Extract from manual:
U. S. Environmental Protection Agency. 1983.
Phosphorus, All Forms. Method 365.1 (Colorimetric, Automated,
Ascorbic Acid). pp.365-1.1 -- 365-1.7. In Methods for
Chemical Analysis of Water and Wastes, EPA-600/ 4-79-020.
U.S.E.P.A., Cincinnati, Ohio, USA.
PHOSPHORUS, ALL FORMS
Method 365.1 (Colorimetric, Automated, Ascorbic Acid)
1. Scope and Application
- Total 00665
- Total Orthophosphate (P-ortho) 70507
- Total Hydrolyzable Phosphorus (P-hydro) 00669
- Total Organic Phosphorus (P-org) 00670
- Dissolved Phosphorus (P-D) 00666
- Dissolved Orthophosphate (P-D, ortho) 00671
- Dissolved Hydrolyzable Phosphorus (P-D, hydro) 00672
- Dissolved Organic Phosphorus (P-D, org) 00673
- Insoluble Phosphorus 00667
- Insoluble orthophosphate00674
- Insoluble Hydrolyzable Phosphorus 00675
- Insoluble Organic Phosphorus 00676
1.1These methods cover the determination of specified forms
of phosphorus in drinking, surface and saline waters, domestic and
2. Summary of Method
1.2 The methods are based on reactions that are specific for
the orthophosphate ion. Thus, depending on the prescribed
pretreatment of the sample, the various forms of phosphorus given in
Figure 1 may be determined. These forms are defined in Section 4.
1.2.1 Except for in-depth and detailed studies, the most
commonly measured forms are phosphorus and dissolved phosphorus,
and orthophosphate and dissolved orthophosphate. Hydrolyzable
phosphorus is normally found only in sewage-type samples.
Insoluble forms of phosphorus are determined by calculation.
1.3 The methods are usable in the 0.001 to 1.0 mg P/l range.
Approximately 20-30 samples per hour can be analyzed.
3. Sample Handling and Preservation
2.1 Ammonium molybdate and antimony potassium tartrate react
in an acid medium with dilute solutions of phosphorus to form an
antimony-phospho-molybdate complex. This complex is reduced to an
intensely blue-colored complex by ascorbic acid. The color is
proportional to the phosphorus concentration.
2.2 Only orthophosphate forms a blue color in this test.
Polyphosphates (and some organic phosphorus compounds) may be
converted to the orthophosphate form by manual sulfuric acid
hydrolysis. Organic phosphorus compounds may be converted to the
orthophosphate form by manual persulfate digestion(2). The developed
color is measured automatically on the AutoAnalyzer.
4. Definitions and Storet Numbers
3.1 If benthic deposits are present in the area being
sampled, great care should be taken not to include these deposits.
3.2 Sample containers may be of plastic material; such as
cubitainers, or of Pyrex glass.
3.3 If the analysis cannot be performed the same day of
collection, the sample should be preserved by the addition of 2 ml
conc. H2SO4 per liter and refrigeration at 4 degrees C.
4.1 Total Phosphorus (P) - all of the phosphorus present in
the sample regardless of form, as measured by the persulfate
digestion procedure. (Storet #00665)
4.1.1 Total Orthophosphate (P-ortho) - inorganic
phosphorus [(PO4)-3] in the sample as measured by the direct
colorimetric analysis procedure. (70507)
4.1.2 Total Hydrolyzable Phosphorus (P-hydro) -
phosphorus in the sample as measured by the sulfuric acid
hydrolysis procedure, and minus predetermined orthophosphates.
This hydrolyzable phosphorus includes polyphosphates [(P207)-4,
(P3O10)-5, etc.] plus some organic phosphorus. (00669)
4.1.3 Total Organic Phosphorus (P-org) - phosphorus
(inorganic plus oxidizable organic) in the sample as measured by
the persulfate digestion procedure, and minus hydrolyzable
phosphorus and orthophosphate. (00670)
4.2 Dissolved Phosphorus (P-D) - all of the phosphorus
present in the filtrate of a sample filtered through a
phosphorus-free filter of 0.45 micron pore size and measured by the
persulfate digestion procedure. (00666)
4.2.1 Dissolved Orthophosphate (P-D, ortho) - as measured
by the direct calorimetric analysis procedure. (00671)
4.2.2 Dissolved Hydrolyzable Phosphorus (P-D, hydro) - as
measured by the sulfuric acid hydrolysis procedure and minus
predetermined dissolved orthophosphates. (00672)
4.2.3 Dissolved Organic Phosphorus (P-D, org) - as
measured by the persulfate digestion procedure, and minus
dissolved hydrolyzable phosphorus and orthophosphate. (00673)
4.3 The following forms, when sufficient amounts of
phosphorus are present in the sample to warrant such consideration,
may be calculated:
4.3.1 Insoluble Phosphorus
(P-I) = (P) - (P-D) (00667)
18.104.22.168 Insoluble orthophosphate
(P-I, ortho) = (P, ortho) - (P-D, ortho) (00674)
22.214.171.124 Insoluble Hydrolyzable Phosphorus
(P-I, hydro) = (P.hydro) - (P- D, hydro) (00675)
126.96.36.199 Insoluble Organic Phosphorus
(P-I, org) = (P. org) - (P-D, org) (00676)
4.4 All phosphorus forms shall be reported as P. mg/l, to the
5.1 No interference is caused by copper, iron, or silicate at
concentrations many times greater than their reported concentration
in sea water. However, high iron concentrations can cause
precipitation of and subsequent loss of phosphorus.
5.2 The salt error for samples ranging from 5 to 20% salt
content was found to be less than 1%.
5.3 Arsenate is determined similarly to phosphorus and should
be considered when present in concentrations higher than phosphorus.
However, at concentrations found in sea water, it does not
5.4 Sample turbidity must be removed by filtration prior to
analysis for orthophosphate. Samples for total or total hydrolyzable
phosphorus should be filtered only after digestion. Sample color
that absorbs in the photometric range used for analysis will also
6.1Technicon AutoAnalyzer consisting of:
6.1.2 Manifold (AAI) or Analytical Cartridge (AAII).
6.1.3 Proportioning pump.
6.1.4 Heating bath, 50 degrees C.
6.1.5 Colorimeter equipped with 15 or 50 mm tubular flow
6.1.6 650-660 or 880 nm filter.
6.1.8 Digital printer for AAII (optional).
6.2 Hot plate or autoclave.
6.3 Acid-washed glassware: All glassware used in the
determination should be washed with hot 1:1 HCl and rinsed with
distilled water. The acid-washed glassware should be filled with
distilled water and treated with all the reagents to remove the last
traces of phosphorus that might be adsorbed on the glassware.
Preferably, this glassware should be used only for the determination
of phosphorus and after use it should be rinsed with distilled water
and kept covered until needed again. If this is done, the treatment
with 1:1 HCI and reagents is only required occasionally. Commercial
detergent should never be used.
7.1 Sulfuric acid solution, 5N: Slowly add 70 ml of conc.
H2SO4 to approximately 400 ml of distilled water. Cool to room
temperature and dilute to 500 ml with distilled water.
7.2 Antimony potassium tartrate solution: Weigh 0.3 g
K(SbO)C4H4O6 x 1/2H20, dissolve in 50 ml distilled water in 100 ml
volumetric flask, dilute to volume. Store at 4 degrees C in a dark,
7.3 Ammonium molybdate solution: Dissolve 4 g (NH4)6Mo7O24 x
4H2O in 100 ml distilled water. Store in a plastic bottle at 4
7.4 Ascorbic acid, 0.1M: Dissolve 1.8 g of ascorbic acid in
100 ml of distilled water. The solution is stable for about a week
if prepared with water containing no more than trace amounts of
heavy metals and stored at 4 degrees C.
7.5 Combined reagent (AAI): Mix the above reagents in the
following proportions for 100 ml of the mixed reagent: 50 ml of 5N
H2SO4 (7.1), 5 ml of antimony potassium tartrate solution (7.2), 15
ml of ammonium molybdate solution (7.3), and 30 ml of ascorbic acid
solution (7.4). Mix after addition of each reagent. All reagents
must reach room temperature before they are mixed and must be mixed
in the order given. If turbidity forms in the combined reagent,
shake and let stand for a few minutes until the turbidity disappears
before processing. This volume is sufficient for 4 hours operation.
Since the stability of this solution is limited, it must be freshly
prepared for each run.
NOTE 1: A stable solution can be prepared by not including
the ascorbic acid in the combined reagent. If this is done, the
mixed reagent (molybdate, tartrate, and acid) is pumped through the
distilled water line and the ascorbic acid solution (30 ml of 7.4
diluted to 100 ml with distilled water) through the original mixed
7.6 Sulfuric acid solution, 11 N: Slowly add 310 ml conc.
H2S04 to 600 ml distilled water. When cool, dilute to 1 liter.
7.7 Ammonium persulfate.
7.8 Acid wash water: Add 40 ml of sulfuric acid solution
(7.6) to 1 liter of distilled water and dilute to 2 liters. (Not to
be used when only orthophosphate is being determined).
7.9 Phenolphthalein indicator solution (5 gal): Dissolve 0.5
g of phenolphthalein in a solution of 50 ml of ethyl or isopropyl
alcohol and 50 ml of distilled water.
7.10 Stock phosphorus solution: Dissolve 0.4393 g of
pre-dried (105 degrees C for 1 hour) KH2PO4 in distilled water and
dilute to 1000 ml. 1.0 ml = 0.1 mg P.
7.11 Standard phosphorus solution: Dilute l00.0 ml of stock
solution (7.10) to 1000 ml with distilled water. 1.0 ml = 0.01 mg P.
7.12 Standard phosphorus solution: Dilute 100.0 ml of
standard solution (7.11) to 1000 ml with distilled water. 1.0 ml =
0.001 mg P.
7.13 Prepare a series of standards by diluting suitable
volumes of standard solutions (7.11) and (7.12) to 100.0 ml with
distilled water. The following dilutions are suggested:
ml of Standard Conc,
Phosphorus Solution (7.12) mg P/l
ml of Standard
Phosphorus Solution (7.1.1) mg P/l
8.1.1 Add 1 ml of sulfuric acid solution (7.6) to a 50 ml
sample and/or standard in a 125 ml Erlenmeyer flask.
8.1.2 Add 0.4 g of ammonium persulfate.
8.1.3 Boil gently on a preheated hot plate for
approximately 30-40 minutes or until a final volume of about 10 ml
is reached. Do not allow sample to go to dryness. Alternately,
heat for 30 minutes in an autoclave at 121 degrees C (15-20 psi).
8.1.4 Cool and dilute the sample to 50 ml. If sample is not
clear at this point, filter.
8.1.5 Determine phosphorus as outlined in (8.3.2) with acid
wash water (7.8) in wash tubes.
8.2 Hydrolyzable Phosphorus
8.2.1 Add l ml of sulfuric acid solution (7.6) to a 50 ml
sample and/or standard in a 125 ml Erlenmeyer flask.
8.2.2 Boil gently on a preheated hot plate for 30 10
minutes or until a final volume of about 10 ml is reached. Do
not allow sample to go to dryness. Alternatively, heat for 30
minutes in an autoclave at 121 degrees C (15-20 psi).
8.2.3 Cool and dilute the sample to 50 ml. If sample is
not clear at this point, filter.
8.2.4 Determine phosphorus as outlined in (8.3.2) with
acid wash water (7.8) in wash tubes.
8.3.1 Add l drop of phenolphthalein indicator solution
(7.9) to approximately 50 ml of sample. If a red color develops,
add sulfuric acid solution (7.6) drop-wise to just discharge the
color. Acid samples must be neutralized with 1 N sodium
hydroxide (40 g NaOH/l).
8.3.2 Set up manifold as shown in Figure 2, AAI or Figure
8.3.3 Allow both calorimeter and recorder to warm up for
30 minutes. Obtain a stable baseline with all reagents, feeding
distilled water through the sample line.
8.3.4 For the AAI system, sample at a rate of 20/hr, I
minute sample, 2 minute wash. For the AAII system, use a 30/hr,
2:1 cam, and a common wash.
8.3.5 Place standards in Sampler in order of decreasing
concentration. Complete filling of sampler tray with unknown
8.3.6 Switch sample line from distilled water to Sampler
and begin analysis.
10. Precision and Accuracy (AAI system)
9.1 Prepare a standard curve by plotting peak heights of
processed standards against known concentrations. Compute
concentrations of samples by comparing sample peak heights with
standard curve. Any sample whose computed value is less than 5% of
its immediate predecessor must be rerun.
10.1 Six laboratories participating in an EPA Method Study,
analyzed four natural water samples containing exact increments of
orthophosphate, with the following results:
Increment as Precision as -------------------
Orthophosphate Standard Deviation Bias, Bias,
mg P/liter mg P/liter % mg P/liter
-------------- ------------------ ------ ----------
0.04 0.019 +16.7 +0.007
0.04 0.014 - 8.3 -0.003
0.29 0.087 -15.5 -0.05
0.30 0.066 -12.8 -0.04
10.2 In a single laboratory (EMSL), using surface water
samples at concentrations of 0.04, 0.19, 0.35, and 0.84 mg P/l,
standard deviations were +/-0.005, +/-0.000, +/-0.003, and +/-0.000,
10.3 In a single laboratory (EMSL), using surface water
samples at concentrations of 0.07 and 0.76 mg p/l, recoveries were
99% and 100%, respectively.
1. Murphy, J. and J. Riley. 1962.
A Modified Single Solution for the Determination of Phosphate in
Natural Waters. Anal. Chim. Acta v.27 p.31.
2. Gales, M., Jr., E. Julian, and R. Kroner. 1966.
Method for Quantitative Determination of Total Phosphorus in
Water. Journ. AWWA, v.58 No.10 p.1363.
3. Lobring, L. B. and R. L. Booth. 1972.
Evaluation of the AutoAnalyzer II; A Progress Report, Technicon
International Symposium, June, 1972. New York, N.Y.
4. Annual Book of ASTM Standards. 1976.
Part 31, "Water", Standard D515-72, p.388.
5. Standard Methods for the Examination of Water and Wastewater. 1975.
14th Edition, p 624, Method 606.