Source of Procedure*. No remedy for its presence is currently
available, but one should be aware that arsenate also responds to this
analysis and can contribute to erroneously high phosphorus values.
*Source of Procedure: Methods for Chemical Analysis of Water and Wastes,
1974, Environmental Protection Agency, Methods Development and Quality
Assurance Research Laboratory, Cincinnati, Ohio, page 249.
Page No. 3-5
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
FLOW SHEET OF OPERATING PROCEDURES
Total Sample (No Filtration)
Direct
Colorimetry
Persulfate
Digestion
Colorimetry
Hydrolysis &
Colorimetry
Filter (through a 0.45 micron filter)
Direct
Colorimetry
Persulfate
Digestion &
Colorimetry
H2S04
Hydrolysis
& Colorimetry
Residue
Sample
Filtrate
Total
Phosphorus
Hydrolyzable &
Orthophosphate
Orthophosphate
Dissolved
Orthophosphate
Dissolved Total
Phosphorus
Dissolved Hydrolyzable
& Orthophosphate
This EMP includes only the material within the dotted line area.
No. 3-6
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
Equipment and Supply Requirements
A. Capital Equipment:
1. Balance, triple-beam, capable of 0.1 gram sensitivity
2. Balance, analytical, capable of weighing to 0.1 mg under a 200 g load
3. Desiccator
4. Hot plate or plates, capable of holding a minimum of ten 125 ml
Erlenmeyer flasks or an
autoclave, capable of 121°C (15-20 psi), with capacity for a minimum of
ten 125 ml Erlenmeyer flasks
5. Oven, drying, for use at 105°C
6. pH meter, electric, equipped with single combination electrode, capable
of + 0.1 pH unit sensitivity
7. Refrigerator, capable of maintaining a 4°C temperature
8. B and L Spectronic 20 (or equivalent) spectrophotometer equipped with
accessory infrared phototube and filter capable of operation at 650
or 880 nm or a
filter photometer, equipped with red filter or a
spectrophotometer, uv-visible, capable of operation at 650 nm or 880 nm
9. Vacuum source or pump drawing 15 inches mercury
B. Reusable Supplies:
1. One apron, laboratory
2. One pound glass beads, 5 mm diameter, for smoothing boiling action
3. One beaker, 250 ml
4. One beaker, 1000 ml
5. One beaker, 1500 ml
6. Two 100 ml bottles, glass or plastic with caps
7. One shallow, open mouthed bottle
8. Three 500 ml bottles, plastic with caps
9. One 500 ml bottle, dark glass with stopper
10. Two 1000 ml bottles, glass with stoppers or caps
11. Two 1000 ml bottles, plastic with caps
12. One 2000 ml bottle, glass with cap
13. One bulb, rubber for pipetting
14. One 25 ml cylinder, graduated
15. One 100 ml cylinder, graduated
16. One 500 ml cylinder, graduated
17. One 1000 ml cylinder, graduated
18. One evaporating dish, porcelain, 100 ml, to contain anmonium persulfate
19. One evaporating dish, porcelain, 35 ml to dry potassium dihydrogen phosphate
20. XXX membrane filter assembly with funnel in a #7 stopper to fit the mouth
of a 500 ml suction flask. One as minimam, faster with nine plus one
for each sample.
21. XXX 500 ml suction flask with side arm—one for each filter assembly
22. XXX 50 ml flasks, volumetric with stoppers, nine + one for each sample
23. One 500 ml flask, vol-umetric with stopper
Page No. 3-7
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
B. Reusable Supplies (Cont'd.):
24.
One
1000 ml flask, volumetric with stopper
25.
XXX
125 ml flasks, Erlenmeyer, graduated, nine plus one for each sample
26.
Two
funnels: 1 glass, powder and 1 to fit 50 ml volumetric flask
27.
One
pair rubber gloves for washing glassware with acid solution
28.
One
pair goggles or safety glasses
29.
XXX
hose lengths for connecting suction flasks to vacuum sources
30.
One
1 ml pi pet, graduated in 0.1 ml
31.
One
1 ml pipet, volumetric
32.
One
3 ml pipet, volumetric
33.
One
5 ml pipet, volumetric
34.
One
10 ml pipet, volumetric
35.
One
20 ml pipet, volumetric
36.
One
30 ml pipet, volumetric
37.
One
50 ml pipet, volumetric
38.
Two
10 ml pipets, graduated (Mohr)
39.
One
pneumatic trough or small pan for cold-water bath
40.
One
respirator if a hood is not available
41.
One
spatula
42.
One
0.4 g measuring spoon, Hach or equivalent (optional)
43.
One
8 inch stirring rod, glass
44.
One
pair tongs
45.
One
wash bottle, squeeze type
C. Consumable Supplies:
NOTE: All reagents must be of high purity, such as "A.C.S.," "reagent grade,"
or "analyzed"
1. Water, distilled (as needed)
2. Hydrochloric acid (HC1), concentrated, 1 pint minimum
3. Sulfuric acid (H^SO^), concentrated, 1 pint minimum
4. Antimony potassium tartrate [K(SbO)C^H^Og¦1/2 H^O] (recommend purchase
of 1 lb. units)
5. Ammonium molybdate [(NH^)g MOyO^^^H^O] (recommend purchase of 1 lb. units)
6. Ascorbic acid (recommend purchase of 5-ounce units)
7. .Ammonium persulfate [ (NH^) 2^2*^81 (recommenc' purchase of 1 lb. units)
8. Potassium dihydrogen phosphate (Kl^PO^) (recommend purchase of 1 lb. units)
9. Sodium hydroxide (NaOH) (recommend purchase of 1 lb. units)
10.*Mercuric chloride (HgCl?)
11.*Sodium chloride (NaCl)
12. Boats, weighing, plastic disposable
*0nly needed if samples must be preserved (i.e. if analysis cannot be performed
on the same day that the sample was collected).
Page No. 3-8
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or-, of
Orthophosphate (as P), Single Reagent Method
13. Filters, 0.45 micron pore size membrane, phosphorus-free, Gelman GA 6
or equivalent
14. Notebook, bound laboratory, for permanently recording data
15. Paper, graph: 8 1/2 inch by 11 inch dimestore school supply
is suitable. Recommend graph paper have seven major divisions
along 8 1/2 inch side and 10 major divisions along 11 inch side.
16. Tape, labeling, one roll (masking tape is suitable)
17. Tissue, lint-free, for wiping colorimeter tubes or cuvettes
Page No.
-------
EFFLUENT MONITORING PROCEDURF.: Determination of Total PHosphorus (as P) or of Orthophosphate (as P), Pa9e No- 3-10
————————— single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
TOTAL PHOSPHORUS (as P)
A. Glassware
Preparation
OR OF ORTHOPHOSPHATE (as P), SINGLE REAGENT METHOD
1. Assemble all necessary
equipment.
2. Heat 500 ml 1:1 HCV.
3. Rinse all glassware to be
used in procedure.
4. Discard all 1:1 HC1 used
in rinsing glassware.
5. Flush away discarded
1:1 HC1.
6. Rinse the glassware with
tap water.
7. Rinse the glassware with
distilled water.
8. Rinse the glassware with
combined reagent.
la. See pages 7-9 for list of necessary equipment.
2a. In a 1000 ml beaker.
2b. Use a hot plate or bunsen burner.
2c. For directions on making 1:1 HC1, See B,"Reagent
Preparation."
2d. CAUTION: Use extreme precautions with hot 1:1
HC1 acid. This solution will cause severe burns.
Wear gloves, apron, goggles, etc., while handling.
Vapor from hot acid is extremely irritating to
eyes and throat. Use a hood or wear a respirator
while using.
3a. Use hot 1:1 HC1.
4a
5a. Use plenty of tap water.
6a. Fill and empty two times.
7a. Use several portions of distilled water
CAUTION: 1:1 HC1 carelessly poured down drains
will quickly eat out traps.
8a. One time.
8b. For directions on making combined reagent, see B,
Reagent Preparation.
(continued)
I
(P. 39)
-------
£FFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P),
—————'—"—— Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Glassware
Preparation
(continued)
9. Check all combined reagent-
rinsed glassware.
10. Rinse the glassware with
distilled water.
8c. The combined reagent will turn blue on contact
with orthophosphates.
8d. The purpose of this cleaning procedure is to
remove all phosphates. Appearance of blue color
on combined reagent-rinsed glassware is indicative
of failure of first cleaning or else phosphate
contamination in the distilled water.
9a. After 10 minutes contact with the combined
reagent.
9b. Look for blue color.
9c. For any glassware showing blue color, repeat
steps 3 through 9 of this Operating Procedure.
9d. For any glassware not responding with blue color
in the combined reagent, proceed to step 10.
9e. If any glassware shows blue color after second
cleaning, have distilled water checked for
phosphates.
10a. Use generous amounts.
B. Reagent Preparation
1. 1:1 hydrochloric
acid
1. Measure out 1000 ml dis-
tilled water.
2. Pour the water into a
clean glass bottle.
3. Measure out 1000 ml con-
centrated hydrochloric
acid (HC1).
Hi
la. Use a 1000 ml (1 liter) graduated cylinder.
2a. Bottle must have a capacity greater than 2 liters.
3a. Use a 1000 ml graduated cylinder.
3b. CAUTION: Hydrochloric acid causes severe burns.
Vapor is extremely irritating. Use care when
handling.
Page No. 3-11
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total PHosphorus (as P) or of Orthophosphate (as P), ^a9e ^°* 3-12
Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
4. Slowly, pour the 1000 ml
4a. Avoid spattering the acid by holding the bottle
(conti nued)
of concentrated HC1 into
at an angle so that the acid runs down the side.
the bottle.
5. Gently swirl the bottle to
mix the contents.
6. Label the bottle "1 :1
6a. The date and initials of preparer should always
Hydrochloric Acid."
be included on the label of any reagent container.
2. 10 N sodium
1. Prepare a shallow cold-
la. In a small pan or pneumatic trough.
hydroxide
water bath.
2. Weigh out about 40 grams
2a. In a tared weighing boat on a triple beam balance.
sodium hydroxide (NaOH)
2b. Sodium hydroxide rapidly picks up moisture.
pellets as rapidly as
possible.
3. Transfer the pellets to a
250 ml beaker.
4. Measure out 100 ml of
4a. Use a 100 ml graduated cylinder.
distilled water.
5. Place the 250 ml beaker
in the prepared cold-water
bath.
6. Slowly pour the 100 ml
of distilled water into
the reagent container.
7. Gently stir the beaker's
7a. Use a glass stirring rod.
contents in the cold-water
7b. To mix the contents and cool the solution to
bath.
room temperature.
-------
_EFFLUENT_MOrn>TORIfNG_PROCEDURE>: Determination of Total Phosphorus (as P) or of Orthophosphate (as P),
Single Reagent Method
OPERATING PROCEDURES
i
STEP SEQUENCE
"I
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TO ft THTMP
i r\Mnuiiu
GUIDE NOTES
B. Reagent Preparation
(continued)
8.
When solution is cool,
remove the beaker from the
water bath and pour the
solution into a reagent
container.
8a. Reagent container must be 100 ml capacity or
greater.
8b. NOTE: Plastic storage containers are preferable
for sodium hydroxide (NaOH) solutions as they will
etch glass over a period of time, resulting in a
loss of strength of the solution.
9.
Label the reagent bottle
"10 N Sodium Hydroxide."
9a. Solution is indefinitely stable if container is
kept tightly capped when not in use to prevent
admittance to atmospheric carbon dioxide (C02)
gas.
3. 0.1 N sodium
hydroxide
1.
Measure out 10 ml of the
10 N sodium hydroxide.
la. Use a 25 ml graduated cylinder,
lb. This is reagent 42, above.
2.
Pour the 10 ml of 10 N
sodium hydroxide into a
reagent container.
2a. The reagent container should be 1 liter capacity
or greater.
2b. A plastic reagent container is preferred, as
sodium hydroxide etches glass.
3.
Measure out 900 ml of
distilled water.
3a. Use a 1000 ml graduated cylinder.
4.
Slowly pour the distilled
water into the reagent
container with the 10 ml
10 N sodium hydroxide.
5.
Swirl the container.
5a. To thoroughly mix the contents.
6.
Label this reagent con-
tainer "0.1 N Sodium
Hydroxide."
6a. This reagent will be, used solely for adjusting
the pH of samples and standards.
Page No. 3-13
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P), Page No. 3-14
Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(continued)
4. Strong acid
1. Measure out 600 ml dis-
la. Use a 1000 ml graduated cylinder.
solution, 11 N
tilled water.
sulfuric acid
2. Pour the distilled water
into a 1500 ml beaker.
3. Measure out 310 ml con-
3a. Use a 500 ml graduated cylinder.
centrated sulfuric acid
3b. CAUTION: Contact with concentrated sulfuric acid
(h2so4).
causes severe burns.
4. Place the 1500 ml beaker
4a. In a small pan or pneumatic trough.
in a cold-water bath.
5. Very slowly pour the 310 ml
5a. Hold the beaker at an angle, so the acid runs
concentrated sulfuric acid
down the side of the container.
into the 1500 ml beaker.
5b. CAUTION: If the acid is added too quickly, the
water will boil and spatter the sulfuric acid.
6. Gently stir the contents
6a. Use a glass stirring rod.
of the beaker in the cold-
6b. To mix the contents.
water bath.
6c. Let the reagent container stand in the cold-water
bath while the solution cools to room temperature.
7. Measure out 90 ml of
7a. Use a 100 ml graduated cylinder.
distilled water.
8. Slowly pour the 90 ml of
distilled water into the
1500 ml beaker.
9. Gently stir the contents
9a. Use a glass stirring rod.
of the 1500 ml beaker.
9b. To thoroughly mix the contents.
-------
-rFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P),
~"" Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
10. When solution is cool,
10a. Reagent container may be either glass or plastic.
(continued)
remove the beaker from the
10b. It must be 1 liter capacity or greater.
water bath and pour the
solution into a reagent
container.
11. Label the reagent contain-
11a. Solution is indefinitely stable.
er "Strong Acid Solution."
5. 1.1 N Sulfuric
1. Measure out 900 ml of
la. Use a 1000 ml graduated cylinder.
acid
distilled water.
2. Pour the distilled water
2a. The reagent container should be glass, 1 liter
into a reagent container.
capacity or greater.
3. Measure out 100 ml of 11 N
3a. Use a 100 ml graduated cylinder.
sulfuric acid.
3b. This is reagent #4, above.
4. Slowly pour the 100 ml of
11 N sulfuric acid into
the reagent container with
the distilled water.
5. Swirl the reagent
5a. To thoroughly mix the contents.
container.
6. Label this reagent con-
6a. This reagent will be used solely for adjusting the
tainer "1.1 N Sulfuric
pH of samples and standards.
Acid."
6. 5 N sulfuric acid
1. Measure about 400 ml dis-
la. Use a 500 ml graduated cylinder.
tilled water.
2. Pour the distilled water
into a 500 ml volumetric
flask.
Page No. 3-15
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P), Page No. 3-16
————————"single Reagent Method
OPERATING PROCEDURES
B. Reagent Preparation
(continued)
STEP SEQUENCE
7. Antimony potassium
tartrate solution
3. Measure out 70 ml concen-
trated sulfuric acid
(h2so4).
4. Place the volumetric flask
in the cold-water bath.
5. Slowly pour the 70 ml con-
centrated sulfuric acid
into the flask.
6. Gently swirl the flask in
the cold-water bath.
7. When solution is cooled to
room temperature, add dis-
tilled water to bring
solution to 500 ml volume.
8. Transfer the solution to a
500 ml plastic storage
container.
1. Weigh out exactly 1.3715
grams of antimony potassium
tartrate
[K(Sb0)C4H406-l/2 Hz0].
2. Quantitatively (that is,
completely) transfer the
1.3715 grams of antimony
potassium tartrate to a
500 ml volumetric flask.
INFORMATION/OPERATING GOALS/SPECIFICATIONS
3a. Use a 100 ml graduated cylinder.
3b. CAUTION: Contact with sulfuric acid causes
severe burns.
4a. In a small pan or pneumatic trough.
5a. Hold the flask at an angle, so the acid runs down
the side of the flask.
5b. CAUTION: If the acid is added too quickly, the
water will boil and spatter the sulfuric acid.
6a. To mix the contents and cool the solution to room
temperature.
TRAINING
GUIDE NOTES
8a. Container should be labeled "5 N Sulfuric Acid."
8b. Prepare this solution weekly.
la. In a weighing boat,
lb. Use an analytical balance,
lc. Observe all handling precautions given on the
reagent bottle label.
2a. Funnel the chemical into the flask, using a
distilled water squirt bottle to wash all traces
of the chemical from the weighing boat and powder
funnel into the flask.
2b. CAUTION: Use minimum amount of distilled water
necessary.
-------
_Lr_FjLU^N^^OTj^TC^RI_NG__PRCiC^DUR^: Determination of Total Phosphorus (as P) or of Orthophosphate (as P),
—"Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
3. Measure out 400 ml of
3a. Use a 500 ml graduated cylinder.
(continued)
distilled water.
4. Pour the 400 ml of dis-
tilled water into the
flask.
5. Swirl the flask gently.
5a. Until the chemical has dissolved.
6. Dilute the contents of the
flask to 500 ml.
7. Transfer the solution to a
7a. Bottle must be 500 ml capacity or greater.
clean storage bottle.
7b. Bottle must be dark and glass-stoppered.
8. Label the storage bottle
8a. Store this solution in the dark at 4°C.
"Antimony Potassium
Tartrate Solution."
8. Ammonium molyb-
1. Weigh out 20 grams
la. In a weighing boat.
date solution
ammonium molybdate
lb. Use a triple-beam (0.1 g sensitivity) balance.
[(NH4)6Mo7024-4H20].
2. Measure out 500 ml of
2a. Use a 500 ml graduated cylinder.
distilled water.
3. Transfer the 20 grams of
3a. Use a powder funnel and distilled water squirt
ammonium molybdate to a
bottle to wash all traces of the chemical from the
plastic storage bottle.
weighing boat and powder funnel into the storage
bottle.
3b. Bottle must be 500 ml capacity or greater.
3c. Use a minimum of distilled water.
4. Rinse any remaining chemi-
4a. Use part of the 500 ml distilled water measured
cal from the weighing boat
out in step 2.
into the plastic storage
bottle.
Page No. 3-17
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P), „ _
Single Reagent Method Pa9e No- 3"18
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(continued)
9. 0.1 H ascorbic
acid
5. Pour the remaining dis-
tilled water into the
plastic storage bottle.
6. Gently swirl the plastic
bottle.
7. Label the bottle "Ammonium
Molybdate Solution."
1. Weigh out 1.76 grams of
ascorbic acid.
2. Measure out 100 ml dis-
tilled water.
3. Transfer the 1.76 g ascor-
bic acid to a storage
bottle.
4. Pour the remaining dis-
tilled water into the
storage bottle.
5. Gently swirl the storage
bottle,
6. Label the bottle "Ascorbic
Acid Solution."
6a. To dissolve the ammonium molybdate.
7a. Store this solution at 4°C.
7b. Prepare this solution weekly.
la. In a weighing boat.
lb. Use an analytical balance.
2a. Use a 100 ml graduated cylinder.
3a. Storage bottle must be 100 ml capacity or greater.
3b. Storage bottle may be either plastic or glass.
3c. Use part of the 100 ml of distilled water measured
out in step 2 to rinse any remaining traces of
ascorbic acid from the weighing boat into the
storage bottle.
5a. To dissolve the ascorbic acid.
6a. Store the solution at 4°C.
6b. Prepare this solution weekly.
-------
EiilUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P), or of Orthophosphate (as P)
Single REagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(continued)
10. Combined reagent
(combination of
reagents 6, 7, 8,
and 9 above)
1. Bring reagents 6, 7, 8,
and 9 to room temperature
before doing the following
steps.
2. Measure 50 ml 5 N sulfuric
acid into a storage con-
tainer.
3. Pi pet 5 ml antimony
potassium tartrate solu-
tion into the storage
bottle.
4. Gently swirl the storage
bottle.
5. Measure 15 ml airmonium
molybdate solution into
the storage bottle.
6. Gently swirl the bottle.
la. It is critical that all solutions used in the
makeup of this combined reagent be at room
temperature before mixing, and that they be mixed
in the order given.
2a. Use a 100 ml graduated cylinder.
2b. Solution must be at room temperature.
2c. Storage container may be either glass or plastic.
2d. Storage container must be 100 ml capacity or
greater.
3a. Use a 5 ml volumetric pipet and a rubber bulb.
3bl Solution must be at room temperature before
addition.
4a. To thoroughly mix the contents.
4b. If any turbidity (cloudiness) is observed, shake
the bottle and allow it to stand for a few minutes
until the turbidity disappears before proceeding
to step 5.
5a. Use a 25 ml graduated cylinder.
5b. Solution must be at room temperature before
addition.
6a. To thoroughly mix the contents.
6b. If any turbidity (cloudiness) is observed, shake
the bottle and allow to stand for a few minutes
until the turbidity disappears before proceeding
to step 7.
Page No. 3-19
-------
EFTLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P), ?a9e No. 3-20
Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS |
TRAINING
GUIDE NOTES
B. Reagent Preparation
(continued)
7.
Measure 30 ml ascorbic
acid solution into the
storage bottle.
7a. Use a 100 ml graduated cylinder.
7b. Solution must be at room temperature before
addition.
8.
Gently swirl the bottle.
8a. To thoroughly mix the contents.
8b. If any turbidity (cloudiness) is observed, shake
the bottle and allow the combined reagent to stand
for a few minutes until the turbidity disappears
before using the combined reagent.
9.
Label the storage bottle
"Combined Reagent."
9a. The combined reagent is extremely unstable and
must be prepared fresh before each use.
9b. This 100 ml of combined reagent is sufficient for
12 determinations. If large numbers of samples
are to be run simultaneously, larger quantities of
the combined reagent may be prepared by using the
same reagent proportions.
11. Ammonium
persulfate
1.
Transfer about 50 grams of
ammonium persulfate
(NH^SjOg into a
container.
la. Use a spatula.
lb. Put into any open-mouthed, shallow container con-
venient to scoop or weigh from,
lc. CAUTION: This is a vigorous oxidizing agent.
2.
Label the container
"Ammonium Persulfate."
2a. Store in a desiccator.
2b. Prevent contact with any combustible material.
V.B.ll.2a
(p- 41)
12. Stock phosphorus
solution
1.
Preheat an oven to 105°C.
la. An oven used for drying suspended solids crucibles
or filters is suitable.
2.
Transfer a few grams of
potassium dihvdrogen phos-
phate (K^PO^) to a suit-
able container.
2a. Use a spatula.
2b. NOTE: Any shallow, open container is suitable as
long as it can withstand 105°C heat. A small
porcelain evaporating dish is handy for the
purpose.
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total PHosphorus (as P) or of Orthophosphate (as P),
Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(continued)
3. Transfer the container of
potassium dihydrogen phos-
phate to the preheated
oven.
3a. Use tongs.
3b. This will drive off atmospheric moisture that the
chemical has picked up and allow accurate weighing.
3c. Dry for a minimum of 1 hour before proceeding to
step 4.
3d. NOTE: Oven door should not be opened during
drying period.
4. Transfer the container of
potassium dihydrogen phos-
phate to a desiccator.
4a. Use tongs.
4b. NOTE: Potassium dihydrogen phosphate may be safe-
ly desiccated with ammonium persulfate.
4c. To cool to room temperature.
4d. About 30-40 minutes should be sufficient.
V.B.12.4
(p. 41)
5. Transfer the container of
potassium dihydrogen phos-
phate to a spot convenient
to the analytical balance.
5a. Use tongs.
6. Weigh out exactly 0.2197
grams of potassium di-
hydrogen phosphate.
6a. In a weighing boat.
6b. On the analytical balance.
6c. NOTE: This step should be accomplished as quickly
as is consistent with best weighing technique to
avoid the pickup of atmospheric moisture by the
chemical during weighing.
7. Completely transfer the
0.2197 grams of potassium
dihydrogen phosphate to a
one liter volumetric flask.
7a. Funnel the chemical into the flask, using a dis-
tilled water squirt bottle to wash all traces of
the chemical from the weighing boat and funnel
into the flask.
8. Fill the volumetric flask
about one-half ful1.
8a. Use distilled water.
9. Gently swirl the flask.
9a. To completely dissolve the potassium dihydrogen
phosphate.
10. Dilute the contents of the
flask to one liter.
,10a. Use distilled water.
Page No. 3-21
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P),
Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(continued)
11. Stopper or cap the flask.
12. Gently invert the flask.
13. Transfer the solution to
a storage bottle.
14. Label the bottle "Stock
Phosphorus Solution."
12a. Do this half-a-dozen times to ensure complete
mixing.
13a. Bottle must be 1000 ml capacity or greater.
13b. Bottle- can be glass or plastic.
14a. 1.0 ml equals 0.05 mg P (50 microgram P).
14b. Solution is stable for a maximum of six months if
stored at 4°C when not in use.
14c. NOTE: Solution must be warmed to room temperature
before use.
C. Preparation of
Standard Phosphorus
Solution
1. Pipet exactly 20 ml of
stock phosphorus solution
into a one liter volumetric
flask.
2. Dilute the stock phos-
phorus solution in the
flask to one liter.
3. Stopper or cap the flask.
4. Gently invert the flask.
5. Label the flask "Standard
Phosphorus Solution."
la. Use a 20 ml volumetric pipet and a rubber bulb.
lb. NOTE: This volume only applies for the Bausch
and Lomb Spectronic 20 (or equivalent) equipped
with the standard 1/2 inch tubes. For other than
1/2 inch tubes this volume must be adjusted.
See Training Guide.
2a. Use distilled water.
4a. Do this half-a-dozen times to ensure complete
mixing.
5a. 1.0 ml equals 1.0 jig P.
5b. This dilute solution is unstable and must be
prepared daily.
Vl.C.lb
(P. «)
-------
EFFUJENT^IONnORIJJG^J^ROCEDUR^: Determination of Total Phosphorus (as P) or of Orthophosphate (as P),
Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
| TRAINING
GUIDE NOTES
Preparation of
Phosphorus Calibra-
tion Standards
If you are preparing a
calibration curve, measure
the amounts of standard
phosphorus solution shown
in Table 1 into nine 50 ml
volumetric flasks. If a
calibration curve has
already been established,
omit this step and steps
2 through 6 and proceed to
step 7.
la. Use volumetric pipets and a rubber bulb,
lb. Label each flask with its appropriate mg/1
phosphorus concentration as given in Table 1.
NOTE: If you will be using spectrophotometer
tubes with a width greater than one half inch,
the concentration of these standards will be
different. See Training Guide,
lc. MOTE: The 40 ml volume of standard phosphorus
solution may require a combination of volumetric
pipets.
TABLE 1
VI.D.lb
(P. 42)
Flask
No.
ml of Standard
Phosphorus Solution
per 50.0 ml
Concentration
of Phosphorus,
mq per liter
1
0
0.00
2
1.0
0.02
3
3.0
0.06
4
5.0
0.10
5
10.0
0.20
6
20.0
0.40
7
30.0
0.60
8
40.0
0.80
9
50.0
1.00
Page No. 3-23
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P), Page No. 3-24
Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Preparation of
Phosphorus Calibra-
tion Standards
(continued)
2. Dilute the various
amounts of standard
phosphorus solution in
the nine flasks to the
50.0 ml mark.
3. Stopper or cap each flask.
4. Gently invert each flask,
5. Pour each of the nine
prepared calibration
standards from their 50 ml
volumetric flasks into a
125 ml Erlenmeyer flask.
6. If you are preparing a
calibration curve, omit
steps 7 through 15 and
proceed to E, "Preparation
of Samples."
7. Pi pet 5 ml of standard
phosphorus solution into a
50 ml volumetric flask.
8. Label the flask "0.10 mg/1
P."
2a. Use distilled water.
2b. NOTE: The 50 ml flask requiring 0 ml of standard
phosphorus solution is a "reagent blank" and will
merely be filled to the 50 ml mark with distilled
water. However, this flask must be carried
through the rest of the steps, being treated
exactly as any sample or calibration standard.
4a. Do this half-a-dozen times to ensure complete
mixing.
5a. Label each 125 ml Erlenmeyer flask with the mg/1 P
concentration corresponding to the particular 50ml
volumetric flask emptied into it.
7a. Use a 5 ml volumetric pipet and a rubber bulb.
8a. This is a "low" calibration standard. It must be
used to check the accuracy of the calibration
curve.
8b. If you will be using spectrophotometer tubes with
a width greater than one half inch, the concen-
tration of this standard will be different. See
Training Guide.
VI.D.8b
(p. 43)
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P),
——— Sl-ng-]e Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Preparation of
9. Pi pet 40 ml of standard
9a. You may have to use a 20 ml volumetric pi pet, 1
Phosphorus Calibra-
phosphorus solution into a
filling it twice and using a rubber bulb.
tion Standards
50 ml volumetric flask.
(continued)
10. Label the flask "0.80
10a. This is a "high" calibration standard. It will
mg/1 P."
be used to check the accuracy of the calibration
curve.
10b. If you will be using spectrophotometer tubes with
VI.D.10b
a width greater than one half inch, the concen-
(p. 43)
tration of this standard will be different. See
Training Guide.
11. Dilute the standard
11a. Use distilled water.
phosphorus solution in the
two flasks to the 50.0 ml
mark.
12. Stopper or cap each flask.
13. Gently invert the flask.
13a. Do this half-a-dozen times to thoroughly mix the
contents. |
14. Empty these flasks into
14a. Label the 125 ml Erlenmeyer flasks with the B
each of two 125 ml
corresponding mg/1 P concentrations. B
Erlenmeyer flasks.
|
115. Pi pet 50 ml of distilled
15a. Use a 50 ml volumetric pipet and a rubber bulb. g
B water into a clean 125 ml
15b. Label this flask "0.00 mg/1 P."
| Erlenmeyer flask.
15c. This is the "reagent blank." It is carried
S
through all the steps, being tested exactly as
I
any sample or calibration standard.
Page No. 3-25
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P), Pa96 N°' 3-26
Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
E. Preparation of
Samples
1. Record the sample identi-
fication information.
2. Shake the sample.
3. Immediately pipet 50 ml
of sample into a 125 ml
Erlenmeyer flask.
Label this 125 ml
Erlenmeyer flask "Sample."
la. Sample should be at hand before continuing with
this test,
lb. Use a laboratory notebook.
1c. Record "location," "identification," "type",
"date and time collected," name of Sample
collector," and "date and time analysis began"
on the data sheet provided.
3a. Use a 50 ml volumetric pipet and a rubber bulb
unless the sample contains large particulate
matter. Then use a 50 ml graduated cylinder.
3b. Measure rapidly since solids may settle in the
sample container while you are filling the pipet
or cylinder.
3c. NOTE: Wastewater samples may contain more than
1.00 mg/liter phosphorus and require dilution.
With a wastewater sample of unknown mg/liter P
concentration, it is desirable to set up addi-
tional flasks containing sample aliquots diluted
to 50.0 ml.
3d. NOTE: If orthophosphate is to be run, any sample
containing appreciable quantities of turbidity or
suspended solids must be filtered through a 0.45
micron phosphorus-free filter. Before attempting
to run orthophosphate on such a sample, refer to
the Training Guide for an explanation of the
required procedure modification. Sample aliquots
on which total phosphorus is to be determined
must not be filtered at this time.
4a. If the sample dilutions are being used, include
the amount of dilution on the label.
4b. Also record the amount of sample dilution on the
data sheet provided.
VII. E. la
(p. 44)
IX.E.lb
(p. 48 )
IX.E.lc
(p. 49)
VII.E.3c
(p. 45)
l/I I.E. 3d
(p. 46)
I I.E.4a
(P- 40)
IX.E.4b
(p. 49)
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P),
Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS |
TRAINING
GUIDE NOTES
E. Preparation of
Samples (continued)
5. If only orthophosphate is
to be determined, adjust
the pH of the sample and
calibration standards to
7.0 + 0.2, then skip
Procedures F and G and
start at Procedure H,
"Preparation of Spectro-
photometer." If total
phosphorus is to be
determined, continue with
Procedure F, "Digestion
Procedure for Total
Phosphorus Determination."
5a. Use an electronic pH meter.
5b. Use the ION and 0.1 N sodium hydroxide and the
strong acid solution (11 N sulfuric acid) and the
1.1 N sulfuric acid to adjust the pH. On any pH
adjustment, begin with the strong acid (11 N) or
base (ION), and use the weaker (1.1 N sulfuric
acid and 0.1 N sodium hydroxide) solutions only
for the final precise adjustments.
5c. If no sample dilution is being used (i.e., you use
50.0 ml of sample) any acid or base used for pH
adjustment will cause a volume error (final volume
will be greater than 50.0 ml), and thus cause low
results. Significant pH adjustment volume errors
on strongly acid or basic samples may be minimized
by very roughly adjusting the pH of the 50.0 ml
aliquot using concentrated (36 N) sulfuric acid or
very strong (10 N) sodium hydroxide dropwise,
followed by precise adjustment using the more
dilute solutions as given in 5b above. Small
volume errors will still be unavoidable.
5d. If a sample dilution is being used, a volume error
from pH adjustment may be avoided by pipetting the
filtered sample aliquot into an Erlenmeyer flask
or beaker, adding distilled water to bring the
volume to approximately 40 ml, performing the pH
adjustment, and then pouring the pH adjusted sample
dilution into a 50.0 ml volumetric flask and add-
ing distilled water as needed to bring the volume
to the 50.0 ml mark.
5e. NOTE: If you are preparing a calibration curve,
there will be nine calibration standards to pH
adjust (prepared in D, steps 1 through 5). If a
calibration curve has already been established,
there will be three calibration standards to pH
adjust (prepared in D, steps 7 through 15).
Page No. 3-27
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P), ^
———————1~~~single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Digestion Procedure
1. Turn on a hot plate, or
la. Let them heat.
for Total Phosphorus
plates.
lb. The surface area of the hot plate(s) must be large
Determi nation
enough to accommodate a minimum of 10-125 ml
(Calibration
Erlenmeyer flasks.
Standards, Reagent
lc. If an autoclave is to be used, omit this step.
Blank, Samples)
2. Add 1 ml of strong acid
2a. Use a 10 ml graduated (Mohr) pipet and a rubber
solution (11 N sulfuric
bul b.
acid) to each 125 ml
2b. NOTE: All standards including the reagent blank
Erlenmeyer flask.
are digested along with the sample.
3. Remove the ammonium per-
sulfate from the
desiccator.
4. Weigh out a 0.4 gram
4a. In weighing boats.
portion of ammonium
4b. Using a triple-beam balance.
persulfate for each
4c. NOTE: If you are using a 0.4 gram Hach measuring
solution in a flask.
spoon (or equivalent), this step may be omitted as
the portions may be scooped as needed.
5. Add 0.4 gram ammonium per-
sulfate to each of the
125 ml Erlenmeyer flasks.
6. Add 3 or 4 glass boiling
6a. This will control bumping (uneven boiling).
beads to each flask.
7. Place the flasks on the
7a. Alternately, the flasks may be autoclaved for
preheated hot plate(s).
30 minutes at 121°C (15-20 psi).
8. Gently boil the flasks.
8a. For 30-40 minutes or until a volume of approxi- j
mately 10 ml is reached. 1
8b. CAUTION: Do not allow any of the flasks to go to j
dryness. This will ruin the determination. 1
-------
l.rLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P),
Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
— l
INFORMATION/OPERATING GOALS/SPECIFICATIONS
T
TKAilNlNb
GUIDE NOTES
G. pH Adjustment of
Digested Calibration
Standards, Reagent
Blank, Samples
1. Set up as many 0.45 micron
filter assemblies as you
have standards, blanks,
and samples.
2. Cool the digestion flasks.
3. Filter each standard,
blank, and sample.
4. Rinse each Erlenmeyer
flask and filter the
rinse water.
5. Pour each filtrate back
into its corresponding
125 ml Erlenmeyer flask.
6. Rinse each filter flask.
7. Adjust the pH of each
standard, blank, and
sample.
la. See the Training Guide Note referenced to this
step for information concerning this type of
assembly and the required phosphorus-free filters.
lb. If you do not have this many filtration assem-
blies, you can rinse out and reuse the equipment.
This requires more time.
2a. Hold them under running tap water or use a very
shallow cold-water bath.
3a. Use a phosphorus-free 0.45 micron pore size filter
and assembly.
4a. Use distilled water,
4b. Use no more than 2,5 ml portions for each flask,
adding each portion, swirling, and then pouring
each portion through the appropriate filter.
5a. A powder funnel may be useful.
5b. For laboratories having only double-electrode pH
meters, labeled 100 ml beakers may be substituted
for the 125 ml Erlenmeyers at this point.
6a. Use one 10 ml portion of distilled water, adding
the rinse water to the 125 ml Erlenmeyer flasks.
6b. Volume in each flask must not exceed 35 ml.
7a. Adjust to pH 7 +_ 0.2.
7b. Use an electric pH meter.
7c. NOTE: When adjusting the pH, add 10 N sodium
hydroxide rapidly using a graduated (Mohr) pipet
or eyedropper until the pH is raised to about 3
(this will require approximately 1 ml). There-
after, add base slowly and dropwise to pH 6,
watching the pH meter carefully. At this point
continue the dropwise addition, but using 0.1 N
sodium hydroxide until the'pH is up to 7.0 + 0.2.
If pH is raised too high, use 1.1 N sulfuric acid
VII.G.la
(p. 47)
dropwise to lower the pH.
Page No. 3-29
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P), Page No' 3-30
Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
G. pH Adjustment of
Digested Calibration
Standards, Reagent
Blank, Samples
(continued)
8. Add 0.1 ml of 11 N sul-
furic acid to each pH-
adjusted standard, blank,
and sample.
9. Pour each standard, blank,
and sample into the 50 ml
volumetric flask in which
it was originally made up.
10. Dilute each flask to the
mark.
11. Stopper each flask.
12. Gently invert each flask.
13. Pour each 50.0 ml solution
back into its correspond-
ing 125 ml Erlenmeyer
flask.
8a. Use a 1 ml pi pet, graduated in 0.1 ml, and a rubber
bul b.
8b. NOTE: This will prevent possible adsorption of
phosphorus on iron, aluminum, manganese or other
metal precipitates.
9a. If there is room in the 50 ml volumetric flask,
add a small volume rinse of the flask or beaker
used to adjust pH.
10a. Use distilled water.
12a. Do this half-a-dozen times to thoroughly mix the
contents.
13a. Each is now ready for the addition of colorimetry
reagents.
H. Preparation of
Spectrophotometer
1. Turn the instrument on.
la. Allow a warm-up period of approximately 20 minutes
(10 minutes minimum).
lb. Use a B & L Spectronic 20 (or equivalent) equipped
with accessory infrared phototube and filter for
use at 880 or 650 nm wavelength,
lc. There is an EMP on "Use of a Spectrophotometer."
V.H.lb
(p. 41)
-------
EFFLUENT MONITORING PROCEDURF: Determination of Total Phosphorus (as P) or of Orthophosphate (as P),
^ Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
I. Color Development
1. Add 8.0 ml of combined
reagent to each 125 ml
Erlenmeyer flask.
2. Gently swirl the flasks.
3. Allow a 10 minute
(minimum) to 30 minute
(maximum) waiting period.
la. Use a 10 ml graduated (Mohr) pipet and a rubber
bulb.
2a. To ensure complete mixing.
3a. For maximum color development.
J. Spectrophotometry
Measurements
1. Adjusting the
i nstrument
1. Consult the manufacturer's
instructions for calibrat-
ing your particular
instrument.
la. Instrument must be warmed up at least 10 minutes,
lb. There is an EMP on "Use of the Spectrophotometer."
2. Adjust the wavelength to
880 nm.
2a. 880 nm is the preferred wavelength, but 650 nm
may also be used.
V.J.1.2a
(P- 41)
3. Check to make sure that
the instrument reads
infinite absorbance with
no sample tube in the
instrument.
3a. If it does not, adjust the instrument so that it
does read infinite absorbance. (See manufac-
turer's instructions).
2. Reading
absorbance
4. Use the reagent blank
(0.00 mg/liter P) to ad-
just the instrument to
zero absorbance.
5. Repeat step 3.
1. Measure and record the
absorbances for each of
the calibration standards.
4a. Spectrophotometer tubes must be cleaned with
1:1 HC1, etc. See Procedure A, Glassware
Preparation.
4b. Use manufacturer's instructions to make the
adjustment.
la. If you are preparing a calibration curve, there
are 8 calibration standards.
lb. If you are running check standards, there are 2.
(continued)
Page No. 3-31
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P), paqe No. 3-32
—————————¦——— Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS'
TRAINING
GUIDE NOTES
J. Spectrophotometry
Measurements
(continued)
lc. In either case, proceed from the lowest to the
highest concentration.
Id. Record the absorbance value next to the corre-
sponding mg/liter P concentration of the cali-
bration or check standards on the data sheet
provided.
IX.J.2.Id
(p. 49)
2. Measure and record the
absorbances for each of
the samples.
2a. In the absorbance column provided for samples on
the data sheet.
IX.J.2.2a
(p. 49)
3. Turn off the
spectrophotometer.
3a. Unless it is to be used for other measurements.
K. Making a Calibration
Curve
1. If a calibration curve
has been established, omit
this Operating Procedure
and proceed to Operating
Procedure L, "Checking
the Calibration Curve."
If a calibration curve has
not been previously es-
tablished, proceed with
step 2 below.
la. Since the standards for a total phosphorus
determination must be digested before colorime-
try, and the standards for an orthophosphate
determination are not digested, one calibration
curve will be needed for the total phosphorus
determination and a separate calibration curve
for the orthophosphate determination. Unless they
are exact duplicates, the two curves must not be
used interchangeably.
2. Obtain an 8 1/2 x 11 inch
piece of graph paper.
2a. The last page of this EMP is a model of graph
paper labeled for this test.
IX
(p. 50)
3. Label the longer side as
the concentration axis.
4. Label the shorter side as
the absorbance axis.
-------
^F£XJJJE^n_MO££IjrORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P),
Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
K. Making a Calibration
Curve (continued)
5. Use the absorbance value
and corresponding con-
centration for each of
the standards to make a
plot of absorbance versus
concentration.
6. Skip Operating Procedure
L, "Checking the Cali-
bration Curve," and
proceed to Procedure M,
"Reading Results from the
Calibration Curve."
5a. This graph should be prepared with utmost care.
5b. The points plotted should form a straight line.
5c. This straight line plot is the calibration curve.
L. Checking the
Calibration Curve
1. Locate the absorbance
value' just recorded for
the 0.10 mg/1iter P
calibration standard.
2. Read its observed con-
centration.
3. Record this curve mg/1iter
P concentration.
4. Compare this observed
mg/1iter P concentration
to its true value of 0.10
mg/1iter P.
la. On the calibration curve for the determination
you are doing—total phosphorus or orthophosphate.
lb. If you adjusted the concentration of your stand-
ards for other than half-inch width spectro-
photometer tubes, the concentration of this
standard is different. See Training Guide.
3a. In the column next to the absorbance column for
check standards on the data sheet provided.
4a. The observed mg/1iter P concentration of the
calibration standard, as read from the cali-
bration curve must be within + 2% of its true
value of 0.10 mg/1iter P.
- 2% of 0.10 is 0.002
- Thus the acceptable range is 0.098 to 0.102
mg/1iter P.
(continued)
VI.L.lb
(p. 42)
IX.L.3a
(p. 49)
Page No. 3-33
-------
£^^E^JMUNinORI>NG_PROCEDIJR^: Determination of Total Phosphorus (as P) or Orthophosphate (as P),
Single Reagent Method
Page No. 3-34
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
L. Checking the
Calibration Curve
5. If the observed concen-
tration is within the
acceptable range of the
true value, proceed to
step 6. If the observed
concentration is not
within the acceptable
range of the true value,
discard the calibration
curve and prepare a new
one by starting at
Procedure D, following all
directions for "If you are
preparing a calibration
curve."
6. Locate the absorbance
value recorded for the
0.80 mg/1iter P cali-
bration standard.
7. Read its observed
concentration.
8. Record this curve mg/1iter
P concentration.
9. Compare this observed nig/
liter P concentration to
its true value of 0.80 mg/
liter P.
4b. See Training Guide if you adjusted the concen-
tration of this standard.
5a, Failure of the observed and true concentrations
to agree within _+ 2% of the true value means
that the calibration curve is no longer suffic-
iently accurate to report mg/1iter P data obtainec
from it.
VI.L.4b
(p. 43)
VII.L.5a
(p. 47)
6a. Again, on the calibration curve for your specific
phosphorus determination.
6b. If you adjusted the concentration of the stand-
ards for other than half-inch width cells, use
the adjusted concentration.
8a. In the column next to the absorbance column for
check standards on the data sheet provided.
9a. The observed mg/1iter P concentration of the
calibration standard, as read from the calibra-
tion curve, must be within ;+ 2% of its true value
of 0.80 mg/1iter P.
- 2% of 0.80 is 0.016
- The acceptable range is therefore 0.784 to
0.816 mg/1iter P.
(continued)
IX.L.8a
(P. 49)
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P),
Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
L. Checking the
Calibration Curve
(continued)
10. If the observed concen-
tration is within the
acceptable range of the
true value, proceed to
Procedure M, "Reading
Results from the Cali-
bration Curve." If the
observed concentration is
not within the acceptable
range of the true value,
discard the calibration
curve and prepare a new
.one by starting at Pro-
cedure D, following all
directions for "If you are
preparing a calibration
curve."
9b. See Training Guide if you adjusted the concen-
tration of this standard.
VI.L.9b
(p. 43)
M. Reading Results
from the Calibration
Curve
1. Use the absorbance value
recorded for each sample
and the standard curve
for your specific phos-
phorous determination to
obtain the mg/liter P
concentration.
2. Record this curve mg/liter
P concentration.
2a. In the column next to absorbance column for
samples on the data sheet provided.
IX.M.2a
(p. 49)
Page No. 3-35
-------
EFFLUENT MONITORING PROCEDURE.: Determination of Total Phosphorus (as P) or of Orthophosphate (as P), Page No. 3-36
Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
N. Calculations
1. Determine the dilution
factor.
2. Record the dilution
factor.
3. Multiply the curve mg/
liter P by the dilution
factor.
4. Record this final mg/liter
P.
5. Sign, the data sheet.
la. The dilution factor for a straight (undiluted)
sample is 1.
lb. The total sample volume is always 50 ml, so 25 ml
of sample diluted to 50 ml in the volumetric
flask would be 25/50 or 1/2 dilution, and the
dilution factor would be 2. For other dilutions,
see the Training Guide.
lc. The data sheet has a section with "Example
Calculations."
2a. In the column provided on the data sheet next to
the curve mg/liter P column.
4a. In the column provided on the data sheet.
5a. On the line provided on the data sheet, "Analyst."
II.N.lb
(p. 40)
IX.N.lc
(p. 49)
I X.N.2a
(p. 49)
IX.N.4a
(p. 49)
IX.N.5a
(p. 49)
0. Reporting Data
1. Report total phosphorus,
mg/liter P, or orthophos-
phate, mg/liter P.
la. On any required record or report sheets.
IX.0.1a
(p. 48)
P. Clean-Up
1. Discard unused combined
reagent and standard
phosphorus solution.
2. Store the other reagents.
la. Combined reagent must be made fresh before each
run.
lb. Standard phosphorus solution may be retained for
other analyses to be performed that same day.
2a. Observe special storage requirements of some
reagents as stated in B, "Reagent Preparation."
-------
- , LUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P),
Single Reagent Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
P. Clean-Up
(continued)
3. Transfer all glassware to
wash area.
4. Clean all glassware.
4a. In readiness for next determination.
4b. According to the steps in Operating Procedure A,
"Glassware Preparation.11
4c. This step may be performed when time permits.
4d. It is desirable, but not mandatory, that all
glassware used in this procedure be maintained as
a separate stock, used only for the phosphorus
determination.
4e. NOTE: Never clean glassware to be used in
phosphorus determinations in commercial detergent,
as the active ingredient is usually a phosphate
compound.
Page No. 3-37
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
TRAINING GUIDE
SECTION TOPIC
I* Introduction
II* Educational Concepts - Mathematics
III Educational Concepts - Science
IV Educational Concepts - Communications
V* Field & Laboratory Equipment
VI* Field & Laboratory Reagents
VII* Field & Laboratory Analysis
VIII Safety
IX* Records & Reports
~Training guide materials are presented here under the headings marked *.
These standardized headings are used through this series of procedures.
Page No. 3-38
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
INTRODUCTION
Section I
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
Sources of phosphates in water besides the geological
include agricultural fertilizers, sewage (human
wastes, synthetic detergents, biological protoplasm)
and various industrial wastes.
Phosphates are a necessary and sometimes growth-
limiting nutrient for microorganisms. In high
concentrations, phosphates can produce nuisance
levels of algae and other photosynthetic aquatic
organisms.
Since the natural phosphorus content of most waters
is quite low, the presence of high phosphate con-
centrations can be an excellent indicator of the
level of pollution. Hence, the phosphate test will
be a common tool of technicians monitoring water
quality for the NPDES system.
The orthophosphate (P0^)E ion is the smallest and
simplest of the phosphorus-oxygen radicals. The
orthophosphate determination as given here is limited
to the inorganic phosphorus (PO^ in the sample
as measured by the direct colorimetric analysis
procedure.
More complex phosphorus compounds are usually com-
josed of linked orthophosphates or of phosphorus
linked to carbon compounds (organic phosphorus). The
total phosphorus determination as given here refers
to all of the phosphorus present in the sample,
regardless of form, as measured by the persulfate
digestion procedure.
The test described in this instruction can be found
in the 1974 EPA Methods Manual on page 249, titled
"hosphorus, All Forms (Single Reagent Method). Other
references which have acceptable procedures for
WDES purposes are: 14th ed. Standard Methods, on
3ages 476 and 481, and 1975 ASTM Part 31 on page 384
Standard Methods for the
xamination of Water and
Wastewater. 14th ed., 1976
APHA, New York, N.Y. p. 466
Griffith, et. al., editors.
Environmental Phosphorus
Handbook. 1973. John Wiley
and Sons, New York, N.Y.
p. 443ff.
Methods for Chemical Analysis
of Water and Wastes. 1974.
EPA, MDQARL., Cincinnati, OH
45268. p. 251.
Ibid, p. 249.
Op. cit. pp 476 and 481.
Annual Book of Standards,
Part 31, Water, 1975, ASTM
Philadelphia, PA, p. 384.
Page No. 3-39
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
EDUCATIONAL CONCEPTS - MATHEMATICS Section II
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
E.4a
N.lb
Since the dilution is only part sample, when the
absorbance reading obtained for it is converted to
a mg/liter P concentration using the calibration
curve, the concentration obtained is only that of
the dilution. To obtain the mg/liter P concentration
of the sample, the mg/liter P concentration of the
dilution must be multiplied times the amount of
dilution factor. For a 1/2 dilution (25 ml sample/
50 ml total volume) the dilution factor would be 2
(the dilution is only half sample). For a 1/5
dilution (10 ml of sample/50 ml total volume) the
dilution factor would be 5. Use of dilution factors
is illustrated for a total phosphorus determination
in the typical data sheet in Section IX at the back
of this Training Guide. Below is a table of common
dilution factors for a 50 ml sample.
ml of Sample per Amount of Dilution
50 ml Total Volume Dilution Factor
25 1/2 2
10 1/5 5
5 1/10 10
1 1/50 50
0.5 1/100 100
0.05 1/1000 1000
The dilution factor for any dilution may be calcu-
lated by dividing the ml of sample used in the
dilution into 50:
Dilution Factor = —i s—^ ^ —
ml sample used in dilution
Example: 2 ml of sample diluted to 50 ml
Dilution Factor = ^ = 25
The dilution factor would be 25,
Page No. 3-40
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
FIELD AND LABORATORY EQUIPMENT
Section v
B.11.2a
B.12.4
H. lb
J.1.2a
Desiccants are hygroscopic materials capable of
absorbing moisture from air. Silica gel (S^) and
calcium sulfate (CaSO^) are two commonly used
desiccants available from laboratory supply compa-
nies. Desiccants must always be dry before use.
The moisture can be removed from them by heating in
an oven (103-105°C).
Ordinarily a wavelength of 880 nm is used for
phosphorus determinations. The second wavelength
(650 nm) may be desirable because of your particular
instrument capabilities or because of unusual inter-
ferences in the sample. If you have such a situation,
test your standards at the 650 nm wavelength to see
if you get a range of responses significant enough
to construct a calibration curve. If you do, you
can use the 650 nm wavelength setting.
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
Page No. 3-41
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
FIELD AMD LABORATORY REAGENTS SectionVI
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
C.lb
D.lb
L. lb
The mg/liter P concentration range that a Spectronic
20 with 1/2 inch colorimeter tubes can detect is
from 0.02 mg/liter P to approximately 1.00 mg/liter
P. This covers the useful working range of absorb-
ance readings from 0.005 to approximately 0.7
(Readings above about 3/4 of full scale deflection,
which is approximately 0.7 absorbance, are inaccu-
rate and should be discarded). Using 20 ml of stock
to prepare 1 liter of standard phosphorus solution
allows the preparation of 8 calibration standards
whose mg/liter P concentration covers the range of
0.02 to 1.00 mg/liter P. If the absorbance of these
same solutions were to be measured in 1 inch
colorimeter tubes, they would give absorbances
ranging from 0.02 to approximately 1.4. Since the
useful working range is from 0.005 to about 0.7,
about half of the standards would be useless, as
they would read off the scale. This is because with
a 1 inch colorimeter tube, you are measuring the
absorbance of twice the thickness of colored solu-
tion, and twice the thickness of a given colored
solution will absorb twice as much light and give
twice the absorbance reading (Beer's Law).
If you are using 1 inch colorimeter tubes, you will
need to use 10 ml of stock phosphorus solution,
rather than 20 ml, to prepare the standard phos-
phorus solution. The various ml of standard
phosphorus solution used in Table 1 (Operating
Procedure D.lc) will then give calibration standards
of the correct concentrations for use with 1 inch
colorimeter tubes. The concentrations as given in
Table 1 will now be inaccurate, however. If you
use 1 inch spectrophotometer tubes, and hence only
use 10 ml of stock phosphorus solution to make up
the standard phosphorus solution, Table 1 will read
as follows:
ml of Standard Phosphorus
Solution per 50.0 ml
Concentration of
Phosphorus, mg/
1 iter
0
0.00
1.0
0.01
3.0
0.03
5.0
0.05
10.0
0.10
20.0
0.20
30.0
0.30
40.0
0.40
50.0
0.50
Page No. 3-42
(continued)
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
FIELD AND LABORATORY REAGENTS
Section VI
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
C.lb
D. lb
L. lb
(continued)
D.8b
D.lOb
L.4b
L. 9b
Notice that since you are using 1 inch colorimeter
tubes that have twice the thickness of 1/2 inch
tubes, the mg/liter P concentrations of the stand-
ards have been halved. Calibration curves and
data sheets made up using these 1/2 strength
standards will need to have these new concentrations
substituted for those given in the typical cali-
bration curve and data sheet at the back of this
Training Guide, as they are examples of data ob-
tained using 1/2 inch colorimeter tube calibration
standards.
If you are using 1 inch colorimeter tubes, the
strength of the calibration curve check standards
will also be different, and hence the acceptable
range of observed concentrations they can have will
be different. For 1 inch tubes the concentration of
the calibration curve check standard using 5 ml of
that standard phosphorus solution will be 0.05 mg/
liter P. Two percent of 0.05 is 0.001, so the
acceptable +_ 2% range will be from 0.049 to 0.051
mg/liter P. The concentration of the calibration
curve check standard using 40 ml of standard phos-
phorus solution will be 0.40 mg/liter P. Two percent
of 0.40 is 0.008, so the acceptable observed
concentration range will be 0.392 to 0.408 mg/
liter P.
Page No. 3-43
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
FIELD AND LABORATORY ANALYSIS
Section VII
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
E.la
COLLECTION OF SAMPLES FOR THIS TEST:
Samples should be collected from a preagreed site by
a preagreed technique known to all parties con-
cerned. You should be familiar with the following
information since you record most of it on your
laboratory data sheet. You may be responsible for
actually collecting the sample; consult your
supervisor.
LOCATION - Plant control and self-monitoring re-
quirements will be the basis for selecting places
to collect samples. Final collection points should
be such that samples drawn there are as representa-
tive of the entire sample source as possible.
Consult your supervisor.
IDENTIFICATION - Each collection location should be
assigned a number or simple identification code.
Use this to label samples from that location and to
record on the lab data sheet.
TYPE - Permit requirements determine whether a grab
or a composite sample will be collected; consult
your supervisor. Mark type on sample container and
on laboratory data sheet.
TIME OF COLLECTION - Mark time and date on sample
container and on lab data sheet.
CONTAINER - The analyst should know what volume
container is required for each sample source. Con-
tainers should be capped, and may be of plastic
material (such as cubitainers) or of Pyrex glass.
Used containers should be rinsed with hot 1:1 HC1,
with tap water (2 times), with distilled water;
checked for phosphate traces with combined reagent,
then rinsed again with tap and distilled water (see
Operating Procedure A, "Glassware Preparation," in
the EMP for specific details).
COLLECTION - Rinse container two or three times with
sample, then collect the sample. If benthic deposits
are present in the area being sampled, great care
should be taken not to include these deposits.
SIGNATURE - Sample collector should sign his name on
the container or label so this information can be
recorded on the lab data sheet.
Standard Methods for the
Examination of Water and
Wastewater. 14th ed., 1976,
APHA, New York, NY, p. 38.
Ibid,
Methods for Chemical
Analysis of Water and
Wastes. 1974. EPA-NERC-
MDQARL, Cincinnati, Ohio
45268. p. 249.
(continued)
Ibid.
Page No. 3-44
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
FIELD AND LABORATORY ANALYSIS
Section VII
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
E.la
(continued)
E. 3c
PRESERVATION - If the analysis cannot be performed
the same day as collection, the sample should be
preserved by the addition of 2 ml concentrated
sulfuric acid (F^SO^) or 40 mg mercuric chloride
(HgC^) per liter and refrigeration at 4°C. If
HgCl2 is used as a preservative, samples should be
spiked with a minimum of 50 mg/liter of sodium
chloride (NaCl) to prevent interference of the HgC^
with samples containing low (less than 50 mg Cl/1)
chloride levels.
HOLDING TIME - Maximum holding time for preserved
samples is seven days. Samples for the orthophos-
phate determination that must be filtered, should
be filtered as soon as practical after collection.
A phosphorus determination on a 50 ml aliquot of any
sample containing over 1.00 mg/liter P will result
in an absorbance outside the range of the calibra-
tion curve. The blue color produced by addition of
the combined reagent will be so strong that the
spectrophotometer will be unable to measure it.
Samples containing over 1.00 mg/liter P concentra-
tions must be diluted. Since the mandatory sample
size is 50 ml, all dilutions will be based on a
lesser amount of sample diluted to 50 ml. The
correct procedure is to use a volumetric pipet to
transfer a volume of sample to a 50 ml volumetric
flask, then to dilute that volume of sample to 50 ml
with distilled water and mix thoroughly. This
dilution may then be used in the procedure.
A natural question arising is, "what amount of dilu-
tion should I use?" The best answer is that only
trial and error experience will show you the best
dilution to use with a given sample. A rule of thumb
is that potable water samples will usually require
little or no dilution. A typical series to run on
a potable water sample of unknown mg/liter P con-
centration might be to prepare one flask containing
50 ml of undiluted sample, one flask containing 25 m
of sample diluted to 50 ml (this would be a 1/2
dilution, 25 ml sample/50 ml total volume) and a
third flask containing 10 ml of sample diluted to
50 ml (this would be a 1/5 dilution, 10 ml sample/
50 ml total volume).
(continued)
Ibid, p. 249-50, 252.
Ibid, p. x and xi
Page No. 3-45
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
FIELD AND LABORATORY ANALYSIS
Section v11
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
E.3c
(continued)
E.3d
Sewage samples may contain 10 mg/liter P concentra-
tions or more, and consequently require dilutions as
high as 1/1000 (.05 ml of sample diluted to 50 ml).
The problem encountered here is that volumes of less
than 1 ml are hard to measure directly with any
accuracy, and obtaining a representative sewage
sample using such a small volume is unlikely. In
making dilutions requiring less than 1 ml of sample,
a good procedure is to use dual dilutions. A dual
dilution means taking a volume of sample, diluting
it, taking a volume of the first dilution, and
diluting it again. To illustrate the use of dual
dilutions, consider a sample requiring a 1/100
dilution to get a mg/liter P concentration inside
the required range of 0.02 mg/liter to 1.00 mg/liter
First, take a 50 ml volumetric flask and pipet into
it 5 ml of sample. Dilute this flask to the mark,
and you have a 1/10 dilution. Each 1 ml of the
contents of this flask contains 0.1 ml of the
original sample. 0.5 ml of the sample is needed to
dilute to 50 ml to achieve a 1/100 dilution, so if
you pipet 5 ml from the first dilution flask into
a second 50 ml volumetric flask, you will have
0.5 ml of sample in a 50 ml flask. When diluted to
the 50 ml mark, this second flask will be a 1/100
sample dilution, ready for determination.
INTERFERENCES - Turbidity or suspended solids inter-
fere with the orthophosphate determination. This is
because a spectrophotometer works by measuring the
amount of light absorbed by color produced in a
sample by the addition of specific reagents.
Turbidity or suspended solids in a sample will
falsely increase the absorbance reading because a
spectrophotometer cannot differentiate between
light absorbed by the color in a sample and that
scattered or blocked by solids.
The interference of turbidity or suspended solids in
the orthophosphate determination may be eliminated by
filtering the sample through a .45 micron membrane
filter before the determination is begun. The
filtration should be done as soon as possible after
sample collection. A change in the name of the
phosphorus fraction reported must be made to signify
that the sample was filtered. An orthophosphate
determination made on a filtered sample must be re-
sorted as Dissolved Orthophosphate. mg P/liter.
(continued)
Ibid., p. 251
Page No. 3-46
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
rIELD AND LABORATORY ANALYSIS
Section VII
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
E.3d
(continued)
G. la
L.5a
A membrane filter assembly typically consists of a
funnel clamped to a fritted (porous) base, holding
between them a .45 micron pore size cellulose
membrane filter. A stopper on the fritted base is
used to hold this assembly upright in the neck of a
500 ml side-arm flask connected to a vacuum source.
A quantity of well-mixed sample is measured into the
funnel. The vacuum is applied, and the sample is
drawn through the filter into the side-arm flask.
This filtered sample will now be free from all
turbidity or suspended solids. It may be decanted
from the side-arm flask and used in subsequent steps
of the determination.
Since the very fine porosity filters clog quickly,
samples containing high levels of particulate
matter may require that 2 or 3 filters be used in
succession to obtain enough filtrate for the de-
termination. In the case of a total phosphorus
determination, the entire digested sample must be
filtered and recovered.
Before use, the membrane filter assemblies must be
cleaned in the same manner as all other glassware
used in the procedure.
The membrane filters must also be phosphorus-free.
This can be accomplished by soaking ordinary .45
micron membrane filters in distilled water: 50
filters per 2 liters distilled water for 1 hour,
changing the water, and soaking an additional
3 hours. Alternately, phosphorus-free filters may
be purchased (Gelman GA6 or equivalent).
In the determination of total phosphorus, low values
have been reported because of possible adsorption of
phosphorus on iron, aluminum, manganese or other
metal precipitates. This can be avoided by fil-
tration before neutralization and re-dissolving the
metal hydroxides that form with 2-3 drops of acid
before color development.
If you find that you must frequently discard your
calibration curve because one or both of the 0.10
and 0.80 mg/liter P calibration curve check standards
fall outside the +_ 2% acceptable range of their true
value, you may find it advisable to run the full set
of calibration curve standards and prepare a new
calibration curve for each batch of samples
determined.
Standard Methods for the
Ixamination of Water and
Wastewater, 14th ed., 1976,
UPHA, New York, NY. p. 472.
"Changes and Errata in
Methods for Chemical Analysis
of Water and Wastes," 1974,
EPA-NERC-MDQARL, Cincinnati,
Ohio 45268
Page No. 3-47
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
RECORDS AND REPORTS Section IX
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
E.lb
All laboratory records must be kept for three years,
preferably in a permanently bound notebook. The
time period is required by regulatory agencies.
Attached as the next two pages are an example data
sheet and a graph which can be used to construct a
calibration curve. These can be used for either a
Total Phosphorus (asP) or an Orthophosphate (as P)
determination.
0.1a
Depending on your organizational set-up, it may be
your job responsibility to enter this data on the
plant operation record, state report form, etc.
Check with your supervisor.
Page No. 3-48
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of
Orthophosphate (as P), Single Reagent Method
RECORDS AND REPORTS Section IX
EXAMPLE DATA SHEET FOR TOTAL PHOSPHORUS OR FOR ORTHOPHOSPHATE, mg/liter P
E.lc
Sampling Location
Final
Effluent
E.lc
Sample Identification
E.S.
Is.
E.lc
Type of Sample
Grab
or Composite
E.lc
Date and Time Collected 1/17/75 9:OC a.m.
E.lc
Sample Collector
Tom Sampler
E.lc
Date and Time Analysis Began 1/17/75 9:30 a.m.
N.5a
Analyst
Dick Analyst
J.2.Id
L.3a
Calibration Standards
mg/liter P
Absorbance
Check Standards
mg/liter P
Absorbance
Curve
mq/1iter P
L.8a
0.02
0.06
0.10
0.10
0.20
0.40
0.60
0.80
0.80
1.00
E.4b
J.2.2a
Amount of
Sample Dilution
Absorbance
Curve
mq/liter P
Dilution
Factor
Final
mg/liter P
M. 2a
N.2a
N.4a
EXAMPLE CALCULATIONS
N.lc
Amount of
Sample Dilution
Absorbance
Curve
mg/liter P
Dilution
Factor
Final
mq/liter P
Straight Sample
off scale
_
1
_
1 (25 ml sample
off scale
2
.
2 50 ml total)
1 (10 ml sample
0.3525
0.520
5
2.60
5 50 ml total)
1 (5 ml sample
0.1775
0.260
10
2.60
10 50 ml total)
1_ (2.5 ml sample
0.0875
0.130
20
2.60
20 50 ml total)
Page No. 3-49
-------
Page No. 3-50
EFFLUENT MONITORING PROCEDURE: Determination of Total Phosphorus (as P) or of Orthophosphate (as P),
Single^ Reagent Method
RECORDS AND REPORTS
SECTION IX
1.00
0.80
DETERMINATION OF TOTAL PHOSPHORUS (AS P)
(DIGESTED PHOSPHORUS STANDARDS)
OR
DETERMINATION OF ORTHOPHOSPHATE (AS P)
(NON-DIGESTED PHOSPHORUS STANDARDS)
CALIBRATION GRAPH
SIGNATURE OF PREPARER:
DATE GRAPH WAS PREPARED:
CO
C£
O
in
co
0.60
0.40
0.20
0.00
0.10 0.20 0.30 0.40 0.50 0.60 0.70
CONCENTRATION OF PHOSPHORUS, mg/liter
0.80
0.90
1.00
-------
A PROTOTYPE FOR DEVELOPMENT OF
ROUTINE OPERATIONAL PROCEDURES
for the
DETERMINATION OF CHEMICAL OXYGEN DEMAND
as applied in
WASTEWATER TREATMENT FACILITIES
and in the
MONITORING OF EFFLUENT WASTEWATERS
National Training Center
Municipal Operations and Training Division
Office of Water Program Operations
U.S. ENVIRONMENTAL PROTECTION AGENCY
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
This operational procedure was developed by:
Name Audrey Donahue
Address EPA, WPO. National Training Center, Cincinnati, Ohio
Position Chemist-Instructor
Education and Technical Background
B.A. Edgecliff College
1 year Industrial Research Chemist
8 years Secondary School Chemistry Instructor
4 years DHEW-DI Water Quality Program Chemist
6 years DI-EPA Chemist-Instructor
Page No. 4-3
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
1. Objective:
To determine the mg/liter Chemical Oxygen Demand of organic and oxidizable
inorganic substances in a wastewater sample.
2. Description of Analysis:
A measured water sample is mixed with a measured volume of potassium dichromate
solution which is a strong oxidizing agent. A volume of concentrated sulfuric
acid equal to the combined volume of sample and oxidizing agent is added to
provide a 50% by volume mixture which particularly promotes oxidation of
organic and oxidizable inorganic substances in the sample.
The mixture is 1n a flask which is then attached to a condenser over a source
of heat. The heat is applied to maintain the mixture at a gentle boiling
temperature of' 145°C for a two hour period. The condenser cools and re-
liquifies materials that vaporize during this period.
In order to determine the amount of sample that is oxidized under these
conditions, the potassium dichromate solution must be added in excess. The
measurement involves titrating any unused oxidizing solution after the oxi-
dation period, and.then calculating the Chemical Oxygen Demand from the
amount of oxidizing solution that was used. A reducing agent, ferrous
ammonium sulfate solution, is used to titrate the unused potassium
dichromate solution in the test mixture. Ferroin is used colqr indi-
cator in this titration.
If there is no potassium dichromate left to titrate after the two hour
oxidation period, the test must be done over using less sample. Water is
added to make up for the missing volume of sample in order to maintain the
50% volume of concentrated sulfuric acid required in the test mixture.
Organic substances are particularly susceptible to oxidation when placed in
the conditions of this test. Even when the best laboratory technique is
used, some organic contamination may be present and will affect test results.
Consequently, a blank using distilled water instead of sample is run with
each group of samples and is titrated with ferrous ammonium sulfate solution.
The results are included in the calculation formula to correct the data for
minor contamination. The titration results for the blank may be of a
magnitude to prompt a check of reagents and/or distilled water as contributors
of excessive organic contamination in the test.
3. Applicability of this Procedure:
a. Range of Concentration:
5 to 50 mg/liter COD
Information is given so the same stepwise procedure can be used for COD
greater than 50 mg/liter.
b. Pretreatment of Samples:
The Federal Register Guidelines do not specify any pretreatment.
Page No. 4-4
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
c. Treatment of Interferences in Samples:
This procedure includes directions for conditioning glassware and in-
formation about checking distilled water to minimize organic contamination.
To minimize loss of volatile materials during the addition of sulfuric
acid, instructions include cooling the test flask in ice water. Addition
of mercuric sulfate to complex routine levels of interfering chlorides is
also part of the procedure. However, if the chloride concentration exceeds
2000 mg/liter, consult the Source of Procedure* for the required modi-
fication of mercuric sulfate addition and of the calculation formula.
No other interferences are noted in the Source of Procedure.*
*Source of Procedure: Methods for Chemical Analysis of Water and Wastes, 1974,
Environmental Protection AGency, Methods Development and Quality Assurance
Research Laboratory, Cincinnati, OH, p. 21.
Page No. 4-5
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
FLOW SHEET:
BLANK
SAMPLE
TITRATION
OXIDATION
CALCULATIONS
FERROUS AMMONIUM SULFATE
STANDARDIZATION
Page No. 4-6
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Equipment and Supply Requirements
A. Capital Equipment:
Balance, with a 0.1 or 0.01 gram sensitivity
2. Balance, analytical with a 0.1 milligram sensitivity
3. Distillation Equipment - Use an all-glass distillation unit if possible.
A metal still is acceptable if all the surfaces that contact the
distillate are heavily coated with pure tin. The still should be
located away from areas where volatile organic solvents are stored
and/or used. DO NOT USE ion - exchange columns or membrane filters
to prepare the water. These treatments can add organic contamin-
ation.
4. Magnetic Stirrer - Hot Plate and Magnetic retriever (pick-up rod). OPTIONAL
5. Oven, laboratory for drying chemicals at 103°C.
6. Specific Conductance Meter and related equipment to test inorganic quality
of distilled water. OPTIONAL
7. Total Organic Carbon Analyzer and related equipment to test organic quality
of distilled water. OPTIONAL
Reusable Supplies:
1. Reflux Apparatus: One flask-condenser-heating surface assembly is re-
quired for each sample or blank to be tested. These should be
permanent assemblies in the laboratory, protected from contamin-
ation by glass wool plugs in the open end of the condensers and
with the flasks connected to the condensers.
Flasks, heat-resistant glass, 500 ml Erlenmeyer or 300 ml round
bottom, with a ground glass neck to fit the condenser of choice.
If the Erlenmeyer type flask is to be used, purchase those having
graduations for approximate volumes contained in the flask.
Condensers, 12 inch Allihn or equivalent with a ground glass joint
to fit into the flask. (24/40 is a commonly used joint size.)
Tubing Connections from cooling water source to condensers.
Heating Surface, flat for Erlenmeyer flasks or heating mantles for
round bottom flasks. Either should have sufficient power to
produce at least 9 watts/square inch to supply the 145°C temper-
ature required. The amount of heat supplied should be adjustable.
NOTE: A 16 amp line is usually required for a series of 6 reflux
set-ups.
2. 2 Automatic dispensers (pipets), glass with delivery settings up to 10 ml.
OPTIONAL
3. Beads, glass about 2 mm diam. 5 for each flask - condenser assembly
Page No. 4-7
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
B. Reusable Supplies: (Continued)
4. 1 Beaker, glass, 250 ml
5. 2 Bottles, brown glass, about 50 ml with dropper pipet in screw cap for
ferroin. Alternatively, use a stoppered reagent bottle and a
medicine dropper.
6. 3 Bottles, glass, screw cap, minimum capacity of 1 liter each to store
reagents.
7. 1 Buret, 50 ml, 0.1 ml graduations, teflon stopcock plug preferred.
8. 1 Clamp, buret, for titration stand
9. Containers, storage, glass or heavy plastic with screw caps for COD
waste test materials containing mercury complexes and significant
amounts of sulfuric acid.
10. 1 Buchner funnel to catch glass beads when test wastes are transferred
from flasks to storage containers.
11. 2 Cylinders, graduated, 25 ml.
12. 2 Cylinders, graduated, 100 ml.
13. 1 Cylinder, graduated, 500 ml.
14. 1 Desiccator to store cooling chemical for reagent preparation.
15. 1 Evaporating dish per sample to separate flask from heating surface.
(Optional)
16. 2 Flasks, Erlenmeyer, wide mouth 500 ml.
17. 3 Flasks, volumetric, 1 liter.
18. 1 Funnel, short stem, diam. about 75 mm (to fill 50 ml buret).
19. 1 Pan for ice water to cool mixtures, about 4 inch depth and about 8 inch
diameter is sufficient.
20. 1 Pipet bulb
21. 1 Pipet, graduated, 10 ml (Omit if an automatic dispenser is used for
the concentrated sulfuric acid).
22. 1 Pipet, volumetric, 10 ml.
23. 1 Pipet, volumetric, 25 ml.
24. 2 Pipets, volumetric, 50 ml.
25. 2 Pipets, volumetric, 100 ml.
26. 1 Reagent bottle, glass with glass stopper. Only required if preparing
less than 9 pounds of the sulfuric acid - silver sulfate solution.
27. 1 Reagent spoon to roughly measure 1 gram of mercuric sulfate.
28. Rings, cork as supports if round bottom flasks are used, 1 per flask.
29. 1 Stand, titration, support for buret.
Page No. 4-8
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
B. Reusable Supplies: (Continued)
30. 1 Stirring rod, glass to use in 250 ml beaker. Omit if ferroin
solution is purchased already prepared or if a magnetic stirrer
is available.
31. Storage containers for distilled water, preferably glass. If only
polyethylene bottles are available, be aware that organic
plasticizers may be leached into water stored in such bottles
over a period of time.
32. 1 Wash bottle, squeeze type 500 ml.
C. Consumable Supplies:
1. Glass wool, to make plugs for condensers, bottle of distilled water, etc.
2. Labels for reagent bottles, at least 7.
3. Laboratory notebook with spaces for information similar to the "Typical
Laboratory Data Sheet" in this EMP.
4. Pencil, wax marking.
5. Towels, paper.
6. Weighing boats, at least 5.
7. Ice to cool flasks during test.
8. Reagents - Quantities for one sample plus one blank:
2 grams mercuric sulfate (HgSO^) reagent grade.
1 1/3 - 9 pound bottles concentrated sulfuric acid^SO^) reagent grade.
23.5 grams silver sulfate (Ag2S04) reagent grade
6.5 liters distilled water, high quality with very low chemical
oxygen demand
14 grams potassium dichromate (I^C^Oy) primary standard grade.
*1.5 grams 1-10 (ortho) phenanthroline with one molecule of water of
hydration (ferroin) (cf-C-^Hg^'^0^ •
*1 gram ferrous sulfate with seven molecules of water of hydration
(Fe S04-7H20)
98 grams ferrous ammonium sulfate with six molecules of water of
hydration [Fe (NH^UQ^-CH^O]
*If ferroin indicator solution is purchased, these reagents are not required.
Page No. 4-9
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Page No. 4-10
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Preparing to
Test the Sample.
1. Assemble all equipment
to be used.
2. Prepare the reagents for
the test.
3. If necessary, condition
any flasks, boiling beads
or condensers to be used
for the test.
4. If necessary, condition
any other glassware to
be used in the test.
5. Record the sample identi-
fication information.
la. Equipment list is on pp.6, 7 and 8.
lb. Flasks, boiling beads and condensers should be
with the chosen heat source in a permanent
assembly.
2a. See Procedure B. Reagent Preparation
3a. Conditioning is necessary if the equipment is
new, if .it has been used for COD mixtures that
turned green during the boiling period, or if
it was used for tests other than COD.
3b. See Procedure D , "Conditioning Flasks, Boiling
Beads and Condensers"
4a. This glassware is included in the equipment
1ist on pp. 6, 7 and 8.
4b. Conditioning is necessary i.f the glassware is
new, if it has been used to measure COD samples,
or if it has been used for tests other than COD.
4c. See Procedure E , "Conditioning Glassware
Other Than Flasks, Boilinq Beads or Condensers".
5a. The sample should be at hand before continuing
with the test.
5b. Use a laboratory notebook with space for inform-
ation similar to the "Typical Laboratory Data
Sheet" in this EMP.
5c. Record "Identification", "Type"(grab or compos-
ite), "Date and Time Collected", and the name
of the "Sample Collector" in one of the columns.
I
(p. 45)
IX.Sheet I
(P. 51)
IX.A.5.
(P- 51)
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
Reagent Preparation
1. Mercuric Sulfate
2. Concentrated
sulfuric acid
3. Sulfuric acid -
silver sulfate
solution
2.
3.
Use a 1 gram reagent spoon
to measure the mercuric
sulfate (HgSO*) at the timq
of the test.
Use concentrated sulfuric
acid (HpSO.) to prepare
other reagents and also as
a reagent in the test.
weighing boat, weigh
grams of silver
sulfate (Ag^SO^).
In
23
Put the weighed chemical
in a 9 pound bottle of
reagent grade, concentrated
sulfuric acid.
Screw the cap onto the
bottle of acid.
la. 'Jse reagent grade powdered mercuric sulfate,
lb. Use one gram for each sample and for the blank.
la. You need reagent grade concentrated sulfuric
acid.
lb. You need 2.5 liters for preparations,
lc. You need 5 ml for each sample and for the
blank.
Id. Since sulfuric acid causes severe burns to the
skin, you may want to put it in an automatic
dispenser for use during the test. Label the
di spenser.
la. Use reagent grade silver sulfate,
lb. You need 70 ml of this solution for each sample
and each blank. If you do this test routinely,
it is easiest to prepare the amount of reagent
as given in this procedure. To make smaller
volumes of the reagent, multiply the ml of reagent
desired by 0.0108 grams to find how many grams
of silver sulfate are needed,
lc. Use a balance with 0.1 or 0.01 gram sensitivity.
2a. To make smaller volumes, measure the acid with
a graduate and carefully pour it into a glass
reagent bottle. Add an amount of silver sulfate
calculated as described above in lb.
V.B2.1d.
(p. 46)
Page No. 4-11
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Page No. 4-12
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(continued)
3. Sulfuric acid -
silver sulfate
solution
(continued)
4. Swirl the mixture in the
bottle every half hour or
so until the silver sulfate
di ssolves.
4a. It will take several hours for the silver sulfate
to dissolve. If you have a magnetic stirrer
assembly, use it to speed up the dissolving.
CAUTION: Sulfuric acid causes severe skin burns.
Be careful not to splash it out of the bottle when
you put the stirring bar in. Also, use a
retriever to get the bar out and thoroughly rinse
the acid off of the retriever and the stirring
bar at once, with water.
5. Label the container.
5a. This is the sulfuric acid - silver sulfate
solution to be used in the test. Also write the
date and your name on the label.
5b. You may want to put some of this solution in an
automatic dispenser for use during the test.
Label the dispenser.
V.B3.5b.
(p. 46)
4. Distilled water
1. Prepare 7 liters of high
quality distilled water with
very low chemical oxygen
demand due to organic or
inorganic contamination.
la. Requirements for distillation equipment are
described on p. 6.
lb. When distilling water, use clean glass wool
packing around delivery tubes to prevent organic
contamination of the distillate,
lc. Requirements for water storage containers are
described on p. 8.
Id. Mark the date of distillation on the water
container.
le. Plug the container of distilled water with clean
glass wool or cover it with a screw cap.
If. Store the container of distilled water away from
areas where organic solvents are stored and/or
used.
lg. You can test the inorganic quality of water with
specific conductance measurements, either in line
or on the distillate. The specific conductance
should be less than 2.0 micromhos at 25°C.
(continued)
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
Reagent Preparation
(continued)
4. Distilled water
(continued)
5.
0.250 N
potassium
dichromate
solution
1. Dry about 14 grams of
potassium dichromate (l<2Cr207
in a laboratory oven for two
hours at 103°C.
2. Remove the chemical from the
oven to a desiccator to cool.
3. In a weighing boat weigh out
12.259 grams of the dried
potassium dichromate.
4. Put the weighed chemical into
a 1 liter volumetric flask.
5. Add about 500 ml distilled
water to the flask.
6. Swirl to dissolve the
potassium dichromate.
7. Add distilled water up to the
one liter mark on the flask
8. Mix the solution by inverting
the flask several times.
lh. The organic quality of water is difficult to
monitor in line. If test blanks indicate
significant organic contamination (See Training
Guide, BLANKS) you could arrange to have total
organic carbon tests done on the distillate. At
least check the still for cleanliness and check
storage procedures.
la. Use primary standard grade potassium dichromate.
lb. A round weighing is sufficient for this step.
2a. Desiccant should be dry.
2b. Allow about 20 minutes for cooling.
VII.B4.1h.
(p. 48)
3a. Use an analytical balance.
5a. Use high quality distilled water with very low
COD (See B.4).
6a. Support the bottom of the flask with your hand
while swirling.
Page No. 4-13
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Page No. 4-14
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(conti nued)
5. 0.250 N
potassium
dichromate
solution,
(continued)
9. Pour the solution into a
screw cap bottle.
10. Label the container.
10a. This is 0.250 N potassium dichromate solution. It
is used for testing samples with COD greater than
50 mg/1i ter.
10b. It is very stable and can be stored at room
temperature for several months.
10c. To use it for COD less than 50 mg/1iter, you must
dilute it to be 0.025 N.
6. 0.025 N
potassium
dichromate
solution.
1. Measure 100.0 ml of 0.250N
potassium dichromate
(K2Cr207) solution.
2. Drain the 100.0 ml into a
1 liter volumetric flask.
3. Add distilled water up to
the one liter mark on the
flask.
4. Label the container.
la. Use a volumetric pipet.
3a. Use high quality distilled water with very low
COD (See B.4).
4a. This is the 0.025 N potassium dichromate solution
to be used for COD less than 50 mg/1iter.
4b. Write the date and your name on the label.
7. Ferroin indicator
solution.
1. In a weighing boat, weigh
1.48 grams of ferroin,
1-10 (ortho) phenanthroline
monohydrate (erCi2^2"t^O).
2. Put the weighed ferroin
into a 250 ml beaker.
la. You can purchase this indicator solution already
prepared.
lb. You can use a balance with 0.01 gram sensitivity.
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(continued)
7. Ferroin indicator
3. In a weighing boat, weigh
3a. Use the same balance as above.
solution (continued)
0.70 grams of ferrous
sulfate with seven mole-
cules of water of
hydration.
4. Put this into the same
250 ml beaker.
5. Measure 100 ml distilled
5a. Use high quality distilled water with very low
water in a graduate.
COD (See B.4).
6. Put the water into the
250 ml beaker containing
the two weighed chemicals.
7. Stir to dissolve.
7a. Use a stirring rod or a magnet and magnetic
stirrer apparatus.
7b. You can speed the dissolving process by heating
the solution until it is just warm.
8. Put the indicator solution
8a. Use brown glass bottles.
into dropper bottles.
8b. You need two bottles of about 50 ml capacity each.
9. Label the container.
9a. This is the ferroin indicator solution to be used
in the test. Also write the date and your name on
the label.
8. 0.250 IN Ferrous
1. In a weighing boat weigh
la. Use reagent grade ferrous ammonium sulfate.
ammoni um
out 98 grams of ferrous
lb. You can use a balance with 0.1 or 0.01 gram
sulfate solution
ammonium sulfate crystals.
sensitivity.
[Fe(NH4)2(S04)2-6H20].
lc. In this section, the letters FAS will be used when
referring to this chemical.
Page No. 4-15
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Page No. 4-16
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(continued)
8. 0.250 N Ferrous
2. Put the weighed chemical
ammoni urn
into a 1 liter volumetric
sulfate solution
flask.
(continued)
3. Fill the flask about two
3a. Use high quality distilled water with very low
thirds full with distilled
COD (See B.4.).
water.
4. Swirl to dissolve the FAS.
4a. Support the bottom of the flask with your hand
while swirling.
5. Measure 20 ml of concen-
5a. CAUTION: Sulfuric acid causes severe burns to the
trated sulfuric acid in a
skin.
graduate.
6. Tilt the 1 liter flask and
6a. The solution may get slightly warm.
slowly pour the acid down
along the inside wall of
the flask and into the
solution.
7. Swirl to mix the acid and
7a. Support the bottom of the flask with your hand
the FAS solution.
while swirling.
8. Add distilled water up to
8a. Use high quality distilled water with very low
the one liter mark on the
COD (See B.4.).
flask.
9. Mix the solution by
inverting the flask
several times.
10. Pour the solution into a
1
screw cap bottle.
|
-------
EFFLUENT MONITORING PRDCEDURF: Determination of Chemical Oxygen Demand
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(continued)
8. 0.250 Ferrous
ammonium sulfate
solution (continued)
9. 0.025 N Ferrous
ammonium sulfate
solution.
11. Label the container.
1. Measure 100.0 ml of
0.250 N ferrous ammonium
sulfate
[Fe(NH4)2(S04)2-6H20]
solution.
2. Drain the 100.0 ml into a
1 liter volumetric flask.
3. Add distilled water up to
the one liter mark on the
flask.
4. Label the container.
11a. This is 0.250 N ferrous ammonium sulfate solution.
It is used for testing samples with COD greater
than 50 mg/liter.
lib. It is unstable and should be stored in a dark
bottle.
11c. When using it for tests, it must be standardized
with potassium dichromate solution (Procedure C.).
lid. To use it for COD less than 50 mg/liter, you must
dilute it to 0.025 N.
la. Use a volumetric pipet.
3a. Use high quality distilled water with very low
COD (See B.4.).
4a. This is the 0.025 N ferrous ammonium sulfate
solution to be used for COD less than 50 mg/liter.
4b. Write the date and your name on the label.
4c. The solution is unstable and should be stored in a
dark bottle.
4d. When using it for tests, it must be standardized
with potassium dichromate solution (Procedure C.).
Page No. 4-17
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Page No. 4-18
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
Standardization of
Ferrous Ammonium
Sulfate Solution
1. Meas-ure 15 ml distilled
water.
2. Pour the water into a
250 ml Erlenmeyer flask.
3. Repeat steps 1 and 2 with
a second flask for a
duplicate test.
4. Prepare an ice bath.
5. Place one flask into the
ice bath.
6. Measure 10.0 ml of the
0.025 N potassium dichro-
mate (I^C^O^) solution.
7. Drain the 10.0 ml into the
250-ml flask in the ice
bath.
8. Swirl the beaker to mix
the contents.
| 9. Let the flask in the ice
bath.
10. Repeat steps 5 through 9
for the duplicate test
flask.
11. Measure 20 ml concentrated
sulfuric acid (h^SO^j.
la. Use a graduate.
lb. Use high quality distilled water with very low
COD.
4a. The depth of the water should be about one inch.
6a. Use a volumetric pi pet.
9a. You want to cool the flask.
11a. Use a graduate or an automatic dispenser checked
for accurate delivery,
lib. CAUTION: Sulfuric acid causes severe burns to the
skin.
V.C.lla.
(p. 46)
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Standardization of
Ferrous Ammonium
Sulfate Solution
(conti nued)
12. Tilt the 250 ml flask and
slowly pour the acid down
along the inside wall and
into the solution.
13. Swirl the flask in the ice
bath to mix the contents.
14. Remove the flask from the
ice bath.
15. Repeat steps 11 through
14 for the duplicate test
flask.
16. Put a buret clamp onto a
titration stand.
17. Rinse and drain the inside
of a clean 50 ml buret
with about 15 ml of the
ferrous ammonium sulfate
[Fe(NH4)2(S04)2-6H20]
solution which is about
0.025 N.
|l8. Put the buret into the
clamp on the stand.
119. Close the stopcock of the
buret.
20. Add about 15 ml of FAS
solution to the buret.
12a. The solution and the flask will get warm.
13a. You want to cool the flask to room temperature.
14a. The bottom of the flask may be slightly warm to
the touch.
17a. In this section, the letters FAS will be used when
referring to this ferrous ammonium sulfate
solution.
7b. Put the FAS in a beaker so you can pour it into
the buret.
20a. Use a funnel.
Page No. 4-19
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand Page No. 4-20
OPERATING PROCEDURES
C. Standardization of
Ferrous Ammonium
Sulfate Solution
(continued)
STEP SEQUENCE
21. Check the tip of the
buret for air bubbles.
22. You may have to add more
FAS solution to the buret.
23. Record the level of the
solution in the buret.-
24. Check that the 250 ml
flask and contents are at
room temperature before
proceeding.
25. Add one drop of ferroin
indicator to the mixture
in the flask.
26. Gently swirl the flask to
mix the contents.
27. Add about 8 ml of ferrous
ammonium sulfate solution
from the buret fairly
rapidly while constantly
swirling the mixture in
the flask.
INFORMATION/OPERATING GOALS/SPECIFICATIONS
21a. If there is an air pocket, swiftly turn the
stopcock in a complete circle to expel it. You
may have to repeat this turning of the stopcock.
22a. You will need at least 10 ml of FAS for the
ti tration.
23a. Use one of the columns on the sheet titled
"Standardization of Ferrous Ammonium Sulfate
(FAS) Solution."
23b. This number is "ml FAS at START of titration."
23c. Use the lowest part of the curve of the liquid
(the meniscus) to take this reading. Some burets
have a color stripe and you can see a colored
point. Record the reading using the line where
the point rests.
24a. You may have to put the flask back into the
container of cold water to get this condition.
25a. The ferroin should be in a dropper bottle. If it
isn't, use a medicine dropper to transfer it.
25b. One drop is used for a 45 ml mixture.
26a. This ensures thorough mixing.
26b. Do not swirl any of the contents out of the
flask.
26c. The mixture is a deep orange color.
27a. You must constantly swirl the flask so the FAS
solution comes into contact and reacts with the
mixture in it.
TRAINING
GUIDE NOTES
IX.Sheet II
(p. 52)
IX.C.23.
(p. 52)
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EFFLUENT MONITORING PROCEDURE:Determination of Chemical Oxygen Demand
OPERATING PROCEDURES
STEP
SEQUENCE
INFORMATION/OP&RATING GOALS/SPECIFICATIONS
v—
TRAINING
GUIDE NOTES
C. Standardization of
Ferrous Ammonium
Sulfate Solution
(continued)
28. Now adjust the stopcock so
the FAS solution in the
buret goes into the flask
more slowly and continue
swirling the flask.
29. Now regulate the stopcock
so the FAS solution in the
buret goes into the flask
one drop at a time.
30. Stop adding FAS when all
the mixture in the flask
is a reddish-brown color.
31. Record the final level of
the solution in the buret.
32. Repeat steps 22 through 31
for the duplicate test
flask.
33. For each column of data,
subtract the recorded "ml
FAS at END of titration"
and record the difference
on your data sheet.
28a. The mixture in the flask gradually changes color
during this titration. Beginning with a deep
orange color, the mixture becomes green, then
blue-green. At that stage, you are very close to
the end point and the end point color of reddish-
brown will appear at the surface of the mixture
in the flask when drops of FAS reach it. When
you observe this reddish-brown color, close the
stopcock.
29a. Swirl the flask after each drop is added. At the
end point, one drop is enough to change the color
of all of the solution to a reddish-brown.
30a. This is the end point of the reaction.in the
flask.
31a. Use the same column as before on the sheet titled
"Standardization of Ferrous Ammonium Sulfate (FAS
Solution."
31b. This number is "ml FAS at END of titration."
33a. Use the same columns on the sheet.
33b. This is the "ml of FAS solution used for the
standardization" reaction.
IX.Sheet II
(p. 52)
IX.C.31.
(p. 52)
IX.Sheet
(p. 52)
IX.C.33.
(p. 5Z)
II
Page No. 4-21
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EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand Page No. 4-22
OPERATING
PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPEC IFICATIONS
TRAINING
GUIDE NOTES
C. Standardization of
Ferrous Ammonium
Sulfate Solution
(continued)
34. The differences found in
step 33 above should agree
within ±0.05 ml.
34a. If the differences do not agree within ±0.05 ml,
repeat steps 1 through 34 (omitting 3, 10, 15, 32'
to get a third difference which should agree with
one of the differences recorded in step 33 within
the ±0.05 ml limit.
35. Divide 0.250 by one of the
"agreeing" ml differences
found in step 34 above.
Your answer should have
four decimal places.
35a. Since the final answer is rounded off, you need
not use averaged ml differences for this division,
35b. The division comes from using this formula:
.. i ., _ /ml potassiuiTK /N potassium-.
Norma l y ( j-ichromate - bichromate ^
ml ferrous ammonium sulfate
0r Nrjr _ (10.0) (0.025)
ml FAS
36. Record this four decimal
place answer.
37. Round off the answer to
the division so the final
answer has three decimal
places.
36a. Use the same column on the sheet.
IX.C.36.
(p. 52)
38. Record this three decimal
place answer.
38a. Use the same column on the sheet.
38b. This is the "Normality of the FAS solution." The
number will be used later to calculate COD.
IX.C.38.
(p. 52)
39. Record the date.
39a. Use the same column on the sheet.
IX.C.39.
(D. 52)
40. Sign the sheet.
40a. Use the same column on the sheet.
IX.C.40.
(p. 52)
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Conditioning Flasks,
Boiling Beads and
Condensers.
1. If flasks, beads or con-
densers are new, if they
were used for COD tests when
the boiling mixture turned
green, or if they were used
for other tests, use these
steps to condition them for
use in COD tests.
2. Measure 50 ml distilled
water.
3. Pour the water into the
flask to be used in the test.
4. Repeat steps 2 and 3 for
each flask to be used in the
test.
5. Measure 25 ml 0.025 N
potassium dichromate (K^CrpOy
solution. '
6. Pour this ir.tc one of the
flasks.
7. Swirl the flask to mix the
contents.
8. Repeat steps 5, 6, and 7 for
each flask to be used in the
test.
la. After conditioning, do not use this glassware for
any other laboratory procedures. Even traces of
organic materials on the glassware will react
during the test and give higher results.
2a. Use a graduate.
2b. Use high quality distilled water with very -low
COD. (See B.4.).
3a. Rouhd bottom flasks can be supported by a heating
mantle or a cork ring during these steps.
5a. Use a graduate.
Page No. 4-23
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Page No. 4-24
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Conditioning Flasks,
9. Measure 75 ml concentrated
9a. Use a graduate.
Boiling Beads and
sulfuric acid (H0SO/,).
9b. CAUTION: Sulfuric acid causes severe burns to the
Condensers.
C.
skin.
(continued)
10. Tilt the flask and slowly
10a. The solution and flask get very hot.
pour the acid down the
inside wal1.
11. Swirl the flask to mix the
contents.
12. Repeat steps 9, 10 and 11
for each flask to be used
in the test.
13. Add 5 glass beads to each
flask containing mixtures.
14. Carefully swirl each flask
14a. CAUTION: You must thoroughly mix the contents of
again.
the flask to avoid an explosion during procedure.
15. Check the heat of each
15a. If the flasks are just warm to the touch, go to
flask.
the next step. If the flasks are very hot, put
them one by one down into a container of cold
water' to get rid of excess heat.
16. Use a paper towel to wipe
off any water droplets on
the outside of the flask.
17. Attach one of the flasks to
17a. The condenser is described in the equipment list,
a condenser.
page 6.
18. Gently twist the flask while
18a. This ensures a good seal.
gently pushing it upward
onto the condenser.
-------
AFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Conditioning Flasks,
Boiling Beads and
Condensers (Continued)
19. Center the flask on/in a
heating surface.
20. Repeat steps 17, 18 and 19
for each flask you are
conditioning.
21. Do not turn on the water to
cool the condensers.
22. Turn on the heat source for
each flask.
23. When the contents of the
flasks begin to boil, keep
looking to see if vapors
come out of the top of the
condenser.
24. Note the time when you see
vapors coming out.
25. Let the boiling continue
5 to 10 minutes.
26. Turn off the heat source for
each flask.
27. Allow flasks to cool.
28. Squirt distilled water into ¦
the opening at the top of
each of the condensers.
19a. Options for heaters are described in the equip-
ment list, page 6.
21a. You want the vapors of this cleaning mixture to
move all the way up inside the condenser.
27a. This takes 10 to 15 minutes.
28a. Use up to 25 ml of high quality distilled water
with very low COD.
28b. This rinses any condensates down the inside walls
and into the flask.
Page No. 4-25
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Page No. 4-26
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Conditioning Flasks,
Boiling Beads and
Condensers (Continued)
29. Lightly plug the top opening
of each condenser with clean
glass wool.
30. Using a twisting motion,
partially disconnect one of
the flasks from the con-
denser.
31. Squirt distilled water over
the condenser tip, allowing
this rinsing to go down into
the flask.
32. Remove the flask from under
the condenser.
33. Squirt distilled water on
the inside of the neck of
the flask, allowing this
rinsing to go down into the
flask.
34. Turn on the cold water in a
sink.
35. Slowly pour the contents of
the flask directly into the
drain.
36. Let the cold water run at
least 5 minutes.
29a. This prevents contamination from air-borne
particles. The plug can be left in the condenser
during the test.
31a Do not touch the condenser tip with your fingers,
paper towels, etc. Organic contamination of the
tip could result.
34a. You will have to dispose of the cleaning mixture.
34b. Plumbing must be able to tolerate acid.
35a. The glass beads should stay in the flask.
35b. You could pour the contents through a Buchner
funnel to catch the glass beads.
36a. This dilutes the acid in the drain.
-------
.-'LUENT MONITORING PROCEDURE: Determination of Chemical Uxygen Demand
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
.'RAINING
GUIDE NOTES
D. Conditioning Flasks,
Boiling Beads and
Condensers (Continued^
37. Use tap water to rinse the
flask 3 times.
38. Use distilled water to rinse
the flask 3 times.
39. Drain the last of the
distilled water from the
flask.
40. Attach the flask to the
rinsed condenser.
41. Repeat steps 30 through 40
for each flask that is
being prepared for use.
37a. The glass beads should stay in the flask for
these rinsings or rinse those in the Buchner
funnel.
38a. Use high quality distilled water with very low
CCD.
38b. The glass beads should stay in the flask for
these rinsings or rinse those in the Buchner
funnel.
39a. The glass beads stay in the flask, if you have
used a Buchner funnel, roll the beads back into
the flask.
40a. The flask should stay there until it is used for
a test.
E. Conditioning Glassware
Other Than Flasks,
Boiling Beads or
Condensers.
1. If the glassware is new, if
it has been used to measure
COD samples, or if it has
been used for tests other
than COD, use these steps
to condition it for use.
la. Glassware is included in the equipment list on
pages 6, 7 and 8.
lb. This section applies to glassware used to prepare
and store reagents as well as to glassware used
during the COD test.
1c. After conditioning, do not use this glassware for
other laboratory procedures. Even traces of
organic materials on the glassware will react
during the test and give higher results.
Page No. <"-27
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EFFLUENT MONIIORING PROCEDURE: Determination of Chemical Oxygen Demand
Page No. 4-28
OPERATING PROCEDURES
STEP SEQUENCE
I INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
E. Conditioning Glass-
ware Other Than
Flasks, Boiling Beads
or Condensers.
(Continued)
i
2. Measure 250 ml distilled
water.
1
3. Pour the water into a clean
bottle.
4. Measure 125 ml of 0.025 N
potassium dichromate
solution.
5. Pour the measured potassium
dichromate(K?Cr„07) into the
same bottle.
6. Put the bottle into an ice
bath.
7. Keeping the bottle in the
ice water, swirl the con-
tents in the bottle.
8. Leave the bottle in the ice
bath.
9. Measure 375 ml of concen-
trated sulfuric acid
(h2so4).
2a. Use a graduate.
2b. Use high quality distilled water with very low
COD. (See B.4.).
3a. The bottle will be used for storage so have one
with a screw cap. You can use a clean acid
bottle.
4a. Use a graduate.
6a. The depth of ice water should be an inch above
the level of acid in the bottle.
7a. You want to mix it.
8a. You want it to get cool.
9a. Use a graduate.
9b. Use reagent grade acid.
9c. CAUTION: Sulfuric acid causes severe burns to
the skin.
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
OPERATING PROCEDURES
STEP SEQUENCE
1
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
E. Conditioning Glassware
Other Than Flasks,
Boiling Beads or
Condensers.
(Conti nued)
10. Tilt the bottle in the ice
bath and slowly pour the
acid down the inside wall.
11. Keeping the bottle in the
ice bath, swirl the con-
tents in the bottle.
12. Check that the bottle is
cool enough to handle.
13. Label the bottle.
14. Use the warm solution to
rinse over the walls of the
glassware.
15. Discard the used solution.
16. Repeat steps 14 and 15 two
more times for each piece
of glassware to be con-
ditioned .
17. Rinse each piece of glass-
ware with tap water 3 times.
18. Rinse each piece of glass-
ware with distilled water
3 times.
ICa. The solution and bottle get hot.
11a. You want to mix it.
13a. This is Conditioning Solution for COD glassware.
Also mark the date and your name.
14a. You can store the solution for future use. In
this case, pour an adequate volume into a beaker
and warm the solution on a hot plate.
14b. CAUTION: The sulfuric acid in the solution causes
severe burns to the skin.
15a. Turn the cold water tap on in a sink.
15b. Slowly pour the acid down the drain.
15c. Let the tap run at least 5 minutes.
17a. CAUTION: The first rinse contains a significant
amount of sulfuric acid.
18a. Use high quality distilled water with low COD.
Page No. 4-29
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Page No. 4-30
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
E. Conditioning Glassware
Other Than Flasks,
Boiling Beads or
Condensers.
(Continued)
19. Let the glassware drain dry,
20. Store the glassware
separately from glassware
used for tests other than
COD.
21. Store unused solution for
future use.
F. Oxidation of the Sample
and Blank
1. Remove two reflux flasks
from COD flask-condenser
asserr.bl ies.
2. Mark the sample identifi-
cation on the outside of
one of the flasks.
3. Mark the word, "Blank" on
the outside of the second
flask.
4. Measure 1 grarr. of mercuric
sul fate(HgSO^)•
5. Place the mercuric sulfate
in the Sample flask.
la. Equipment is described on pages 6, 7 and 8.
lb. Each flask should have 5 glass beads in it.
lc. All flasks, glass beads and condensers should
have been used previously.for CCD tests. If any
flask, beads or condenser is new or has been used
for other tests,each must be conditioned according
to Procedure D. in this EMP.
2a. Use a wax marking pencil.
2b. See the label on the sample bottle for an identifi-
cation code.
2c. In this procedure we will call this the Sample
flask.
3a. You will prepare a blank and test it in the same
manner as the sample.
3b. In this procedure, we will call this the Blank
flask.
4a. Use a 1 gram reagent spoon.
5a. Round bottom flasks can be supported by a heating
mantle or a cork ring during these steps.
VII.F.3a.
(p. 48)
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EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
'. Oxidation of the
Sample and Blank
(continued)
6. Repeat steps 4 and 5 for
the Blank flask.
7. Shake the bottle of sample.
8. Draw 50.0 ml of sample
into a pi pet.
8a. Use a clean volumetric pipet.
8b. Use a pipet bulb.
8c. For samples that turn green during the test and
which cannot be titrated to an acceptable end
point, you may need to dilute the sample to a
final volume of 50.0 ml at this step. You won't
know you have to do this until you have run
50.0 ml of the sample through the test up to H.
Quantification, Step 12. To avoid this
uncertainty, you can prepare dilutions now. See
Training Guide.
VII.F.8c.
(p. 49)
9. Deliver the 50.0 ml into
the Sample flask.
9a. Record "S, ml Sample Used" on the "Typical
Laboratory Data Sheet" in this EMP.
IX.F.9.
(p. 51)
10. To prepare the blank,
draw 50.0 ml of distilled
water into another pipet.
10a. Use high quality distilled water with very low
COD.
10b. Use a clean volumetric pipet.
10c. Use a pipet bulb.
11. Deliver the 50.0 ml
distilled water into the
Blank flask.
12. Draw 5.0 ml concentrated
sulfuric acid (H„S0,) into
a pipet.
12a. CAUTION: Sulfuric acid causes severe skin burns.
12b. Use a clean 10 ml graduated pipet and a pipet
bulb, or else an automatic dispenser checked for
5.0 ml delivery.
V.F.12b.
(p. 46)
13. Deliver the 5.0 ml of acid
into the Sample flask.
13a. Tilt the flask and deliver the acid down along the
inside wal1.
Page No. 4-31
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EFFLUENT MONITORING PROCEDURE:Determination of Chemical Oxygen Demand
Page No. 4-32
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Oxidation of the
Sample and Blank
(continued)
14. Rinse off the outside of
the pipet at a sink.
15. Swirl the contents of the
flask.
16. Repeat steps 12 through 15
to add 5.0 ml of acid to
the Blank flask and rinse
the pipet.
17. Prepare an ice bath.
18. Place the Sample flask
into the ice bath.
19. Draw 25.0 ml of 0.025 N
potassium dichromate
(K^Cr^O^) solution into a
pi pet.
20. Swirl the flask as you
slowly add the 25.0 ml of
0.025 N potassium dichro-
mate solution.
21. Let the flask in the ice
bath.
22. Measure 70 ml of sulfuric
acid-silver sulfate
(H^SO^-Ag^SO^) solution.
23. Tilt the flask in the ice
bath and swirl to continu-
ously mix as you slowly add
the acid-silver sulfate
solution down the inside
wall of the flask.
14a. Use tap water to rinse any acid into the sink.
14b. Let the water continue to run for a few minutes.
15a. Most of the mercuric sulfate dissolves.
17a. The depth of the water should be about one inch.
19a. Use a clean volumetric pipet.
19b. Use a pipet bulb.
21a. You want to cool the flask.
22a. CAUTION: Sulfuric acid causes severe skin burns.
22b. Use a clean 100 ml graduate or use an automatic
dispenser checked for accurate delivery.
23a. If the acid-sulfate solution is added too rapidly,
heat at the surface of the solution can cause
spattering upward.
V.F.22b.
(p. 46)
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
r. Oxidation of the
24. Swirl the flask in the ice
24a. You want to cool the flask.
Sample and Blank
bath.
(continued)
25. Remove the flask from the
25a. The bottom part of the flask may still be warm.
ice bath.
26. Wipe the water off the
26a. Use a paper towel.
outside of the flask.
27. Carefully swirl the flask
27a. CAUTION: You must thoroughly mix the contents of
again.
the flask to avoid an explosion during reflux.
27b. CAUTION: Do not swirl so vigorously that the
contents come out of the flask.
28. Repeat steps 18 through 27
to add potassium dichromate
and sulfuric acid-silver
sulfate solutions to the
Blank flask.
29. Attach the Sample flask to
29a. The condenser is described in the equipment list,
a condenser.
page 6.
30. Gently twist the flask
30a. This ensures a good seal.
while gently pushing it
upward ont6 the condenser.
31. Center the flask on/in a
31a. Choices for heaters are described in the
heating surface.
equipment list, page 6.
32. Repeat steps 29 through 31
to attach the Blank flask
to a condenser.
33. Start the circulation of
cooling water through the
two condensers.
Page No. 4-33
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Page No. 4-34
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Oxidation of the
Sample and Blank
(continued)
34. Turn on the heat source
for each flask.
35. When the contents of the
flasks begin to boil,
record the date and time.
36. Regulate the heat sources
if necessary.
35a. Use the "Typical Laboratory Data Sheet."
35b. Use the columns for the sample(s) and blank.
36a. Adjust the heat to maintain a gently rolling boil
in each flask.
IX.F35.
(p. 51)
37. Reflux the contents of the
flasks for two hours.
37a. The mixtures in the flasks are usually a dark
orange color during this period. If some turn
to a green color, the potassium dichromate may
be completely reacted. Continue the test for
such flasks, though, because there may be enough
potassium dichromate left to titrate later on in
Procedure H. Quantitation.
37b. If the samples are known to require less time for
complete oxidation, less reflux time is
acceptable.
VII.F37b.
(p. 50 )
G. Rinsing and Removing
Flasks from
Condensers (continued)
1. Turn off the heat under
the flask-condenser
assemblies.
2. Allow the flasks to cool.
3. Squirt distilled water in-
to the opening at the top
of the condenser which is
attached to the flask
containing the sample.
la. The contents of the flasks should have gently
boiled for 2 hours.
2a. This takes 10 to 15 minutes.
2b. Placing an evaporating dish upside down between
the flask and the heating surface makes for
faster cooling.-
3a. Use high quality distilled water with very low
COD.
3b. You want to rinse any condensates down the inside
walls and into the flask.
3c. Use up to 25 ml of water.
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
G. Rinsing and Removing
Flasks from
Condensers (continued)
4. Using a twisting motion,
partially disconnect the
flask from the condenser.
5. Squirt distilled water
over the condenser tip,
allowing this rinsing to
go down into the flask.
6. Remove the flask from
under the condenser.
7. Squirt distilled water on
the inside of the neck of
the flask, allowing this
rinsing to go down into
the flask.
8. Squirt distilled water
down along the inside of
the wal1s of the flask.
9. Add enough distilled
water to the flask con-
taining the sample to
bring the final volume
to about 300 ml.
4a. Point the lower tip of the condenser down into
the flask.
4b. Be very careful to avoid adding organic contimina-
tion to the joint and into the inside of the neck
of the flask. Do not touch these parts with your
fingers, paper towels, etc.
7a. You want to rinse any condensates down into the
flask.
8a. If a 300 ml/round bottom flask has been
used, transfer the mixture to a 500 ml Erlenmeyer
flask. Squirt distilled water down the inside
walls of the original flask and pour the rinsing
into the Erlenmeyer flask. Repeat this rinsing
of the original flask three times.
9a. If volumes are marked on the flask, add distilled
water to the 300 ml mark.
9b. If volumes are not marked on the flask, estimate
the amount of water needed to bring the volume
to 300 ml, measure it in a graduate and add it
to the flask. (The original mixture totaled
150 ml and rinsings of the condenser, joint and
flask would range from 40 to 70 ml.)
Page No. 4-35
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand Page No. 4-36
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
G. Rinsing and Removing
Flasks from
Condensers (continued)
10. Put this flask near the
titration stand.
11. Squirt distilled water in-
to the opening at the top
of the condenser which is
attached to the flask
containing the distilled
water blank.
12. Repeat steps 4 through 10
above to rinse inner walls
of this condenser and
flask and to bring the
final volume to about
300 ml.
11a. Use high quality distilled water with very low
COD.
lib. You want to rinse any condensates down the
inside walls and into the flask.
11c. Use up to 25 ml of water.
H. Quantification:
Titration of Sample
and Blank
1. Rinse and drain the inside
of a clean, 50 ml buret
with about 15 ml of
ferrous ammonium sulfate
[Fe(NH4)2(S04)2-6H20]
solution of known
normali ty.
2. Put the buret into the
clamp on the titration
stand.
3. Close the stopcock of the
buret.
4. Add about 15 ml of the
ferrous ammonium sulfate
solution.
la. Ferrous ammonium sulfate solution is unstable and
must be standardized on the day you use it so the
normality is known. The procedure to do this is
described in "C. Standardization of Ferrous
Ammonium Sulfate Solution."
4a. Use a funnel.
4b. In this section, the letters FAS will be used
when referring to the ferrous ammonium sulfate
solution of known normality.
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
H. Quantification:
Titration of Sample
and Blank (continued)
5. Check the tip of the buret
for air bubbles.
6. Add more FAS solution to
the buret.
7. Record the level of the
solution in the buret.
8. Check that the flask con-
taining the sample is at
room temperature before
proceeding.
9. Gently swirl the contents
of the flask containing
the sample.
10. Add 10 drops of ferroin
indicator solution to the
mixture in the flask.
11. Again, gently swirl the
contents of the flask.
12. Add FAS solution from the
buret fairly rapidly,
while constantly swirling
the mixture in the flask.
5a. If there is an air pocket, swiftly turn the stop-
cock in a complete circle to expel it. You may
have to repeat this turning of the stopcock.
6a. You will need up to 25 ml of FAS for each sample
and blank.
7a. On the "Typical Laboratory Data Sheet" in the
column with the sample identification information
7b. This is the "ml FAS at START of titration".
8a. You may have to put the flask into a pan of cool
water to get this condition.
9a. This ensures thorough mixing.
9b. Do not swirl any of the contents out of the flask.
10a. The ferroin should be in a dropper bottle. If it
isn't, use a medicine dropper to transfer it.
10b. Ten drops are used for a 300 ml mixture.
11a. This ensures thorough mixing.
lib. The mixture is a deep orange color.
12a. You must constantly swirl the receiving flask so
that the FAS solution comes into contact and
reacts with all the mixture in it.
12b. The mixture in the flask will gradually change
color becoming green, then blue-green. When the
addition of FAS solution makes a reddish-brown
color at the surface of the sample solution,
close the stopcock.
(continued)
IX.Sheet I.
(p. 51)
IX.H.7.
(p. 51)
Page No. 4-37
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Page No. 4-38
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
H. Quantification:
Titration of Sample
and Blank (continued)
13.
14.
15.
16.
17.
Now adjust the stopcock so
the FAS solution in the
buret goes into the flask
one drop at a time.
Stop adding FAS solution
when all the mixture in
the flask is a reddish
brown color.
12c. If a sample had turned a green color during the
2-hour boiling period, there may be no potassium
dichromate solution left in the flask. If you
add up to 22 ml of FAS solution to such a
mixture and cannot observe the color changes
described above in 12b., stop the titration.
You should do the test over, using a smaller
volume of sample. (See Training Guide). Also,
the flask, boiling beads and condenser used for
that sample will have to be conditioned before
re-use. (See Procedure D.)
13a. Swirl the flask after each drop is added. At the
end point, one drop is enough to change the color
of all the solution to a reddish brown.
14a. This is the end point of the reaction in the
flask.
VII.H.12c.
(p. 49)
Record the final
the FAS solution
buret.
level of
in the
Subtract the recorded "ml
of FAS at beginning of
titration "from" ml of FAS
at end of titration" and
record the difference on
your data sheet.
To titrate the blank,
first check the level of
the FAS solution in the
buret.
15a.
On the data sheet,
thi s
is "ml FAS at END of
titration".
15b.
Use the column for
thi s
sample.
16a.
On the data sheet,
thi s
is "B, ml. FAS used to
titrate the Sample1
l
16b.
Use the column for
thi s
sample.
17a. You will need up to 25 ml of FAS solution to
titrate the blank. Add more FAS if necessary.
IX.H.15
(p. 51)
IX.H.16.
(P- 51)
-------
EFFLUENT MONITORING PROCEDURF: Determination of Chemical Oxygen Demand
OPERATING PROCEDURES
STEP SEQUENCE
IN FORMAT ION/OPERATING GOALS/SP ECIFICATIONS
TRAINING
GUIDE NOTES
H. Quantification:
Titration of Sample
and Blank (continued)
18. Record the level of the
FAS solution in the buret.
19. Check that the flask
containing the blank is at
room temperature before
proceedi ng.
20. Gently swirl the contents
of the flask containing
the distilled water
blank.
21
18a.On line one of a column on the data sheet, write
"Blank" as the identification.
18b. In that column, record the level of FAS as "ml
FAS at START of titration".
19a. You may have to put the flask into a pan of cool
water to get this condition.
20a. This ensures thorough mixing.
20b. Do not swirl any of the contents out of the flask
Repeat steps 10 through 14
above to add the FAS solu-
tion from the buret until
all the mixture in the
flask is reddish brown.
22.
23.
Record the final
the FAS solution
buret.
level of
in the
Subtract the recorded "ml
of FAS at beginning of
titration" from "ml of FAS
at end of titration" and
record the difference on
your data sheet.
IX.Sheet
(p. 51)
IX.H.18.
(p.51)
22a. On the data sheet, this is "ml FAS at END of
titration."
22b. Use the column for the blank.
23a. On the data sheet, this is "A, ml FAS used to
titrate the Blank".
23b. Use the column for the blank.
IX.H.22.
(P. 51)
IX.H.23.
(p. 51)
Page No. 4-39
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Page No. 4-40
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
I. Clean Up
Carefully pour the
contents of both the
Sample and the Blank
flasks through a cleaned
Buchner funnel and into a
storage container.
2. Use tap water to rinse
each flask 3 times.
3. Use distilled water to
rinse each flask 3 times.
4. Wipe any wax markings off
the outside of the flasks.
5. Use tap water to rinse
the beads 3 times.
6. Use distilled water to
rinse the beads 3 times.
7. Transfer 5 glass beads to
each rinsed reflux flask.
8. Attach each flask to a
condenser used and rinsed
during the test.
la. The glass beads should stay in the funnel.
lb. The storage container should be glass or thick
plastic with a screw cap.
1c. These mixtures have up to 25% concentrated
sulfuric acid so handle and store them with
caution.
Id. These mixtures also contain mercury complexes
which need special treatment for disposal.
2a. If the flask contained a mixture which turned
green during the 2 hour boiling period, the
flask, beads and the condenser will have to be
conditioned before re-use for a COD test.
(See Procedure D.)
3a. Use high quality distilled water with very low
COD (See B.4.)
5a. The beads are held in the Buchner funnel
6a. The beads are still in the funnel.
7a. Do not contaminate the beads at this step. Use
a spatula to roll the beads from the edge of the
funnel to the flask or use forceps to make the
transfer.
8a. The flasks should stay there until they are used
agai n.
VI.I.Id.
(p. 47)
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
OPERATING PROCEDURES
STEP SEQUENCE
|
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
I. Clean Up (continued)
9. Drain any ferrous ammonium
sulfate solution out of
the buret.
10. Use tap water to rinse
the inside of the buret
3 times.
11. Use high quality distilled
water to rinse the buret
3 times.
12. Put the buret back in the
titration stand but upside
down with the stopcocK
open.
13. Other glassware (pipets,
etc.) used during the test
should be rinsed with tap
water.
14. As soon as possible, this
other glassware should be
cleaned using Procedure E.
9a. This can be put directly down the drain of a sink,
12a. The buret can drain completely.
12b. This buret should be used only for the COD test.
Even traces of organic materials from other
solutions may result in errors in future COD
ti trations.
Page No. 4-41
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Page No. 4-42
OPERATIC PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
J. Calculations
1. Use the following steps to
calculate COD, rug/liter:
3.
4.
Subtract "B, ml FAS used to
titrate the Sample" on line
10 from "A, ml FAS used to
titrate the Blank" on line
9.
Write the difference on
line 11 of the data sheet.
Record "N, normality of
FAS" on line 12 of the
data sheet.
la. The calculation formula is:
Chemical Oxygen Demand, mg liter =
(A-B) N x 8000
S
Where:
A= ml FAS to titrate the Blank
B= ml FAS to titrate the Sample
N= normality of FAS
S= ml Sample Used
8000 converts to COD, mg/liter
lb. The "Typical Laboratory Data Sheet" has the steps
and an example for doing this calculation.
lc. Numbers used in the examples below are from the
example in the last columns on the "Typical
Laboratory Data Sheet".
2a. Example on data sheet:
line 9 : 23.55
line 10: 15.00
Difference= 8.55
3a. This has been done for the example on the data
sheet.
4a. This number is calculated as shown by the
example on the sheet titled "Standardization of
Ferrous Ammonium Sulfate (FAS) Solution.''
4b. The example number, 0.024, from C.38 on that
sheet has been recorded on line 12 of the data
sheet.
IX.Sheet I
(p. 51)
IX.Sheet I
(P. 51)
IX.J.3.
(p. 51)
IX.J.4.
(p. 51)
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
J. Calculations
(continued)
5. Multiply the difference on
line 11 by "N, normality of
FAS" on line 12.
5a. Example on data sheet:
8.55 x 0.024 = 0.2052
6. Record the product on line
13.
6a. This has been done for the example on the data
sheet.
IX.J.6.
(p. 51)
7. Divide 8000 by "S, ml Sample
Used" which is recorded on
1ine 6.
7a. Example on data sheet:
8000 - ifin
50.0 " 160
8. Record the answer on line 14,
8a. This has been done for the example on the data
sheet.
IX.J.8.
(p. 51)
9. Multiply line 13 by line 14.
9a. Example on data sheet!
0.2052 x 160 = 32.8320
10. Record the product on line
15.
10a. This has been done for the example on the data
sheet.
IX.J.10.
(p. 51)
11. Round off the number on line
15 to the nearest whole
number of mg/liter.
11a. 32.8320 becomes 33.
lib. If your answer for a sample is greater than
50 mg/liter, you should start using the 0.250 N
solutions of potassium dichromate and ferrous
ammonium sulfate for samples from that same source.
See the Training Guide.
I.J.lib.
(p. 44)
12. Record this number on line
16.
12a. This has been done for the example on the data
sheet.
IX.J.12
(p. 51)
13. Sign the data sheet on line
17.
13a. This has been done for the example on the data
sheet.
IX.J.13.
(p. 51)
Page No. 4-43
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
TRAINING GUIDE
SECTION
TOPIC
I*
Introduction
II
Educational Concepts-Mathematics
III
Educational Concepts-Science
IV
Educational Concepts-Communications
V *
Field & Laboratory Equipment
VI *
Field & Laboratory Reagents
VII *
Field & Laboratory Analysis
VIII
Safety
IX *
Records & Reports
*Training guide materials are presented here under the heading marked*.
These standardized headings are used throughout this series of procedures.
Page No. 4-44
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Introduction
Section I
TRAINING GUIDE MOTE
REFERENCES/RESOURCES
The Chemical Oxygen Demand (COD) Test provides an
estimate of the proportion of sample matter suscept-
ible to oxidation by rigorous oxidation conditions.
Some inorganic compounds may be oxidized but most of
the reaction involves organic"compounds. Thus the
COD Test provides a commonly used estimate of organic
materials in water samples.
The procedure described in this EMP is for the range
of 5 to 50 mg/liter COD one expects in treatment
plant effluents. If you use this EMP procedure and
get results greater than 50 mg/liter COD, you should
do the test in the same manner as described in the
EMP but use more concentrated solutions (0.250 N
instead of 0.025 N) of potassium dichromate and of
ferrous ammonium sulfate. For "B. Reagent Prepara-
tion", you would not make number 6 (0.025 N potassium
dichromate solution) nor number 9 (0.025 N ferrous
ammonium sulfate solution). Any place in the
procedure that refers to 0.025 N concentrations of
either of these solutions should be read as 0.250 N.
All other instructions and information are to be
followed as written.
The Test described in this instruction can be found
in the 1974 EPA Methods Manual on Page 21, entitled
Chemical Oxygen Demand (Low Level). Other references
which have acceptable procedures for this test for
NPDES purposes are: 14th ed. Standard Methods on
page 550 and 1975 ASTM Part 31 on page 472.
Methods for Chemical
Analysis of Water and
Wastes, 1974, EPA, MDQARL
Cincinnati, Ohio 45268
p. 21.
Standard Methods for the
Examination of Water and
Wastewater, 14th ed., 1976,
APHA, Mew York, N.Y. p. 550
Annual Book of Standards,
Part 31, Water, 1975, ASTM,
Philadelphia, PA, p. 472
Page No. 4-45
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Field and Laboratory Equipment
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
AUTOMATIC DISPENSERS:
Since sulfuric acid causes severe burns to the skin,
you may choose to use a glass, automatic dispenser
(pipet) to store and measure the two reagents
involving this acid. Use the manufacturer1s instruc-
tions to fill and prime the dispenser and to make the
initial setting of the delivery volume. Sulfuric
acid is heavier than water so this delivery volume
must be checked. Do this by delivering the acid into
a clean, dry graduate. Allow the acid to "settle"
in the graduate, then read the volume. If that
volume is more or less than it should be (see below),
adjust the delivery setting on the dispenser accord-
ingly. Then check the new setting
delivery, using another clean, dry
tinue this procedure until you are
the delivery volume is accurate.
for accurate
graduate. Con-
satisfied that
The final volumes required for the concentrated
sulfuric acid reagent are 5 ml and 20 ml so adjust
a dispenser to deliver 5 ml. Then dispense the 5 ml
four times for the 20 ml requirement.
The final volume required for the sulfuric acid -
silver sulfate reagent is 70 ml. In this case,
adjust a dispenser to deliver 10 ml. Then dispense
the 10 ml seven times for the 70 ml requirement.
Page 4-46
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Field and Laboratory Reagents Section VI
TRAINING GUIDE MOTE
REFERENCES/RESOURCES
I.Id.
DISPOSAL OF MERCURY-CONTAINING WASTES:
These wastes can be treated to convert the soluble
mercury complexes to some insoluble formation.
Some refiners of mercury are willing to accept
shipments of such precipitates to recycle the metal.
Ask your City, County, or State Pollution Control
Agency for specific instructions on how you are to
dispose of COD test wastes.
Dean, Williams, Wise:
"Disposal of Mercury
Wastes from Water
Laboratories," Environmental
Science and Technology
Vol. 5, No. 10, 1971.
p. 1044
^aag and Hecker:
"Recovery of Mercury in
Solution," Journal of
Environmental Quality,
Vol. 1, No. 2, 1972, p. 192.
Page No. 4-47
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Field and Laboratory Analysis
Section VII
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
BLANKS:
You run a blank by using, distilled v/ater instead of
sample water and testing that distilled water in the
same way you test samples. By doing this, you are
checking the COD of the disti-lled water and of the'
reagents used in the test. You use the titration
results for the blank in the calculation formula to
correct your COD values for samples.
Some contamination is expected to show up and affect
the titration results. However, if the blank
requires less than 90% of the ml of ferrous
ammonium sulfate solution that would be required to
titrate a situation of "no contamination," it is
a signal to you that the distilled water or reagents
are contributing contamination and should be
checked.
EXAMPLE - If 25.0 ml of 0.025 N potassium dichromate
solution are used"to try to oxidize a blank con-
taining no oxidizable contamination, it would take
25.0 ml of 0.025 N ferrous ammonium sulfate solution
to react with the remaining potassium dichromate
during the Quantitation Titration Procedure. 90% of
25.0 ml of FAS would be 22.5 ml. If you use less
than 22.5 ml of FAS for two or more blanks, a check
of the distilled water and of the reagents is
advisable.
See "B. Reagent Preparation, Procedure 4. Distilled
Water" for information about checking the quality
of distilled water and about storing it.
To check reagents, consult your laboratory records
to see which reagent was made most recently. Using
the "E. Conditioning..." Procedure, clean the glass-
ware required and then- prepare a fresh supply of
that reagent. Use the fresh reagent and run a blank.
If the blank results are still too high, you should
purchase a new supply of the chemical to make your
reagent solution. If you always purchase reagent
grade chemicals and take care not to contaminate
them with dirty spatulas, etc., you should not have
problems with them.
Page No. 4-48
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Field and Laboratory Analysis Section VII
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
F.8 c.
H.12c.
SMALLER VOLUMES OF SAMPLE FOR THE TEST:
If you cannot titrate a 50.0 ml sample to an accept-
able end point during "H". Quantitation step 12,"
you will have to re-run the test .beginning with
"F. Oxidation of the Sample and Blank". At F. Step
8, you will have to use less sample and then add
dis-tilled water to make the 50.0 ml volume. The
workable proportions can only be determined by trial
and error. You might prepare two mixtures at this
step such as:
Add 10.0 ml sample, then 40 ml distilled water to
the flask
Add 25.0 ml sample, then 25 ml distilled water to
the fl-ask.
(1) Use a pi pet to measure the sample,
(2) Use a graduate to measure the water,
(3) Use high quality distilled water with low COD.
(4) If you get results for both dilutions, use
the results for the 25.0 ml sample.
(5) For future tests of. samples from the same
source, at step F8 use the dilution proportions
you found workable..
(6) Do not change the volumes of any other solutions
in the test. The volumes as given in the EMP
are critical conditions of the test.
(7) If you cannot titrate as low as 10.0 ml of
sample (with 40 ml water added to the test
mixture) during "H. Quantitation, s'tep 12",
you will have to do the test using the 10.0 ml
sample and more concentrated potassium
dichromate and ferrous ammonium sulfate
solutions (the 0.250 N solutions are used).
See section I in the Training Guide for a
discussion of this.
Page No. 4-49
-------
EFFLUENT MONITORING PROCEDURE: Determination of Chemical Oxygen Demand
Field and Laboratory Analysis
Section VII
TRAINING GUIDE MOTE
REFERENCES/RESOURCES
F.37b.
TWO HOUR OXIDATION (BOILING) PERIOD:
Some samples contain materials that can be oxidized
in the COD test conditions within a very short time
period. If your samples always have the same
materials in them, you may want to check them and
see if you might use a shorter oxidation (boiling)
period. Prepare two test mixtures from the same
sample. Boil one for two hours, boil the other for
say 30 minutes. Complete the test as usual and
calculate COD results for each. Do 6 other such
duplicate tests on 6 other samples. These should
be done over a period of time on samples collected
from the same source over a period of time. Compare
the results from the seven tested by using the
usual 2 hours with the results from the seven tested
by using a shorter oxidation (boiling) period. If
the results are the same or if they agree within +
4 mg/liter COD, you may use the shorter oxidation
(boiling) time for future samples from the same
source. About once every 10 times you perform the
test on such samples you should check that_they
continue to be the same composition. Do this by
preparing a duplicate test mixture, using a two hour
boiling period for the second mixture, and comparing
the results for agreement as above.
Methods for Chemical
Analysis of Water and
Wastes, 1974 EPA, MDQARL
Cincinnati, OH 45268, p. 23.
Handbook for Analytical
Quality Control in Water
and Wastewater Laboratories.
1971, EPA, MDQARL,
Cincinnati, Ohio 45268
6-1.
Page No. 4-50
-------
tv currT t
Typical Laboratory Data Sheet for Chemical Oxygen Demand, mg/liter
Name of Plant
A.5
Identification
Blank
EFF #1
1
A.5
Type (grab, composite)
Composi te
2
A.5
Date and Time Collected
3/17/75
0600-1200
3
A.5
Sample Collector
Tom Sampler
4
F. 35
Date and Time Boiling Began
3/17/75
1300
5
F. 9
RECORD: S, ml Sample Used
50.0
6
H.15
H. 22
ml FAS * at END of titration
38.55
20.00
7
H. 7
H. 18
ml FAS * at START of titration
15.00
5.00
8
H. 23
A, ml FAS* used to titrate the Blank
23.55
23.55
9
H. 16
B, ml FAS * used to titrate the Sample
15.00
10
J.3
SUBTRACT B (line 10) from A (line (9)
8.55
11
J.4
RECORD: N, normality of FAS *
(Calculated on Standardization Sheet,C.29)
0.024
12
J.6
MULTIPLY ml Difference of FAS * (line 11)
by Normality of FAS (line 12)
0.2052
13
J.8
DIVIDE 8000 by S, ml Sample Used
(See line 6)
160
14
J.10
MULTIPLY line 13 by line 14
32.8320
15
J.12
ROUND OFF line 15 to the nearest
whole number of mg/liter
33
16
J.13
Si gnature
Jim Analyst
17
* FAS means Ferrous Ammonium Sulfate Solution CALCULATION FORMULA: COD, mg/liter = (A-B)N x 8000
S Page No. 4-51
-------
Page No. 4-52
STANDARDIZATION OF FERROUS AMMONIUM SULFATE (FAS) SOLUTION IX SHEET II
Flask 1
Duplicate
C. 31
ml FAS at END of titration
24.60
35.15
1
C. 23
ml FAS at START of titration
14.00
24.60
2
C. 33
—
ml FAS used for Standardization
(SUBTRACT ml FAS at START on line 2
from ml FAS at END on line 1)
10.60
1
10.55 5 3
i
1
C. 36
DIVIDE 0.250* by the ml difference
on line 3 to a 4 decimal place answer.
i
0.0236 j 4
C. 38
Normality of the FAS solution
(ROUND OFF line 4 to 3 decimal places)
!
0.024 : 5
r
1
C. 39
Date
1
3/17/75 | 6
1
C.40
Signature
1
Jim Analyst ! 7
* From the formula:
(10.0 ml potassium) (0.025 N potassium)
Normality FAS = dichromate dichromate
ml FAS
-------
A PROTOTYPE FOR DEVELOPMENT OF
ROUTINE OPERATIONAL PROCEDURES
for the
DETERMINATION OF TOTAL KJELDAHL NITROGEN
as applied in
WASTEWATER TREATMENT FACILITIES
AND IN THE
MONITORING OF EFFLUENT WASTEWATERS
National Training Center
Municipal Operations and Training Division
Office of Water Program Operations
U.S. Environmental Protection Agency
CH.N.EMP.lb.3.76
Page No. 5-1
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
This Operational Procedure was developed by:
NAME William T. Engel
ADDRESS Charles County Community College
P. 0. Box 910
LaPlata, Maryland 20646
POSITION Assistant Professor of Chemistry
EDUCATION & TECHNICAL BACKGROUND
BS - Saint Francis College, Loretto, Pennsylvania
MS - Xavier University, Cincinnati, Ohio
6 years Instructor:
Instructor - Associate Professor (Chemistry)
Page No. 5-3
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
1. Objective:
To determine the Total Kjeldahl Nitrogen content of an effluent.
2. Description of Analysis:
The procedure converts nitrogen components of biological origin such as amino
acids, proteins, and peptides to ammonia. Two alternatives are listed for the
determination of ammonia after distillation: the titrimetric method which is
applied to concentrations above 1 mg N/liter and the colorimetric metFiocl
which is applicable to concentrations below 1 mg N/liter.
3. Applicability of this Procedure:
a. Range of Concentration:
Colorimetric Method - 0.03 to 1.0 mg NH-j-N/liter
Titrimetric Method - 1.0 to 25 mg NH^-N/liter
(The range of these methods may be extended for samples by dilution.)
NOTE: A range from 0.05 to 1400 mg NhL-N/liter is available by using
an ammonia selective ion electrode. A separate EMP on this
method is available.
b. Pretreatment of Samples:
The Federal Register Guidelines do not specify any pretreatment.
c. Treatment of Interferences in Samples:
The Source of Procedure* does not note any interferences to this
determination.
*Source of Procedure: Methods for Chemical Analysis of Water and Wastes, 1974,
Environmental Protection Agency, Methods Development and Quality Assurance Research
Laboratory, Cincinnati, Ohio, page 175.
Page No. 5-4
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
FLOW CHART
DIGEST
DISTILL
SAMPLE
DISTILL
TITRIMETRIC
METHOD
TITRIMETRIC
METHOD
NESSLERIZATION
METHOD
NESSLERIZATION
METHOD
LESS THAN
1 mg/1
AMMONIA
LESS THAN
1 mg/1
AMMONIA
AMMONIA
NITROGEN
RESULTS
GREATER
THAN 1 mg/1
AMMONIA
GREATER
THAN 1 mg/1
AMMONIA
TOTAL KJELDAHL
NITROGEN (TKN)
RESULTS
It should be mentioned that the Ammonia determination listed on the right side
of the flow chart follows the same procedure as the left side from DISTILL down
to the completion.
The Organic Nitrogen may be calculated as follows:
Organic Nitrogen = Total Kjeldahl Nitrogen - Ammonia Nitrogen.
Page No. 5-5
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
Equipment and Supply Requirements
A. Capital Equipment - Macro or Micro Determinations
1. Digestion Apparatus and Distillation Apparatus:
The pieces of equipment required to assemble a system for digestion
and distillation will differ according to whether a 500 ml sample
(macro determination) or a 50 ml sample (micro determination) is
analyzed. See the diagrams at the end of these listings to identify
the items required for your choice of determinations.
2. Balance, analytical, capable of weighing to 0.1 mg at a 200 g load
3. Balance, triple beam, capable of weighing to 0.1 g at a 500 g load
4. Spectrophotometer for use at 400-425 nm with a light path of 1 cm or
longer
5. Water Still and an anion-cation exchange system to produce ammonia-
free water
B. Reusable Supplies - Macro or Micro Determinations
1. One 50 ml beaker, graduated
2. One 100 ml beaker, graduated
3. Two 150 ml beakers, graduated
4. One 250 ml beaker, graduated
5. One 50 ml bottle, glass with stopper
6. Three 150 ml bottles, glass with dropper tops
7. One 500 ml bottle, glass
8. Two 1000 ml bottles, glass with tops
9. One 50 ml buret
10. One 10 ml cylinder, graduated
11. One 100 ml cylinder, graduated
12. One 500 ml cylinder, graduated
13. One 50 ml Erlenmeyer flask, graduated
14. One 125 ml Erlenmeyer flask, graduated
15. Four 1000 ml Erlenmeyer flasks, graduated
16. Five 1000 ml volumetric flasks with stoppers
17. Glass beads, 4 mm
18. Nine Nessler tubes, scored at 50 ml
19. One Nessler tube support
20. Two 10 ml pi pets, Mohr, graduated
21. One 10 ml pi pet, volumetric
22. One 25 ml pi pet, volumetric
23. One 50 ml pipet, volumetric
24. One ring stand
25. One buret holder
26. One #3 or #6 rubber stopper or a cap to fit Nessler tubes
NOTE: All beakers and flasks should
Page No. 5-6
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
C. Consumable Supplies - Macro or Micro Determinations
1. 4 g ammonium chloride, NH^Cl, reagent, granular
2. 20 g boric acid, H^BOg,
3. 700 ml ethyl alcohol, C2H,-0H, reagent,denatured
4. 0.5 g methyl orange, indicator (for titration method)
5. 200 mg methyl red, reagent (for titration method)
6. 200 mg methylene blue (for titration method)
7. 4 g mercuric oxide, HgO, reagent powder, red
8. 100 g mercuric iodide, Hg^
9. 5 g phenolphthalein powder
10. 70 g potassium iodide, KI, reagent powder
11. 134 g potassium sulfate, I^SO^, reagent powder
12. 5 g sodium carbonate, r^COg, reagent powder (for titration method) anhydrous
13. 660 g sodium hydroxide, NaOH, reagent pellets
14. 25 g sodium thiosulfate pentahydrate, Na2S202*5^0, reagent grade
15. 223 ml sulfuric acid, h^SO^, reagent grade
16. 14 weighing boats
The following reagents may be purchased commercially thus alleviating
several sections under reagent preparations and some of the above
chemical requirements.
*1. Nessler reagent (100 g mercuric iodide, 70 g potassium iodide,
160 g sodium hydroxide)
*2. Phenolphthalein indicator solution, 1% (500 ml ethyl alcohol,
5 g phenolphthalein)
*3. Digestion reagent [i.e. Kel-Pac^(Olin-Matheson)] [4 g mercuric oxide (red),
134 g potassium sulfate, 220 ml sulfuric acid]
*4. Sulfuric acid (0.02N) (5 g sodium carbonate, 3 ml sulfuric acid)
Commercially prepared reagents may be used in analyses for NPDES
purposes if the solutions have been prepared according to the
reagent section of the approved methods cited in the Federal
Register. It is strongly recommended that purchased reagents be
verified by initially checking them against a quality control check
sample available through your Regional EPA Analytical Quality
Control Coordinator (from 3/21/75 EPA-MDQARL memo).
Page No. 5-7
-------
* t„>»i Kieldahi Nitrogen
Scbo KJEtom"determ.nat.on
digestion APPARATUS
kjeldahl
SPRAY TRAP-
800 ml
KJELDAHL
flask
CONDENSER
{[ 500 ml
' ERLENMEYER
receiving
flask
distillation APPARATUS
Page Ho. 5-8
-------
EFFLUENT MONITORING PROCEDURE: Deterwtnatlon of Total Kjeldahl Nitrogen
MICRO KJELDAHL DETERMINATION
-0-2.-5;
DIGESTION APPARATUS
SAFETY TUBE
50 ML BURET
RUBBER
TUBE —
RUBBER
SLEEVE
PINCH'
CLAMP
18/9
18/9
TRAP
29/42
CONDENSERS
3 L FLASK
KJELDAHL
FLASK
100 ML
50 ML
ERLENMEYER
FLASK
GAS
STEAM DISTILLATION APPARATUS
SAFETY TUBE
I D 6mm
LENCiTH 120 cm.
C#- CORNING
RUBBER
STOPPER
SEPARATORY
- FUNNEL
C#94810
TYGON
TUBE
SUCTION FLASK
CffS340
RUBBER B/S 28/15
FITTING 1
TAPERED
FITTING
24/40
GLASS
TUBING
CONDENSER
C891630
SPECIAL
ASSEMBLY
KJELDAHL
FLASK
W 24/40 NECK
GAS
STEAM DISTILLATION APPARATUS
Page No. 5-9
-------
EFFLUENT MONITORING PROC_EDURF: Determination of Total Kjeldahl Nitrogen
Page No. 5-10
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
DETERMINATION OF TOTAL KJELDAHL NITROGEN
A. Equipment
Preparation
1. Glassware Washup
2. Balance Inspection
3. Spectrophotometer
Inspection
4. Still Cleaning
1. Clean all glassware in
suitable detergent.
1. Clean balance
1. Clean spectrophotometer.
2. Turn main power on by
rotating the zero control
clockwise.
3. Select wavelength by
rotating the knob at the
extreme right on the top
of the instrument
either clockwise or
counterclockwise.
4. Zero the instrument.
5. Use an empty cell and ad-
just the light control to
100% T.
1. Add a 1:1 mixture of
ammonia-free distilled
water and sodium hydroxide-
sodium thiosulfate solution
to each of Kjeldahl flasks
to be used.
la. Distilled water drains without leaving any
droplets.
la. Free of dust and dirt.
la. Free of dust and dirt.
2a. Pilot lamp on.
3a. 425 nm.
4a. Meter needle reads zero % T.
5a. To be sure that the instrument can achieve 100% T.
la. See Equipment and Supply Requirements Section for
diagrams of Kjeldahl Apparatus. (Pages 5-8 & 5-9)
lb. Glass beads should be added to each flask,
lc. At least 400 ml of solution should be used for
macro equipment. Use 40 ml for micro equipment.
Id. See B, Reagent Preparation #5.
I
(p. 36 )
V.A.I .la
(P. 39)
V.A.3.la
(p. 39)
V.A.3.4a
(p. 39)
V.A.3.5a
(p. 39)
V.A.4.la
(p. 39)
-------
^r'FLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Equipment
Preparation
(continued)
2. Using the appropriate
apparatus, distill half of
this solution.
3. Add 1 ml of Nessler's
Reagent to the distillate.
2a. The distillate should be checked colorimetrically
to insure that it is ammonia-free. The Nessler
reagent that is used for the ammonia determination
can be used at this point. (Reagent #15)
3a. If the distillate remains colorless, the glassware
is not contaminated with ammonia. If the dis-
tillate turns yellow, distill another half and
repeat step 3.
B. Reagent Preparation
1. Distil led Water
1. Prepare at least four (4)
liters of distilled water.
This water should be free
from ammonia.
la. All solutions must be made with ammonia-free
water. It is best to have an ion exchange
system in conjunction with a suitable water still
to insure high quality water. An anion-cation
exchange resin should be used.
VI.B
(p. 40)
VI.B.l.la
(P. 40)
2. Sulfuric
Solution
volume)
Acid
(20% by
1. Measure 50 ml of distilled
water in a 125 ml
Erlenmeyer flask.
2. Add 20 ml of concentrated
sulfuric acid (H?S0.) and
mix.
3. Dilute the solution to
100 ml.
la. This solution is used in Reagent Preparation #3.
2a. Flask should be tilted to avoid splattering.
2b. Solution may be diluted directly in the Erlenmeyer
flask.
3. Mercuric
Solution
Sulfate
1. Weigh 4 grams of red
mercuric oxide (HgO) in a
weighing boat.
2. Dissolve the mercuric oxide
in 25 ml of the 20% sul-
furic acid solution.
la. This solution is used in Reagent Preparation #4.
2a. A 100 ml beaker may be used.
Page No. 5-11
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
Page No. 5-12
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(continued)
4. Digestion Reagent
3. Dilute the solution to
50 ml with distilled water
1. Weigh 134 grams of potas-
sium sulfate (K2S04).
2. Add 650 ml of distilled
water to a 1 liter
Erlenmeyer flask.
3. Add 200 ml of concentrated
sulfuric acid (HpSO-) and
mix.
la. A 150 ml beaker is suitable for this weighing,
3a. Erlenmeyer flask should be tilted to avoid
splattering.
3b. Caution: Solution and flask tend to become warm.
A cold water bath may be used to keep the tempera-
ture down.
4. Dissolve the potassium
sulfate in this solution.
5. Add 25 ml of the mercuric
sulfate solution (reagent
3) to the solution and
mix.
5. Sodium Hydroxide-
Sodium Thiosul-
fate Solution
6. Dilute the solution to 1
liter.
Weigh 500 grams of sodium
hydroxide (NaOH) and 25
grams of sodium thiosul-
fate pentahydrate
in a 1
(Na2S20.j
5H 20)
liter Erlenmeyer flask.
6a. Store in glass container.
6b. The solution should be kept at about 14°C to
prevent crystallization.
6c. If crystals form, warm the solution in the flask
on a hot plate and stir/swirl to re-dissolve the
crystals.
la. Exercise caution with such a large amount of
sodium hydroxide since this is a very caustic
substance.
lb. Double check for pyrex glassware.
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(continued)
2. Add approximately 700 ml
of distilled water and
dissolve the reagents.
3. Dilute to 1 liter with
distilled water.
2a. Fumes will be given off. Therefore use a suitable
venting device.
3a. Cool to room temperature before diluting to
final volume.
6. Phenolphthalein
Indicator
Solution (0.5%)
1. Weigh 5 grams of phenolph-
thalein in a weighing boat.
2. Dissolve in 500 ml of 95%
ethyl alcohol in a glass
bottle or container.
3. Add 0.02 N NaOH until a
faint pink color appears.
4. Store in a glass or plastic
bottle.
3a. Dissolve 0.4 g NaOH in 500 ml of ammonia-free
distilled water to make 0.02 N NaOH. Very exact
weighing is not necessary.
7. Methyl Red
Indicator
Solution (0.2%)
1. Weigh 200 mg of methyl red
indicator in a 150 ml
beaker.
2. Add 100 ml of 95% ethyl
alcohol and dissolve the
indicator.
2a. This solution will be used to prepare #9, mixed
indicator which is required for the titrimetric
method to determine ammonia.
8. Methylene Blue
Indicator
Solution (0.2%)
1. Weigh 200 mg of methylene
blue indicator in a 150 ml
beaker.
2. Add 100 ml of 95% ethyl
alcohol and dissolve the
indicator.
2a. This solution will be used to prepare #9, mixed
indicator.
Page No. 5-13
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
Page No. 5-14
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(continued)
9. Mixed Indicator
10. Methyl Orange
Indicator Solution
11. Boric Acid
Solution
12. Ammonium Chloride
Stock Solution
1. Mix 100 ml of the methyl
indicator solution with 50
ml of the methylene blue
indicator solution.
1. Weigh 100 mg of methyl
orange indicator in a
150 ml beaker.
2. Add 100 ml ammonia-free
distilled water.
3. Stir to dissolve the
indicator.
4. Store in a 150 ml glass
bottle with dropper top.
1. Weigh 20 grams of boric
acid (H..B0,) in a weighing
boat. J J
2. Transfer to a 1 liter
Erlenmeyer flask and
dilute the acid to 1 liter.
1. Weigh 3.819 grams of
ammonium chloride (MH^Cl)
in a weighing boat.
la. The mixed indicator is required for the titrimet-
ric method to determine ammonia,
lb. This solution should be prepared fresh every
30 days.
la. This indicator is required for the titrimetric
method to determine ammonia.
3a. If the solution is cloudy, filter it.
-------
liFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(continued)
2. Dissolve the ammonium
chloride in ammonia-
free distilled water
in a 1 liter volumetric
flask.
3. Dilute to 1 liter.
3a. 1 ml - 1.0 mg Ammonia Nitrogen (NH^-N).
13. Ammonium Chloride
Standard Solution
1. Dilute 10.0 ml of the
stock solution to
1 liter in a volumetric
flask.
la. Use a volumetric pi pet.
lb. 1 ml - 0.01 mg ammonia nitrogen (NH^-N).
14. Sodium Hydroxide
Solution
1. Weigh 160 grams of sodium
hydroxide (NaOH) in a
1 liter Erlenmeyer flask.
2. Add 500 ml of ammonia-
free, distilled water.
3. Dissolve the sodium
hydroxide, and cool to
room temperature.
la. This solution is used in Reagent Preparation #15
which is required for the colorimetric (Nessler)
method to determine ammonia.
15. Nessler Reagent
1. Weigh 100 grams of
mercuric iodide (HgI2)
and 70 grams of
potassium iodide (KI)
together in a 250 ml
beaker.
Page No. 5-15
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
Page No. 5-16
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(continued)
2. Add enough distilled
water to dissolve the
mixture.
3. Add this mixture slowly
with stirring to the
sodium hydroxide solution
(#14).
4. Dilute the mixture to
1 liter.
2a. Approximately 50 ml should be sufficient.
4a. The solution is stable for at least one year in a
pyrex bottle out of direct sunlight.
C. Preparation and
Standardization of
0.02.N Sulfuric Acid
Titrant
1. Sulfuric Acid,
Approximately
0.1 N
2. Sulfuric Acid,
Approximately
0.02 N
1. Add 3 ml of concentrated
sulfuric acid (F^SO^) to
600 ml of carbon dioxide-
free water in a 1 liter
volumetric flask.
2. Dilute this solution to
1 liter.
1. Dilute 200 ml of the 0.1 N
sulfuric acid solution to
1 liter in a volumetric
flask.
Ca. This entire procedure is required only if you are
using the titrimetric method to determine
ammonia.
la. Heat 2 liters of distilled water for 15 minutes to
drive off the carbon dioxide (CO2).
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Preparation and
Standardization of
0.02 N Sulfuric Acid
Titrant (continued)
3. Sodium Carbonate
Standard,
0.0200 N
1. Dry 5 grams of sodium
carbonate (Na2C03) at
140°C for 2 hours.
2. Cool in a desiccator.
3. Weigh 1.060 grams in a
weighing boat.
4. Transfer to 1 liter
volumetric flask.
5. Dissolve the salt and
dilute to 1 liter with
carbon dioxide-free water.
2a. Thirty minutes is recommended.
3a. Use the analytical balance.
5a. See C.l.la for preparation of carbon dioxide -
free water.
4. Standardization of
the Sulfuric Acid
Solution
1. Fill a 50 ml buret with
approximately 0.02 N
sulfuric acid solution.
2. Transfer 25.0 ml of the
sodium carbonate solution
to a 125 ml Erlenmeyer
flask.
3. Add 2 drops of a methyl
orange indicator to the
flask.
2a. Transfer with 25.0 ml volumetric pipet.
2b. A beaker may be used, if a magnetic stirrer
is available.
Page No. 5-17
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
Page No. 5-18
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Preparation and
Standardization of
0.02 N Sulfuric Acid
Titrant (continued)
4. Add the approximately
0.02 N sulfuric acid
solution to the sodium
carbonate solution until
the color changes from
yellow to orange.
5. Record the ml of sulfuric
acid used.
6. Calculate the normality of
the sulfuric acid titrant.
7. Record the correct normal-
ity on the storage bottle.
4a. A pink color indicates the titration has gone too
far and should be repeated.
6a. Example Calculation
If the number of ml used is 20.8, the
calculations would be as follows:
NH2S04 = NNa2C03 x VNa2C03
vh2so4
NHoS0„ = 0.0200 N X 25.0
c 20.8
NH9S0. = 0.500
2 4 20.8
NHnS01 = 0.0240 N
7a. For example, the above 0.0240 N value should be
recorded on the storage bottle for the sulfuric
acid titrant.
-------
EFFLUENT MONITORING PROCEDURF: Determination of Total Kjeldahl Nitrogen
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPEC IFICATIONS
TRAINING
GUIDE NOTES
D. Analysis Using Macro
Apparatus (800 ml
Flasks)
D.a. See diagrams of Macro Apparatus in the Section on
Equipment and Supply Requirements. (Page 5-8).
See Procedure E for
Analysis Using Micro
Apparatus (100 ml
Flasks), page 23
1. Measurement of
Sample
1. Place a measured amount of
well-shaken sample into an
800 ml Kjeldahl flask.
la. Sample size can be determined from the following
table:
Kjeldahl Nitrogen Sample Size
in Sample; mq/liter ml
0-5 500
5-10 250
10-20 100
20-50 50.0
50-500 25.0
lb. A normal effluent should have an organic nitrogen
concentration between (0) and (1) mg/liter. If
it is known that the concentration is greater than
1 mg/liter, the sample volume should be adjusted
appropriately.
lc. Record information about the sample and the "ml
sample used" on an appropriate data sheet. See
Training Guide.
IX.D.1.1c.
(p. 41)
2. If the sample size is less
than 500 ml, dilute to 500
ml with distilled water.
2a. Use a graduated cylinder to measure the
difference in volume.
3. Add several glass beads.
3a. Glass beads should prevent bumping in the flask.
Page No. 5-19
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EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
Page No. 5-20
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Analysis Using Macro
Apparatus (800 mi
Flasks) (continued)
2. Reagent Addition
3. Digestion
Add 100 ml of the diges-
tion reagent to the flask.
Evaporate the mixture in
the Kjeldahl apparatus
until sulfur trioxide
(S03) fumes are given off
and the solution turns
pale yellow.
la. Use a graduated cylinder for the digestion
reagent prepared in B.4.
lb. If commercially available packets are used, then
1 packet (for macro Kjeldahl digestions) would be
added in place of the reagent.
la. See diagram in Section on Equipment and Supply
Requirements for proper position in digestion
rack. (Page 5-8)
lb. SOjfumes will be indicated when white smoke begins
rising from the solution,
lc. Sulfur trioxide (SOg) fumes are extremely toxic.
Therefore extreme caution should be observed.
VI.D.2.la
(p. 40)
4. Distillation
2. Continue heating for 30
additional minutes.
3. Cool the residue.
1. Add 300 ml of ammonia-
free, distilled water to
the digested mixture in
the Kjeldahl flask.
2. Add 0.5 ml of the
phenolphthalein
indicator solution.
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Analysis Using Macro
Apparatus (800 ml
Flasks) (continued)
3. Add 50 ml of the 2% boric
acid to a 500 ml
Erlenmeyer receiving
flask.
4. Position the Erlenmeyer
flask so that the tip of
the condenser (or an ex-
tension of the condenser
tip) is below the level of
the boric acid solution in
the receiving flask. (See
diagram next to Step 6
below)
5. Tilt the flask and care-
fully add 100 ml of the
sodium hydroxide-
thiosulfate solution to
form an alkaline layer at
the bottom of the flask.
(See diagram at right).
3a. Before using the flask, measure 350 ml of ammonia-
free distilled water in a graduate, pour it into
the flask and make a mark at 350 ml on the out-
side. You will need this marking for a later
step.
KJELDAHL FLASK
SODIUM HYDROXIDE- ^
SODIUM THIOSULFATE \ S \
SOLUTION "^7
ALKALINE LAYER
5a. The lower layer should be red.
5b. Do not agitate the digestion flask until it is
connected to the distillation apparatus, since
free arranonia may be liberated too soon.
Page No. 5-21
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
Page No. 5-22
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Analysis Using Macro
Apparatus (800 ml
Flasks) (continued)
6. Connect the Kjeldahl flask
to the condenser. (See
diagram at right)
7. Turn on the heat source.
8. Distill up to the 350 ml
mark on the Erlenmeyer
flask at the rate of
6-10 ml/min into the boric
acid solution.
9. Remove the receiving flask
10. Put a small beaker under
the condenser tip.
11. Remove the heat source.
KJELDAHL
SPRAY TRAP-/
800 ml
KJELDAHL
FLASK-
CONDENSER
500 ml
ERLENMEYER
RECEIVING
FLASK
10a. To receive any additional distillate.
11a. To stop the distillation.
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Analysis Using Macro
Apparatus (800 ml
Flasks) (continued)
5. Determining
Ammonia
12. Dilute the distillate to
500 ml by adding ammonia-
free distilled water up
to the 500 ml mark on the
flask.
1. If the sample is a normal
effluent, the anticipated
nitrogen concentration of
0-1 mg/liter requires the
Colorimetric Method
presented as Procedure F.
2. If it is known that the
nitrogen concentration is
greater than 1 mg/liter,
use the Titrimetric
Method presented as
Procedure G.
12a. Record 500 ml on the data sheet as "B. ml total
distillate, including boric acid (H-B0-) and
dilution water."
IX.D.4.12a
(p. 41)
E. Analysis Using Micro
Apparatus (100 ml
Flasks)
1. Measurement of
Sample
1. Place a measured amount of
well-shaken sample into a
100 ml Kjeldahl flask.
Ea. See diagram of Micro Apparatus in the Section on
Equipment and Supply Requirements. (Page 5-9)
la. Sample size can be determined from the following
table:
Kjeldahl Nitrogen Sample Size
in Sample; mq/1 ml
0-5 50
5-10 25
10-20 10
20-50 5
50-500 2
(continued)
Page No. 5-23
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
Page No. 5-24
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
E. Analysis Using Micro
Apparatus (100 ml
Flasks) (continued)
2. Reagent Addition
3. Digestion
2. If sample size is less
than 50 ml, dilute to
50 ml with distilled
water.
3. Add several glass beads.
1. Add 10 ml of the digestion
reagent to the flask.
1. Evaporate the mixture in
Kjeldahl apparatus until
sulfur trioxide (SO^)
fumes are given off and
the solution turns pale
yellow.
2. Continue heating for an
additional 30 minutes.
3. Cool the residue.
lb. A normal effluent should have an organic nitrogen
concentration between (0) and (1) mg/1. If it is
known that the concentration is greater than
1 mg/1, the sample volume should be adjusted
appropriately.
1c. Record information about the sample and the "ml
sample used" on an appropriate data sheet. See
Training Guide.
2a. Use a graduated cylinder to measure the
difference in volume.
3a. Glass beads should prevent bumping in the flask.
la. Use a graduated cylinder for the digestion
reagent prepared in B.4.
lb. If commercially available packets are used, then
1 packet (for micro Kjeldahl digestion) would be
added in place of the reagent.
la. See diagram in Section on Equipment and Supply
Requirements for proper position in digestion
rack, (page 5-9)
lb. SOj fumes will be indicated when white smoke
begins rising from the solution.
1c. Sulfur trioxide (S0g) fumes are extremely toxic.
Therefore extreme caution should be observed.
IX.E.1.1c
(p. 41)
VI.E.2.la
(p. 40)
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
OPERATING PROCEDURES
STEP SEQUENCE
1
INFORMATION/OPERATING GOALS/SPECIFICATIONS
—
IKAllNlNb
GUIDE NOTES
E. Analysis Using Micro
Apparatus (100 ml
Flasks) (continued)
4. Steam Distillation
1. Add 30 ml of anmonia-free
distilled water to the
digested mixture in the
Kjeldahl flask.
2. Add 2 drops of the
phenolphthalein indicator
solution.
3. Connect the Kjeldahl flask
to the ground glass joint
of the Micro steam
distillation apparatus.
4. Add 5 ml of the 2% boric
acid to a 50 ml Erlenmeyer
receiving flask.
5. Position the receiving
flask so that the tip of
the condenser (or an
extension of the condenser
tip) is below the level of
the boric acid solution in
the receiving flask.
6. Carefully add 10 ml of the
sodium hydroxide-
thiosulfate solution from
the dropping funnel.
7. Turn on the heat source.
3a. Diagrams of this apparatus are in the section on
Equipment and Supply Requirements, (page 5-9)
4a. A 50 ml short-form Nessler tube also may be used.
4b. Before using the flask or Nessler tube, measure
35 ml of ammonia-free distilled water in a
graduate, pour it into the receiving container
and make a mark at 35 ml on the outside. You
will need this marking for a later step.
6a. The mixture in the Kjeldahl flask should be red.
Page No. 5-25
-------
EFFLUENT MONITORING PROCEDURF: Determination of Total Kjeldahl Nitrogen
Page No. 5-26
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
E. Analysis Using Micro
Apparatus (100 ml
Flasks) (continued)
5. Determining
Ammonia
8. Distill up to the 35 ml
mark on the receiving
flask at the rate of 6-10
ml/min, into the boric
acid solution.
9. Remove the receiving flask.
10. Put a small beaker under
the condenser tip.
11. Remove the heat source.
12. Dilute the distillate to
50 ml by adding ammonia-
free distilled water up
to the 50 ml mark on the
Nessler tube.
1. If the sample is a normal
effluent, the anticipated
nitrogen concentration of
0-1 mg/liter requires the
Colorimetric Method
presented as Procedure F.
2. If it is known that the
nitrogen concentration is
greater than 1 mg/liter,
use the Titrimetric Method
presented as Procedure G.
8a. Exercise caution when working with this steam
apparatus.
9a. If an Erlenmeyer receiver was used, transfer the
distillate to a 50 ml Nessler Tube now.
10a. To receive any additional distillate.
11a. To stop the distillation.
12a. Record 50 ml on the data sheet as "B. ml total
distillate, including boric acid (H-B0.J and
dilution water."
IX.E.4.12a
(p. 41)
F. Colorimetric Method
1. Color Development
of Standards and
Sample
1. Place nine Nessler tubes in
the Nessler support rack.
2. Label the tubes (1-9).
2a. Use small stick-on labels.
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Colorimetric Method
(continued)
3. The Nessler tubes will be
used for preparing the
standards for the
determination. The table
on the right lists the
volumes of standard
ammonia solution to be
added to each tube. The
volumes of standard should
be measured with a Mohr
pi pet.
3a.
Tube #
ml of standard
Ammonia Solution
mg of Ammonia
Nitrogen per 50.0 ml
1
0.0
0.0
2
0.5
0.005
3
1.0
0.010
4
2.0
0.020
5
4.0
0.040
6
5.0
0.050
7
8.0
0.080
8
10.0
0.10
4. Add ammonia-free distilled
water to each tube, di-
luting each to the 50 ml
line.
5. Into tube #9 place 50 ml
of the sample taken from
the receiving flask con-
taining distillate.
5a.
5b.
5c.
If Macro apparatus was used, pour the distillate
from D.4.12 into tube #9 up to the 50.0 ml mark.
If Micro apparatus was used, the distillate was
either collected in a 50 ml Nessler tube or
else transferred to one after distillation.
The distillate was diluted to 50 ml in
step E.4.12. Label this Nessler tube as #9
for these steps.
In either case, record 50 ml on the data sheet
as "C. ml distillate taken for Nesslerization."
IX.F.l.5c
(P- 41)
Page No. 5-27
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
Page No. 5-28
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Colorimetric Method
(continued)
2. Spectrophoto-
metry
Measurements
6. Add 1 ml of Nessler
Reagent to each standard
and sample.
7. Mix the solution by plac-
ing a cap on the tube and
inverting three times.
8. Place the tubes back into
the rack and let sit for
20 minutes.
1. Arrange 9 spectrophoto-
metry tubes (1/2'') in a
test tube rack and label
1-9.
2. After the twenty minute
time span, transfer the
appropriate standards and
sample to these tubes.
3. Place tube (#1) in the
sample holder of the
instrument.
4. Using the light control
turn the knob until
the meter needle reads
100% on the transmittance
(T) scale.
6a. Use a Mohr Pipet.
7a. A #3 or a #6 rubber stopper may be used instead of
a cap.
7b. Rinse and dry the cap or stopper after each use
with a tube.
8a. During this time span, the spectrophotometer
should be double checked for proper operation,
(see Spectrophotometric Inspection in A.3.)
la. If you do not have a matched set of tubes for your
spectrophotometer, a single tube can be used. It
should be rinsed with distilled water, then with
the solution to be put into the instrument. The
procedure is presented in the EMP, "Use of a
Spectrophotometer".
3a. The wavelength should be set at 425 nm.
-------
Eh FLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Colorimetric Method
(continued)
5. Place tube §2 in the
sample holder and record
the absorbance value.
5a. This value will be found on the bottom scale as
shown below.
% TRANSMITTANCE H
Place tubes #3 through #9
in the sample holder,
recording each absorbance
value.
ABSORBANCE H4+
>2.0
+H
+1
100
1111111111111111
0.3
For example the transmittance was 50%, the
absorbance should be recorded as 0.3 A.
6a. Values should be recorded in a notebook or on an
appropriate data sheet. See Training Guide.
6b. EXAMPLE RECORD OF ABSORBANCE VALUES
IX.F.2.6a
(p. 42)
Tube #
Concentration
Absorbance
(mg NH3-N/50.0 ml)
1
0.0
0.0
2
0.005
0.04
3
0.010
0.08
4
0.020
0.16
5
0.040
0.32
6
0.050
0.41
7
0.080
0.64
8
0.10
0.82
9 To
be determined from
0.52
calibration curve.
Page No. 5-29
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
Page Mo. 5-30
OPERATING PROCEDURES
F. Col orimetric Method
(continued)
3. Plotting and use
of the calibration
curve.
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
1. Plot the absorbance values
for the standards obtained
in F2. above vs. the
concentration of ammonia
nitrogen in the standards
as in the Table in
F.l,3.3a.
2. Draw the best straight line
through all the points to
produce a calibration
curve.
3. Use the absorbance value
for the sample (Tube #9)
obtained in F.2.6.6b. above
to draw a dotted line from
the absorbance line over
to the calibration curve.
4. From that point on the
calibration curve, draw a
perpendicular line down to
the concentration line.
la. Following, below, there is an example calibration
curve using the example absorbances from the
table in F.2.6b above vs the concentration of
ammonia nitrogen in the standards as in the Table
in F.l.3.3a. The absorbance value used for the
sample is 0.52.
TRAINING
GUIDE NOTES
u
z
<
CO
BC
O
IS>
CO
<
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
r
<>
/
s
V
y
_ .
. *-
__
__
m
—T
SAMPLE (TUBE #9)
NABSORBANCE = 0.52
s
A
d
r
A
<
V
A
J
6
A
J
(concentration of sample
r
&
,0.063 mg NH3-N/50.0ml J
y
V
V
.01 .02 .03 .04 .05 .06 .07 .08 .09 .10
CONCENTRATION
(mg NH3 —N/50.0ml)
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Colorimetric Method
(continued)
4. Final Calculation
for Macro Analysis
(See next proce-
dure, #5, for
Final Calculation
for Micro Analysis)
5. Record the concentration
value at this point for
sample as mg NH^-N/BO.Oml.
1. Using the formula at the
right, compute the Total
Kjeldahl Nitrogen
concentration.
5a. Record this on the data sheet as "A, mg NFL-N/50.0
ml from curve."
5b. In this example, the concentration for the sample
is 0.063 mg NH3-N/50.0 ml.
la. TKN, mg/1 = A X 1000 v E
ml sample C
Where:
A = mg NH^-N (ammonia nitrogen)/50.0 ml from curve
B = ml total distillate, including boric acid
(H^BO^) and dilution water
C = ml distillate taken for Nesslerization
ml sample = ml of original sample taken
An example calculation using a value from a
calibration curve would be:
TKN, mg/1 = A X 1000 v B
ml sample C
A = 0.044
B = 500 ml (300 ml distillate + 50 ml boric acid +
150 ml dilution water)
C = 50 ml
ml sample = 500 ml
2 10
TKN, mg/1 = 0.044 X 70P0 v ?00
m * ~W
1 1
= 0.044 X 2 X 10
= 0.044 X 20
= 0.88
IX.F.3.5a
(P. 41)
TKN = 0.88 mq/1
Page No. 5-31
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
Page No. 5-32
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Colorimetric Method
(continued)
5. Final Calculation
for Micro Analysis
1. Using the formula at the
right, compute the Total
Kjeldahl Nitrogen
concentration.
la. TKN, mg/1 = A X 1000 „ B
ml sample C
Where:
A = mg NH3-N (ammonia nitrogen)/50.0 ml from curve
B = ml total distillate, including boric acid
(H3B03) and dilution water
C = ml distillate taken for Nesslerization
ml sample = ml of original sample taken
An example calculation using a value from a
calibration curve would be:
TKN, mg/1 = A X 1000 „ B
ml sample C
A = 0.045
B = 50 ml (30 ml distillate + 5 ml boric acid +
+ 15 ml dilution water)
C = 50 ml
ml sample = 50 ml
20 1
TKN, mq/1 = 0.045 X 7000 „ 20
00 A W
1 1
= 0.045 X 20 X 1
= 0.045 X 20
= 0.90
TKN = 0.90 mg/1
-------
t.rLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
G. Titrimetric Method
1. Titration
2. Calculations
1. Transfer the contents of
the distillate receiving
flask (from D.4.12 or
E.4.12)to the next
largest volume Erlenmeyer
titration flask.
2. Add 3 drops of mixed
indicator to the flask
and its contents.
3. Set up a buret for
titration.
4. Fill the buret with a
0.020 N sulfuric acid
(H2S0^) standard solution.
5. Add the sulfuric acid
titrant until the color of
the solution changes from
green to purple.
1. The Total Kjeldahl Nitro-
gen (TKN) would be calcu-
lated by the formula to
the right.
2a. If ammonia nitrogen is present the color of the
solution will be green.
3a. Use a 50 ml buret.
5a. A blank can be analyzed also, so that the true
color can be seen.
5b. A blank contains all necessary reagents except
distilled water is substituted for the sample.
la. TKN mg/1
= (A-B)N X F X 1000
S
A = ml of standard H^SO^ used in titrating sample
B = ml of standard F^SO^ used in titrating blank
N = normality of sulfuric acid (Procedure C.)
F = 14 (the millequivalent weight of nitrogen)
S = ml of sample digested
If the Normality (N) of the Sulfuric Acid is
exactly 0.020 N then the formula may be reduced to
TKN mg/1 = (A-B) X 280
S
where A, B, S, refer to the same terms as above.
Page No. 5-33
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EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
Page No. 5-34
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
G. Titrimetric Method
(continued)
2. Given the following
sample data, the compu-
tation would be made as
shown at the right.
A = 19.2 ml
B = 0.4 ml
N = 0.021
F = 14
S = 500 ml
2a. EXAMPLE CALCULATION:
TKN, mq/1 = (A-B)N X F X 1000
S
= (19.2 - 0.4) X 0.021 X 14 X 1000
500 2
= 18.8 X 0.021 X 14 X 7000
J500
1
= 18.8 X 0.021 X 14 X 2
= 18.8 X 0.021 X 28
= 18.8 X 0.588
=11.1 mg/1
TKN, mg/1 = 11.1 mq/1
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
TRAINING GUIDE
SECTION
TOPIC
I*
Introduction
II
Educational Concepts-Mathematics
III
Educational Concepts-Science
IV
Educational Concepts-Communications
V*
Field and Laboratory Equipment
VI*
Field and Laboratory Reagents
VII
Field and Laboratory Analysis
VIII
Safety
IX*
Records & Reports
^Training guide materials are presented here under the headings marked*.
These standardized headings are used throughout this series of procedures.
Page No. 5-35
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EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
INTRODUCTION
Section I
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
Nitrogen has long been recognized as a very im-
portant element in regard to pollution control
analysis. Nitrogen can exist in several forms.
These forms are related by what is called the
nitrogen cycle (shown below).
Chemistry for Sanitary
Engineers, Sawyer and
McCarty, 2nd Edition,
McGraw-Hill, 1967.
6^CTER!AL0X|04r
NITRITE
NOo
ATMOSPRjs
FECAL
MATTER
ORG. N
URINE
UREA
NITRATE
N03
ANIMAL
PROTEIN
ORGANIC N
PLANT
PROTEIN
FOO^i^UFFW^'C N
THE NITROGEN CYCLE
Page No. 5-36
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
INTRODUCTIO
N
Section I
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
Looking at the diagram one can see that the
atmosphere contains a large amount of nitrogen
existing as (N2). The atmospheric nitrogen is
converted to N2O,. in an electrical storm. This
nitrogen pentoxide is subsequently converted to
nitrates, (NOg)", as a result of the mixing with
water. These nitrates serve as fertilizer for
plants and are subsequently converted to plant
protein.
Animals and human beings utilize plant protein for
the growth and repair of muscle tissue as well as
energy. These nitrogen compounds are subsequently
discharged as waste products (fecal matter and
urine). Bacterial decomposition of fecal matter
as well as hydrolysis of urine will produce ammonia.
The bacterial decomposition may be accomplished
under aerobic or anaerobic conditions.
The ammonia formed by this process may now further
undergo bacterial oxidation (aerobic conditions)
to form nitrites, (NO2)", and eventually nitrates,
(NOg)", which can be used as fertilizer for plants
etc.
It should be noted that several changes may occur
that will modify the fate of a certain compound in
the cycle. For example the system suddenly turns
anaerobic upon nitrate, (NOg) , formation. This
would cause bacterial reduction to occur. Several
other examples are shown in the cycle.
The treatment plant utilizes the nitrogen cycle
in its processes. The raw sewage will have some-
what high Organic Nitrogen content. As it moves
through the treatment process, it is converted to
ammonia, nitrites and finally nitrates. An example
of the Nitrogen Transformation in a typical treat-
ment system is shown on the next page.
Page No. 5-37
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
INTRODUCTION
Section I
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
o 10
4 5 6
TIME (DAYS)
Isolating a certain volume of raw sewage on Day I
(Point A), one can see that the Organic Nitrogen
is relatively high but decreases and is converted
to ammonia (Point B). Subsequent bacterial
oxidation produces nitrites (Point C) and finally
nitrates (Point D).
It therefore can be seen that the treatment plant
simply follows the nitrogen cycle, and with proper
monitoring procedures, (analysis of these 4
parameters), one can very easily measure the
efficiency of the treatment process.
The test described in this instruction can be found
in the 1974 EPA Methods Manual on page 175. Another
reference with an acceptable procedure for NPDES
purposes is 14th ed. Standard Methods on page 437.
Methods for Chemical
Analysis of Water and
Wastes, 1974, EPA, MDQARL,
Cincinnati, OH 45268,
p. 175.
Standard Methods for the
Examination of Water and
Wastewater, 14th ed., 1976,
APHA, New York, NY, p. 437.
Page No. 5-38
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
INTRODUCTION
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.I.la
A.3.la
A.3.4a
A.3.5a
.4.1a
Most glassware can be cleaned simply by washing
them first with detergent such as Alconox. Next
rinse with tap water and finally with distilled
water. For glass stopcocks, wrap the glass plug
in tissue during storage.
If a film or droplets appear on the glassware, use
an Acid-Potassium Dichromate cleaning solution.
The EMP written for the operation of this instru-
ment should be consulted for further detail. The
"Spectronic 20" operates on the principle that
visible light broken down into all wavelengths may
be used in quantitative determinations.
After 10 minutes warm-up time, the zero control
may be adjusted to bring the meter needle to "0" on
the percent transmittance (%T) scale.
Later on during the procedure a reagent blank is
used for the final adjustment of the light control.
The 0&M manual for the appropriate digestion rack
should be consulted before operation.
A macro Kjeldahl distillation apparatus utilizes an
800 ml flask which requires 500 ml of sample. A
micro Kjeldahl apparatus utilizes a 100 ml flask
which requires 50 ml of sample. Either Apparatus
is acceptable.
U.S. EPA Handbook for
Analytical Quality Control
in Water and Wastewater
Laboratories, 1972, AQCL-
NERC, Cincinnati, Ohio
"Guidelines to the Care and
Use of Analytical Glassware
and Apparatus" Hudson
Champlain Project
Page No. 5-39
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
LABORATORY REAGENTS
Section VI
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.I. la
D.2.1a
E.2.la
If Organic Nitrogen concentrations are known to be
below 1 mg/1 then the following reagents may be
eliminated from procedure.
1. Methyl Red Indicator Solution - B.7
2. Methylene Blue Indicator Solution - B.8
3. Mixed Indicator - B.9
4. Methyl Orange Indicator Solution - B.10
5. Sulfuric Acid Titrant - All of Section C
High Ammonia (NH,) concentration in distilled water
could possibly influence the expected low values
for normal nitrogen content of effluents.
Disposal of mercury-containing samples is a
recognized problem; research investigations are
under way to replace it as a preservative.
Dean, Williams, Wise:
"Disposal of Mercury Wastes
from Water Laboratories,"
Environmental Science and
Technology, Vol. 5, No. 10,
1971. p. 1044
Maag and Hecker:
"Recovery of Mercury in
Solution," Journal of
Environmental Quality,
Vol. 1, No. 2, 1972, p. 19k.
Page No. 5-40
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
RECORDS AND REPORTS Section IX
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
Typical Laboratory Data Sheet
for
Total Kjeldahl Nitrogen, mg/1
Name of Plant
D.I.1c.
E.l.lc.
Sampling Location
Type of Sample
Date and Time Collected
Sample Collector
Date and Time Analysis Began
Analyst
Method Used (Macro or Micro)
ml. sample used
D.4.12a
E.4.12a
B. ml total distillate including boric acid (00.,)
and dilution water
F.1.5c.
C. ml distillate taken for Nesslerization
F.3.5a.
A. mg NH-^N/50,0 ml,from curve
Use this formula in calculating the results for the colorimetric method:
TKN mg/1 = Ax 1000 B
ml sample C (See pp. 5-31 and 5-32)
If Organic Nitrogen (mg/1) is needed and a separate ammonia analysis has been
performed, use the following equation to determine this.
Since: TKN = Organic/N + Ammonia/N;
Then: Organic/N = TKN - Ammonia/N
Final Results
TKN mg/1
NH3-N, mg/J
Org-N, mg/1
Page No. 5-41
-------
EFFLUENT MONITORING PROCEDURE: Determination of Total Kjeldahl Nitrogen
RECORDS AND REPORTS
Section IX
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
F.2.6a
Values from Nesslerization Procedure
Tube #
Concentration
Absorbance
Absorbance
Absorbance
mg NH3-N/50.0 ml
1
0.0
2
0.005
3
0.010
4
0.020
5
0.040
6
0.050
7
0.080
8
0.10
9
Sample
10
Sample
11
Sample
Page No. 5-42
-------
0.50
0.40
CO
ce
o
to
2 0.30
T.KFLUCNT
MONITORING
PROCEDURE:
Determination
of Total
Kjeldahl
Nitrogen
-
-
RECORDS
AND
REPORTS
1
SECTION
IX :
nPTFDMTNflTTflW
nr
AMMONIA NITROGEN
CALIBRATION
GRAPH
ilbNrtlUKt
ui-
rKtrAKtK:
nflTF
GRAPW UAS
PRFPARFIV
——
0.20
0.10
0.00
0.01
0.02 0.03 0.04 0.05 0.06 0.07
CONCENTRATION OF AMMONIA NITROGEN mg/50.0 ml
0.08
0.09
0.10
Page No, 5-43
-------
A PROTOTYPE FOR DEVELOPMENT OF
ROUTINE OPERATIONAL PROCEDURES
for the
NITROGEN, AMMONIA DETERMINATION
as applied in
WASTEWATER TREATMENT FACILITIES
and in the
MONITORING OF EFFLUENT WASTEWATERS
National Training Center
Municipal Operations and Training Division
Office of Water Program Operations
U.S. Environmental Protection Agency
CH.N.am.EMP.la.9.75
Page No. 6-1
-------
EFFLUENT MONITORING PROCEDURE: Nitrogen, Ammonia Determination
This operational procedure was developed by:
NAME Paul F. Hall bach
ADDRESS EPA-WPO-National Training Center, Cincinnati, Ohio
POSITION Chemist Instructor
EDUCATION AND TECHNICAL BACKGROUND
B.S. Chemistry
14 years Industrial Chemist
16 Years HEW-FWPCA-EPA-Chemi st
Page No. 6-3
-------
EFFLUENT MONITORING PROCEDURE: Nitrogen, Ammonia Determination
1. Objective:
To determine the nitrogen (as ammonia) content of an effluent
2. Brief Description of Analysis:
The sample is buffered at a pH of 9.5 with a borate buffer and is then
distilled into a solution of boric acid. For samples containing ammonia
concentration of less than one milligram per liter, the ammonia concen-
tration can be determined colorimetrically. For samples containing
higher concentrations (1.0 to 25 mg/liter) the ammonia concentration is
determined by a volumetric titration procedure.
3. Applicability of this Procedure:
a. Range of Concentration:
Colorimetric Method - 0.03 to 1.0 mg NH^-N/liter
Titrimetric Method - 1.0 to 25 mg NHg-N/liter
(The range of these methods may be extended for samples by dilution.)
NOTE: A range from 0.05 to 1400 mg NH^-N/liter is available by using
an ammonia selective ion electrode. A separate EMP on this
method is available.
b. Pretreatment of Samples:
This procedure includes the manual distillation of the sample at
pH 9.5 as specified in the Federal Register Guidelines.
c. Treatment of Interferences in Samples:
This procedure includes addition of sodium thiosulfate to remove
residual chlorine. If samples contain volatile alkaline compounds
or mercury salts (sometimes used as preservatives), consult the
Source of Procedure* for appropriate treatments.
~Source of Procedure: Methods for Chemical Analysis of Water and Wastes,
1974, Environmental Protection Agency, Methods Development and Quality
Assurance Research Laboratory, Cincinnati, Ohio, p. 159
Page No. 6-4
-------
EFFLUENT MONITORING PROCEDURE: Nitrogen, Ammonia Determination
Equipment and Supply Requirements
A. Capital Equipment
1. Analytical balance, 200 g capacity
2. Trip balance, 500 g capacity
3. Meter, pH
4. Spectrophotometer and cuvettes
B. Reusable
1. Eurner, Meker type, gas
2. Safety glasses
3. Laboratory apron
4. Pipettes, volumetric, 1, 2, 5, 25, 50 ml
5. Graduated cylinder, 100 ml
6. Kjeldahl flask, 800,-ml
7. Condenser, Allihn, 600 ml
8. Kjeldahl spray trap,
9. Support, tripod base, 10 x 24 inch
10. Clamps, two, utility
11. Beaker, 600 ml
12. Flask, Erlenmeyer, 500 ml
13. Reagent bottles, 200 ml, 500 ml
14. Plastic squeeze bottle, 500 ml
15. Nessler tubes, 50 ml
C. Consumable
1. Concentrated sulfuric acid
2. Boiling chips
3. Boric acid
4. Methyl red indicator
5. Ethyl alcohol or denatured (3A or 30)
6. Methylene blue
7. Mercuric iodide
8. Potassium iodide
9. Sodium tetra borate
10. Sodium thiosulfate
11. Sodium hydroxide
12. Ammonium chloride
All reagents should be high quality.
Page No. 6-5
-------
EFFLUENT MONITORING PROCEDURE: Nitrogen, Ammonia Determination
Page No. 6-6
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
NITROGEN, AMMONIA DETERMI
A. Sample Preservation
1. Addition of
preservative
NATION
1. Add 2 ml of concentrated
sulfuric acid (l^SO^) or
40 mg of mercuric chloride
(HgC^) per liter and store
at 4° centigrade.
la. Because organic nitrogen is progressively
ammonified by biologic activity, the determination
of ammonia is best made on a fresh sample,
lb. The use of mercuric chloride is discouraged.
(p. 17)
I.A.la.
(P. 17)
B. Equipment Preparation
1. Glassware wash-up
2. Still Cleaning
1. Clean all glassware in
suitable detergent.
1. Add 500 ml of ammonia-free
water to an 800 ml Kjeldahl
flask.
2. Add a few boiling chips.
3. Set up the still assembly.
4. Ignite the burner under the
flask and apply heat cau-
tiously so that the water
boils slowly.
5. Test the distillate by adding
about 5.0 ml of Nessler's
reagent.
la. Distilled water drains without leaving any
droplets.
la. Use deionized distilled water. Shake 4 liters of
distilled water with 10 grams of Ionac C-101
cation exchange resin, available from Ionac
Chemical Company, Birmingham, NJ*.
2a. The addition of boiling chips which have been
previously treated with dilute sodium hydroxide
will prevent bumping.
3a. Assembly consists of gas burner, distillation
flask, condenser and receiving flask.
5a. If the distillate remains colorless, the glassware
is not contaminated with ammonia.
*Cation exchange resins are available from many
manufacturers. This recommendation is not an
endorsement this particular product.
-------
EFFLUENT MONITORING PROCEDURE: Nitrogen, Ammonia Determination
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Reagent Preparation
1. Boric Acid
Solution
2. Mixed Indicator
Solution
3. Nessler Reagent
1. Dissolve 20 grams of boric
acid (H_B03) in distilled
water and ailute to one
liter with distilled water.
1. Dissolve 200 mg methyl red
indicator in 100 ml 95%
ethyl alcohol.
2. Dissolve 100 mg methylene
blue in 50 ml of 95% ethyl
alcohol.
3. Transfer the above two
solutions into a dispensing
glass bottle.
1. Dissolve 100 grams of
mercuric iodide and 70 grams
of potassium iodide in about
300 ml of distilled water.
2.
Add the above mixture slowly
to a cooled solution of
160 grams of sodium hydroxide
previously dissolved in
500 ml of distilled water.
la. This is a 2 percent solution of boric acid.
la. Specially denatured ethyl alcohol conforming to
formula 3A or 30 of the U.S. Bureau of Internal
Revenue may be substituted for 95% ethanol.
3.
Dilute
1 iter.
the mixture to 1
3a. This solution should be prepared fresh every
30 days.
la. Mercuric iodide dissolves after potassium
iodide is added.
2a. Use a glass rod for stirring or a magnetic stirrer
when the mixture is being added.
3a. Store the reagent in a pyrex glass bottle. Keep
out of direct sunlight. It will remain stable
for a period of up to one year.
Page No. 6-7
-------
EFFLUENT MONITORING PROCEDURE: Nitrogen, Ammonia Determination
Page No. 6-8
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
4. Borate Buffer
1. Add 10 ml IN sodium
hydroxide to 50 ml of
distilled water and dilute
to the mark with distilled
water.
2. Add 4.75 grams of sodium
tetraborate (Na2B407.10^0)
to about 300 ml distilled
water in a 500 ml volumet-
ric flask.
3. Dissolve and dilute to the
500 ml volume with
distilled water.
4. Add 88 ml of the 0.1 N NaOH
(Step 1) to a 1 liter
flask.
5. To the same flask add
500 ml of the 0.025 M
sodium tetraborate (Step 3)
6. Swirl to mix and dilute to
the 1 liter volume with
distilled water.
la. Use a 100 ml volumetric flask,
lb. This solution will have a concentration of 0.1 N.
1c. See reagent #6 for 1 N sodium nydroxlde
preparation.
3a. This solution will have a concentration of 0.025 M
6a. This is the borate buffer solution.
-------
EFFLUENT MONITORING PROCEDURE: Nitrogen, Ammonia Determination
OPERATING PROCEDURES
5. Sulfuric Acid
Stock Solution
Approximately 0.
IN
6. Sodium Hydroxide
IN
7. Sodium Thiosulfate
(1/70N)
8. Stock arrmonium
chloride (1 ml =
1.0 mg of ammonia
nitrogen)
STEP SEQUENCE
Add 3 ml of concentrated
sulfuric acid (specific
gravity 1.84) to about
800 ml of CO,
tilled water.
free dis-
Mix wel1
and dilute to 1000 ml with
CO2 free distilled water.
2. Dilute 200 ml of this solu-
tion to one liter with C02
free distilled water.
1. Dissolve 40 grams of sodium
hydroxide (NaOH) in am-
monia-free water and dilute
to one liter.
1. Dissolve 3.5 grams of
sodium thiosulfate
pentahydrate in about
300 ml of distilled water
and dilute to one liter
with distilled water.
1. Dissolve 3.819 grams of
NH-C1 in water and dilute
to 1 liter.
INFORMATION/OPERATING GOALS/SPECIFICATIONS
la. Use a 3 ml pipette. A pipette bulb must be used,
lb. Use a 1000 ml volumetric flask.
2a. Use a 1000 ml volumetric flask. The concentration
of this solution should be about 0.02N.
2b. Standardize according to the procedure prescribed
in the EMP "Determination of Total Kjeldahl
Nitrogen."
la. Use a volumetric flask,
lb. Transfer reagent to pyrex reagent bottle fitted
with a rubber stopper.
la. This solution can be used to remove residual
chlorine from the sample prior to distillation,
lb. One ml of this solution will remove 1 mg/liter
of residual chlorine in 500 ml of sample.
lc. Use sodium thiosulfate pentahydrate
Na2S20g
5H20
la. Wherever water is mentioned it refers to
ammonia-free water.
TRAINING
GUIDE NOTES
Page No. 6-9
-------
EFFLUENT MONITORING PROCEDURE: Nitrogen, Airmonia Determination
Page No. 6-10
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
9. Working ammonium
chloride solution
(1 ml = 0.01 mg
of ammonia
nitrogen)
1. Dilute 10.0 ml of the
NH^Cl to 1 liter with
water.
la. Use the stock ammonium chloride solution
(reagent #8).
D. Procedure (Sample
contains 1.0 to
25.0 mg/1 ammonia
nitrogen)
1. Add 500 ml of ammonia-free
water to an 800 ml Kjeldahl
flask.
2. Add a few boiling chips to
the flask.
3. Set up the still assembly
as before.
4. Ignite the burner and steam
out the distillation
apparatus.
5. Continue the cleaning
process until you are
assured that no traces
of anmonia are present.
6. Transfer a 400 ml aliquot
of sample into a 600 ml
beaker..
la. Use a graduate cylinder.
2a. The addition of boiling chips which have been
previously treated with dilute sodium hydroxide
will prevent bumping during the distillation
process.
3a. Cooling water to condenser turned off.
4a. Periodically check the distillate in the re-
ceiving flask by adding a few milliliters of
Nessler's reagent. If the distillate remains
colorless, the apparatus is not contaminated with
any trace of ammonia.
6a. If chlorine is present in the sample it must be
removed prior to the distillation by adding 1 ml
of sodium thiosulfate for each 1 mg/1iter of
residual chlorine in 500 ml of sample.
VII.D.6a.
(p. 18)
-------
EFFLUENT MONITORING PROCEDURE: Nitrogen, Ammonia Determination
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
7. Add sodium hydroxide solution
(1NJ until the pH is raised
to 9.5.
8. Transfer the sample to the
previously steam-cleaned
800 ml Kjeldahl flask.
9. Add 25 ml of the borate
buffer.
10. Attach the flask and connect
the still assembly.
11. Add 50 ml of 2 percent
boric acid to the 500 ml
receiving flask, and
position the flask under
the condenser tip.
7a. Use a magnetic stirrer or a glass stirring rod for
stirring. Use a dropping bottle for the addition
of sodium hydroxide.
7b. Check the pH during the addition with the use of a
pH meter or by the use of short range pH paper.
VII.D.7a.
(P. 18)
10a. Turn on water to cooling condenser.
11a. The condenser tip should be adjusted so that it
is below the surface of the liquid.
12. Ignite the burner and distill
300 ml at the rate of 6 to
10 ml per minute.
13. Remove the receiving flask
and turn off the burner.
14. Add 3 drops of mixed indi-
cator to the receiving flask*
and its contents.
Page No. 6-11
-------
EFFLUENT MONITORING PROCEDURE: Nitrogen, Ammonia Determination
Page No. 6-12
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
15. Set up a titration burette
and fill it with 0.02 N
sulfuric acid standard
solution.
16. The color change at the end
point in the titration shoulc
match the color change pro-
duced at the end point when
a plain distilled water
sample is run through the
same procedure using- all
reagents that would be used
for a sample.
15a. Use a 50 ml burette.
E. Calculations for
Titration Procedure
(Sample contains 1.0
to 25.0 mg/1 ammonia
nitrogen)
1. The amount of ammonia
nitrogen present can be
determined with the use of
the formula to the right.
2. There is a calculation sheet
page 19.
la. NH-.-N mq/1 = A x 0.28 x 1000
J S
where:
A - will equal the milliliters of 0.02 N
sulfuric acid used in the titration
S - will equal the milliliters of sample
used in the test
An example of the use of this formula for the
analysis of a wastewater sample follows:
1. Sample size for the analysis = 400 ml
2. ml of 0.02 N sulfuric acid = 17.0
3. NH,N mg/1 = 17.0 x 0.28 x 1000
400
NH^N mg/1 = 11.9 mg/1
-------
EFFLUENT MONITORING PROCEDURE: Nitrogen, Airanonia Determination
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
3. If the sample contains more
than 25 mg/liter of ammonia,
an appropriately smaller
sample size must be used.
4. If the sample contains
1.0 mg or less ammonia
nitrogen per liter, the
following procedure should
be used.
F. Procedure
(Sample contains
0.05 to 1.0 mg
ammonia nitrogen
per liter)
1. Transfer a 400 ml sample into
a 600 ml beaker.
2. Add 1N_ NaOH with an eye
dropper until the pH is 9.5.
3. Transfer the pH 9.5 sample
to a steam cleaned 800 ml
Kjeldahl flask and add
25 ml of borate buffer.
4. Distill 300 ml at the rate
of 6-10 ml/minute into 50 ml
of 2 percent boric acid
contained in a 500 ml glass
stoppered Erlenmeyer
flask.
5. Dilute to 500 ml.
6. Into 50 ml Nessler tubes
pi pet the following volumes
of the working ammonium
chloride solution: 0.0, 0.5,
1.0, 2.0, 3.0, 4.0, 5.0, 8.0,
and 10.0 ml.
2a. Use a pH meter or pH paper and stir the solution
during the addition of sodium hydroxide.
3a. Use a 50 ml graduate for the buffer addition.
5a. Use ammonia-free water.
Page No. 6-13
-------
EFFLUENT MONITORING PROCEDURE: Nitrogen, Ammonia Determination
Page No. 6-14
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE KOTES
7. Add sufficient ammonia free
water to bring the volume
to 50 ml.
8. Pi pet into three Messier
tubes 2, 25, and 50 ml of
the distilled sample from
step 5 above. Dilute with
water to 50 ml.
9. Pipette 1.0 ml of the
Nessler reagents into each
standard and sample tube
and mix.
10. Transfer appropriate aliquots
into cuvettes for measure-
ment of the color intensity
in the spectrophotometer.
11. Read the absorbance of all
tubes after 20 minutes at
425 nanometers against the
the 0,0 standard.
8a. Use a volumetric pipette. The purpose of using
three aliquots is to ensure that when the colors
are developed, one of the three will produce a
color which lies within the range of the calibra-
tion curve.
9a. Use a volumetric pipette.
11a. There is an EMP on "Use of a Spectrophotometer."
G. Calculations for
Colorimetric Pro-
cedure (Sample
contains 0.05 to
1.0 mg ammonia
nitrogen per liter)
1. Prepare a calibration curve
of absorbance values of the
standards versus mg of
arrcnonia nitrogen. For
example: if 2.0 ml of the
working NH.C1 are used, and
its concentration is 0.01 mg
of NhU-N/ml, then 0.02 mg is
the value plotted on the
calibration curve versus the
corresponding absorbance.
la. There is an EMP on "Preparation of Calibration
Graphs."
-------
EFFLUENT MONITORING PROCEDURE: Nitrogen, Ammonia Determination
OPERATING PROCEDURES
STEP SEQUENCE
|
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
2. Determine the amount of
NH^-N present in the sample
from the calibration curve.
3. Determine the mg of
NH^-N/liter of sample using
the formula:
mg/1 NH3-N = A XD1000 x £
3a. A = mg NH3~N read from standard curve
B = ml total distillate collected, including boric
acid and dilution
C = ml distillate taken for nesslerization
D = ml of original sample taken
An example calculation using a value from a
calibration curve would be:
A = 0.015 mg (read from standard curve)
B = 500 ml (300 ml distillate + 50 ml boric acid
+ 150 ml dilution water)
C = 25 ml (distillate which was diluted for
nesslerization)
D = 400 ml (ml of original sample taken)
/t mi. m Ax 1000 B
mg/1 NH^-N = ^ x ^
50 20
_ 0.015 x 1000 500
400 x n
20 1
1
= 0.015 x 50
mg/1 NH3-N = 0.75
There is a calculation sheet on page 20.
Page No. 6-15
-------
EFFLUENT MONITORING PROCEDURE: Nitrogen, Ammonia Determination
TRAINING GUIDE
SECTION TOPIC
I* Introduction
II Educational Concepts - Mathematics
III Educational Concepts - Science
IV Educational Concepts - Communications
V Field and Laboratory Equipment
VI Field and Laboratory Reagents
VII* Field and Laboratory Analysis
VIII Safety
IX Records and Reports
*Training guide materials are presented here under the headings marked *
These standardized headings are used through this series of procedures.
No. 6-16
-------
EFFLUENT MONITORING PROCEDURE: Nitrogen, Ammonia Determination
INTRODUCTION Section I
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.la.
The compounds of nitrogen are of interest because
of the importance of nitrogen in the life processes
of all plants and animals. Chemists analyzing
sewage and freshly polluted waters learned that most
of the nitrogen is originally present in the form of
organic (protein) nitrogen and ammonia. As time
progresses, the organic nitrogen is gradually
converted to ammonia nitrogen, and later on, if
aerobic conditions are present, oxidation of ammonia
to nitrites and nitrates occurs. Waters that contain
mostly organic and ammonia nitrogen are considered
to be recently polluted and therefore of great
potential danger. Waters in which most of the nitro-
gen is in the form of nitrates are considered to
have been polluted a long time previously and
therefore are not dangerous to the public health.
Since the treatment plant is an accelerated version
of the natural process of converting nitrogen from
one compound to another, the monitoring of the
ammonia concentration is an effective means of
determining the efficiency of the biodegradation.
Sawyer, C. N. and
McCarty, P. L. Chem.
for San. Eng., 2nd Ed.,
McGraw-Hill, 1967
The test described in this instruction can be found
in the 1974 EPA Methods Manual on page 159, entitled
Nitrogen, Ammonia (Distillation Procedure). If the
distillation is done at pH 9.5, another reference
which contains an acceptable procedure for this test
is on page 410 of the 14th edition of Standard
Methods.
Methods for Chemical
Analysis of Water and
Wastes, 1974, EPA,
MDQARL, Cincinnati,
Ohio 45268, p. 159.
Standard Methods for
the Examination of
Water and Wastewater,
14th ed., 1976, APHA,
New York, New York,
p. 410
Page No. 6-17
-------
EFFLUENT MONITORING PROCEDURE: Nitrogen, Ammonia Determination
FIELD AND LABORATORY ANALYSIS Section VII
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
VII.D.6a.
At some water treatment plants ammonia is added in
the combined residual chlorination of water. Where
the free residual chloririation process is employed,
ammonia nitrogen will react with chlorine in ratios
which vary with the nitrogen concentration. At
low ammonia concentrations (0.1 mg/liter nitrogen)
the ratio approximates 1 to 10, while at higher
ammonia concentrations the ratio approaches 1 part
of ammonia nitrogen to 7.59 parts of chlorine. .If a
sample contains residual chlorine, then monochlora-
mine, dichloramine, or trichloramine may be present.
Dechlorination prior to analysis will convert these
substances to ammonia.
Standard Methods
13th Ed., p. 223
VII.D.7a.
Ammonia recovery from preliminary distillation will
be low on water samples containing more than
250 mg/liter calcium unless the pH is properly
adjusted before distillation is undertaken. The
calcium and the phosphate buffer react to precipitate
calcium phosphate, releasing hydrogen ions and
lowering the pH.
Standard Methods
13th Ed., p. 223
Page No. 6-18
-------
I AOrtOATnOV MTA (UtCT
tnt/uiviiuiM un i n jiilli
Nitrogen, Ammonia Determination
(Sample contains 1.0 to 25.0 mg/1 NH^-N)
Sample No. Date/Time Sampled Sample Point
(Sulfuric acid 0.02N ml ) (0.28)(1000)
jng/liter NH^-N
Sample ml
Date
Analyst
Page No. 6-19*
-------
Page No. 6-20
LABORATORY DATA SHEET
Nitrogen, Ammonia Determination
(Sample Contains 0.05 to 1.0 mg/liter NH^-N)
Sample No. Date/Time Sampled Sample Point
(mq of NH--N )(1000) (Total Distillate* Collected ml )
— 3 x = = mg/1 NH3-N
(Sample ml ) (Distillate Taken for Nesslerization ml )
Date
Analyst
*Include boric acid plus dilution water
-------
A PROTOTYPE FOR DEVELOPMENT OF
ROUTINE OPERATIONAL PROCEDURES
for the
DETERMINATION OF NITRATE-NITRITE NITROGEN AND
OF NITRATE NITROGEN, CADMIUM REDUCTION METHOD
as applied 1n
WASTEWATER TREATMENT FACILITIES
and 1n the
MONITORING OF EFFLUENT WASTEWATERS
National Training Center
Municipal Operations and Training Division
Office of Water Program Operations
U.S. Environmental Protection Agency
CH.N.n/n.EMP.la.3.76
Page No. 7-1
-------
EFFLUENT MONITORING PROCEDURE: Deternrination of Nitrate-Nitrite Nitrogen
and of Nitrate Nitrogen, Cadmium Reduction
Method
This operational procedure was developed by:
NAME Don Roach
ADDRESS Miami-Dade Community College, South Campus, 11011 S.W. 104 Street,
Miami, Florida 33176
POSITION Chairman - Chemistry Department
EDUCATION AND TECHNICAL BACKGROUND
B.S. - Chemistry
M.S. - Chemistry
PhD. - Analytical Biochemistry
1 year Commercial Laboratory Chemist
10 years College Chemistry Instructor
7 years Chemical Consultant to Industry
Page No. 7-3
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen
and of Nitrate Nitrogen, Cadmium Reduction
Method
1. Objective:
To determine the nitrate-nitrite nitrogen and the nitrate nitrogen content
of an effluent.
2. Brief Description of Analysis:
The procedure converts nitrate nitrogen to nitrite nitrogen when the
nitrate is passed through a column containing copper-cadmium granules.
Nitrate is almost quantitatively reduced to nitrite by this process.
The resulting nitrite is determined by reacting the effluent with sul-
fanilamide and coupling with N - (1-napthyl) - ethylenediamine dihydro-
chloride to form a highly colored dye which can then be determined
colorimetrically. A correction must be made for any nitrite initially,
present in the sample since the method determines total nitrite. The
concentration of nitrite originally present in a sample can be determined
by omitting the initial copper-cadmium reduction and carrying out the
remainder of the procedure. Separate nitrate-nitrite values for a sample
may be obtained by analyzing two aliquots of the same; one with the
copper-cadmium reduction step and one without the initial reduction step.
3. Applicability of this Procedure:
a. Range of Concentration:
0.01 to 1.0 mg NO^-f^ N/1 iter
(The range may be extended for samples by dilution.)
b. Pretreatment of Samples:
The Federal Register Guidelines do not specify any pretreatment.
c. Treatment of Interferences in Samples:
This procedure includes directions for removal of turbidity and/or of
grease and oil from samples. It also includes addition of EDTA to
eliminate interferences from metals. No other interferences are noted
in the Source of Procedure.*
* Source of Procedure: Methods for Chemical Analysis of Water and Wastes, 1974,
Environmental Protection Agency, Methods Development and Quality Assurance
Research Laboratory, Cincinnati, Ohio, page 201.
Page No. 7-4
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen
and of Nitrate Nitrogen, Cadmium Reduction
Method
FLOW SHEET:
SAMPLE
TURBIDITY REMOVAL
(if necessary)
OIL AND GREASE REMOVAL
(if necessary)
ADJUST pH TO BETWEEN 5
AND 9 (if necessary)
RESULTS: NITRATE N PLUS
ORIGINAL NITRITE N
MEASUREMENT OF ABSORBANCE AT
540 nm
REDUCTION OF NITRATE TO
NITRITE BY PASSING THROUGH
CADMIUM REDUCTION COLUMN
COLOR DEVELOPMENT BY REACTION
OF NITRITE WITH SULFANILAMIDE
AND WITH N-(l- NAPTHYL) -
ETHYLENEDIAMINE DIHYDROCHLORIDE
The above procedures determine nitrate N plus nitrite N. The initial nitrite
concentration of the samples should be determined prior to reduction. Thus,
the nitrate concentration can be determined by:
Nitrate N = Total Nitrite N - Nitrite N before reduction
Page No. 7-5
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen
and of Nitrate Nitrogen, Cadmium Reduction
Method
Equipment and Supply Requirements
A. Capital' Equipment:
1. Balance, analytical, 160 g capacity, precision j^O.l mg
2. Balance, triple beam, 500 g capacity, precision + 0.25 g
3. pH meter/combination electrode, range 0-14 pH
4. Refrigerator, temperature range 2° - 10°C
5. Spectrophotometer, wave length range 325-825 nm
6. Still and de-ionizing cartridges (or other means of distilling and
de-ionizing water)
B. Reusable Supplies:
1. One apron, laboratory
2. One 100 ml beaker
3. Four 250 ml beakers (3 for buffer solutions)
4. One 400 ml beaker
5. One 1 liter beaker
6. One 2 liter beaker
7. Two bottles, Barnes with stoppers and two droppers, small gauge
8. One 150 ml bottle, dropper
9. One 250 ml bottle, plastic wash
10. One 100 ml bottle, storage with screw-on cap (storage of 6N HC1)
11. Seven 1 liter bottles, storage, brown with screw-on caps or rubber stoppers
12. Two 5 gallon bottles, water with bottom spout
13. One brush, camel hair (cleaning analytical balance)
14. Two brushes, bottle (cleaning glassware)
15. One bulb, propipet type
16. One buret holder, double clamps (reduction column support)
17. Two columns, reduction (see Figure 1 at the end of this section)
18. Three cuvettes
19. One 25 ml cylinder, graduated
20. One 50 ml cylinder, graduated
21. One 100 ml cylinder, graduated
22. One 500 ml cylWer, graduated
23. One 1 liter cyl/nder, graduated
24. One 50 ml flask, volumetric with stopper (dilution of sample)
25. Twelve 100 ml flasks, volumetric with stoppers (for standards)
26. X 100 ml flasks, volumetric with stoppers (for samples - 1 flask
per sample)
27. Twelve 250 ml flasks, Erlenmeyer with stoppers (for standards)
28. X 250 ml flasks, Erlenmeyer with stoppers (for samples-! flask per sample)
29. One 1 liter flask, Erlenmeyer, or a large, empty chemical bottle
(for Cd washings)
30. Three 1 liter flasks, volumetric with stoppers
31. Two 2 liter flasks, volumetric with stoppers
32. One filter funnel for 0.45 u filter (turbidity removal)
Page No. 7-6
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen
and of Nitrate Nitrogen, Cadmium Reduction
Method
B. Reusable Supplies (Continued)
33. One funnel, powder
34. One funnel, large powder with large filter paper (for Cd washings)
35. One 250 ml funnel, separatory (oil and grease removal)
36. One pair glasses, safety
37. Two hoses, rubber, 3" strip, 4 cm I.D. with screw type clamp
38. One notebook (recording data)
39. Two 100 ml volumetric pipets (construction of reduction columns)
40. One C.5 ml pipet, volumetric
41. One 1 ml pipet, volumetric
42. One 2 ml pipet, volumetric
43. One 5 ml pipet, volumetric
44. One 10 ml pipet, volumetric
45. One 25 ml pipet, volumetric
46. One 50 ml pipet, volumetric
47. One rod, stirring (6" or 12")
48. One sieve, 40 mesh
49. One sieve, 60 mesh
50. One spatula (scoopula )
51. Two stands, ring (support funnel, and reduction column)
52. One support, ring, small (support funnel)
C. Consumable Supplies:
1. Glasswool, wad
2. Membrane filter, 0.45 v
3. Notebook (recording data)
4. Pen or pencil (recording data, marking flasks)
5. Soap
6. Sponges (for cleaning)
7. Tissues, soft (wiping cuvettes and electrodes)
8. Towels, paper
9. Twelve weighing boats
10. 26 g ammonium chloride, NH^Cl
*11. 100 ml ammonium hydroxide, NH^OH
*12. 150 ml buffer solution, STD pH 4
*13. 600 ml buffer solution, STD pH 7
*14. 450 ml buffer solution, STD pH 10
**15. 25 g cadmium granules, 40-60 mesh
16. 55 ml chloroform, CHCl^
17. 20 g copper sulfate, pentahydrate, CuSO^-Sh^O
18. 3.4 g disodium ethylenediamine tetraacetate, C^gH^N2Na20g
19. 1 g N-(l-napthyl) - ethylenediamine dihydrochloride, C-|2^-j4N2*^HCl
*20. 200 ml hydrochloric acid, concentrated, HC1
21. 100 ml hydrochloric acid, dilute (6N), HC1
22. 100 ml phosphoric acid, concentrated, HgPO^
*23. Potassium dichromate (cleaning solution), I^C^O^
24. 7.218 g potassium nitrate, KNO^
Page No. 7-7
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen
and of Nitrate Nitrogen, Cadmium Reduction
Method
C. Consumable Supplies (Continued)
25. 6.072 g potassium nitrite, KN0£
26. 240 g sodium hydroxide, pellets, NaOH
27. 10 g sulfanilamide, CgHgN202S
*28. Sulfuric acid, concentrated, (cleaning solution) H2S04
29. 100 g zinc sulfate, heptahydrate, ZnSO^h^O
30. Labels, package, 1 1/2 x 1 inch
31. Paper, graph 8 1/2 x 11, package
All reagents should be reagent grade.
The above amounts do not allow for spillage or mistakes.
*These amounts will vary
**This metal can be purchased from EM Laboratories, Inc.,
500 Executive Boulevard, Elmsford, New York,10523, Cat. 2001 cadmium,
coarse powder
Page No. 7-8
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen
and of Nitrate Nitrogen, Cadmium Reduction
Method
10 cm
80-85 ml
25 cm
-3 cm I.D.
3.5 mm I.D.
Cu/Cd
18.5 cm
100 ml
volumetric
pipet
Cut
GLASS WOOL PLUG
CLAMP
TYGON TUBING
W
'Cut
Figure 1. Reduction column
Page No. 7-9
-------
EFFLUENT MONITORING PROCEDURF: Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
Page No. 7-10
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
DETERMINATION OF NITRATE-NITRITE NITROGEN AND OF NITRATE NITROGEN, mg/liter
A. Equipment
Preparation
1. Glassware Wash-Up
2. Balance Inspection
3. Spectrophotometer
Inspection
1. Clean all glassware in
suitable detergent.
1. Clean balance.
1. Clean spectrophotometer.
2. Turn power on by rotating
the power control
clockwise.
3. Select wavelength by
rotating the wavelength
control knob either
direction until the proper
wavelength is reached.
4. Zero the instrument by
bringing the meter needle
to "0" on the percent
transmittance scale.
5. Use an empty cell and
adjust the light control
to 100% T.
la. Distilled water drains without leaving any
droplets on surfaces,
lb. Use chromerge if necessary.
la. Free of dust and dirt.
la. Free of dust and dirt.
2a. Pilot lamp on.
2b. Directions are for Spectronic 20.
3a. 540 nm on the wavelength scale.
4a. Meter needle reads zero.
I
(p. 41 )
5a. To be sure that the instrument can achieve 100% T.
-------
r'LUENT MONITORING PROCEDURE: Determination of Nitrate-i,. ,,-ite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
1. Distilled Water
2. Concentrated
Ammonium Chloride
EDTA Solution
1. Prepare approximately ten
(10) liters of highly pure
water.
1. Weigh 26 g of ammonium
chloride, NH^Cl, in a
weighing boat and wash
into 2.0 liter graduated
beaker.
2. Weigh 3.4 g of disodium
ethylenediamine tetra-
acetate, C-j QH^N2Na20g,
and wash into the same
beaker.
3. Add enough distilled water
to bring the total volume
to approximately 1800 ml.
4. Use a pH meter to adjust
the pH of the solution to
8.5 by the dropwise addi-
tion of concentrated
ammonium hydroxide,
nh4oh.
5. After the pH has been ad-
justed, transfer the
solution to a 2 liter
volumetric flask.
la. An ion exchange column in conjunction with a still
provides an adequate source of highly pure water.
lb. This water will be used for all reagent prepara-
tion and washing of equipment.
1c. The pH of the water must be between 5.5-7.5.
la. Distilled water should be used for all phases of
solution preparation including water used in
washing a solid into a container.
4a. Mix the solution thoroughly by stirring, after
the addition of each drop of NH^OH.
5a. Whenever a solution is transferred, the container
from which the transfer is made should be washed
and the washings added to the container to which
the transfer was made.
Page No. 7-11
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
Page No. 7-12
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(Continued)
3. Dilute Ammonium
Chloride EDTA
Solution
4. Color Reagent
6. Dilute to volume with
distilled water.
7. Label the bottle in which
the solution is stored.
1. Measure 1200 ml of the
concentrated ammonium
chloride-EDTA solution,
using a graduated
cylinder.
2. Pour the measured solution
into a 2.0 liter volume-
tric flask.
3. Dilute to volume with
distilled water.
4. Store in a labeled
container.
1. Add 800 ml of distilled
water to a 1 liter flask.
2. Add 100 ml of concentrated
phosphoric acid, H^PO^, to
the same flask.
3. Mix thoroughly.
4. Weigh 10 g of sulfanilamide
(C6H8^2S^ in a wei9'11'n9
boat.
6a. The solution is stable for several months.
7a.
Include the name of the solution, your name and
the date of preparation.
4a.
la.
lb.
Both the concentrated and dilute ammonium
chloride-EDTA solutions are stable for several
months.
Use a graduated cylinder.
Use a 1 liter volumetric flask.
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(Continued)
5. Zinc Sulfate
Solution
5. Use a wash bottle and
funnel to wash the
sulfanilamide into the
1 liter flask containing
phosphoric acid solution.
6. Weigh 1 g N-(l-napthyl)-
ethylenediamine dihydro-
chloride, Marshall's
Reagent, and wash into
same flask.
7. Dilute to volume with
distilled water.
8. Store in a labeled
container.
2.
Weigh 100 g of zinc sul-
fate heptahydrate,
ZnS0^-7H20, in a weighing
boat.
Wash into a 1 liter flask
using a wash bottle and a
funnel.
3. Add sufficient distilled
water to dissolve all of
the solid.
8a.
8b.
8c.
Container should be dark 1 liter plastic reagent
bottle.
Store at 4°C when not in use.
Use at room temperature.
8d. The solution is stable for several months.
8e. A very faint pink color may show up in this
color reagent. You may still use the reagent.
If a precipitate forms in the reagent, though,
discard it.
la. This reagent is used if flocculation is employed
as an alternative to filtration if the sample
requires removal of turbidity.
2a. Use a volumetric flask.
Page No. 7-13
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and of
'—————— titrate Nitrogen, Cadmium Reduction Method
Page No. 7-14
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(Continued)
4. Dilute to volume with
distilled water.
5. Store in a labeled
container.
5a. This solution is stable for at least one year.
6. Sodium Hydroxide
Solution (6N)
1. Rapidly weigh 240 g of
solid sodium hydroxide,
NaOH, pellets in a 1 liter
graduated beaker.
2. Add 500 ml distilled
water to dissolve the
sodium hydroxide.
3. Dilute to a total volume
of 1 liter.
4. Store in a glass bottle or
jug and stopper with a
rubber stopper.
5. Label the container.
la. This reagent is used if flocculation is employed
as an alternative to filtration if the sample
requires removal of turbidity.
lb. Sodium hydroxide picks up moisture from the air
quite readily.
2a. The water should be added with constant swirling
to avoid fusing.
3a. The solution should be allowed to cool to room
temperature before the dilution is made.
4a. Sodium hydroxide slowly etches glass causing
glass stoppers to stick.
4b. The solution is stable for at least a year.
7. Ammonium
Hydroxide
1. A 100 ml supply should be
available.
2. Place in a Barnes
(dropper) bottle.
la. Drop quantities may be required for pH
adjustment.
8. Hydrochloric
Acid, (6N)
1. Add 50 ml of distilled
water to a 400 ml beaker.
la. A 100 ml graduated cylinder is suitable for
measuring the volume of the distilled water and
the volume of the acid.
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(Continued)
9. Copper Sulfate
Solution (2%)
10. Nitrate Stock
Solution
2. Slowly add 50 ml of
concentrated hydrochloric
(HC1) acid (12 N) to the
same beaker.
3. Mix thoroughly.
4. Store in a 100 ml bottle.
5. Label the container.
1. Weigh 20 g of copper
sulfate pentahydrate,
CuS04-5H20, in a weighing
boat.
2. Wash copper sulfate into a
one liter volumetric
flask.
3. Add sufficient distilled
water to dissolve the
sol id.
4. Dilute to volume with
distilled water.
5. Store in a labeled
container.
1. Carefully weigh 7.218 g of
potassium nitrate, KNO^,
in weighing boat
3a. About 500 ml of water should be sufficient.
4a. This solution is stable for at least one year.
la. An analytical balance should be used.
Page No. 7-15
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
Page No. 7-16
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(Continued)
11. Nitrate Standard
Solution
2. Transfer the solid to a
1 liter volumetric flask
equipped with a powder
funnel.
3. Use wash bottle to wash
the solid into the flask.
4. Add sufficient distilled
water to dissolve the
sol id.
5. Dilute to volume with
distilled water and
thoroughly mix.
6. Store in a labeled glass
bottle.
7. Preserve
adding 2
CHC13.
the solution by
ml of chloroform,
1. Carefully pipet 10.0 ml of
nitrate stock solution
into a 1 liter volumetric
flask.
2. Dilute to volume with
distilled water.
2a. This is best achieved by washing the solid onto
the funnel with a wash bottle.
3a. The weighing boat should be rinsed three times
and all of the rinse water should be added to
the flask.
4a. About 500 ml is sufficient.
7a. The solution prepared,stored and preserved in
this manner should be stable for at least
6 months.
7b. The nitrate stock solution contains 1.00 mg of
nitrate nitrogen (NOg-N) in each 1.00 ml of
solution.
la. This nitrate standard solution should be prepared
fresh for each use.
lb. The nitrate stock solution should be at room
temperature before using,
lc. Use a 10 ml volumetric pipet.
-------
EFFLUENT MONITORING PROCEDURF.: Determination of Nitrate-Nitrite Nitrogen and of
——————————— Nitrate Nitrogen, Cadmium Reduction Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(Continued)
12. Nitrite Stock
Solution
3. Store in a labeled
container.
1. Weigh 6.072 g of
potassium nitrite, KNO
in a weighing boat.
2'
2. Transfer the solid to a
1 liter volumetric flask
using a powder funnel.
3. Use wash bottle to wash
the solid into the flask.
4. Add sufficient distilled
water to dissolve the
sol id.
5. Dilute to volume and mix
thoroughly.
6. Store in a labeled glass
bottle.
7. Preserve the solution by
adding 2 ml of chloroform
for each 1 liter of solu-
tion and refrigerate when
not in use.
3a. Use within one hour of preparation.
3b. The nitrate standard solution contains 0.01 mg
of nitrate nitrogen (N0--N) in each 1.0 ml of
solution.
la. An analytical balance should be used for all
weighings involving standards.
3a. The weighing boat should be washed three times
and the washings added to the flask.
4a. About 500 ml is sufficient.
7a. The solution should be stable for at least
3 months when preserved this way and stored
at about 4°C when not in use.
7b. The nitrite stock solution contains 1.00 mg of
nitrite nitrogen (N0?-N) in each 1.0 ml of
solution.
Page No. 7-17
-------
EFFLUENT MONITORING PRQCEDURR: Determination of Nitrate-Nitrite Nitrogen and of
""————Nitrate Nitrogen, Cadmium Reduction Method
Page No. 7-18
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(Continued)
13. Nitrite Standard
Solution
1. Pipet 10.0 ml of nitrite
stock solution into a
1 liter volumetric flask.
2. Dilute to volume with
distilled water.
3. Store in a labeled
container.
la. This nitrite standard solution should be prepared
fresh for each use.
lb. The nitrite stock solution should be at room
temperature before using.
1c. Use a 10 ml volumetric pipet.
3a. Use within 1 hour of preparation.
3b. The nitrite standard solution contains 0.01 mg of
nitrite nitrogen (N0,>-N) in each 1.0 ml of
solution.
C. Reduction Column
Preparation
1. Preparation of the
Glass Column
1. Construct a glass column
by joining a 10 cm length
of 3 cm ID glass tubing
with a 25 cm length of
3.5 mm ID tubing using
figure 1 as a guide.
2. Loosely plug the delivery
tip of the column with
glass wool.
la. Figure 1 is at the end of the Equipment and
Supply Requirements Section,
lb. The column shown in Figure 1 was constructed by
cutting both ends off a 100 ml volumetric pipet
as indicated,
lc. Fire polish all cut surfaces.
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Reduction Column
Preparation
(Continued)
2. Preparation of
Copperized
Cadmium for
Packing the
Glass Column
1. Weigh about 2 0 g of
cadmium granules in a
weighing boat.
2. Transfer the cadmium to a
400 ml beaker.
3. Add enough dilute (6N)
hydrochloric acid to
cover the granules.
4. Swirl the contents of the
beaker.
5. Pour off the acid while
retaining the granules
in the beaker.
6. Add enough distilled water
to cover the granules.
la. This will be enough for one column.
lb. Granulated cadmium (40-60 mesh) can be purchased.
1c. Alternatively, file sticks of pure cadmium metal
(reagent grade) with a coarse metal hand file
(about second cut) and collect the fraction which
passes a sieve with 10 mesh openings and is re-
tained on sieves with 40, then 60 mesh openings.
Id. Handling cadmium is hazardous, thus filing should
be conducted under a hood using rubber gloves and
mask.
2a. A scupula and wash bottle with water is good for
this.
5a. All decanting should be done into a container
equipped with a large funnel and filter paper
so as to catch all the small cadmium particles.
5b. Use this filter paper for any subsequent cadmium
washings.
VIII.C.2.Id
(p. 46 )
Page No. 7-19
-------
iEJFFJLUiEiNi^^Orn>TOR_I_NG__PROCiEDUiR^: Determination of Nitrate-Nitrite Nitrogen and of
""Nitrate Nitrogen, Cadmium Reduction Method
Page No. 7-20
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Reduction Column
Preparation
(Continued)
7. Pour off the water while
retaining the granules in
the beaker.
8. Repeat steps 6 and 7,
above, two more times so
that the granules receive
a total of three dis-
tilled water washings.
9. Add 100 ml of the 2%
copper sulfate solution to
the granules and swirl for
five minutes or until the
blue color of the copper
sulfate fades.
10. Carefully decant off the
solution leaving the
copperized cadmium
granules in beaker.
11. Repeat steps 9 and 10
until a brown colloidal
(very fine) precipitate
of metallic copper does
form.
12. Wash the copper-cadmium
at least 10 times with
distilled water.
13. Place the washed copper-
cadmium on the 60 mesh
sieve.
9a. A brown colloidal (very fine) precipitate of
metallic copper may form.
10a. Also decant off through the filter paper any
precipitate that formed.
12a. All of the brown precipitated copper should be
removed by washing 10 times but continue to
wash if any remains.
-------
lUENT MONITORING PROCEDURE: Determination of Nitrate-Ni rte Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
GUIDE NOTES
C. Reduction Column
Preparation
(Continued)
14. Pour water over the
granules at least three
times so that all the
small particles will
wash through the 60 mesh
screen.
15. Return meshed granules to
the beaker.
16. Decant off excess water
used to transfer the
cadmium.
17. Close the clamp on the
column delivery tube.
18. Fill the column almost to
the top of the cup part
with ammonium chloride-
EDTA solution.
19. Loosely fill the reduction
column with copper cadmium
granules to a level about
2 cm below the broad,
cup-like section as shown
in Figure 1.
14a. Hold the sieve over the filter paper during
these washings.
15a. Use a scupula and the wash bottle.
18a. Use a graduated cylinder and very slowly pour the
solution down the inside wall of this column so
air pockets do not form.
19a. Avoid tight packing of granules by allowing the
granules to "float" down through the solution
of ammonium chloride-EDTA.
19b. A glass stirring rod may be used to transfer the
cadmium to the column.
19c. For regeneration of column see training guide.
19d. When column is not in use, fill it with ammonium
chloride-EDTA solution so that the granules are
covered with about 2.5 cm of solution above them.
VII.C.2.19c
(P- 43 )
Page No. 7-21
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
Page No. 7-22
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Reduction Column
Preparation
(Continued)
20. Open the screw clamp and
measure the flow rate of
ammonium chloride-EDTA
solution through the
column.
21. When the flow rate can be
maintained between 7 ml
and 10 ml/minute, drain
off the ammonium chloride-
EDTA solution until it is
about 2.5 cm above the
top of the granules.
22. Close the screw clamp.
20a. To calculate the flow rate, place a short 50 ml
graduated cylinder under column and measure the
amount of fluid collected in one minute.
20b. The flow rate should be between 7 ml and
10 ml/minute.
20c. If the flow rate is too fast, tighten the screw
clamp. If the clamp must be so tight that control
is lost, add more copper cadmium granules to the
column.
20d. If the flow rate is too slow, decrease the length
of the copper cadmium column until a flow rate of
7-10 ml/minute is achieved.
21a. When the column is not in use, the copper cadmium
granules should be covered with ammonium chloride-
EDTA solution so they do not dry out.
D. Removal of
Interferences
1. Turbidity Removal
(If Necessary)
1. Prior to analysis, remove
turbidity from samples
by filtering through a
0.45 u membrane filter.
la. If the turbidity is not removed by filtration,
proceed as follows: Add 1 ml of the zinc
sulfate solution to 100 ml of sample. Add
enough 6 N sodium hydroxide to bring the pH to
10.5, (about 8 to 10 drops is usually sufficient).
Let the treated sample stand for 15 minutes.
Filter through a 0.45 p membrane filter,
lb. Suspended solids can clog the reduction column.
VI. D
(P- 42)
-------
Lm-UJENT MONITORING PROCEDURF: Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Removal of
Interferences
(Continued)
2. Oil and Grease
Removal (If
Necessary)
1. Prior to analysis,
measure 100 ml of the
sample (filtered sample
if the original sample
was turbid) into a 400 ml
beaker.
2. By dropwise addition, add
sufficient concentrated
hydrochloric acid (12 N)
to bring the pH down to 2.
3. Place the sample in a
250 ml separatory funnel.
4. Add 2.5 ml of chloroform.
5. Shake gently to extract
the oils and grease into
the chloroform layer.
6. Allow the separatory
funnel to stand until all
of the chloroform layer
settles to the bottom.
7. Open the stopcock and
allow the bottom (chloro-
form) layer to pass into
a 400 ml beaker.
la. Oil and grease can clog the reduction column
and coat the Cu/Cd granules.
2a. Use a pH meter in adjusting the pH to 2.
2b. Standardize using standard buffer of pH = 4.00.
5a. Carefully release the pressure after shaking
gently so that no sample is lost. This can be
accomplished by inverting the separatory funnel
and slowly opening the stopcock away from face
and other people.
6a. Place funnel in ring stand.
6b. Remove stopper while layer is settling.
7a. Grease and oils are extracted into chloroform
layer leaving a grease-oil free sample which is
used for analysis.
Page No. 7-23
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
Page No. 7-24
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Removal of
Interferences
(Continued)
8. Repeat steps 4, 5, 6, and
7 with 25 ml of fresh
chloroform.
8a. The second chloroform extract is added to the
same beaker as the first extract.
E. Preparation of
Nitrate Working
Standards
1. Nitrate Working
Standards
1. Prepare nitrate working
standards by respectively
pipetting the following
volumes of nitrate
standard solution into
each of six 100 ml
volumetric flasks.
Add This For This
Volume of Concentra-
Nitrate tion of
To Flask Standard NO3-N in
No. Solution mg/1
1 0.0 ml 0.00
2 0.5 ml 0.05
3 1.0 ml 0.10
4 2.0 ml 0.20
5 5.0 ml 0.50
6 10.0 ml 1.00
2. Dilute each of the flasks
to volume with distilled
water.
la. Label flasks.
lb. Use appropriate volumetric pipets (0.5 ml, 1.0 ml,
2.0 ml, 5.0 ml, 10.0 ml).
lc. The 0.00 solution which contains no nitrate (or
nitrite) serves as the reagent blank for the
nitrate samples and standards which are passed
through the reduction column.
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
E. Preparation of
Nitrate Working
Standards (Continued)
3. Use the working standards
immediately after their
preparation.
F. Reduction of Nitrate
to Nitrite
1. Adjustment of pH
2. Activation of
Column
Use a pH meter to adjust
the pH of each of the
working standards to
between 5 and 9 either
with concentrated hydro-
chloric acid or with
concentrated ammonium
hydroxide.
1. Pipet 25.0 ml of working
standard #6 to a small
Erlenmeyer flask.
2. Add 75 ml of the dilute
ammonium chloride-EDTA
solution to the same
flask.
3. Mix the working standard
thoroughly by swirling
the contents of the flask.
4. Place a 250 ml beaker
under the reduction
column.
la.
lb.
Use a beaker small enough for this volume of
standard to cover the pH electrode(s).
Make sure that the pH meter is calibrated within
this range.
lc. Use buffer solutions pH 4, pH 7, pH 10 to
calibrate and check the meter.
Id. This pH adjustment is necessary to insure that
the pH is approximately 8.5
(Mo pH adjustment is necessary if the pH is
already between 5 and 9.)
la.
Activation of column is necessary to prepare
surfaces of Cu-Cd granules for reduction process.
This standard is 1.00 mg NO^-N/liter
concentration,
lc. A 250 ml flask is good for this purpose.
lb
2a. A 100 ml graduated cylinder is good for this
purpose.
4a. You will collect the reduced working standard
in this beaker.
Page No. 7-25
-------
EFFLUENT MONITORING PROCEDURF:
Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
Page No. 7-26
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Reduction of Nitrate
to Nitrite
(Continued)
5. Check that the level of
ammonium chloride-EDTA
solution in the column is
down to the top of the
granules.
6. Pour the prepared nitrate
working standard into the
reduction column.
7. Using the screw clamp
(see Figure 1) adjust the
collection rate to
7-10 ml per minute.
8. Collect the reduced
working standard until
the level of solution is
0.5 cm above the top of
the granules.
9. Close the screw clamp to
stop the flow.
10. Discard the entire re-
duced working standard.
11. Measure about 40 ml of
ammonium chloride-EDTA
solution.
5a. If the level is too high, drain the excess into
the beaker.
6a. Since the column will not hold the total amount,
add the final amount after the first 15 ml has
passed through the column.
7a. The clamp should be slowly opened until a
collection rate of 7-10 ml per minute is
achieved.
7b. A collection rate of 7-10 ml of solution per
minute should be carefully maintained throughout
the collection process to assure complete re-
duction of nitrate in the sample.
10a. The column is now activated.
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Reduction of Nitrate
to Nitrite
(Continued)
Reduction of
Working Standards
12. Pour the 40 ml into the
column.
13. Repeat steps 8 and 9.
1. Pipet 25.0 ml of the
lowest concentration of
nitrate working standard
into a small Erlenmeyer
flask.
2. Add 75 ml of the dilute
ammonium chloride-EDTA
solution to the same
flask.
3. Mix nitrate working stand-
ard thoroughly by swirling
the contents of the flask.
4. Place a short graduated
cylinder under the reduc-
tion column.
5. Pour the prepared nitrate
working standard into the
reduction column.
6. Using the screw clamp
(see Figure 1) adjust the
collection rate to 7-10 ml
per minute.
13a. The nitrate standard should now be "washed off11
the column.
la. A 250 ml flask is good for this purpose,
lb. Label the flask.
lc. Begin with the 0.00 mg/liter solution.
2a. Use a 100 ml graduated cylinder.
4a. You need to measure 25 ml of solution in the
graduate.
5a. Since the column will not hold the total amount,
add the final amount after the first 15 ml has
passed through the column.
6a. The clamp should be slowly opened until a
collection rate of 7-10 ml per minute is
achieved.
6b. A collection rate of 7-10 ml of solution per
minute should be carefully maintained throughout
the collection process to assure complete re-
duction of the nitrate in the nitrate working
standard.
Page No. 7-27
-------
EFFLUENT MONITORING PROCEDURE: Determination of Citrate-Nitrite Nitrogen and of
~titrate Nitrogen, Cadmium Reduction Method
Page No. 7-28
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Reduction of Nitrate
to Nitrite
(Continued)
7. Discard the first 25 ml
of solution which is
collected.
8. Replace the graduate with
the rinsed,air-dried
flask used for this
standard.
9. Collect the remaining
portion of the reduced
standard in the original
flask.
10. Analyze the reduced
standard immediately
after collection from
the reduction column.
11. Repeat steps 1 through 10
for each of the prepared
working nitrate standards.
7a. This discard portion serves to "wash off"
solution remaining in the column from any
previous pass-through.
9a. Close the screw clamp when the level of solution
is about 0.5 cm above the granules.
9b. About 70 ml should be in the flask.
10a. While one solution is passing through the column
you should proceed to color development of the
previous solution that has already been reduced.
Color development (Section G) must begin within
15 minutes after reduction.
11a. Proceed from the least concentrated to the most
concentrated standard.
lib. Label each receiver flask.
G. Color Development of
Reduced Nitrate
Working Standards
1. Use a 50.0 ml pi pet to
remove a 50.0 ml aliquot
from flask #1 (0.00 mg/
liter NO^-N).
la. By using a propipet the aliquot can remain in
the pipet during the next two steps,
lb. Aliquots of each of the working standards should
have been passed through the reduction column as
described in the previous section (Section F).
1c. The reduced working standards should be analyzed
as soon as possible after the reduction and in
no case should they be allowed to stand for more
than 15 minutes after reduction before color
development is begun.
-------
l. . lUENT MONITORING PROCEDURE: Determination of Nitrate-NiU ite Nitrogen and of
——————Nitrate Nitrogen, Cadmium Reduction Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
G. Color Development of
Reduced Nitrate
Working Standards
(Continued)
2. Discard the remainder of
the nitrate reduced
working standard.
3. Shake flask dry.
4. Add the 50.0 ml working
standard back to same
flask from which it was
removed.
5. Add 2.0 ml of the color
reagent to the 50.0 ml
of working standard.
6. Mix thoroughly by
swi rling.
7. Allow the working standard
to stand until color
develops.
8. Repeat steps 1 through 7
for each of the reduced
working standards.
3a. Do not rinse the flask.
4a. If you find the technique in steps 1-4 too
difficult, transfer the 50.0 ml to a different
flask.
5a. Use a 2.0 ml volumetric pipet.
7a. The reduced working standard should be allowed
to stand for at least 10 minutes but not more
than two hours before doing Procedure L,
Spectrophotometry Measurements.
8a. Start with least concentrated solution and
proceed to most concentrated.
8b. Rinse the 50.0 ml pipet thoroughly after each
standard.
H. Analysis of Samples
for Nitrate Reduced
to Nitrite
1. Dilution of
Samples (if
necessary)
1. Pipet 25.0 ml of unknown
sample into 50 ml volu-
metric flask.
la. Potable water samples will usually require no
dilution, while sewage samples may require
dilution.
Page No. 7-29
-------
EFFLUENT^MONITORING PROCEDURF.: Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
Page No. 7-30
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
H. Analysis of Samples
for Nitrate Reduced
to Nitrite
(Continued)
2. Adjustment of pH
3. Reduction of
Nitrate to Nitrite
in Samples
4. Color Development
in Samples
2. Dilute to volume with
distilled water.
1. Use a pH meter to adjust
the pH of each sample to
between 5 and 9 either
with concentrated hy-
drochloric acid or with
concentrated ammonium
hydroxide.
1. Aliquots of each of the
samples should be passed
through the reduction
column as described in
Procedure F.3, "Reduction
of Working Standards."
1. Follow the steps in
Procedure G, "Color
Development."
2a. If you need to dilute a sample, you must apply a
dilution factor to the concentration found from
a standard curve.
la. Put the 50 ml of sample in a small beaker so
the pH electrode(s) is covered with solution.
lb. Make sure that pH meter is calibrated within
this range.
lc. Use buffer solutions pH 4, pH 7, pH 10 to cali-
brate and check the meter.
Id. This pH adjustment is necessary to insure that
the pH is approximately 8.5
(No pH adjustment is necessary if the pH is
already between 5 and 9.)
VII.H.l.2a
(p. 44 )
I. Preparation of
Nitrite Working
Standards
1. Nitrite Working
Standards
1. Prepare nitrite working
standards by respectively
pipetting the following
volumes of nitrite stand-
ard solution into each of
six 100 ml volumetric
flasks.
la. Label flasks.
lb. Use appropriate volumetric pipets (0.5 ml, 1.0 ml,
2.0 ml, 5.0 ml, 10.0 ml),
lc. The 0.00 solution which contains no nitrite (or
nitrate) serves as the reagent blank for the
nitrite standards and samples that are not passed
through the column.
-------
l.- . lLIENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
I. Preparation of
Nitrite Working
Standards
(Continued)
Add This
For This
Volume of
Concentra-
Nitrite
tion of
To Flask
Standard
NO2-N in
No.
Solution
mg/1
1
0.0 ml
0.00
2
0.5 ml
0.05
3
1.0 ml
0.10
4
2.0 ml
0.20
5
5.0 ml
0.50
6
10.0 ml
1.00
2. Dilute each of
the flasks
to volume with distilled
water.
2. Adjustment of pH
3. Use the working standards
immediately after their
preparation.
1. Use a pH meter to adjust
the pH of each of the
working standards to
between 5 and 9 either
with concentrated hydro-
chloric acid or with
concentrated anmonium
hydroxide.
la. Use a beaker small enough for this volume of
standard to cover the pH electrode(s).
lb. Make sure that pH meter is calibrated within this
range.
lc. Use buffer solutions pH 4, pH 7, pH 10 to
calibrate and check the meter.
Id. This pH adjustment is necessary to insure that
the pH is approximately 8.5
(No pH adjustment is necessary if the pH is
already between 5 and 9.)
Page No. 7-31
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
Page No. 7-32
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPEC IFICAT IONS
TRAINING
GUIDE NOTES
J. Color Development of
Nitrite Working
Standards
1.
Pi pet 25.0 ml of each of
the nitrite working
standards into each of
six clean 250 ml
Erlenmeyer flasks.
la. Use a 25.0ml volumetric pipet.
lb. Label each flask.
lc. The nitrite working standards are not passed
through the reduction column.
2.
Add 75 ml of dilute
ammonium chloride-EDTA
solution to each of the
nitrite working standards.
2a. Use a 100 ml graduated cylinder.
3.
Mix each thoroughly by
swirling each flask.
4.
Use a 50.0 ml pipet to
remove a 50.0 ml aliquot
from flask #1 (0.00 mg/
1 iter N02~N).
4a. By using a propipet the aliquot can remain in the
pipet during the next two steps.
5.
Discard the remainder of
the standard from the
flask.
C.
Shake the flask dry.
6a. Do not rinse the flask.
7.
Add the 50.0 ml nitrite
working standard back to
the same flask from which
it was removed.
8.
Add 2.0 ml of the color
reagent to each nitrite
working standard.
8a. Use a 2.0 ml volumetric pipet.
9.
Mix thoroughly by
swirling.
-------
|LU^N^_MON^TOK_ING_PiROCEiDUR^: Determination of Nitrate-h,. „rite Nitrogen and of
—~~~~Nitrate Nitrogen, Cadmium Reduction Method
~ 1
OPERATING PROCEDURES
-i
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
J. Color Development of
Nitrite Working
Standards (Continued)
10. Allow the working stand-
ards to stand until color
develops.
11. Repeat steps 4 through 10
for each of the nitrite
standards.
10a. At least 10 minutes but no more than 2 hours
should be allowed before doing Procedure L,
Spectrophotometri c Measurements.
11a. Proceed from the least concentrated to the most
concentrated standard,
lib. Rinse the 50.0 ml pipet thoroughly after each
standard.
K. Analysis of
Non-reduced
Samples for
Nitrite
1. Dilution of
Samples (if
necessary)
2. Adjustment of pH
3. Color Development
1. Pipet 25.0 ml of unknown
sample into 50 ml volu-
metric flask.
2. Dilute to volume with
distilled water.
1. Use a pH meter to adjust
the pH of each sample to
between 5 and 9 either
with concentrated hydro-
chloric acid or with
concentrated ammonium
hydroxide.
1. Pipet 25.0 ml of sample
into a clean 250 ml
Erlenmeyer flask.
la. NOTE: Potable water samples will usually
require no dilution, while sewage samples
may require dilution.
2a. If you need to dilute a sample, you must apply a
dilution factor to get a final answer.
la. Put the 50 ml of sample in a small beaker so the
pH electrode(s) is covered with solution.
lb. Make sure that pH meter is calibrated within
this range.
lc. Use buffer solutions pH 4, pH 7, pH 10 to
calibrate and check the meter.
Id. This pH adjustment is necessary to insure that
the pH is approximately 8.5
(No pH adjustment is necessary if the pH is
already between 5 and 9.)
la. Use a 25.0 ml volumetric pipet.
lb. Label the flask.
lc. The sample is not passed through the reduction
column.
VII.K.1.2a
(P- 44)
Page No. 7-33
-------
EFFLUENT MONITORING PRQCEDURF: Determination of Nitrate-Nitrite Nitrogen and of
——————— titrate Nitrogen, Cadmium Reduction Method
Page No. 7-34
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
K. Analysis of
Non-reduced
Samples for
Nitrite (Continued)
2. Add 75 ml of the dilute
ammonium chloride-EDTA
solution to the same
flask.
3. Mix the sample thoroughly
by swirling.
4. Use a 50.0 ml pipet to
remove a 50.0 ml aliquot
from flask.
5. Discard the remainder of
the solution from the
flask.
6. Shake flask dry.
7. Add the 50.0 ml of sample
back to same flask from
which it was removed.
8. Add 2.0 ml of the color
reagent to the same flask.
9. Mix the sample thoroughly
by swirling.
10. Allow the sample to stand
until color develops.
11. Repeat steps 1 through
10 for each sample.
2a. Use a 100 ml graduated cylinder.
4a. By using a propipet the aliquot can remain in
the pipet during the next two steps.
6a. Do not rinse the flask.
8a. Use a 2.0 ml volumetric pipet.
10a. At least 10 minutes but no more than 2 hours
should be allowed before doing Procedure L,
Spectrophotometry Measurements.
11a. Rinse the 50.0 ml pipet thoroughly after each
sample.
-------
,-LUENT MONITORING PROCEDURE: Determination of Nitrate-^ i crite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
L. Spectrophotometry
Measurements
1. Adjusting the
Instrument
2. Reduced Nitrate
Standards and
Sample(s)
3. Non-reduced
Nitrite Stand-
ards and
Sample(s)
1. Consult the manufacturer's
instructions for cali-
brating your particular
instrument.
2. Adjust the wavelength to
540 nm.
3. Check to make sure that
the instrument reads
infinite absorbance with
no sample cell in the
instrument.
1. Use the reduced nitrate
reagent blank to adjust
the instrument to zero
absorbance.
2. Measure and record the
absorbance of each re-
duced nitrate working
standard.
3. Measure and record the
absorbance for each
reduced sample.
1. Use the nitrite reagent
blank (non-reduced) to
adjust the instrument to
zero absorbance.
la. Instrument must be warmed up for at least
10 minutes.
lb. There is an EMP on "Use of a Spectrophotometer."
3a. If it does not read infinite absorbance with no
sample cell in it, adjust the instrument so that
it does read infinite absorbance (see manu-
facturer's instructions).
3b. Use on and off switch to calibrate infinite
absorbance.
la. Use 0.00 nitrate working standard reagent blank
which has been passed through the column.
lb. Adjust to zero absorbance using the calibration
knob.
2a. Use the nitrate working standards which have
been passed through the column.
2b. Use data sheet provided.
3a. Use data sheet provided.
la. Use 0.00 nitrite working standard reagent blank,
lb. Adjust to zero absorbance using the calibration
knob.
IX.L.2.2b
(P. 47)
Page No. 7-35
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and of
—————-——— Nitrate Nitrogen, Cadmium Reduction Method
Page No. 7-36
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
L. Spectrophotometry
Measurements
(Continued)
2. Measure and record the
absorbance of each non-
reduced nitrite working
standard.
3. Measure and record the
absorbance for each non-
reduced sample.
2a. Use data sheet provided.
3a. Use data sheet provided.
IX.L.3.2a
(P- 47)
M. Preparation of
Calibration Curve
1. Obtain an 8 1/2 x 11 inch
piece of graph paper.
2. Label the longer side as
the concentration axis.
2a. See Training Guide for an example of labeling
the axis on a calibration curve.
VII.M.2a
(p. 45)
3. Label the shorter side
as the absorbance axis.
4. Use the absorbance value
and its corresponding ni-
trate concentration for
each of the nitrate working
standards to make a plot of
absorbance versus
concentration.
4a. Use the absorbances and concentrations recorded
on the data sheet in Column B, "Total N02+N03-N."
4b. This will be the standard curve for reduced
samples.
IX.M.4a
(p. 47)
5. On another piece of graph
paper follow steps 1, 2, 3,
and 4 using absorbance val-
ues and the corresponding
nitrite concentrations for
each of the nitrite working
standards.
5a. Use the absorbances and concentrations recorded
on the data sheet in Column D, "NO2-N."
5b. This will be the standard curve for non-reduced
samples.
IX.M.5a
(P. 47)
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
N. Checking Column
Efficiency
1. Divide the absorbance
value for the 1.00 mg/
liter NITRATE (N03)
working standard by the
absorbance for the 1.00
mg/liter NITRITE (NOg)
working standard to
obtain the column
efficiency as follows:
abs of 1.00 mg/liter NO^ std
la. The abbreviation, abs is used to stand for
absorbance.
x 100 = % efficiency
3a. The average value for the column efficiency
should be between 96% and 104%. If the average
% efficiency does not fall in this range,
another cadmium reduction column should be
prepared and tested until the average column
efficiency does fall in this range.
3b. For regeneration of a column, see Training
Guide.
VII.N.3b
(P. 43)
abs of 1.00 mg/liter N0£ std
2. Divide the absorbance
values for each of the
other NITRATE (NO3)
working standards by the
absorbance value for the
corresponding NITRITE
(N02) working standard to
obtain a column efficiency
value in each case as was
done in the previous step.
3. Calculate the average
value for the column
efficiency.
Page No. 7-37
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and of
Nitrate Nitrogen, Cadmium Reduction Method
Page No. 7-38
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
0. Determination of
mg/liter Nitrite
Nitrogen Plus Nitrate
Nitrogen in a Sample
1. Use the absorbance for
the reduced sample and
the standard curve for
reduced samples ("Total
NO^+NO^-N") to obtain
the mg/liter of nitrite-N
plus nitrate-N in the
sample and record it in
Column (A) on the data
sheet provided.
la. If the sample was not diluted (25 ml of sample
is used), the mg/liter result is read directly
from the nitrate standard curve,
lb. If the concentration of nitrate in the sample
is too high for analysis, the sample must be
diluted. The procedure is described in H.l and
involves diluting the sample to a 50 ml volume.
In this case, the mg/liter result from the nitrate
standard curve must be multiplied by a dilution
factor which would be:
Dilution Factor = —< < 50ml— —
ml sample used in dilution
lc. The reduction process converts the nitrate-N
initially present in the sample to nitrite
nitrogen and the species analyzed is nitrite
nitrogen.
Id. Any nitrite nitrogen initially present in the
sample remains as nitrite nitrogen after the
reduction. Thus the total nitrite analyzed is
the sum of the nitrite initially present and
the nitrite which has been formed by reduction
of nitrate.
IX.0.1a
(p. 47)
VII.0.lb
(p. 44)
P. Determination of
mg/liter Nitrite
Nitrogen in a Sample
1. Use the absorbance for
the non-reduced sample
and the standard curve
for non-reduced samples
("N02-N") to obtain the
mg/liter of nitrite-N in
the sample and record it
in Column (C) on the data
sheet provided.
le. If the sample was not diluted (25 ml of sample is
used), the mg/liter result is read directly from
the nitrite standard curve,
lb. If the sample was diluted to a 50 ml volume (as
given in H.l), the mg/liter result read from the
nitrite standard curve must be multiplied by a
dilution factor which would be:
Dilution Factor = 50 ml
ml sample used in dilution
IX.P.la
(p. 47)
VI I. P.lb
(P- 44)
-
-------
cFFLUENT MONITORING PROCEDURF.: Determination of Nitrate-Nitrite Nitrogen and of
——————————-— Nitrate Nitrogen, Cadmium Reduction Method
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
Q. Calculation of
mg/liter Nitrate
Nitrogen in a Sample
1. Subtract the mg/liter of
nitrite-N in the sample
from the mg/liter of
nitrite-N plus nitrate-N
in the sample to obtain
the concentration of
nitrate-N.
2. Record the answer in
Column (E) on the data
sheet provided.
la. Since the procedure measures the total nitrite
concentration in a sample, the nitrite concen-
tration of samples must be determined before
reduction and after reduction. The nitrate
concentration of a sample is then determined by:
NOo-N = (NO^+NOt-N) TOTAL - (NO.-N) BEFORE
* AFTER <¦ REDUCTION
RE-
DUC-
TION
These concentrations were recorded on the data
sheet in Columns (A) and (C) respectively.
IX.Q.la
(p. 47)
R. Calculation of
mg/liter Nitrate
in Sample
1. Multiply the value found
for nitrate-nitrogen
(N03~N) by a factor of
4.43.
2. Record the answer in
Column (F) on the data
sheet provided.
la. (N03-N) x (4.43) = mg/liter Nitrate in sample,
lb. NO^-N value was calculated in Procedure Q and
recorded in Column (E).
IX.R.lb
(p. 47)
S. Calculation of
mg/liter Nitrite
in Samples
1. Multiply the value found
for nitrite-nitrogen
(NO^-N) by a factor of
3.29.
2. Record the answer in
Column (G) on the data
sheet provided.
la. (NO2-N) x (3.29) = mg/liter Nitrite in sample.
lb. NO2-N value is found by using the calibration
curve for non-reduced samples as in Procedure P
and recorded in Column (C).
IX.S.lb
(p. 47)
Page No. 7-39
-------
TRAINING GUIDE
SECTION TOPIC
I* Introduction
II Educational Concepts - Mathematics
III Educational Concepts - Science
IV Educational Concepts - Communications
V Field and Laboratory Equipment
VI* Field and Laboratory Reagents
VII* Field and Laboratory Analysis
VIII* Safety
IX* Records and Reports
*Training guide materials are presented here under the headings marked*.
These standardized headings are used throughout this series of procedures.
Page No. 7-40
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and Nitrate
Nitrogen, Cadmium Reduction Method
INTRODUCTION
Section i
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
The cadmium reduction procedure for nitrate-nitrite
nitrogen provides a sensitive method for the deter-
mination of nitrate singly, or nitrite and nitrate
combined in drinking, surface, and saline waters.
The method is commonly used to determine both
nitrate-N and nitrite-N in water samples.
The procedure described in this EMP is applicable
for range of 0.01 to 1.0 mg/liter of nitrate-
nitrite nitrogen. However, the range may be
extended by appropriate sample dilution.
The test described in this instruction can be found
in the 1974 EPA Methods Manual on page 201, entitled
Nitrogen, Nitrate-Nitrite (Cadmium Reduction Method)
Another reference which contains an acceptable
procedure for this test is on page 423 of the 14th
edition of Standard Methods.
The major sources of nitrogen entering the environ-
ment are: through the heavy application of nitrogen
ous fertilizers which cause agricultural runoffs, as
the end products of aerobic stabilization of organic
nitrogen, in domestic sewage, through animal and
plant processing wastes, in animal manure, through
the atmosphere and in various types of industrial
effluents.
While nitrogen is essential to our survival (as in
the make-up of amino acids and proteins), when it
exists as nitrate and nitrite it can be toxic. A
limit of 10 mg/1 nitrate-N and 1 mg/1 nitrite-N is
recommended for public water sources. The desirable
criteria is virtually 0 mg/liter.
In ruminant animals (i.e. cows) nitrates may be
internally reduced by bacteria present in the rumen
to nitrites. The nitrites have been found to be tox
ic to these animals. Dr. Joptha E. Campbell, (Chief,
Food Chemistry Unit, Milk and Food Research, Environ-
mental Sanitation Program, Public Health Service,
U.S. Department of H.E.W., Cincinnati, Ohio, 1968)
has reported methemoglobinemia in cattle receiving
water containing 2.790 mg/liter of nitrate.
Nitrates in high concentrations have also been found
to stimulate vegetative growth under favorable con-
ditions. Heavy undesirable growth in fresh water can
lead to eutrification of important waterways.
Methods for Chemical
Analysis of Water and
Wastes, 1974, EPA-
MDQARL, Cincinnati,
Ohio 45268, p. 201.
2. Standard Methods for the
Examination of Water and
Wastewater, 14th ed.,
1976, APHA, New York,
New York, p. 423.
3. Federal Water Pollution
Control Administration
Water Quality Criteria,
U.S. Government Printing
Office, Washington, D.C.
1968.
Page No. 7-41
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and Nitrate
Nitrogen, Cadmium Reduction Method
FIELD AND LABORATORY REAGENTS
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
D.
Samples should be analyzed for nitrate nitrogen as
soon as possible after sampling to avoid any change
in nitrogen balance due to biological activity. If
analysis can be made within 24 hours, the sample
should be preserved by refrigeration at 4°c.
Samples should be preserved with sulfuric acid if
they are to be held more than 24 hours. To pre-
serve samples for analysis, add 2.0 ml of con-
centrated sulfuric acid per liter of sample and
store at 4°C.
Page No. 7-42
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and Nitrate
Nitrogen, Cadmium Reduction Method
FIELD AND LABORATORY
ANALYSIS
Section v11
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
C.2.19c
N.3b
Check the column efficiency when it is suspected
that column efficiency is decreasing, as indicated
by suspected low concentration levels. Prepare
working standard nitrate solutions, and pass them
through the column. (Begin at E. Preparation of
Nitrate Working Standards.) If the absorbance for
the known concentration does not give an average
between 96% and 104% of your standard curve value
for reduced nitrate standards of equivalent concen-
tration, the column must be reactivated.
REACTIVATION OF COLUMN
1.
Empty cadmium granules from column into a clean
beaker.
2.
Wash with distilled water 3 times.
3.
Add enough dilute HC1 to cover granules.
4.
Swirl contents.
5.
Decant HC1.
6.
Wash with distilled water 3 times.
7.
Add 100 ml CuSO^ solution to granules.
8.
Swirl contents of beaker for approximately 5
minutes until the blue color fades to colorless.
9.
Decant liquid leaving the granules.
10.
Repeat steps 7,8, and 9 until a very fine
brown-red precipitate forms.
11.
Wash granules with distilled water (approximately
10 times) until precipitate is removed.
12.
Place granules on the 60 mesh sieve.
13.
Shake to remove the small particles (the particles
which remain on the sieve are the ones you want.;
14.
Repack column (packing must be loose).
15.
Standard curve using nitrate working standards
must be re-established.
16.
Check column efficiency as described in N,
Checking Column Efficiency.
Page No. 7-43
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and Nitrate
——— ~~ Nitrogen, Cadmium Reduction Method
FIELD AND LABORATORY ANALYSIS Section VII
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
H.1.2a
K.1.2a
0.1b
P.lb
Since a dilution is only part sample, when the
absorbance reading obtained for it is converted to
a concentration using a calibration curve, the
concentration obtained is only that of the dilution.
To obtain the mg/liter concentration of the sample,
the mg/liter concentration of the dilution must be
multiplied times the amount of dilution (must be
multiplied times the dilution factor). For a 1/2
dilution (25 ml sample/50 ml total volume) the
dilution factor would be 2 (the dilution is only half
sample). For a 1/5 dilution (10 ml of sample/50 ml
total volume) the dilution factor would be 5. Below
is a table of some dilution factors when the sample
is diluted to a 50 ml volume.
ml of Sample per Amount of Dilution
50 ml Total Volume Dilution Factor
25 1/2 2
10 1/5 5
5 1/10 10
1 1/50 50
0.5 1/100 100
0.05 1/1000 1000
The dilution factor for any dilution may be calcu-
lated by dividing the ml of sample used in the
dilution into 50:
Dilution Factor - |n1 samp1e ysed ,n j1)ution
Ex. 2 ml of sample diluted to 50 ml
50 _ or
~Sf" 25
The dilution factor for this dilution would be 25.
Page No. 7-44
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and Nitrate
Nitrogen, Cadmium Reduction Method
FIELD AND LABORATORY ANALYSIS Section VII
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
M.2a
A calibration curve is prepared by plotting the
measured absorbance of each of the workinq
standard versus the concentration in the working
standard as shown below.
LU
CC
CONCENTRATION OF N03 or NO2 - N, mg/liter
Page No. 7-45
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and Nitrate
Nitrogen, Cadmium Reduction Method
SAFETY Section VIII
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
C.2.1d
Cadmium metal is highly toxic thus caution must be
exercised in the use of cadmium. Cadmium metal
should never be handled directly since cadmium has
been shown to have cumulative effects. Rubber
gloves should be used whenever cadmium must be
handled. A mask should be worn during the filing of
cadmium and the filing should be done in a hood.
The waste cadmium should be disposed of in an
appropriate manner which conforms to Federal, State
and local pollution control regulations.
Page No. 7-46
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and Nitrate
Nitrogen, Cadmium Reduction Method
RECORDS AND REPORTS
Section IX
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
L.2.2b
M.4a
L.3.2a
M. 5a
0. la
P.la
Q.la
R.lb
S.lb
You will need the following Key to use the Example
Data Sheet found on the next page:
KEY TO DATA SHEET
(B) Record the absorbances of the column-reduced
nitrate working standards and of the column-
reduced sample(s) in Column (B).
(D) Record the absorbances of the non-reduced
nitrite working standards and of the non-reduced
sample(s) in Column (D).
(A) Read the mg/liter (concentration) of
Total NOg+NO^-N in the column-reduced sample(s)
from the corresponding calibration curve and
record the answer(s) in Column (A).
(C) Read the mg/liter (concentration) of NO^-N in
the non-reduced sample(s) from the corresponding
calibration curve and record the answer(s) in
Column (C).
(E) Subtract: Value (A) - Value (C) = Value (E)
(F) Multiply: Value (E) x 4.43 = Value (F)
(G) Multiply: Value (C) x 3.29 = Value (G)
Page No. 7-47
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and Nitrate
Nitrogen, Cadmium Reduction Method
RECORDS AND REPORTS Section IX
EXAMPLE DATA SHEET
See Key on Page No. 7-47
mg/1iter
TOTAL
NOo+NO.-N
ABSORBANCE
OF TOTAL
NO„+NO,-N
SAMPLE
NUMBER
mg/1iter
N0o-N
ABSORBANCE
N0o-N
mg/1iter
NOn-N
mg/1iter
mg/1iter
Reduced Nitrate
Working Standards
0.05
0.05
0.22
0.10
0.10
0.44
0.20
0.20
0.89
0.50
2.22
0.50
1.00
1.00
4.43
Reduced Sample(s)
Non-reduced Nitrite
Working Standards
0.16
0.05
0.33
0.10
0.20
0.66
1.65
0.50
3.29
1.00
Non-reduced Sample(s)
Page No. 7-48
-------
EFFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nitrite Nitrogen and of Nitrate Nitrogen,
Cadmium Reduction Method
RECORDS AND REPORTS
Section IX
TOTAL
Ut 1 tKrll mH 1 iUIV
OF
NU„TIU
3
-N
u
(Reduced
Nitrate Standards;
GRAPH
LMLldKMIiUN
SIGNATURE
OF
DDFDflPFD•
DATE
uKArri
KKtrttKLU
i
i
1
1
|
l
1
1
! 1
i 1
1
|
1
1
I
1
1
1
-
I
i
I
1
I
1.00
0.80
0.60
0.40
0.20
n nn
0.20
0.30 0.40 0.50 0.60 0.70
CONCENTRATION OF NITRATE NITROGEN, mg/liter
0.80
0.90
1.0C
Page No. 7-49
-------
Page No. 7-50
E-FFLUENT MONITORING PROCEDURE: Determination of Nitrate-Nfitrite Nitrogen and of Nitrate Nitrogen,
Cadmium Reduction Method
RECORDS AND REPORTS
SECTION IX
DETERMINATION OF N02-N
(Non-reduced Nitrite Standards)
CALIBRATION GRAPH
SIGNATURE OF PREPARER:
DATE GRAPH WAS PREPARED:
_1_L
+
0.10
0.20 0.30 0.40 0.50 0.60 0.70
CONCENTRATION OF NITRITE NITROGEN, mg/liter
0.80
0.90
1.00
-------
A PROTOTYPE FOR DEVELOPMENT OF
ROUTINE OPERATIONAL PROCEDURES
for the
DETERMINATION OF OIL AND GREASE
as applied in
WASTEWATER TREATMENT FACILITIES
and in the
MONITORING OF EFFLUENT WASTEWATERS
National Training Center
Municipal Operations and Training Division
Office of Water Program Operations
U.S. Environmental Protection Agency
CH.oq.EMP.lb.12.75
Page No. 8-1
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil and Grease
This operational procedure was developed by:
NAME Charles R. Feldmann
ADDRESS EPA-WPO-National Training Center, Cincinnati, OH 45268
POSITION Chemist-Instructor
EDUCATION AND TECHNICAL BACKGROUND
B.S. - Chemistry
M.S. - Chemistry
1-1/2 years Industrial Chemist
4 years additional Graduate School
4 years college Chemistry Instructor
1-1/2 years DHEW - Air Pollution Program, Chemist
4-1/2 years DI - EPA, Chemist-Instructor
Page No. 8-3
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil and Grease
1. Analysis Objectives:
The operator will be able to perform an oil and grease determination on
a sewage sample.
2. Brief Description of Analysis:
The sample is shaken in a separatory funnel with 1,1,2-trichloro-l ,2,2-
trifluoroethane, CpF^CU, Freon TF or Genolsov D. The solvent and water do
not dissolve in each otner, and after the shaking, they separate and form
two layers, with the solvent on the bottom. Duringthe shaking, the oil
and grease are taken from the water layer into the "solvent layer, because
the oil and grease are more soluble in the solvent than in water. The solvent
is transferred to a previously weighed distilling flask. This process
of shaking the water with solvent, the taking of the oil and grease into
the solvent, and the separation of the solvent, is called extraction! The
extraction is repeated two more times. All three solvent portions are
combined in the distilling flask and evaporated. The flask is again
weighed. The increase in weight is due to the oil and grease in the
sample.
The method cannot distinguish between oil and grease, because both are
soluble in the solvent, The two components are treated as one. Other solvent
soluble materials may also be present and contribute to a result higher
than it should be.
3. Applicability of this Procedure:
a. Range of Concentration:
5 to 1000 mg/liter extractable material
b. Pretreatment of Samples:
The Federal Register Guidelines do not specify any pretreatment.
c. Treatment of Interferences in Samples:
The Source of Procedure* does not note any interferences to this
determination.
*Source of Procedure: Methods for Chemical Analysis of Water and Wastes, 1974,
Environmental Protection Agency, Methods Development and Quality Assurance
Research Laboratory, Cincinnati, Ohio, p. 229
Page No. 8-4
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil and Grease
General Description of Equipment Used in the Process
A. Capital
1. Analytical balance (200 g capacity)
2. Still, or other source of distilled water
3. Source of vacuum (water aspirator or vacuum pump)
4. Hot water bath (80°C temperature needed)
5. Oven (103°C temperature needed)
6. Refrigerator, 4°C (for storing samples which will not immediately
be analyzed after collection)
7. Hot plate (must have continuous setting between its lower and
upper limit; cannot have only low, medium and high settings)
8. Steam bath (large enough to accommodate at least 1 distilling
flask, 125 ml size)
B, Reusable
1. Brushes (for cleaning glassware)
2. Brush (for cleaning balance)
3. Laboratory apron
4. Safety glasses
5. Pen or pencil
6. Notebook (for recording data)
7. Centigrade thermometer (for taking readings at 70°C and R0°C)
8. Distilling flask, 125 ml, with a 24/40 ground glass neck (Corning
number 4100 is an example) One flask is used for each determination.
9. Glass stoppered bottle, 1 liter
10. Grease pencil (for marking bottle)
11. Desiccator (large enough to hold at least one 125 ml distilling
flask)
12. Crucible tongs (may be used in place of lintless tissues)
13. Graduated cylinders, 10 ml and 50 ml
14. Erlenmeyer flask, 125 ml
15. Glass stoppered bottle, 50 ml capacity
16. Ring stand
17. Funnel, 60°, 100-150 mm
18. Ring (to support the funnel)
19. Separatory funnel with Teflon stopcock, 2 liter
20. Ring (to support the separatory funnel)
21. Clamp (to fit neck of distilling flask)
22. Rubber stopper and glass tubing for preparing suction device;
see figure 2
23. Beakers, 1000ml (1), 100-150 ml (2)
24. Glass Stoppered bottle (for storing cleaning solution if prepared)
25. Beaker, 250 ml (for preparing cleaning solution)
Page No. 8-5
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil and Grease
B. Reusable (Cont'd.)
26. Rubber stopper to fit the distilling flask, item 8 above; see
figure 2.
27. Fifteen inches of pyrex glass tubing (6 mm size); see figure 2
28. Gas and laboratory burner to bend the glass tubing; see figure 2
29. File to cut the glass tubing; see figure 2
C. Consumable
1. Concentrated sulfuric acid, H0SO., or concentrated hydrochloric
acid, HC1. (Either acid may bemused in the determination. Con-
centrated sulfuric acid, HpSO., may be needed for cleaning glassware.)
2. Sodium dichromate, fla?Cr?07 (for cleaning glassware)
3. Detergent (for cleaning glassware)
4. 1,1,2-trichloro-l,2,2-trifluoroethane*
5. Desiccant (enough to cover the bottom of the desiccator)
6. Lintless tissues (may be used in place of crucible tongs)
7. Whatman number 40 filter paper (to fit the funnel in B.17)
8. pH sensitive paper (for measurement at pH 2)
9. Anhydrous sodium sulfate, Na2S04
10. Matches
*Freon 113 is a general name used by E. I. DuPont de Nemours, Inc.,
for the above solvent. TF and PCA are two specific grades of
Freon 113. TF is the better of the two. Genosolv D is the name
used by Allied Chemical Company for the above solvent. Either
Freon TF or Genosolv D may be used in the determination.
Page No. 8-6
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil and Grease
2 LITER
SEPARATORY
FUNNEL
RING
TEFLON
STOPCOCK
FILTER
PAPER
SMALL
FUNNEL
DISTILLING
FLASK
125ml
RING
STAND
FIGURE 1
Page No. 8-7
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil and Grease
TO
GLASS
TUBING
RUBBER
DISTILLING
FLASK
125ml
FIGURE 2
Page No. 8-8
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil and Grease
liebig condenser
holder
clamp
water out
rubber tubing
water in
rubber tubing
ring stand
erlenmeyer
flask
3 prong
extension
clamp
clamp holder
adapter
adapter
extension
clamp
125 ml
distilling
flask
!§gjjjp|j# (;
;;( 600 ml
j I beaker
FIGURE 3
Page No. 8-9
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil and
OPERATING PROCEDURES
STEP SEQUENCE
A. Equipment
Preparation
1. Cleaning of
glassware
2. Sample container
1. Clean the 125 ml distilling
flask and all other
glassware.
2. Rinse all of the glassware
with distilled water.
3. Allow all of the glassware
to drain dry.
4. Rinse the 125 ml distilling
flask with 20 ml of
1,1,2-trichloro-l,2,
2-trifluoroethane (Freon
TF or Genosolv D.
5. Allow the flask to drain
dry.
1. Pour 1 liter of distilled
water into a 1 liter glass
stoppered bottle.
2. Place a grease pencil mark
on the outside of the
bottle at the 1 liter level.
3. Empty the water.
Page No. 8-10
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
I
(p. 24)
V.A.I.1
(p. 25)
For the remainder of this procedure the symbol
TF/D will be used to mean this particular solvent
Use a graduated cylinder to measure the TF/D.
Proceed with preparation of the sample container
and desiccator while waiting.
Measure the water with a graduated cylinder,
500 ml or 1000 ml size.
V.A.I.4
(p. 26)
There must be at least 1 inch space between the
bottom of the glass stopper and the surface of
the water.
So that no oil will be lost by clinging to the
stopper.
And to allow room for the addition of reagents
later in the determination.
If there is less than 1 inch space, a larger
bottle (marked at the 1 liter level) must be used.
-------
EFFLUENT MONITORING PROCEDURR: Determination of Oil and Grease
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Equipment Preparation
(continued)
4. Allow the bottle to drain
thoroughly.
c
5. Rinse the glass stoppered
bottle with about 20 ml of
TF/D.
6. Hold the bottle upside down,
7. Lean the bottle up against
some part of the laboratory
bench top.
5a. This TF/D may be disposed of by pouring it into a
small beaker and allowing it to evaporate in a
well ventilated area.
7a. Make sure the bottle will not fall over.
7b. The bottle should not be standing vertically.
7c. The heavy TF/D vapors must be able to escape
from the mouth of the bottle.
3. Desiccator
1. Prepare a desiccator for
use.
la. It must be large enough to hold at least one
125 ml distilling flask,
lb. The desiccator size will depend on the number
of flasks to be held.
1c. One flask is required for each sample and blank
determination.
4. Distil 1ing flask
1. Wipe the clean dry 125 ml
distilling flask thoroughly
with lintless tissues.
2. Dry the flask in an oven.
3. Cool the flask in a
desiccator.
la. To remove all finger prints,
lb. From this step on, until the determination has
been completed, always handle the flask witTi
lintless tissues or crucible tonqs.
2a. For 1 hour at 103°C.
3a. For 30 minutes.
3b. Store the flask in the desiccator until needed.
5. Stopper and glass
tubing suction
fitting
1. Drill 2 holes in the rubber
stopper.
la. To accommodate the 6 mm glass tubing; see figure
2.
Page No. 8-11
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil and Grease
Page No. 8-12
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
A. Equipment Preparation
(continued)
2. Cut, bend, and fire polish
the glass tubing.
3. Insert it through the
holes in the rubber
stopper.
2a. See figure 2.
3a. See figure 2.
B. Reagent Preparation
1. Sulfuric acid,
H2S04, 50% by
volume
1. Measure 10 ml of distilled
water.
2. Pour it into a 125 ml
Erlenmeyer flask.
la. Use a graduated cylinder.
-------
... rLUENT MONITORING PROCEDURE: Determination of Oil and Grease
OPERATING PROCEDURES
1
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Reagent Preparation
(continued)
3. Measure 10 ml of concen-
trated sulfuric acid,
1^2^0/1 •
4. Pour about 1/2 of the acid
slowly down the inside of
the Erlenmeyer flask.
5. Gently swirl the flask to
mix.
6. Pour the rest of the acid
into the flask.
7. Gently swirl the flask to
mix.
8. Allow the mixture to cool
to room temperature.
9. Store the 50% sulfuric acid,
HpSO-, in a small glass
stoppered bottle.
3a. Use a graduated cylinder.
3b. Concentrated hydrochloric acid, HC1, may be
substituted for the concentrated sulfuric acid,
H2SV
4a. Caution: Heat will be generated.
6a. Caution: Heat will be generated.
9a. Of about 50 ml capacity.
9b. Five ml are needed for each determination.
9c. Larger quantities of the 50% acid (hydrochloric
may be substituted) may be prepared if needed.
C. Sample
1. Collection
1. Fill the glass stoDpered
bottle to the 1 liter mark
with sample.
2. Measure 5 ml of 50% by vol-
ume sulfuric acid, H^SO^.
3. Add the acid to the sample
bottle.
4. Gently swirl the bottle to
Mix the acid and sample.
la. Collect the sample directly in the bottle so as to
minimize loss of oil/grease by the use of an
intermediate container.
2a. Use a graduated cylinder.
2b. Fifty percent by volume hydrochloric acid, HC1,
may be substituted.
Page No
V.C.I,1
(p. 26)
. 8-13
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil and Grease
Paqe No. 8-14
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Sample
(continued)
2. Preservation
5. Check the pH of the acidi-
fied sample.
6. If the pH is not 2 or less,
add 5-10 more drops of the
50% acid.
7. Swirl the bottle and again
check the pH as before.
1. If the analysis will not be
done immediately, store the
acidified sample in a re-
friqerator at 4°C.
5a. Use pH sensitive paper.
5b. The pH must be 2 or less.
7a. With the stopper off.
7b. Repeat the acid addition, mixing, and pH check
until the pH is 2 or less.
la. For no longer than 24 hours. Otherwise, the
analytical result may be unreliable.
D. Procedure
1. Extraction
1. Mount a 2 liter separatory
funnel on a ring stand.
2. Tighten the screw or clamp
which holds the stopcock in
place.
3. Close the stopcock.
4. Pour the acidified sample
into the separatory funnel.
5. Measure 30 ml of TF/D.
6. Pour it into the sample
bottle.
7. Swirl the sample bottle.
la. The separatory funnel should have a Teflon
stopcock.
lb. Use a ring.
2a. A loose stopcock can cause loss of the sample
by leakage.
4a. Use a funnel of about 75 mm diameter.
5a. Use a graduated cylinder.
7a. To thoroughly rinse the inside of the bottle
with the TF/D.
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil end Grease
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Procedure
(continued)
8. Pour the TF/D from the
sample bottle into the
separatory funnel.
9. Stopper the funnel.
10. Holding one hand over the
stopper, lift the funnel
out of the ring stand.
11. Carefully turn the funnel
upside down.
12. Slowly open the stopcock.
13. Close the stopcock.
14. Shake the funnel gently for
about 5 seconds.
15. Slowly open the stopcock.
16. Close the stopcock.
17. Shake the.flask gently for
about 5 seconds.
18. Slowly open the stopcock.
19. Close the stopcock.
20. Shake the funnel vigorously
for 2 minutes.
21. Place the separatory funnel
back in the ring stand.
8a. Pour the TF/D carefully so that any solids
present are transferred to the separatory
funnel.
11a. The stopper is pointed down. Be sure the tip
of the funnel is not pointed toward your face.
12a. A hissing sound may be heard.
15a. A hissing sound may be heard.
18a. A hissing sound may be heard.
Page No. 8-15
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil and
OPERATING PROCEDURES
STEP SEQUENCE
D. Procedure
(continued)
22. Remove the stopper.
23. Allow the TF/D and water
layers to separate.
24. While the layers are
separating, weigh the
125 ml distilling flask.
25. Mount a 60° funnel (about
50 mm size) under the tip
of the separatory funnel.
26. Fold a piece of Whatman
number 40 filter paper to
fit into the small funnel.
27. Place it in the funnel.
28. Place a 100-150 ml beaker
under the tip of the small
funnel.
29. Pour about 10 ml of TF/D
into a second 100-150 ml
beaker.
30. Slowly pour the TF/D into
the small funnel.
Page No. 8-16
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
The TF/D layer will be under the water layer.
There may be some bubbles at the point where the
water and TF/D layers meet.
These bubbles should break after a few minutes
standing.
If the shaking was extremely vigorous, an emulsion
may have formed; that is, the water and TF/D
molecules are so well mixed that they will sepa-
rate only after long standing.
Which had been stored in the desiccator.
See the example data sheet on page 27.
Use an analytical balance to weigh the flask.
The tip of the separatory funnel should extend
down about one-half inch into the separatory
funnel.
The size of the filter paper will depend on the
size of the funnel.
The entire
wet.
The TF/D will evaporate from the filter paper
rapidly.
ire surface of the filter paper must be
-------
¦UENT MONITORING PROCEDURE: Determination of Oil and Grease
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
Training
GUIDE NOTES
D. Procedure (continued)
31. When all of the TF/D has
drained through the small
funnel, the TF/D may be
disposed of.
32. Place the previously
weighed distilling flask
under the tip of the
small funnel.
33. Examine the separatory
funnel and note whether the
TF/D and water layers have
separated so to form a
sharp line between the two
layers.
34. If they have not, pour
about 1 g. of anhydrous
sodium sulfate, Na2S0^,
into the small funnel.
35. Open the stopcock on the
separatory funnel slowly.
36. When the water layer is
about to enter the hole
through the stopcock, close
the stopcock.
31a. By evaporation in a well ventilated area.
32a. The tip of the small funnel should extend down
into the neck of the flask about 1 inch (see
figure 1).
33a. No clear answer can be given as to how long the
layers may take to separate. As little as a few
minutes may suffice.
33b. About one-half hour would be the longest practical
time one should wait before deciding to use the
anhydrous sodium sulfate, Na^SO^ (see step 34).
|34a. Estimate the 1 g.
I34b. Omit step 34 if the two layers have separated.
34c. If there is doubt as to whether or not the two
layers have separated properly, use the anhydrous
sodium sulfate, Na^SO^.
35a. The TF/D should flow slowly from the separatory
funnel into the small funnel, through the sodium
sulfate (if used), through the filter paper, and
into the distilling flask.
36a. A drop or two of the TF/D should remain in the
funnel with the sample.
36b. There may be some scum clinging to the inside walls|
of the separatory funnel. It should be left in
the separatory funnel.
Page No. 8-17
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil and
OPERATING PROCEDURES
D. Procedure
(continued)
STEP SEQUENCE
37. Repeat steps 5 through 23.
38. Place the small funnel (the
same one used before) and
filter paper under the tip
of the separatory funnel.
39. Place the distilling flask
under the tip of the small
funnel.
40. Repeat steps 35 and 36.
41. Repeat steps 5 through 23.
42. Repeat steps 38 and 39.
43. Repeat steps 35 and 36.
44. Pour about 10 ml of TF/D
into a small beaker.
45. Pour a few drops of the
Freon on the tip of the
separatory funnel.
46. Pour the rest of the TF/D
slowly around the inside of
the small funnel.
Page No. 8-18
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
The sample is still in the separatory funnel
Na2S0^!
was used
If anhydrous sodium sulfate,
in the first filtration, it is not necessary
to remove it from the small funnel, even if
the TF/D and water layers have cleanly separated.
If anhydrous sodium sulfate, Na-SO. was not used
in the first filtration, it may be necessary to
use it now, if the TF/D and water layers have
not cleanly separated.
The tip of the small funnel should extend down
into the distilling flask about 1 inch.
The TF/D from the first extraction is still in
the flask.
The sample is still in the separatory funnel.
The TF/D from the second extraction is also
still in the flask.
The distillation flask now contains the TF/D frorr
all three extractions. [
The same one used in step 28 or 29-
To rinse down any TF/D which may contain oil and
grease.
The filter paper and sodium sulfate, if used, will
be washed.
The washings will pass into the distilling flask.
-------
UENT MONITORING PROCEDURE: Determination of Oil and Gr
OPFDflTTKin Dcnrcnnnrc
wi b i m i i inu I J
STEP SEQUENCE
D. Procedure
47. The sample, sodium sulfate,
(continued)
Na2S0^, (if used), and
filter paper may now be
discarded.
2. TF/D removal
1. Fill a 1 liter beaker half
full with tap water.
2. Place the beaker on a hot
plate.
3. Turn on the hot plate.
4. Adjust the hot plate so the
temperature of the water is
70°C.
5. Support the flask in the
70°C water.
6. While the TF/D is evapo- 1
rating, set a steam bath atj
80°C. I
47a.
6a.
6b.
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
The sample remaining in the separatory funnel
after extraction can be discarded now.
The TF/D removal may be done in one of two ways.
The method described in the remainder of this
procedure involves evaporation, and therefore
loss, of the TF/D.
If the TF/D is distilled off, the TF/D may be
recovered for reuse. The source of heat for the
distillation should be a beaker of 70°C water on
a hot plate. While the TF/D is distilling off,
proceed with step 6 (see figure 3).
In a hood or other extremely well ventilated area.
A hood is preferable because of the danger of
inhaling TF/D fumes.
Check the temperature with a thermometer.
Because of air currents, it will probably not be
possible to maintain the temperature at exactly
70°C.
Use a clamp and ring stand.
The lower third of the flask should be in the
water.
The TF/D will begin to boil and evaporate.
If several determinations are being done at
once, a larger water bath will be required.
It will take about 30 minutes for the TF/D to
evaporate at 70°C.
Use a thermometer to check the temperature.
Page No. 8-19
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil and Grease
Page No. 8-20
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Procedure
(continued)
7. After the TF/D has evapo-
rated at 70°C, place the
flask in the 80°C steam
bath.
8. Heat the flask for 15
minutes.
9. Remove the flask from the
steam bath.
10. Support the flask by means
of a clamp and ring stand.
11. Attach the stopper with
glass tubing (see figure 2).
12. Apply suction to the flask
for 1 minute.
13. Wipe the outside of the
flask thoroughly with
lintless tissues.
14. Place the flask in a
desiccator to cool.
7a. Because of air currents, it will probably not be
possible to maintain a temperature of exactly
80°C.
7b. Only the lower third of the flask should be
heated.
12a. While the flask is still warm.
13a. To remove grease which may have been in the water
of either of the two baths.
14a. For 30 minutes.
E. Final Weighing
1. Remove the flask from the
desiccator.
2. Weigh it.
2a. Use the same balance as before.
-------
t. _UENT MONITORING PRQCEDURF: Determination of Oil and Grease
OPERATING PROCEDURES
'
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Blanks
1.
2.
3.
Clean a 125 ml distilling
flask.
Rinse it with TF/D.
Wipe it with lintless
tissues.
la. The same type as was used in the procedure,
lb. Steps 1 through 11 should be carried out in same
manner as was used in the procedure.
4.
Dry it at 103°C.
4a. For 30 minutes.
4b. Stand the flask upside down in the oven so the
heavy TF/D vapors will escape.
5.
Cool it in a desiccator.
6.
Weigh it.
6a. Use an analytical balance.
7.
Measure 100 ml of TF/D.
7a. Use a graduated cylinder.
8.
Pour it into the distilling
flask.
9.
Evaporate the TF/D.
9a. Use the same technique as for the sample.
10.
Cool the flask in the
desiccator.
11.
Weigh it.
11a. The initial and final weights should be within
0.0002 g of each other. (This difference was
suggested by the EPA laboratory which wrote the
1974 EPA oil and grease method of analysis.)
lib. If the two weights are not within 0.2 mg of each
other, check for faulty laboratory techniques.
Page No. 8-21
-------
EFFLUENT MONITORING PROCEDURR: Determination of Oil and Grease
Page No. 8-22
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Blanks (continued)
12. Calculate the value of the
blank.
12a. Blank, D = E-F
D = value of the blank in grams
E = weight of the flask after evaporation of the
100 ml of TF/D (in grams)
F = weight of the empty flask (in grams)
12b. Example calculation:
Weight of the flask after evaporation of the
100 ml of TF/D' (E) = 54.6961 g
Weight of the empty flask (F) = 54.6959 g
Blank, D = 54.6961 g-54.6959 g = 0.0002 g
G. Calculations
1. Calculate the mg of oil and
grease per liter of sample.
la. mg of oil and grease per liter of sample =
[(A-B)-D] x 1000 x 1000/C
lb. A = the weight of the distilling flask + the
oil/grease residue (1n grams)
B = the weight of the empty distilling flask
(in grams)
1000 = a conversion factor to change milliliters
to liters
1000 = a conversion factor to change grams to
mi 11igrams
C = milliliters of sample
D = value of blank (in grams); see F.12 for the
calculation
lc. Example calculation:
Weight of flask and the oil/grease
residual = 54.7803 g (A)
Weight of empty flask = 54.6961 g (B)
Volume of sample = 1000 ml (C)
Value of blank = 0.0002 g (D)
r(54.7803-54.6961)-0.00021 x 1000 x 1000 „
1000 = 84-°
-------
EFFLUENT MONITORING PROCEDURES: Determination of Oil and Grease
TRAINING GUIDE
SECTION TOPIC
I* Introduction
II Educational Concepts - Mathematics
III Educational Concepts - Science
IV Educational Concepts - Communications
V* Field and Laboratory Equipment
VI Field and Laboratory Reagents
VII Field and Laboratory Analyses
VIII Safety
IX Records and Reports
"''Training guide materials are presented here under the headinqs marked
These standardized headings are used through this series of procedures
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil and Grease
INTRODUCTION
Section I
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
The terms oil and grease are not clearly defined.
The definition depends on the procedure used. For
example, the solvent used to extract the grease and
oil, and the presence of extractables which are
neither grease nor oil, will affect the results.
Hydrocarbons, esters, oils, fats, waxes and high
molecular weight fatty acids feel greasy and are
associated with grease problems in wastewater
treatment plants. Gasoline, heavy fuel and
lubricating oils and asphalts are included in the
term oil.
Oil and grease interfere with wastewater treatment
by coating particles of organic matter, thus in-
hibiting oxygen transfer and stabilization by
micro-organisms.
They can coat equipment, reducing its efficiency,
and can cause a safety hazard on walkways and
ladders.
The test dexcribed in this instruction can be found
in the 1974 EPA Methods Manual on page 229. Another
reference which has an acceptable procedure for this
test for NPDES purposes is 14th ed. Standard Methods
on page 515.
Methods for Chemical
Analysis of Water and
Wastes, 1974, EPA, MDQARL
Cincinnati, Ohio 45268,
p. 229.
Standard Methods for the
Examination of Water and
Wastewater, 14th ed.,
1976, APHA, New York, NY,
p. 515.
Page No. 8-24
-------
EFFLUEN 1 MONITORING PROCEDURE: Determination of Oil and Grease
FIELD AND LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
If the glassware is especially dirty and cannot be
cleaned with ordinary detergents, chromic acid
cleaning may be required".
1. Pour 35 ml of distilled water in a 250 ml beaker.
2. Add about 1/8 teaspoon (simply estimate this
quantity) of sodium dichromate, Na?Cr?07, to
the water.
3. Swirl the beaker until the sodium dichromate has
dissolved.
4. Keep repeating steps 2 and 3 until no more
sodium dichromate will dissolve.
5. Pour the solution into a 2 liter beaker.
6. Slowly pour 1 liter of concentrated sulfuric
acid, H^SO^, into the 2 liter beaker.
Caution: Use eyeglasses and protective clothing.
7. Stir the mixture thoroughly.
8. Store it in a glass stoppered bottle.
9. The cleaning solution should be at a temperature
of about 50°C when it is used.
10. It may therefore be necessary to warm the
cleaning solution.
11. When using the warm cleaning solution, fill the
piece of glassware with the solution.
12. Allow it to soak for 2-3 minutes (or longer).
13. Pour the cleaning solution back into the storaqe
bottle.
14. Rinse the piece of glassware ten times with tap
water.
15. The cleaning solution may be reused until it turns
green.
16. It should then be discarded.
13th standard Methods,
p. 135, section 2.c.2
Page No. 8-25
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil and Grease
FIELD AND LABORATORY EQUIPMENT Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
A.1.4
Toward the end of this determination, TF/D will be
evaporated from the distilling flask, and therefore
lost. The TF/D may, however, be distilled from the
flask and recovered for later reuse. This is the
reason for using a distilling flask. (See fig. 3)
C.l.l
Depending on how the plant outfall is constructed,
there will probably be several ways in which the
sample can be collected in the bottle. Whichever
method is chosen, make sure that it is done in the
same manner each time.
Page No. 8-26
-------
EFFLUENT MONITORING PROCEDURE: Determination of Oil and Grease
Blank Determination
Weight of distilling flask after evaporation
of the 100 ml of TF/D = E = grams
Weight of the empty distilling flask used to
determine the blank = F = grams
Value of blank', D = E-F
Sample Deterniination
Weight of distilling flask + the oil/grease
residue = A = grams
Weight of empty distilling flask used for
the sample = B = grams
Volume of sample = C = milliliters
Milligrams of oil/grease residue per liter sample = *^1000 x 1000
Page No. 8-27
-------
OTHER APPROVED ANALYTICAL PROCEDURES
-------
A PROTOTYPE FOR DEVELOPMENT OF
ROUTINE OPERATIONAL PROCEDURES
for the
DETERMINATION OF AMMONIA
BY AN AMMONIA SELECTIVE ION ELECTRODE
as applied in
WASTEWATER TREATMENT FACILITIES
and in the
MONITORING OF EFFLUENT WASTEWATERS
Developed by the
National Training Center
Municipal Operations and Training Division
Office of Water Program Operations
U.S. Environmental Protection Agency
CH.N.am.EMP.2.5.76
Page No. 9-1
-------
EFFLUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia
Selective Ion Electrode
This Operational Procedure was developed by:
NAME John D. Pfaff
ADDRESS EPA, OWPO, National Training Center, Cincinnati, Ohio 45268
POSITION Chemist-Instructor
EDUCATION AND TECHNICAL BACKGROUND
B.S. Chemistry
3 years - Research Chemist
13 years - Training Instructor
Page No. 9-3
-------
EFFLUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia
Selective Ion Electrode
1. Objective:
To place an Orion** ammonia electrode and specific ion meter into operation
to make a determination of the ammonia concentration in an effluent sample.
2. Brief Description of Analysis:
Following a manual distillation of the sample at a pH of 9.5 the ammonia
concentration is determined using an ammonia selective electrode and a specific
ion meter. The procedure includes electrode assembly, membrane installation,
and calibration of the meter.
3. Applicability of this Procedure:
a. Range of Concentration:
0.03 to 1.0 mg NH3~N/liter
Information is given so the same stepwise procedure can be used for NH--N
concentrations up to 1400 mg/liter.
b. Pretreatment of Samples:
The Federal Register Guidelines specify manual distillation of the sample
at pH 9.5 unless sufficient acceptable proof exists to show that non-
distilled samples yield comparable data. The distillation procedure is
not included in this write-up because the step-wise directions are in the
EMP, "Nitrogen, Ammonia Determination."
c. Treatment of Interferences in Samples:
Two interferences are listed in the Source of Procedure*. It notes that
volatile amines in samples contribute to high results. However, no remedy
is given so treatment for this interference is not included in this procedure.
The other interference is the presence of mercury which forms a complex with
ammonia to give low results. The Training Guide in this EMP includes remedies
for this interference.
~Source of Procedure: Methods of Chemical Analysis of Water and Wastes, 1974,
Environmental Protection Agency, Methods Development and Quality Assurance
Research Laboratory, Cincinnati, Ohio, p. 165
and
Instruction manual for Probe and Meter, Orion Research Inc., Cambridge, MA 02139.
**Mention of a particular brand name does not constitute endorsement by the
U.S. Environmental Protection Agency
Page No. 9-4
-------
NITROGEN SERIES DETERMINATION FLOW SHEET
-o
OI
U3
m
10
cn
MINUS
MINUS
TOTAL KJELDAHL VALUE
AMMONIA VALUE
ORGANIC NITROGEN VALUE
NITRITE VALUE
NITRATE VALUE
TKN
DISTILL
DIGEST
CADMIUM
RED. OF
NITRATE TO
NITRITE
AMMONIA
SAMPLE
ORGANIC
NITROGEN
DISTILL
NITRATE
COLORIMETRIC
AS
AMMONIA
POTENTIO-
METRIC
AS
AMMONIA
COLORIMETRIC
DETERMINATION
TOTAL NITRATE
AND NITRITE
COLORIMETRIC
DETERMINATION
NITRITE ONLY
TITRIMETRIC
AS
TKN
COLORIMETRIC
AS
TKN
POTENTIO-
METRIC
AS
TKN
TITRIMETRIC
AS
AMMONIA
o
TO
T3
za
o
o
m
o
a
TO
(SI O
ft) n>
(V
r> -j
< 3
01
o o
3 3
n>
o
r+ 3
S 3
o o
Q.3
(D ->•
a>
cr
<<
o>
3
O
3
-------
EFFLUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia
Selective Ion Electrode
Equipment and Supply Requirements
A. Capital Equipment:
1. Orion Specific Ion Meter, Model 401, 407, or 407A
2. Orion Ammonia Electrode, Model 95-10
3. Magnetic stirrer
4. Analytical balance, 200 g capacity
5. Trip balance, 500 g capacity
6. Water still and ion exchange column containing a strongly acidic cation
exchange resin mixed with a strongly basic anion exchange resin
7. Still for distillation of samples (For details see the EMP, "Determination
of Total Kjeldahl Nitrogen," which contains this procedure.)
B. Reusable Supplies:
1. XXX beakers, 150 ml, two plus one for each sample
2. One cylinder, graduated, 100 ml
3. One flask, Erlenmeyer, graduated, 1000 ml
4. Three flasks, volumetric, 1000 ml
5. One flask, volumetric, 250 ml
6. One pipet, volumetric, 1 ml
7. Two pi pets, volumetric, 10 ml
8. One pipet, volumetric, 25 ml
9. One pipet, volumetric, 100 ml
10. One pipet bulb
11. One plastic wash bottle
12. One pair safety glasses
13. One spatula, medium size
14. One laboratory apron
C. Consumable Supplies:
1. Sodium hydroxide, NaOH, reagent grade, 1 lb. unit
2. Ammonium chloride, NH.C1, analytical grade, 4 oz. unit
3. Brushes and soap to clean glassware
4. Wax marking pencil
5. Disposable paper wipers
6. Two plastic weighing boats
Page No. 9-6
-------
j-j_r^U^jTMMOT'n(TO^I_NG_^ROCEiDUR^: Determination of Ammonia by an Ammonia Selective
Ion Electrode
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
DETERMINATION OF AMMONIA
A. Sample Preservation
1. Collection
2. Addition of
preservative
1. Collect a minimum of 400 ml
in a plastic or glass
container.
2. Cool to 4°C.
1. If more storage time is
needed, 2 ml of concen-
trated sulfuric acid,
H^SO^, per liter may be
added before cooling.
la. Because organic nitrogen is progressively formed
by biological activity, the determination of
ammonia is best made on a fresh sample.
2a. Sample may be held for 24 hours.
la. When acid is added there exists the possibility
of breakdown of organic nitrogen to form ammonia.
This addition is done only if storage time in
excess of 24 hours is expected.
I
(p. 23)
B. Equipment Preparation
1. Glassware
2. Still cleaning
3. Specific ion meter
preliminary check
1. Clean all glassware in
suitable detergent.
2. Rinse with ammonia-free
distilled water.
1. Clean the still until the
distillate shows no trace
of ammonia.
1. Check meter zero.
la. Distilled water should drain without leaving any
droplets.
2a. See section C.l.la.
la. For this procedure consult the Training Guide
or the EMP, "Determination of Total Kjeldahl
Nitrogen."
la. With the instrument turned off the needle on the
meter should point to the center of the scale.
If not, a screw adjustment is located on the meter
face.
I.B.2
(p. 23)
V.B.2.la
(p. 28)
Page No. 9-7
-------
EFFLUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia Selective
Ion Electrode
Page No. 9-8
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
B. Equipment Preparation
(continued)
4. Specific ion meter
operation check
2. Turn function switch to
the BATT position. See
Figure 4.
3. Replace batteries if
necessary.
1. Insert shorting strap in
electrode connectors.
2. Turn function switch to any
measuring position (not
battery test).
2a. Figure 4 is in the Training Guide.
2b. The needle should swing past the green BATT OK
area on the right side of the meter face. If
the needle fails to pass the green area, replace
the batteries.
3a. Replace with two 4.5 volt alkaline type
batteries—NEDA #1306A (Mallory-#MN-1306,
Everready-#523 or Burgess #AL 133).
3b. Place instrument face down and remove four
recessed screws. Lift off rear panel and remove
batteries. Check connections for corrosion and
remove any if it exists. Replace batteries
matching the marked polarity. Repeat battery
test and if okay, replace panel.
la. The shorting strap is a single wire with the
same type connectors that are on the electrode,
one on each end.
lb. Insert large connector into large input jack on
the instrument panel and small connector into
small red input jack.
2a. If the needle is not on scale, turn calibration
control to bring the needle on scale.
2b. If after coming to rest in one position the
needle does not remain stable, the instrument is
not functioning properly and should be serviced.
V.B.3.2a
(p. 26)
C. Reagent Preparation
1. Distilled water
1. Prepare about six (6)
liters of distilled water.
This water should be free
from ammonia.
la. Pass distilled water through an ion-exchange
column containing a strongly acidic cation ex-
change resin mixed with a strongly basic anion
exchange resin.
-------
E. .JENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia Selective
Ion Electrode
1
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Reagent Preparation
(continued)
2. Ammonium chloride
stock solution
(1000 mg
NH3-N/liter)
1. Weigh out 3.819 g of
ammonium chloride (NH^Cl).
2. Transfer the chemical to a
1 liter volumetric flask.
3. Add about 500 ml of water
to the flask.
4. Dilute to the volume mark
with water.
la. Use an analytical balance.
3a. Unless otherwise specified the term water means
ammonia-free water.
4a. Label flask as ammonium chloride 1000 mg
NH3-N/liter.
4b. Mix well by shaking.
3. Ammonium chloride
intermediate
solution
(10 mg NH^-N/liter)
1. Add about 500 ml of water
to a 1 liter volumetric
flask.
2. Pi pet 10 ml of stock
(1000 mg NH^-N/liter)
ammonium chloride solution
into the flask.
3. Dilute to the volume mark
with water.
2a. Use a 10 ml volumetric pipet.
3a. 1.0 ml = 0.01 mg NHj-N.
3b. Label flask as ammonium chloride
10 mg NH3-N/ liter.
3c. Mix well by shaking.
Page No. 9-9
-------
EFFLUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia Selective
~™"—l—ion Electrode
Page No. 9-10
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Reagent Preparation
(continued)
4. Ammonium chloride
standard solution
(1 mg NH^-N/liter)
1. Add about 500 ml of water
to a 1 liter volumetric
flask.
2. Pi pet 100 ml of the inter-
mediate (10 mg NH^-N/liter)
ammonium chloride solution
into the flask.
3. Dilute to the volume mark
with water.
2a. Use a 100 ml volumetric pipet.
3a. Prepare dilution fresh daily.
3b. 1.0 ml = 0.001 mg NH3~N.
3c. Label flask as ammonium chloride
1 mg NH3-N/liter.
3d. Mix well by shaking.
5. Ammonium chloride
standard solution
(0.1 mg NH3-N/
1. Add about 150 ml of water
to a 250 ml volumetric
fl ask.
1 iter)
2. Pi pet 25 ml of the
standard (1 mg NH3-N/liter)
ammonium chloride solution
into the flask.
2a. Use a 25 ml volumetric pipet.
-------
Ei.lUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia Selective
Ion Electrode
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
C. Reagent Preparation
(continued)
6. Sodium hydroxide
solution, 10 M
3. Dilute to the volume mark
with water.
1. Weigh out 400 g of sodium
hydroxide (NaOH).
2. Dissolve the 400 g of
sodium hydroxide in 800 ml
of water in a 1 liter
Erlenmeyer flask.
3. Cool to room temperature.
4. Dilute to the 1000 ml
volume line with water.
3a. Prepare dilution fresh daily.
3b. 1.0 ml = 0.0001 mg NH-j-N.
3c. Label flask as ammonium chloride 0.1 mg NH,-N/
liter.
3d. Mix well by shaking.
la. CAUTION: This is a strong base and should be
handled with care. Use safety glasses.
lb. Use a trip balance.
2a. CAUTION: A large amount of heat is liberated
during dissolution.
3a. Allow cold tap water to run on the side of the
flask.
4a. This solution should be kept in a plastic
container.
4b. Label container as sodium hydroxide, 10 M.
D. Assembly of Electrode
1. Unscrew the top portion of
the electrode through
which the wire passes.
2. Lift out top and attached
inner body of electrode.
la. See Figure 1 in the Training Guide.
V.D.I.la
(p. 24)
Page No. 9-11
-------
EFFLUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia Selective
Ion Electrode
Page No. 9-12
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Assembly of Electrode
(continued)
3. Place inner body on flat
clean surface
4. Unscrew bottom portion of
electrode outer body.
5. Remove 0-ring, spacer, and
old membrane.
6. Remove a membrane from the
the container with the
tweezers.
7. Place the new membrane in
the bottom cap.
5a. If this is the first use of the electrode, there
will be no old membrane in place. The electrode
is shipped dry and without a membrane.
5b. The 0-ring is a red rubber ring.
5c. The spacer is a black plastic ring with a black
0-ring recessed in a notch at one end around its
inside diameter. The end which has the 0-ring
is placed toward the bottom of the bottom cap.
6a. The membranes are packaged with a blue packing
paper between each membrane. Discard the blue
packing paper.
6b. The membrane should not be handled.
7a. With the 'dimpled" side facing upward toward the
inner body and the patterned side facing down
toward the sample solution. See Figure 2 below.
I.D.6a
(p. 23)
DIMPLED PATTERNED
FIGURE 2
-------
^^-UENT MONITORING PROCEDURF.: Determination of Anmonia
Ion Electrode
OPERATING PROCEDURES
STEP SEQUENCE
D. Assembly of Electrode
(continued)
8. Replace spacer.
9. Replace 0-ring.
10. Screw outer body into
bottom cap.
11. Fill outer body with fill
ing solution provided by
the manufacturer.
12. Screw top cap and inner
body onto outer body.
13. Place assembled electrode
into holder attached to
rod on the meter.
14. Plug the electrode cable
into the meter.
by an Ammonia Selective
INFORMATION/OPERATING GOALS/SPECIFICATIONS
8a. With its recessed 0-ring down.
10a. Do this by turning the outer body, not the
bottom cap.
TRAINING
GUIDE NOTES
11a.
lib.
13a.
13b.
It is best to put the filling spout on the bottle.
This spout is provided with but not on the fill-
ing solution.
Fill the outer body with filling solution to
about 1 cm above the joint between the outer
body and the bottom cap. If the outer body is
overfilled, the excess will flow out of the vent
hole when the inner body is replaced.
Electrode must be held at a 20° angle with re-
spect to the vertical to prevent air bubble
entrapment under the electrode.
Orion Research Incorporated sells a holder
(Cat. No. 920001A) which has the proper angle
and will work with their Model 400 series
Specific Ion Meters. See Figure 3 in Training
Guide.
14a.
The electrode cable ends with an input jack and
a pin jack. They should be connected to the
input connector and reference electrode connector:
respectively of the specific ion meter.
The ammonia electrode does not require an
external reference electrode.
14c. See Figure 4 in Training Guide.
V.D.I 3b
(p. 25)
14b,
|V.D.14C
(p. 26)
Page No. 9-13
-------
EFFLUENT MONITORING PROCEDURF: Determination of Ammonia by an Ammonia Selective
Ion Electrode
Page No. 9-14
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
D. Assembly of Electrode
(continued)
15. Lower electrode into about
100 ml of 0.1 M ammonia
chloride solution.
16. Allow electrode to stand
for about one-half hour
before use.
15a. The solution can be put into a small beaker.
E. Electrode Operation
Check
1. Transfer 100 ml of 0.1 mg
NHg-N/liter standard
solution to a 150 ml
beaker.
2. Place the beaker on the
stir plate and add the
stir bar to the beaker.
3. Lower the electrode into
the standard solution.
4. Turn on stirrer.
5. Add 1 ml of the 10 M
sodium hydroxide solution.
la. Use a 100 ml graduated cylinder.
lb. This is Reagent C.5.
2a. Samples and standards should be stirred using a
magnetic stirrer. Some magnetic stirrers
generate sufficient heat to change solution
temperature. This effect can be minimized by
placing a piece of insulating material on the
stirrer (for example a piece of cork or a
plastic petri dish).
2b. Samples and standards should be at the same
temperature.
3a. The solution should at least cover the joint
between the bottom cap and the outer body.
3b. Make sure the stir bar does not hit the
electrode.
4a. Provide a good mixing rate. However, do not stir
solutions at so fast a rate as to cause a vortex
to be formed.
5a. The sodium hydroxide should be added at 1 ml of
10 M sodium hydroxide per 100 ml of (neutral pH 1\
solution.
(continued)
-------
E.ri-lUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia Selective
————————¦ ion Electrode
1
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
E. Electrode Operation
Check (continued)
5b. The pH of any solution to be tested with the
electrode must be above 11 after the addition
of the sodium hydroxide.
5c. Caution: This is a caustic solution. Do not
allow contact with skin.
5d. Use a 1 ml pi pet.
5e. Do not add prior to electrode immersion.
6. Turn the specific ion
meter function switch to
MV EXP (Millivolts Ex-
panded Scale).
6a. This is read from the meter on the blue scale on
the #401; on the black scale on the #407 and
407A. The expanded mode has a + 70 mv range.
See Figure 5 in Training Guide.
V.E.6a
(p. 27)
7. After 30 seconds adjust
the meter to the center
scale.
7a. Turn the CALIB (calibration) knob and adjust
the meter to obtain a reading of 0 (center scale)
on the millivolt scale. See Fiqure 4 in Traininq
Guide.
V.E.7a
(p. 26)
8. Turn the function switch
to off.
9. Raise the electrode out of
the sodium hydroxide
solution.
10. Rinse the electrode with
distilled water and blot
dry with tissue.
11. Transfer 100 ml of 1 mg
NH^-N/liter standard
11a. This is Reagent C.4.
solution to a 150 ml
beaker.
Page No. 9-15
-------
EFFLUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia Selective
—————————ion Electrode
Page No. 9-16
OPERATING PROCEDURES
STEP SEQUENCE
E. Electrode Operation
Check (continued)
12. Place the beaker on the
stir plate and add stir
bar to the beaker.
13. Lower the electrode into
the standard solution.
14. Add 1 ml of the 10 M
sodium hydroxide solution.
15. Turn the function switch
to MV EXP.
16. After 30 seconds read the
meter.
17. Turn function switch to
off.
INFORMATION/OPERATING GOALS/SPECIFICATIONS
12a. The stir bar should be rinsed with distilled
water between uses.
13a. Make sure the stir bar does not hit the electrode.
14a. Do not add prior to electrode immersion.
14b. Use same pipet as in the previous procedure.
16a. The reading should be taken from the same
millivolt scale that was used to set the previous
concentration.
16b. The reading should show a change of approximately
59 mv. This change (59 mv) will occur for every
tenfold change in concentration because of the
electrode make-up.
16c. If an mv reading near 59 mv is not obtained,
check all standard dilutions and repeat all
steps in section E.
16d. If continued failure to obtain an mv change near
59 mv occurs, contact the electrode manfacturer.
17a. Always set this position before lifting any
electrode from the solution and when the meter
is not actually measuring. This will extend the
life of the batteries and protect the meter.
TRAINING
GUIDE NOTES
-------
Et-ri.UENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia Selective
Ion Electrode
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Calibration
1. Setting mid-scale
range
1. Transfer 100 ml of the
0.1 mg NH^-N/liter
standard solution to a
150 ml beaker.
2. Place the beaker on the
stir plate and add stir
bar to the beaker.
3. Lower electrode into the
standard solution.
4. Turn on stirrer.
5. Transfer 1.0 ml of the
10 M sodium hydroxide
solution to the same
beaker.
6. Use appropriate range pH
paper to check if the pH
is greater than 11.
7. Turn the instrument func-
tion switch to the mono-
valent anion position.
After the meter stops
drifting, read the meter.
la. The standards chosen should span the range that
the sample concentration is expected to be in.,
lb. With the standards prepared in this EMP a range
will be covered from 0.01 to 1.0 mg NH,-N/liter
(0.01 to 1.0 ppm).
lc. Use a 100 ml graduated cylinder.
V.F.I.la
(p. 28)
3a. Make sure the stir bar does not hit the
electrode.
5a. Do not add prior to electrode immersion.
5b. Use a 10 ml graduated pipet.
6a. If not,add more sodium hydroxide until pH is
greater than 11.
7a. This position is labeled differently on the
various meters in the Orion 400 series. On the
401 and 407A it is marked x~. On the 407 it is
F". See Figure 4 in Training Guide.
8a. Response of the electrode will be faster for
higher concentrations of ammonia and slower for
lower concentrations. They can vary from less
than 30 seconds to about 10 minutes.
(continued)
V.F.I.7a
(p. 26)
Page No. 9-17
-------
EFFLUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia Selective
" Ion Electrode
Page No. 9-18
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Calibration
(continued)
8b. The reading should be taken from the concen-
tration scale which is usually color coded to
match the color of the monovalent position on
the function switch. The scale is usually the
topmost one and is logarithmically divided.
9. Use the calib (calibra-
tion) control and adjust
the meter needle to read
at center scale.
9a. The marking at center scale is different for the
various meters. It is 100 for the 401 and 407
and 1 for the 407A. See Figure 5 in Training
Guide.
9b. After the adjustment has been made, this center
position will represent a concentration of 0.1 mg
NHg-N/liter.
V.F.I.9a
(p. 27)
10. Turn function switch to
off.
11. Raise the electrode.
12. Rinse the electrode with
distilled water and blot
dry with tissue.
2. Setting high-scale
range
1. Transfer 100 ml of the
1.0 mg NH3-N/1iter
standard solution to a
150 ml beaker.
2. Place the beaker on the
stir plate and add stir
bar to the beaker.
3. Lower electrode into the
standard solution.
4. Turn on stirrer.
la. Use a 100 ml graduated cylinder.
3a. Make sure the stir bar does not hit the
electrode.
-------
L -UENT MONITORING PROCEDURE: Determination of Airenonia by an Ammonia Selective
Ion Electrode
OPERATING PROCEDURES
l
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Calibration
(conti nued)
5.
Transfer 1.0 ml of the
10 M sodium hydroxide
solution to the same
beaker.
5a. Do not add prior to immersion.
5b. Use a 10 ml graduated pipet.
6.
Use appropriate range
pH paper to check if the
pH is greater than 11.
6a. If not, add more sodium hydroxide until pH is
greater than 11.
7.
Turn the function switch
to the monovalent anion
position.
8.
After the meter stops
drifting, read the meter.
8a. Use same uppermost scale as used for previous
concentration.
9.
Use the "Temp °C" control
and adjust meter needle
to read at far right
position.
9a. For location of this control knob, see Figure 4
in Training Guide.
9b. Again the marking will vary by instrument. It
will be 1000 on the 401 and 407 and will be 10
on the 407A. See Figure 5 in Training Guide.
9c. This position now represents a concentration of
1.0 mg NH^-N/liter and the instrument has been
adjusted to represent 0.01 mg to 1.0 mg
NHg-N/liter over the full scale of the meter
face.
9d. Values below 0.03 mg NH^-N/liter should be
disregarded because of a deviation from normal
response curve.
V.F.2.9a
(p. 26)
V.F.2.9b
(p. 27)
10.
Turn function switch to
off.
11.
Recalibrate as needed.
11a. It is advisable to standardize electrodes 3 or
4 times a day by carrying out steps in
section F.
(continued)
Page No. 9-19
-------
EFFLUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia Selective
Ion Electrode
Page No. 9-20
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
F. Calibration
(continued)
lib. Always use fresh solutions of the standards
for recalibration.
G. Procedure
1. After the calibration has
been completed, read each
sample concentration by
doing the following
steps.
2. Rinse electrode.
3. Add 1.0 ml or more of
sodium hydroxide solution
after immersion of
electrode until pH is
greater than pH 11.
4. Read after drifting has
stopped.
la. Use 100 ml volumes in 150 ml beakers.
4a. Read concentration directly in mg NH-j-N/liter
from the concentration scale.
4b. Do not adjust calib (calibration) or Temp °C
(temperature compensator) controls after
calibration. If they are changed, recalibrate
instrument.
H. Storage
1. Between readings
1. Immerse electrode in
alkaline standardizing
solution.
la. You can use one of the standardizing solutions
with 10 M sodium hydroxide which you used in F,
calibration.
lb. The electrode should be iimiersed between
measurements,
lc. Do not store in air.
v.H.i.ic
(p. 28)
-------
FLUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia Selective
Ion Electrode
OPERATING PROCEDURES
STEP SEQUENCE
INFORMATION/OPERATING GOALS/SPECIFICATIONS
TRAINING
GUIDE NOTES
H. Storage
(continued)
2. Overnight
1. Immerse electrode in
ammonium chloride stock
solution (1000 mg
NH3-N/liter).
la. Without sodium hydroxide.
3. Prolonged time
1. Disassemble electrode
completely.
2. Rinse with distilled
water.
3. Dry and reassemble.
2a. Rinse inner body, outer body and bottom cap.
3a. Without filling solution or membrane.
3b. Discard membrane.
4. Membrane
replacement
1. Follow steps under pro-
cedure D, Assembly of
Electrode.
V.H.4
(p. 28)
Page No. 9-21
-------
EFFLUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia
Selective Ion Electrode
TRAINING GUIDE
SECTION TOPIC
I* Introduction
II Educational Concepts - Mathematics
III Educational Concepts - Science
IV Educational Concepts - Communications
V* Field and Laboratory Equipment
VI Field and Laboratory Reagents
VII Field and Laboratory Analysis
VIII Safety
IX Records and Reports
*Training guide materials are presented here under the headings marked *.
These standardized headings are used through this series of procedures.
Page No. 9-22
-------
EFFLUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia
Selective Ion Electrode
INTRODUCTION
Section I
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
Ammonia has been of interest to both water and
wastewater treatment plants for years. The content
of ammonia in the effluent waters of a wastewater
plant car indicate to the operator the efficiency
of operation at which the plant is being run.
Furthermore, ammonia can have a significant effect
on the disinfection of water with chlorine. Con-
sequently, monitoring the concentration of ammonia
should be routine. The analysis of ammonia con-
centrations is also the basis for routine determi-
nations of total nitrogen or of organic nitrogen
in effluent samples.
The test described in this instruction can be
found in the 1974 EPA Methods Manual on page 165.
No other reference is cited in the Federal Register
Guidelines. However, the referenced EPA Methods
Manual in turn refers the analyst to the manu-
facturer's operating manual for the specific ion
meter being used.
Methods for Chemical
Analysis of Water and
Wastes, 1974, EPA,
MDQARL, Cincinnati,
Ohio, 45268, p. 165.
B.2
Distillation is not required if comparability data
on representative effluent samples are on file to
show that this preliminary distillation step is not
necessary. However, manual distillation will be
required to resolve any controversies.
New Federal Register
Guidelines, (1976),
note if4.
If the determination is to be run as part of the
total Kjeldahl nitrogen determination, distillation
must be carried out. Any mercury in the sample
will form a complex with the ammonia and act as an
interference. This will be taken care of in the
distillation procedure by the addition of the
sodium thiosulfate.
Methods for Chemical
Analysis of Water and
Wastes, 1974, EPA,
MDQARL, Cincinnati,
Ohio 45268, pg. 175.
(If mercury is present and the sample is not to be
distilled, add 0.2 g sodium thiosulfate to the
sample to complex the mercury before the determi-
nation of ammonia).
op. c1t.
p. 1(56
D.6a
The ammonia electrode uses a membrane which will not
allow water or ionic species to migrate across it.
However, the ammonia dissolved in the sample can
diffuse through the membrane until the concentratior
of the ammonia is the same on both sides of the
membrane. The reference element, contained in the
ammonia electrode itself, is the same as used in
the chloride specific ion electrode. It senses the
fixed level of the chloride in the ammonia chloride
internal filling solution, thereby acting as a
reference electrode for the arranonia electrode.
Instruction Manual for
Ammonia Electrode Model
95-10, Orion Research,
Inc., Cambridge, MA 02139
Page No. 9-23
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EFFLUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia
Selective Ion Electrode
FIELD & LABORATORY EQUIPMENT
Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
0.1.1a
ASSEMBLY INSTRUCTIONS
outer body
red O-ring
spacer (black O-ring
down)
membrane
bottom cap
bottom cap
FIGURE 1
Page No. 9-24
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EFFLUENT MOMTORING PROCEDURE: Determination of Ammonia by an Ammonia
Selective Ion Electrode
lELD & LABORATORY EQUIPMENT Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
D. 13b
FIGURE 3
Page No. 9-25
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EFFLUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia
Selective Ion Electrode
FIELD & LABORATORY EQUIPMENT Section \j
TRAINING GUIDE NOTE
B.3.2a
D. 14c
E.7a
F.1.7&
F.2.9a
REFERENCES/RESOURCES
bicordI
REIEASI
[c&UB
FIGURE 4
Page No. 9-26
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EFFLUENT MONITORING PROCEDURE: Determination of Armonia by an Ammonia
Selective Ion Electrode
.•IELD AND LABORATORY EQUIPMENT
Section v
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
E.6a
F.1.9a
F.2.9b
MODEL 401
100
4?0 CONCENTRATION SCALE
700 -0+ 30o
MILLIVOLT SCALE
MODEL 407
100
CONCENTRATION SCALE
CXD
6a
40
MILLIVOLT SCALE
MODEL 407A
< CONCENTRATION SCALE
oo
200 -0+ jqq
"Oq
MILLIVOLT SCALE
FIGURE 5
Page No. 9-27
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EFFLUENT MONITORING PROCEDURE: Determination of Ammonia by an Ammonia
Selective Ion Electrode
FIELD AND LABORATORY EQUIPMENT Section V
TRAINING GUIDE NOTE
REFERENCES/RESOURCES
B.Z.la
Add a 1 + 1 mixture of ammonia-free distilled water
and sodium hydroxide-sodium thiosulfate solution to
each Kjeldahl flask to be used. Add glass beads
and, using appropriate apparatus, distill 50 ml of
this solution. The distillate should be checked to
insure that it is ammonia-free. This can be done
with the ammonia probe and meter or by use of the
Nessler's color reagent.
F.l.la
In using a specific ion meter the calibration range
is arbitrary within the appropriate range of the
method. Should the samples to be run fall outside
the range of 0.01 to 1.0 mg NH^-N/liter, a new range
can be set on the instrument. This is done in the
same manner as set down in section F but using a
tenfold concentration increase. For example, using
the 1.0 mg NHg-N/liter solution in step F.l.l and
a 10 mg NH3-N/liter solution in step F.2.1 gives a
range of 0.1 to 10 mg NH^-N/liter.
H.l.lc
If the electorde is accidentally left in the air,
rather than in a solution, that portion of the
internal filling solution between the inside of the
membrane and the sensing element will dry out. To
restore the electrode to operation hold the elec-
trode by the outer body and,grasping the electrode
cable directly above the cap, pull on the cable
so as to lift the sensing element off the membrane.
Fresh internal filling solution will now flow under
the membrane. The electrode will now be ready for
use.
Instruction Manual
Ammonia Electrode
Model 95-10, Orion
Research, Inc.,
Cambridge, MA 02139.
H.4
Membrane failure is characterized by a shift in
electrode potential, drift and poor response. Mem-
brane failure may be apparent on visual inspection
as dark spots or discoloration of the membrane.
Handling the membrane during installation may ad-
versely affect it and shorten its life. Handle the
membrane with the tweezers provided. A membrane
will last from one week to several months depending
on usage.
Ibid
Page No. 9-28
^11 5 GOVERNMENT PRINTING OFFICE: 1976-657-695/5't53 Region No.
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