# 2.3: Day 3 - Preparation of Phosphate Calibration Curve and Analysis of Water Samples for Quantitation of Orthophosphate

### Experimental Background for Colorimetric Orthophosphate (PO43-) Determination30,31

Natural waters contain a combination of phosphorous compounds including soluble inorganic orthophosphates (PO4) 3-, dissolved larger types of phosphorous compounds called polyphosphates (P2O7)4- and (P3O10)5-, and phosphorous that is attached to organic matter. The exact form of the phosphate depends to some extent on the pH. The polyphosphates can all be hydrolyzed into the simpler soluble reactive form of orthophosphate. Phosphate is the principal nutrient responsible for algae growth in inland environments. One of the top problems facing our rivers is eutrophication caused directly by the excessive amounts of nutrients getting into our waterway systems. It can kill our fish and aquatic organisms, produce nasty odors along the shoreline, and impose limitations on our recreational swimming, fishing and boating. Most algae growth in rivers is a direct result of increased phosphorous dumping from municipal wastewater treatment plants, agricultural run-off, and industrial sources of pollutants. Leaves and grass clippings can be another source of phosphorous release into our rivers. The leaves and grass clippings end up along the shoreline and in gutters and are summarily washed into the river. Cutting the grass along the Charles looks quite innocent yet the clippings can have a major impact on the phosphate levels in the river. Soil erosion is another big contributor of phosphates during wind and rainstorms; the soil particles falling into the river carry with them their attached soil-bound phosphates. Presently there really are no strict regulations only a list of suggested recommendations from the EPA. Although this is changing, in November, 2009 the EPA established its first national standards containing numeric limitations on stormwater discharges.32

Phosphorous quantitation requires the conversion of the various forms of phosphorous into soluble reactive orthophosphates followed by colorimetric determination of the soluble dissolved phosphate. Samples must be collected in acid washed bottles and pre-treatment involves filtering off any suspended matter or particles. The larger solid phosphates must first be broken down into detectable orthophosphates (PO43-) as the UV-VIS colorimetric analysis of phosphorous only works for orthophosphates, the soluble inorganic form of phosphorous. The exact phosphate ions that are usually present in the river will run the gambit, the structure heavily dependent on pH, although orthophosphate is the principal form found in natural waters. pH is an important parameter for most natural waters. The river will generally show a variable pH range somewhere between 6.5 and 8.5. The larger the amount of phosphate pollution, the greater the pH. This makes sense as phosphate pollution is usually equated with increased activities such as photosynthesis and a loss of H+ ions resulting in an increase in pH. pH is generally higher during the daytime and periods of dense algae blooms and growth in the springtime. The pH of the river can also be influenced directly by discharges of municipal and industrial waste into the river. Natural rivers contain buffers to absorb sudden changes that might cause a drastic increase or decrease in pH. The natural buffers allow the pH to change slowly over time. As part of this lab we will take temperature and pH readings during our collection at the site.

To analyze the filtered river water for the presence of orthophosphate we will use a modified Molybdate Blue method that was proposed by Strickland and Parsons for Seawater in 1968.33 This involves treating the sampled water with a color developing mixture of chemicals consisting of ammonium molybdate, sulfuric acid, ascorbic acid, and potassium antimonyl-tartrate, which reacts with soluble phosphate to form a phosphomolybdic acid. The phosphomolybdic acid is then subsequently reduced by the ascorbic acid to a blue complex:

Phosphate + Molybdate  Phosphomolybdic Acid

Phosphomolybdic Acid + Ascorbic Acid →  Reduced Phosphomolybdate complex

The reduced phosphomolybdate complex can be observed at 880 nm in the near IR region using a UV-VIS spectrometer. The technique is based on the measurement of the orthophosphate, which is the soluble form of phosphorus present. Digestion of both dissolved organic as well as polyphosphate phosphorous compounds is important for determining the total P present which is sometimes referred to as phosphate or orthophosphate. It’s this soluble form of phosphate that makes itself available to organisms for growth. The concentration is assessed by the reduced molybdate-ascorbic acid complex absorbance at 880 nm. The intensity of the blue color is proportional to the concentration of phosphate present in solution. It has been shown that in dilute acidic solutions with an excess of molybdate present, Beers law is obeyed with respect to orthophosphate.

According to the Lambert-Beer law, the amount of light transmitted by an absorbing sample is given by the following equations:

$\rm % T = I / I_o = 10^{-A} \space\space\space\space\space A = \epsilon c l$

Where, the absorbance A is proportional to the concentration (c, in mol/L) of the solute, the length of the path the light travels through the sample (l, in cm), and the constant of proportionality, ε, called the molar absorptivity coefficient (L mol-1 cm-1) or molar extinction coefficient. Once the Beer-Lambert law is confirmed, a plot of absorbance v. concentration will give a straight line, the slope of the line is the molar absorptivity, (εxl). Aqueous solutions of the blue complex show absorption of light at 880 nm. The intensity of the blue color at 880 nm is directly proportional to the phosphate concentration in the solution. The solutions are analyzed with a UV-VIS spectrometer and the concentration of the orthophosphate ion is determined from a calibration curve.

Day #3: Preparation of Phosphate Calibration Curve and Analysis of Charles River Water Samples for Quantitation of Orthophosphate

### TAs Prepare Color Developing Solutions34

Prepare a 2.6M Sulfuric Acid solution by pouring 140 mL of concentrated sulfuric acid into approximately 200 mL of Milli-Q water in a one Liter volumetric flask. Dilute to 1 Liter volume with Milli-Q water. Transfer into glass storage bottles this solution should be stable for months. Sulfuric acid is extremely corrosive and can cause severe burns. This operation should be conducted in the hood with proper gloves and goggles worn at all times. Always add the acid to water never the reverse.

Ammonium Molybdate solution is prepared by dissolving 40 grams of Ammonium Molybdate tetrahydrate (NH4)6Mo7O24•4H2O into approximately 0.5 Liters of Milli-Q water in a 1 Liter volumetric flask, dilute to 1 Liter with Milli-Q water and transfer to dark amber bottles. Store the solution in the refrigerator at 4°C. Generally this solution will be stable if stored properly however, any evidence of a ppt could be an indication that the solution is breaking down and should be freshly prepared.

Potassium Antimonyl-Tartrate solution is prepared by dissolving 0.680 grams of C8H4K2O12Sb2 • 3H2O in 500 mL of Milli-Q water. The solution can be stored at room temperature and should be stable for the entire semester.

Ascorbic Acid35 solution is prepared on the day of the lab and must be used the same day. Dissolve 27.0 grams of ascorbic acid in approximately 200 mL of Milli-Q water in a 500 mL volumetric flask. Dilute to 500 mL with Milli-Q water. This solution is stable only for the duration of the laboratory and should be discarded at the close of the lab.

TAs should set up four burette dispensing stations for each of the above solutions under the hood. The solutions should be clearly labeled.

### TAs Prepare 10% HCl Solution

TAs prepare a 10% HCl solution from stock and treat BOD bottles, beakers and volumetric flasks that your students will use for the lab about 1 hour prior to the start of the lab. All treated BOD bottles and beakers should be triply rinsed with Milli-Q water and placed into the racks to dry. Racks should be brought out into the lab for students to pick up glassware that they will need at the start of lab and all glassware should be rinsed out with distilled water by students and returned to the racks at the close of lab.

### Preparation of Sample to be analyzed

Students will not go to the river until after the preparation of the Phosphate standards in this lab. Once at the river please obtain samples at the designated sampling site. The samples will be collected in 300 mL BOD bottles that have been rinsed with a 10% dilute HCl solution and finally rinsed several times with Milli-Q water.36 Bottles are then air dryed on a rack in preparation for the lab. Students will collect water samples as directed by the TAs making sure that no trapped air enters the collection bottle. Upon returning to the lab allow the water samples to sit on the lab bench for five minutes undisturbed letting the turbidity and solids settle out. Samples should be analyzed immediately before coming to room temperature. Once allowed to settle take out 50.0 mL of the collected sample and pipette 10.0 mL into five separate small beakers. These will represent the unknown samples. If these end up being too concentrated or fall outside of the standard curve below, you will need to dilute accordingly.

### TAs Preparation of Primary Standard Solution

Prepare a stock solution by taking out 1.0 mL of KH2PO4 out of a reagent grade 1.0 M potassium phosphate monobasic solution and dilute with approximately 200 mL of Milli-Q water into a 1 Liter volumetric flask. Then dilute with Milli-Q water to 1 liter.

Transfer these solutions to amber glass stock bottles and add 1 mL of chloroform to each.37

### Student Preparation of the Phosphate Working Standard Stock Solution

Using a biological 1.00 mL adjustable pipette transfer 1.0 mL from the TAs Primary Standard Solution (1x 10-3M) to a 100 mL volumetric flask previously rinsed with 10% HCl solution and several times with Milli-Q water. Bring the 100 mL flask to the mark with Milli-Q water resulting in a 1x10-5M working solution.

### Student Preparation of Diluted Phosphate Standards from Stock Working Solution

Set up on the lab bench 12 50 mL beakers. Place a few sheets of white paper under the beakers for labeling. It’s easier to mix the solutions with gentle swirling in the beakers as opposed to test tubes, which may be difficult to mix uniformly. The beakers should have been previously washed with 10% dilute HCl solution and then rinsed several times with Milli-Q water and allowed to dry. Label each beakers position on the sheets of white paper. Prepare a fresh set of Phosphate standards by diluting the KH2PO4 stock working standard solution as illustrated below:

 Volume of KH2PO4 Stock (mL) Volume of Milli-Q H2O to Add (mL) Final PO43- Concentration (µM) 0.00 10.00 A- 0.00 0.50 9.50 B- 0.50 1.00 9.00 C- 1.00 2.00 8.00 D- 2.00 4.00 6.00 E- 4.00 6.00 4.00 F- 6.00 8.00 2.00 G- 8.00

Pipette the correct aliquots of each standard + Milli-Q Water for a total volume of 10.00 mL into the first seven beakers, pipette 10.00 mL of the unknown samples from the BOD collection bottle into the next five beakers. Your 0.00 µM standard will also serve as the blank for the experiment.

### Student Prepares Color Developing Reagent38

Take a small Erlenmeyer flask to the hood area and add to the clean flask the following specified volumes of reagents in the following order (This solution should be obtained just prior to when you are going to use it) TAs will have these solutions set up in labeled burettes under the hood:

 Reagent Volume (mL) Ammonium Molybdate 5.0 Sulfuric Acid 12.50 Ascorbic Acid 5.0 Potassium Antimonyl-Tartrate 2.5 Total Volume 25.0

### Student Prepares the Samples with Addition of Color Developing Reagent

Before adding the color-developing reagent to your samples check with the TA to make sure a UV will be available for your run. UVs will be assigned to each team when they have shown the TA that they have the color developer ready to add to their samples. Now using a clean automatic pipette with a disposable tip Pipette 1 mL of the color developing solution into each of the 12 beakers including the blank. Flick the tubes gently or swirl the beakers carefully allowing the samples and reagents to mix thoroughly. Allow the solutions to sit for at least 20 minutes to fully develop the color then run the solutions in the UV. The solutions should be good as long as they are run within an hour after adding the color-developing reagent. Set up twelve 4.0 ml cuvettes for spectrophotmetric analysis and fill each cuvette with the twelve prepared samples. Measure the absorbance of each solution at 880 nm following the UV-VIS instructions in the appendix attached to this experiment. If the absorption of your unknowns does not fall within the range of your calibrated standard, prepare either a more dilute or more concentrated sample. We will be using an automatic cell changer and recording the absorbance readings in one run as prompted by the computer.