Phosphate Analysis

By: Elizabeth Abrams

In measuring phosphate, testing looks specifically for the form orthophosphate (PO₄^3-), which is the simplest form of phosphorus found in nature. This form is taken up, used, and made by organisms. To test for orthophosphate in LGNC water and beyond, an ascorbic acid method for UV-Visible Spectroscopy is used. The method used combined reagents that, when mixed with samples and standards, bind to orthophosphate forming phosphomolybdic acid which turns a molybdenum blue with the inclusion of ascorbic acid. It is with this color that UV-Vis can be utilized.

UV-Visible Spectroscopy

UV-Visible Spectroscopy, or UV-Vis, is an instrumentation method that uses select wavelengths to measure the absorbance of samples when compared to a reference solution.

Reagents

In order to get absorbance readings to determine phosphate concentrations, reagents must be used to make compounds that can produce absorbance readings. Orthophosphate alone does not absorb on the UV or Visible spectra, so reagents are used to form a colored compound that can be detected. More specifically, 5N (2.5 M) sulfuric acid and ammonium molybdate interact with phosphate present to form phosphomolybdic acid which, in the presence of potassium antimonyl tartrate, turns molybdenum blue at the introduction of ascorbic acid. The absorbance, and concentration by extension, of molybdenum blue can be used to determine the concentration of phosphate.

Standards

Figure 4: Erlenmeyer flasks containing reagent-treated standards at 0 ppm, 0.0625 ppm, 0.125 ppm, 0.25 ppm, 0.5 ppm, 0.75 ppm, and 1 ppm.

Standards are solutions with known amounts of analyte, phosphate in this case, at a range of concentrations that can be used to attribute instrument readout to analyte concentration. For the purpose of water analysis for LGNC, standards at 0 ppm phosphate (blank), 0.0625 ppm, 0.125 ppm, 0.25 ppm, 0.35 ppm, 0.5 ppm, and 0.75 ppm. All of the standards had reagent added to form the molybdenum blue with the blank (0 ppm) standard having only water and reagent. All absorbance values were corrected with the blank by subtracting the absorbance output from the blank to ensure the only absorbance being observed was the molybdenum blue from the phosphate.

Glassware Preparation

Before beginning phosphate analysis, all glassware was washed with hot 6M hydrochloric acid and rinsed with Millipore water to ensure to outside contamination.

Reagents

5N Sulfuric Acid

• 208.33 mL of 6M sulfuric acid was diluted with Millipore water to make 500 mL of 5N sulfuric acid.
• The finished solution was stored in a plastic bottle and kept with other acids.

Potassium Antimonyl Tartrate Solution (C₈H₄K₂O₁₂Sb₂ · 3H₂O)

• 0.0549 g of potassium antimonyl tartrate trihydrate was dissolved in Millipore water and diluted to 100 mL.
• The finished solution was stored in a volumetric flask.

Ammonium Molybdate Solution ((NH₄)₂MoO₄)

• 4 g of ammonium molybdate was dissolved in Millipore water and diluted to 100 mL.
• This solution took a long time to dissolve, so additional solution was made on to 500 mL scale to prepare for continued phosphate analysis.
• The finished solution(s) were stored in (a) volumetric flask(s).

Ascorbic Acid Solution

• 1.76 g of L-ascorbic acid was dissolved in Millipore water and diluted to 100 mL.
• This solution was stored and kept refrigerated in a volumetric flask and was remade fresh weekly.

Combined Reagent

The combined reagent was made by combining all of the above reagents in a specific order in specific amounts:

• 25 mL 5N sulfuric acid
• 2.5 mL potassium antimonyl tartrate solution
• 7.5 mL ammonium molybdate solution
• 10 mL ascorbic acid solution

The solution was swirled at the addition of each reagent. Once complete, the combined reagent was usable for up to 4 hours.

Standards

In order to make standards, stock solutions of 100 ppm and 10 ppm PO₄^3- concentration had to first be made. The 100 ppm solution was made using monobasic potassium phosphate (KH₂PO₄).

Dilution	Amount of Solute	Final Volume
100 ppm PO43- stock	0.0143 g of KH2PO4	100 mL
10 ppm PO43- stock	2500 µL from 100 ppm stock	25 mL
0.75 ppm standard	1875 µL from 10 ppm stock	25 mL
0.5 ppm standard	1250 µL from 10 ppm stock	25 mL
0.35 ppm standard	875 µL from 10 ppm stock	25 mL
0.25 ppm standard	625 µL from 10 ppm stock	25 mL
0.125 ppm standard	313 µL from 10 ppm stock	25 mL
0.0625 ppm standard	156 µL from 10 ppm stock	25 mL

Instrument Preparation

Before using the UV-Vis, an approximately 5 ppm methylene blue solution was used to collect an absorbance spectrum. Because there is a lot of literature that shows a consistent methylene blue absorbance spectrum, scanning the methylene blue sample proved to be a reliable way to check that the UV-Vis was calibrated and working as expected.

Calibration

To make the calibration curve, 10 mL of each standard was placed into separate 50 mL Erlenmeyer flasks, with an additional flask holding 10 mL of Millipore water to be used as the reagent blank. 1.6 mL of combined reagent was then added to each flask before all standards were left to sit for 10 minutes. Once the reagent had been added, the solutions were usable for testing for a total of 30 minutes.

Each sample, including the reagent blank, were then placed in the instrument to measure absorbance at 889.4 nm. These values were then used to produce a table and a calibration curve.

Sample Preparation and Measurement

Each sample was filtered and separated into three flasks, each containing 10 mL of each sample to allow for testing in triplicate. An additional reagent blank was also made. Each sample was then treated with one drop of phenolphthalein indicator to ensure the samples were not too basic. While these samples did not change color, 5N sulfuric acid would have been added dropwise to dispel any color if the samples had not remained colorless. Each sample was then treated with 1.6 mL of combined reagent and allowed to sit for 10 minutes. Once the reagent had been added, the samples were usable for testing for a total of 30 minutes. Samples were transferred to quartz cuvettes.

Absorbance values of all samples were measured using a Perkin Elmer Lambda 35 UV-Vis Spectrometer at 889.4 nm, and corrected using the reagent blank.