TABLE OF CONTENTS
Total Phosphorus
Page
Section 1: Introduction to Total Phosphorus................................................................................ 2
Section 2: Glossary.................................................................................................................... 2
Section 3: Safety and Hygiene.................................................................................................... 2
Section 4: Sampling................................................................................................................... 3
Section 5: Sample Preservation.................................................................................................. 3
Section 6: Sample Containers..................................................................................................... 3
Section 7: Interpretations............................................................................................................ 3
Quiz 8.1...................................................................................................................................... 4
Section 8: Total Phosphorus....................................................................................................... 4
Section 9: Equipment and Reagents............................................................................................ 4
Section 10: Laboratory Procedure............................................................................................... 4-5
Section 11: Total Phosphorus..................................................................................................... 5
Section 12: Equipment and Reagents.......................................................................................... 5
Section 13: Laboratory Procedure............................................................................................... 5-6
Section 14: Calibration Curve...................................................................................................... 6
Section 15: Calculations............................................................................................................. 6-7
Section 16: Interferences............................................................................................................ 7
Section 17: QA/QC..................................................................................................................... 7-8
Quiz 8.2...................................................................................................................................... 8
Answers to Quizzes..................................................................................................................... 8-9
Appendix A: References
Appendix B: Phosphorus Reagents
Appendix C: Sample Bench Sheet
Appendix D: Preparation of a Calibration Curve
Appendix E: Methods Checklist
Appendix F: Method Checklist - Reagent
Appendix G: Method Checklist Digester
Appendix H: Phosphorus Ascorbic Acid (Single Reagent)
TOTAL
PHOSPHORUS
Section 1: INTRODUCTION TO TOTAL PHOSPHORUS
Wastewater
is relatively rich in phosphorus compounds.
Phosphorus is a nutrient used by organisms for growth. It occurs in natural water and wastewater
bound to oxygen to form phosphates.
Phosphates come from a variety of sources including agricultural
fertilizers, domestic wastewater, detergents, industrial process wastes and
geological formations.
The
discharge of wastewater containing phosphorus may cause algae growth in
quantities sufficient to cause taste and odor problems in drinking water
supplies. Dead and decaying algae can
cause oxygen depletion problems which in turn can kill fish and other aquatic
organisms in streams. For this reason,
phosphorus removal is an essential role of wastewater treatment plants and
testing for phosphorus in the plant effluent is critical.
Phosphates
are classified as orthophosphates, polyphosphates and organic phosphates. In this procedure, orthophosphates can be
determined directly by colorimetric analysis.
Other types require a digestion step to convert the “combined” phosphate
to the ortho form for analysis. This
gives the Total Phosphorus result.
Section 2: GLOSSARY
Blank: A preliminary analysis omitting only the
sample to provide an unbiased reference point or baseline for comparison. The Blank is usually run on distilled water.
Nutrient: Any substance used by living things that
promotes growth. The term is generally
applied to nitrogen and phosphorus in water, but is also applied to other
essential and trace elements.
Phosphate curve: A calibration curve that plots concentrations
of known standards against the instrument’s response (color intensity,
absorbance, millivolts, etc.).
Section 3: SAFETY AND HYGIENE
Whenever
samples of wastewater are handled, it is very important that operators wash his
or her hands before eating or smoking.
While some laboratory chemicals are not dangerous, many of them are
poisonous or harmful to skin and clothing.
Rubber gloves and safety glasses should be used. It is important to wash thoroughly with soap
and water after handling laboratory chemicals, especially if chemicals come
into contact with the skin. Keep bench
areas free of clutter and clean bench surfaces with disinfectant after testing.
Read
the labels carefully and know what to do in case of an accidental spill. Always clean up spills quickly and in the
safest possible manner using disposable rags or towels.
Acids
and bases can be corrosive. Care should
be taken when handling them. Never add
water to acid. Always add acid to water
very slowly because acids and bases can generate heat when mixed with water.
The
Total Phosphorus test involves heating samples in the digestion step. To avoid injuries caused by heat, use gloves
or tongs to handle hot or potentially hot glassware. Never assume that a beaker or flask is cool.
Section 4: SAMPLING
Samples
used for the determination of phosphorus can be either grab or composite. The type of sample used will depend on the
monitoring requirements, plant operating procedures and the testing and sample
storage capabilities of individual plants.
Samples should be collected from well-mixed areas in the process flow so
that they are representative of the total flow.
Section 5: SAMPLE PRESERVATION
If
Total Phosphorus is the only determination to be made, acidify the sample to
less than pH 2 with sulfuric acid and cool to 4°C. Acidified and cooled samples may be held for
up to 28 days. If the test is run immediately,
no preservative is required.
Samples
to be tested for dissolved reactive phosphorus, dissolved acid-hydrolyzable
phosphorus, or total dissolved phosphorus must be filtered. See the 18thth
Edition of “Standard Methods for the Examination of Water and Wastewater” for
information on these tests.
Section 6: SAMPLE CONTAINERS
Special
sampling devices and storage containers are not necessary for phosphorus
testing. Sampling devices should draw
from well-mixed areas of tanks or pipes, be made of resistant materials that
will not rust or corrode, be capable of taking samples that are proportional to
the plant’s flow, and be easily and thoroughly cleaned.
Storage
containers should be made of corrosion resistant material which can stand
repeated refrigeration. These containers
should have leak-proof tops. Do not
store low concentration samples in plastic bottles since phosphorus may be
adsorbed on the container walls.
Rinse
all sampling containers with diluted (1 + 1) hydrochloric acid. Then rinse 3 times with distilled water.
Never
use commercial detergents for cleansing any glassware used in the storage or
analysis of samples for phosphorus determination.
Section 7: INTERPRETATIONS
Because
of its effect on receiving streams, testing for phosphorus is critical to
wastewater treatment plants. Ranges for
phosphorus concentrations are given below.
Sample type range
Influent 5 - 10 mg/L
Primary Effluent 4 - 9 mg/L
Activated Sludge Effluent 3 - 8 mg/L
Trickling Filter Effluent 4 - 8 mg/L
Advanced Treatment Effluent 0.1 - 2.0 mg/L
What
is normal for one plant is not necessarily normal for another. Each operator must determine what range
reflects normal conditions for his or her plant. Values outside the established normal range
indicate that a condition exists which could yield poor effluent quality.
Quiz
8.1
1. Why
is phosphorus removed from wastewater?
2. What
are the three basic forms of phosphorus found in wastewaters?
3. What
must be done before polyphosphates can be measured?
Section 8: TOTAL PHOSPHORUS
DESCRIPTION OF DIGESTION STEP
Since
phosphorus exists in several distinct forms in wastewater samples and the
approved test method measures only the orthophosphate form, pretreatment
methods have been developed to convert the various forms of
phosphate-phosphorus to the orthophosphate form. If the only determination to be made is Total
Phosphate-Phosphorus, the sample is digested to convert both the polyphosphate
and the organic phosphate to the ortho form at the same time. If the analyst must test for the various
types of phosphate, an acid-hydrolysis must be performed. Since this is not required for most
facilities, it is not included in this text.
Section 9: EQUIPMENT AND REAGENTS
EQUIPMENT
1. Hot plate
2. Tongs or gloves
3. Scoop (0.4 g capacity)
4. 125 mL Erlenmeyer flasks (acid washed)
5. 50 mL graduated cylinders (acid washed)
REAGENTS
1. Phenolphthalein indicator
2. Sulfuric acid solution
3. Ammonium persulfate, crystal
4. Sodium hydroxide, 1 N
See Appendix B for the procedures for
preparation of the reagents used in this method.
Section 10: LABORATORY PROCEDURE
1. Measure 50 mL or an appropriate amount of
sample diluted to 50 mL with distilled water.
2. Add 1 drop phenolphthalein indicator. If a red color develops, add sulfuric acid
solution until color just disappears.
3. Add 1 mL of sulfuric acid solution and 0.4
g of ammonium persulfate.
4. Boil gently for 30 to 40 minutes or until
the total volume is 10 mL.
5. Cool, add 1 drop of phenolphthalein and
neutralize to a faint pink color with 1 N sodium hydroxide.
6. Make up to 50 mL with distilled water. The digested sample is then tested for total
phosphate as outlined in Section 12.
NOTE: If
precipitate forms, do not filter.
Section 11: TOTAL PHOSPHORUS
ASCORBIC ACID PROCEDURE
An
accepted method for determining phosphate-phosphorus is the Ascorbic Acid
Procedure. The procedure is suitable for
concentrations of 0.01 to 6 mg/L PO4-P.
Ammonium
molybdate and potassium antimonyl tartrate react in acidic solution with
orthophosphate to form a heteropolyic acid (phosphomopydbic acid) which can be
reduced by ascorbic acid to form an intense blue color.
Section 12: EQUIPMENT AND REAGENTS
EQUIPMENT
1. Spectrophotometer with an infrared
phototube for use at 880 nm with a light path of at least 2.5 cm (1 in)
2. Acid washed glassware
3. An analytical balance capable of weighing
to 0.1 mg accuracy
REAGENTS
1. Sulfuric acid solution 5 N
2. Potassium antimonyl tartrate solution
3. Ammonium molybdate Solution
4. Ascorbic acid solution
5. Combined reagent
6. Standard phosphate solution
See
Appendix B for the procedures for preparation of the reagents used in this
method.
Section 13: LABORATORY PROCEDURE
1. Pipette 50.0 mL or an appropriate amount
diluted to 50 mL of digested sample into an acid cleaned, dry 125 mL Erlenmeyer
flask.
NOTE: If
only orthophosphate is to be determined, an undigested sample is used.
2. Add 1 drop of phenolphthalein
indicator. If a red color develops, add
5 N sulfuric acid until the color disappears.
3. Add 8.0 mL of combined reagent and mix
thoroughly.
4. Allow at least 10 minutes (but not more
than 30 minutes) for color development.
5. Measure absorbance at 880 nm using a
reagent blank to zero the spectrophotometer.
The reagent blank is made using 50 mL
of distilled water carried through the digestion step and ascorbic acid
procedure.
In highly colored or turbid samples,
prepare a sample blank by adding all the reagents to the sample except the
ascorbic acid and potassium antimonyl tartrate.
Subtract the absorbance of this blank from the absorbance of the sample.
6. Check the sample’s absorbance against the
calibration curve and determine the concentration. Correct for dilution.
Section 14: CALIBRATION CURVE
Since
the phosphate concentration is measured as a function of absorbance, a standard
curve of absorbance versus known phosphate concentrations must be
prepared. Six standard phosphorus
concentrations and a distilled water blank are treated with the same digestion
procedures as the samples. These 6
values are used to plot absorbance versus phosphate concentration to give a
straight line passing through the origin.
Prepare
6 dilutions of the 5.0 mg/L Phosphate standard to result in the following final
concentrations:
final conc. volume of std. final vol. in flask
0.1 mg/L 1.0 mL 50.0 mL
0.2 mg/L 2.0 mL 50.0 mL
0.4 mg/L 4.0 mL 50.0 mL
0.6 mg/L 6.0 mL 50.0 mL
0.8 mg/L 8.0 mL 50.0 mL
1.0 mg/L 10.0 mL 50.0 mL
Perform
the Total Phosphorus procedure including the digestion step and color
development. Record the absorbances and
plot the curve. At least one standard
phosphate concentration must be included with each batch of samples as a check
on the calibration curve. Prepare a new
curve every six months.
See
Appendix D for more information on the preparation of calibration curves.
Section 15: CALCULATIONS
Determine
the final concentration of the sample using the following equation:
mg/L
P =
mg/L from the curve x 50 mL
divided
by initial volume used (mL)
For
example, if 5 mL of Influent gives an absorbance of 0.35 which represents 0.558
mg/L on the calibration curve, the corrected concentration of
phosphate-phosphorus is:
mg/L
P =
0.558 x 50 mL divided by 5 mL = 5.58 mg/L
Section 16: INTERFERENCES
Arsenates
react with the molybdate reagent to produce a blue color similar to that formed
in the phosphate determination.
Concentrations as low as 0.1 mg As/L interfere with the analysis. Hexavalent chromium and nitrite interfere to
give low results.
Chapter 17: QA/QC
A
Quality Assurance/Quality Control program is required by the NPDES permit. Quality Assurance (QA) is a set of operating
principles that must be followed during sample collection and analysis. Lab bench sheets must be maintained that
document when the sample was collected, how it was preserved, and what results
were obtained.
Quality
Control (QC) includes any testing which is done to prove that the results are
reliable. One of every ten samples
analyzed should be a QC check. This may
include duplicate samples, spike samples, reagent blank analyses and known QC
samples obtained from outside sources.
Duplicate
sample analysis involves analyzing the same sample twice and comparing the
results. The closer the results, the
more accurate the analysis. Results
should not differ by more than 10%. Spike sample analysis involves adding known
amounts of analyte to a sample and calculating the percent recovery. These are discussed further in Chapter 10.
In
Phosphorus analysis, a distilled water blank must be run with every batch of
samples tested. It is used both to zero
the spectrophotometer and to show that the glassware and reagents are not
contaminated with phosphates.
At
least one check standard must also be run with each batch. If a variety of samples are being tested with
results expected to cover a wide range of concentrations, test two or more
standards. The absorbencies of the
standards must agree closely with the true value or a new curve must be drawn.
Spike
samples are run by adding the 5.0 mg/L Phosphate standard to a sample. To simplify the calculation, use the same
amount of standard in the spike sample as you are using in your check standard.
For
example, an Effluent sample is spiked with 2 mL of the 5.0 mg/L Phosphate
standard yielding the following results:
Effluent sample result:
0.116 mg/L
Spiked Effluent sample result: 0.310 mg/L
Check standard (2 mL) result: 0.197 mg/L
Expected spike result = sample result + standard result
= 0.116 + 0.197
= 0.313
Percent
recovery = spike result divided by expected result x 100%
= 0.310 divided by 0.313 x 100%
= 99%
QC
samples with known concentrations of Phosphate can be purchased from chemical
supply companies. The results from
duplicate, spike and outside QC samples should be recorded in a QC notebook.
Sample
bench sheets are included in Appendix C.
Quiz 8.2
1. What four reagents are used to make the
combined reagent for the phosphorus test?
What is the storage time of this reagent?
2. What sample volume would you start with if
the estimated phosphorus content of the sample was 1 mg/L? 4 mg/L?
10 mg/L?
Answers
to Quizzes
Quiz 8.1
1. Why is phosphorus removed from wastewater?
Phosphorus is removed from wastewater because it provides a
nutrient or food source for algae. Dead
algae can cause serious oxygen depletion problems in receiving streams which in
turn can kill fish and other aquatic life.
Also, algae can cause taste and odor problems in drinking water
supplies.
2. What are the three basic forms of
phosphorus found in wastewaters?
The
three basic forms of phosphorus found in wastewaters are Orthophosphates
Polyphosphates, and Organic phosphates.
3. What must be done before polyphosphates can
be measured?
The samples must be acid hydrolyzed (or digested) to convert
the polyphosphates to the orthophosphate form for measurement.
Quiz 8.2
1. What four reagents are used to make the combined reagent for the phosphorus test? What is the storage time of this reagent?
The
four reagents that are used to make the combined reagent for the phosphorus
test are:
5 N
Sulfuric acid solution;
Potassium
antimonyl tartrate solution;
Ammonium
molybdate Solution; and,
Ascorbic
acid solution.
The
storage time is 4 hours.
2. What sample volume would you start with if the estimated phosphorus content of the sample was 1 mg/L? 4 mg/L? 10 mg/L?
20
to 30 mL for 1 mg/L
5
to 10 mL for 4 mg/L
1 to
4 mL for 10 mg/L
APPENDIX A
References
Standard
Methods for the Examination of Water and Wastewater,
18thth Edition, AWWA, APHA, WPCF; Water Pollution
Control Federation, Washington, DC, 1992.
Methods
for Chemical Analysis of Water and Wastes, U.S. EPA -
600/4-79-020, March 1979.
A
Field Study Program, Operation of Wastewater Treatment Plants,
Kerri, Kenneth et al, University of California, Sacramento.
NOTES:
APPENDIX
B
Phosphorus Reagents
Digestion
reagents:
1. Sulfuric acid digestion reagent
Carefully add 300 mL concentrated
Sulfuric acid to approximately 600 mL distilled water and dilute to 1 L with
distilled water.
2. Sodium hydroxide 1 N
Dissolve 40 g of Sodium hydroxide
pellets in distilled water. Cool and
dilute to 1 L.
Ascorbic
acid procedure:
1. Sulfuric acid solution 5 N
Dilute 70 mL of concentrated Sulfuric
acid to 500 mL with distilled water (add
acid to water, CAREFULLY).
2. Potassium antimonyl tartrate solution
Dissolve 1.3715 gm of Potassium
antimonyl tartrate in 400 mL of distilled water in a 500 mL volumetric flask
and dilute to 500 mL. Store in a glass
stoppered bottle.
3. Ammonium molybdate solution
Dissolve 20 g of Ammonium molybdate in
500 mL of distilled water. Store in a
glass stoppered bottle.
4. Ascorbic acid solution 0.1 M
Dissolve 1.76 g of Ascorbic acid in 100
mL of distilled water. Store at 4°C and
discard after 1 week.
5. Combined reagent
To prepare 100 mL of combined reagent,
combine the following reagents and mix thoroughly after each addition.
50
mL 5 N Sulfuric acid
5
mL Potassium antimonyl tartrate solution
15
mL Ammonium molybdate solution
30
mL Ascorbic acid solution
Reagents must be added in the order
listed. All reagents must be at room
temperature before mixing. If turbidity
occurs, shake the reagent and let stand until turbidity clears.
The combined reagent is stable for only
4 hours.
6. Stock Phosphate Solution
Dissolve 219.5 mg of anhydrous
potassium dihydrogen phosphate in distilled water and dilute to 1 L. This yields a concentration of 50 mg/L.
7. Standard Phosphate Solution
Dilute 10 mL of the stock solution to
100 mL with distilled water. This yields
a concentration of 5.0 mg/L.
APPENDIX C
Sample Bench Sheet
Phosphate Analysis
Sample collection Date: _______ Time: _______ By: _______
Sample preserved? yes no Preservative: ____________
Analysis run Date: _______ Time: _______ By: _______
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Sample
Type
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Initial
Volume
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Final
Volume
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Absorbance
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Conc.
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Corrected
Conc.
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APPENDIX D
Preparation of a Calibration Curve
In
several of the analyses presented in this manual, there is a need for the
preparation of a standard calibration curve.
The curve establishes the relationship between absorbance or percent
transmittance and the concentration of the desired parameter. Using this curve, samples can be tested by
the measurement of their absorbance or percent transmittance and the
corresponding concentration can be read off the graph.
Rules
for preparing calibration curves.
1. Prepare all standards in duplicate or
triplicate. Use the average of absorbance
or transmittance for preparing the curve.
2. Process the standards in the same manner as
the samples will be analyzed. If the
samples will
undergo a digestion, the standards must
be digested as well.
3. A distilled water sample (blank) must be
processed in the same manner as the standards.
This blank will be used as the reagent blank for setting the
spectrophotometer to read zero absorbance or 100% transmittance.
4. A minimum of one standard should be
processed daily to check the accuracy of the calibration curve and the quality
of the reagents.
5. When new reagents are prepared, the
calibration curve should be checked by at least 3 standards.
6. If the curve appears to differ from a
straight line, the curve may be straightened to approximate a straight line by
plotting the data on semi-logarithmic graph paper.
7. Plot data with the absorbance or percent
transmittance readings on the vertical axis and concentration on the horizontal
axis.
8. Select graph paper with appropriate
graduations. Do not use graduations
which require constant estimation of readings.
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