Chem 223: Experiment 10

Spectrophotometric Determination of Total Phosphate in Water

Reference
1. Harris. 3rd Edition: Ch. 18, Sect. 2-4
2. Harris. 2nd Edition: Ch. 17, Sect. 2-4
3. Shimadzu Application News- Spectrophotometric analysis. No A 354
4. J, Murphy and J. P. Riley. J. Mar. Biol. Ass. U.K. (1958) 37, 9-14
5. Standard method for the examination of water and waste water (1997). 4500-
P- Phosphorous.
6. C. C. Kircher and S. R. Crouch, "KINETICS OF THE FORMATION AND
DECOMPOSITION OF 12-MOLYBDOPHOSPHATE," Anal. Chem. 55(2),
242-248 (1983).

I. Purpose of the experiment:

Phosphorus, widely found in various products such as fertilizer, agricultural pesticides, dyes,
processed food, and alkaline detergent, has greatly enriched our lives. However, it also
contaminates environmental waters by flowing into rivers and lakes along with residential and
industrial waste water, or agricultural runoff. Phosphorous and nitrogen, which enhance the
growth of plankton in lakes, are used as indices of eutrophication. Consequently,
measurement of phosphorus concentration is important for water quality management.
Phosphorus occurs in 3 compounds in natural waters:
• inorganic, dissolved ortho-phosphate
• dissolved organic phosphorus compounds
• particulate phosphorus (bound in biomass or attached to particles),
which add up to the total of phosphorus content P-Total, an important parameter in
monitoring wastewater treatment plant effluents.
In this experiment, students will become familiar with the two standard approaches for the
determination of anions using spectrophotometry.

II. Introduction

There are two common spectrophotometric methods available for determining phosphate or
phosphorus concentrations:
• Molybdenum blue method
• Vanadate/molybdate method (yellow method)
Both techniques are based on the measurement of ortho-phosphate. Digestion of both
dissolved organic as well as particulate phosphorus compounds is therefore mandatory for
determining the total P content. In addition, an unfiltered sample must be acquired in order to
include all solid matters in the digestion process. Digestion is usually performed by heating
the sample with peroxodisulfate and sulfuric acid.

Inorganic phosphorus as orthophosphate, PO43-, is analyzed by reacting the phosphate with

ammonium molybdate under acid conditions to form a heteropoly acid, molybdophosphoric
acid. In the presence of vanadium, yellow vanadomolybdophosphoric acid (max=690 nm) is
formed. The intensity of the yellow color is proportional to the phosphate concentration
present in the unknown sample. The analysis will be performed on a double-beam
spectrophotometer.
In order to improve the sensitivity for phosphate, ascorbic acid is added to reduce Mo(VI) to
Mo(V) and blue vanadomolybdophosphoric acid ( max= 880 nm) is formed. In retreatment of
the unknown samples, in addition to the above, a high-pressure steam sterilizer is used to
decompose substances in the solution that contain phosphorus (P) under high temperature and
high pressure, so that all the phosphorus is in the form of phosphate ions. (This process is
unnecessary for the phosphorus standard solutions used to generate the calibration curve.)
“Total phosphorus” expresses the total amount of phosphorus (P) contained in the compounds
in an aqueous solution, and is measured and expressed in the form of phosphate ions.

III. Chemical Reagents

Provided:
1. Mixed Color Reagent (vanadate molybdate color reagent): Dissolve 3.60 grams of
sodium vanadate in 800 ml of cold water, then add 48.0 grams of sodium molybdate
dehydrate. Filter if necessary and dilute to 1 liter.
2. Ammonium molybdate - ascorbic acid mixed solution: Mix thoroughly 125 mL of 2.5M
sulphuric acid and 37.5 mL of 4% ammonium molybdate solution. Add 75 ml of ascorbic
acid solution and dilute to 250 mL. The mixed reagent does not keep well and should be
prepare within an hour of using.
3. Potassium peroxodisulfate solution (40g/L)
4. Water sample with unknown phosphate

To be prepared: All glassware used in this experiment should be washed with phosphate free
MICRO cleaner, then rinsed with: DI water, 0.1 M HCl (prepared by mixing about 4 mL
of 6 M HCl with about 250 mL water), and again with DI water.

Preparation of standard phosphate stock solution

(1) Weigh about 0.1 gram of KH2PO4 to the nearest 0.0001 g and dissolve in 500.0 mL
water (volumetric flask).
(2) Calculate the ppm P in this stock solution to the appropriate number of significant
figures.

IV. Experimental Procedure

IV.A. Determination of phosphate in yellow heteropoly acid (using calibration curve)

1. Preparation of standards for the calibration curve:

(1) Pipet 0.25; 0.5; 1.25, and 2.5 mL aliquots of the phosphate stock into separate 25.0 mL
volumetric flasks then dilute to the mark with DI water.
(2) Calculate the ppm P in each standard to the appropriate number of significant figures.

2. Preparation of yellow vanadomolybdophosphoric acid

(1) Pipet 25 mL aliquots of each standard, the unknown sample (group members will
analyze their own samples), and DI water into separate 50 mL volumetric flasks. The
flask containing the DI water serves as the blank.
(2) Pipet 10 mL of the vanadate molybdate color reagent into each flask, including the
blank.
(3) Dilute each flask to the mark with DI water, mix well, then wait ten minutes for color
development to occur.
Caution: the color reagent contains NH4VO3 , a suspected mutagen. Wear gloves
when pipeting the color reagent.
3. Spectrophotometric analysis.

(1) Be sure the UV 1601 spectrophotometer is turned on at the beginning of the lab period
to provide enough time for warm up.
(2) In the “Scan" mode, under “Setup”, set the parameters as per Teaching Assistant’s
instructions. Zero the instrument with blank in both cuvettes, and then obtain a spectrum
from 700 nm to 300 nm of the most concentrated standard. Find the wavelength
corresponding to the maximum absorbance. Obtain a printout of the spectrum.
(3) In the "Concentration" mode set the wavelength to the maximum wavelength
recorded in step (2). Set all of the other parameters in Setup according to the Teaching
Assistant’s instructions. The exact concentrations of the standards will have to be
measured. Choose the “linear direct” fit type for the least square analysis. This will force
the curve through zero. Zero the instrument as in step (2). Then, run the standards and
unknowns.
(4) Print out the results and calibration curve.

4. Data analysis

The computer analyzed the data from the standards using a "Beer's Law" fit, i.e. no
intercept. The computer uses the absorbance for the unknown sample to calculate the
concentration of phosphorus in the unknown. You need to do your own linear regression
analysis using the absorbance data for the standards and their concentrations. After you
calculate the slope (m) and the intercept (b), calculate the phosphorus concentration in the
unknown. Thus, you should have two calibration curves and two values for the
phosphorus concentration: one from the computer and the other from your own linear least
square analysis.

Students should also measure the blank samples 10 times and find out the LOD and LOQ
based on the 3 rule.

5. Determining Dynamic Range

Students need to establish the limit of linearity (LOL) in concentration. This quantitation
could be performed using the UV-1601 should be determined based on the calibration curve
generated from measurement of the phosphorus standard solutions (the estimated results from
the calibration curve result were at about 0.05mg/mL- 3mg/mL; the slit width was set to 5 nm,
and a 10 mm square cell was used). In this case, unknown samples were not measured.

IV.B. Determination of Total phosphate in blue heteropoly acid (using a second standard
method)
(1) Take six decomposition flasks
(2) Transfer V(ml) (V< 40 ml) of unknown sample to each decomposition flask,
(3) Transfer phosphorus standard solution (P: 5ppm) of V’ mL (0mL, 0.5 mL, 1.25 mL,
2.5 mL, 5.0 mL and 10 mL respectively) to each decomposition flask, and add
(50-V-V’) mL of deionized water to these decomposition flasks, respectively.
(4) Add 10mL of potassium peroxodisulfate (40g/L), stopper and mix.
(5) Place in a high-pressure steam sterilizer, heat to about 120°C and continue
decomposition heating for 30minutes.
 (6) Remove the decomposition flask, let it cool, transfer 25mL of supernatant to a stopper-
equipped test tube.
(7) Add 2mL of ammonium molybdate – ascorbic acid mixed solution, shake and set aside
for about 15 minutes at 20 - 40°C.
(8) Transfer a portion of solution to an absorption cell, and measure absorbance near
880nm.
(9) As a blank, use 25mL of water, perform steps (6) and (7), measure absorbance, and
use this value to correct the absorbance values of the samples (subtract the blank
value).
(10) The concentration value is determined by applying 6/5 (correction from (3)) to the
concentration obtained from the additional calibration curve.

FOR YOUR REPORT

- Report all the requirements in each experiment.
- Think about the interferences to the determination of phosphate in 2 methods.
- Figure out the main advantages of blue heteropoly acid compared to yellow
heteropoly
- List the substances can be used for reduction of yellow heteropoly acid to blue one