Colorimetric (Spectrophotometric) Determination of Phosphate in Seawater

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1 Colorimetric (Spectrophotometric) Determination of Phosphate in Seawater Background Introduction Living things need phosphorus. Since P is normally a limiting reagent for life, changes in its spatial distribution can lead to important shifts in populations. In this laboratory you will determine the amount of phosphate in a sample of seawater by using a colorimetric method. Phosphorus occurs in several forms. orthophosphate or PO 4 3- condensed phosphates O 3 P-O-(PO 3 ) X -PO 3 organic phosphates, (RO) X PO 4-X. Here R represents an organic group such as CH 3 and X is an integer from 0 to 4. To understand the underlying similarity of these species, draw their Lewis structures. Our analytical method works only for the orthophosphate anion; it does not detect "phosphate" in other forms. For our purposes, this characteristic of the method does not matter much because the concentrations of condensed and organic phosphates are usually very low in sea water. Nonetheless, careful environmental scientists and oceanographers often take pains in the laboratory to convert all phosphorus to orthophosphate (which we will not do) and then report a quantity called P-PO When read aloud, the words are: "phosphorus as phosphate." The quantity has units of mg P/(liter solution) and it reminds us that the orthophosphate measured may include phosphorus that originally had a different form in the sample. In this laboratory, for simplicity, we will work with molarity of orthophosphate until the very end and only then convert to the environmentalists' P-PO The conversion is shown below P-PO 4 3- (mg P/(liter solution)) = Molarity PO 4 3- x 1 mole P/(1 mole PO 4 3- ) x g P/(mole P) x 1000 mg/g = x Molarity PO 4 3-2/14/07 1

2 Chemistry In an acidic environment, ammonium molybdate and antimonyl tartrate combine with any phosphate (PO 4 3- ) present to form an antimony-phosphate-molybdate complex. Reduction of this complex using ascorbic acid produces a deep blue complex with a complex structure that is not well understood to this day. The concentration of the complex can be determined by measuring its absorbance at a wavelength of 880 nm by using an instrument called a spectrophotometer. Specifically, the absorbance of the unknown is compared with the absorbances of solutions of known concentrations. Samples Overview of the laboratory procedures The concentration of phosphate in seawater is too low to measure directly. For this reason, you will concentrate the phosphate (in each of two samples of filtered sea water as explained below) by precipitating it and re-dissolving it in a smaller volume. After chemical treatment, each solution should develop a pleasing blue color. You will measure the absorbance at 880 nm with an instrument called a spectrophotometer. To translate the measured absorbance into a concentration, you will make up several standards containing known concentrations of phosphate, treat them to make the blue complex, and then measure their absorbances. You will also prepare what is known as a procedural blank, that is, a control sample that should contain no or very little phosphate. In sum, during the course of the laboratory you will measure the absorbance of the following solutions. Time-line 1. Two samples of filtered sea water; these samples are your unknowns 2. One procedural blank sample; this sample will tell you whether the chemical procedures introduce any extraneous phosphate; the procedural balnk will be used with the seawater samples. 3. Standards; these samples will enable you to translate measured absorbances into concentrations; one of the standards has no added phosphate and is called a reagent blank, which is different from a full procedural blank. The reagent blank will be used with the standards. Preparation and Acid Washing of Glassware: 30 to 60 minutes Part 1: Co-precipitation (fast), centrifugation (about 1 hour), re-dissolution (fast), and evaporation of solvent of phosphate in samples 1, 2, and 3 (about 1 hour - use this time to carry out most of part 2) Part 2: Preparation of standards (about 1 hour) and measurements of absorbance (about 45 minutes) 2/14/07 2

3 Part 3: Absorbance measurements for sea water samples and procedural blank (about 45 minutes) This schedule allows about 30 to 60 minutes for preparation and clean up, but be prepared, unless you are well organized, time will be very tight. You will also benefit from studying the (separate) instructions for operation of the spectrophotometer. Because of time restraints, work with a partner so that you can divide the different tasks. Maximize your efficiency by working out the details with your partner. Working with a partner will prepare you to work in a team environment. Some of you may work in a different lab since your work has to be performed in the hood. Genreally, there are two hot plates per hood. Reagents Procedure Sodium hydroxide solution, 6M NaOH Mg 2+ solution for procedural blank, ~1 mg Mg 2+ / L Standard phosphate solution 8.07 x 10-5 M PO 4 3- = 2.5 mg / L P-PO 4 3- (the exact number will be provided to you in the lab. Please leave space in your lab notebook so that you do not forget to write down the concentration of stock solution. Record the dilution factor. Combined Reagent contains the following solutions in the volume ratios 10:1:3:6 Sulfuric acid, 2.5 M Ammonium molybdate, 20 g (NH 4 ) 6 Mo 7 O 24 / 500 ml Antimonyl potassium tartrate, g K(SbO)C 4 H 4 O 6 / 500 ml Ascorbic acid, 1.76 g ascorbic acid / 100 ml Combined reagent will be available about 1.5 hours after the start of the laboratory. Try calculating the molarities of the substances in the combined reagent. 2/14/07 3

4 Equipment Ultraviolet-Visible (UV) spectrophotometer - To be turned on about 30 minutes in advance of measurements. Centrifuge Volumetric glassware Standard laboratory glassware 100 ml pipet Automatic pipet Contamination Because of the affinity of phosphate for glassware and the fact that you are working with very low concentrations of phosphate in your sample, it is necessary to acid-wash your glassware to remove any phosphate already present. You will need to acid-wash the following items Two 100-mL volumetric flasks Three 10-mL volumetric flasks Three 250-mL beakers; label them 1, 2, and 3 followed by your initials Three 100-mL beakers; label them 1, 2, and 3 followed by your initials Six 15-mL plastic centrifuge tubes; label them 1a, 1b, 2a, 2b, 3a, and 3b followed by your initials Six 15-mL test tubes; label them A, B, C, D, E, and F followed by your initials Two 50-mL beakers labeled, respectively, "PO 4 3- " and "Combined Reagent" followed by your initials (You can dry the beakers in the oven) Procedure for Acid Washing of Glassware Set up your glassware and rinse them with the following: Rinse with ~25 ml of 8M HNO3, swirl, discard Rinse twice with Deionized Water Rinse with ~25 ml of 1M HCl, swirl, discard Rinse four to six times with Deionized Water To work efficiently in the lab, you can use the same rinse for all your glassware. Rinse one item and then use the wash for the next item etc Discard the waste per your lab protocol. Part 1. Co-precipitation, centrifugation, re-dissolution, and evaporation of phosphate in samples 1, 2, and 3 Scavenging phosphate by co-precipitation 2/14/07 4

5 Rinse a 100-mL volumetric flask with 5-10 ml of Millipore-filtered seawater. Invert the volumetric flask a couple of times to make sure the flask is completely rinsed. Fill to the mark with Millipore-filtered seawater and transfer it to 250-mL beaker 1. To make sure transfer is complete, rinse the volumetric flask with ~ 5 ml of de-ionized water and transfer the rinse to beaker 1. Repeat this procedure with the same 100-mL volumetric flask, but transfer the filtered seawater sample to 250-mL beaker 2. Rinse the second 100-mL volumetric flask with 5-10 ml of the solution containing 1 mg/ml of Mg 2+. Shake dry. Fill to the mark with 100 ml of the solution containing 1 mg/ml of Mg 2+ and transfer it to beaker 3. To make sure transfer is complete, rinse with ~ 5 ml of de-ionized water and transfer the rinse to beaker 3. This solution will be the procedural blank. Ordinarily, this level of care should not be necessary in preparing a procedural blank. Why not? To each of these three beakers add 20 ml of the 6N NaOH solution and mix thoroughly. A white precipitate will begin appearing immediately. What is the precipitate? This fine and gelatinous precipitate is most easily recovered through centrifugation. After waiting five minutes for the precipitate to form, begin centrifuging the solutions. Assign centrifugation tubes 1a and 1b to beaker 1, 2a and 2b to beaker 2, and 3a and 3b to beaker 3. Swirl the beakers to have uniform suspension. Fill centrifugation tubes 1a and 1b to a level approximately 1 cm below the top of the tube with liquid and solid from beaker 1; place the tubes at opposite positions in the centrifuge - but don't turn on the centrifuge yet. Why is it important that the volumes of solutions in the two centrifuge tubes that occupy opposite positions be nearly the same? Repeat for the liquids/solids in beaker 2 and in beaker 3. You will now have six labeled tubes in the centrifuge. Did you remember to label the centrifugation tubes? Centrifuge for two to three minutes. Let the centrifuge slow down by itself - you're more likely to lose time than gain it by stopping the rotor manually. Have ready three labeled plastic transfer pipets (the transfer pipets are located in the kit that you received from the stockroom). We suggest that the labels match the labels on 2/14/07 5

6 the beakers. The idea here, of course, is that you will use one and only one plastic transfer pipet for each sample. The liquid above the precipitate is called the supernatant. By using a labeled plastic transfer pipet, carefully pipet and discard the supernatant liquid from matched pairs of centrifugation tubes. Note: If the precipitate is so easily disturbed that you cannot transfer much of the supernatant without losing the precipitate, then increase the centrifugation time to 4 minutes. With care, you should be able to get the level of the liquid down to within a few mm of the level of the precipitate. After removal of the clear supernatant, refill each tube by continuing to transfer the solution and precipitate from the appropriate beaker and centrifuge again. Between uses of the plastic transfer pipets be sure to put them down in a way that avoids contamination. One way is to lay them across the top of a (clean) beaker. Continue with this procedure, being careful to keep track of which samples go where, until you have centrifuged all the solution in each beaker and have all the precipitate collected in the centrifugation tubes. Rinse each beaker with some deionized water to ensure quantitative transfer all of the precipitate to the centrifugation tube for the final centrifugation. If all goes well, the centrifugation will take about one hour. Redissolution of phosphate To each centrifuge tube, add ~ 2 ml of concentrated HCl (check the label carefully, make sure you use concentrated HCl). For this purpose, we recommend that you "calibrate" a clean plastic transfer pipette by filling it with 2 ml of de-ionized water (from, say, a graduated cylinder) and marking the level of the water on the plastic pipet. Using the appropriate plastic transfer pipet from part 1 (not the one you used for transferring HCl), mix each solution until the precipitate completely dissolves. When dissolution is complete, transfer each matched pair of solutions to the appropriately numbered 100 ml beaker using a little (~ 1 ml) deionized water as a rinse. You should now have three solutions in labeled 100 ml beakers. Place these beakers on the hot plates under the hoods. There are two hot plates per hood. If the hood is not turned on, ask your instructor to turn on the hood. It is necessary to evaporate these solutions to dryness without loss of material, which means that temperature should be low enough to avoid boiling over or splattering of the solution. At an appropriate temperature, the evaporation will take about one hour. 2/14/07 6

7 Caution: Please be careful with the boiling. When the solution is close to boiling, it can splatter. During this time begin work on Part Two. Part 2. Preparation of the calibration curve 1. In separate, clean beakers, obtain 15 ml of the phosphate standard solution (mg / L P-PO 4 3- ) and 25 ml of the "combined reagent." (You may have to take a clean and dry beaker with a watch glass to the stockroom personnel to obtain your standard phosphate solution. Set up the six test tubes in a rack. Using an automatic pipet (Finnpipet that you used before), measure out each of the following volumes into one of the test tubes. 2. A) 0.0 ml standard, 8.6 ml de-ionized water = reagent blank (not to be confused with procedural blank) B) 0.5 ml standard, 8.1 ml de-ionized water C) 1.0 ml standard, 7.6 ml de-ionized water D) 2.0 ml standard, 6.6 ml de-ionized water E) 3.0 ml standard, 5.6 ml de-ionized water F) 4.0 ml standard, 4.6 ml de-ionized water Please read this paragraph all the way through before you begin following the instructions. Using the automatic pipet, add 1.4 ml of the combined reagent to the reagent blank and to each standard and mix thoroughly. Note the total volume is 10.0 ml. Record the time at which you add the combined reagent. Space the additions in intervals of about 5 minutes so that later, you will be able to measure the absorbance of each sample after a uniform time has elapsed from mixing to measurement. 3. Zero the spectrophotometer by using distilled water. Save the cuvet with the distilled water so that you can reuse it. (Consult the separate instructions for operating the spectrophotometer.) 4. Wait min after mixing for the color to develop. Measure the absorbance of each solution between 15 to 20 minutes after mixing. Record the time at which you measure the absorbance and enter the data in your lab notebook in a Table similar to the following Table. 5. Re-zero the spectrophotometer between readings by using de-ionized water. Test Tube # A (Reagent Blank) B C D E F Clock Time When combined reagent is added Time Absorbance is Measured Absorbance Corrected Absorbance For Reagent Blank 0 2/14/07 7

8 6. Prepare a table in your notebook that records the concentration, the absorbance and the absorbance corrected for reagent blank for each of the calibration standards. 7. To make sure you are on track, sketch roughly the calibration curve by plotting in your notebook reagent blank-corrected absorbance against concentration for each of the standards. Part 3. Absorbance measurements for sea water samples and procedural blank By now your samples should be dry enough to continue, that is, white and not just translucent. When this point has been reached, redissolve each sample in a minimum of deionized water (~3-5 ml) and transfer it quantitatively to an acid-washed, 10 ml volumetric flask (The stockroom will provide you with 10 ml volumetric flasks if you do not have them in your drawer). Noting the time and using the automatic pipet, add 1.4 ml of combined reagent to the first flask. Dilute to the 10 ml mark with deionized water, swirling to ensure good mixing. Space the additions in intervals of about 5 minutes so that later, you will be able to measure the absorbance of each sample after a uniform time has elapsed from mixing to measurement. To record the data, your Table may look as follows: Sample Procedural Blank Seawater #1 Seawater #2 Time Combined Reagent is added Time Absorbance is Measured Absorbance Corrected Absorbance For Procedural Blank 0 Measure the absorbance of each solution. The perceptive student will have noted that the sample and standard solutions are not prepared in identical ways in this laboratory. The samples are brought to 10 ml in volumetric flasks (the right way to do things) while the standards are brought to volume with the automatic pipet. We have compromised on accuracy here because the volumes of different kinds of solutions are not strictly additive. For example, 1 ml of M NaCl and ml of distilled water will not make exactly ml of 0.5 M NaCl. For the best accuracy, you would have made all the samples by using just one volumetric flask. Unfortunately, to do so, you would also have had to acid-wash that flask 9 times, which would have taken too much time. 2/14/07 8

9 Report Your report should include: 1. Abstract. Maximum 100 words. Include the calculated average concentration of phosphate in sea water both as a molarity (mol PO 4 3- /L sea water) and as the environmentalists' P-PO 4 3- (mg P/(liter sea water)). Please be careful with units: there will be penalties for leaving out these concentrations out and for using the wrong units. Please follow the instructions to get full credit. 2. Introduction. Maximum 3 sentences. State the purpose of the measurement. 3. Experimental methods. None. 4. Results and discussion. About 1.5 pages. In preparing your report, you should by all means use the spreadsheet that you developed for your pre-lab assignment. Please note, however, that the concentration of phosphate in the solution placed in the spectrophotometer is NOT the same as its original concentration in the seawater. The original concentration in the seawater was smaller by a factor that you can calculate from the procedure. HINT: The original concentration of phosphate in the seawater can be found by taking the molarity of phosphate in the solution placed in the spectrophotometer and dividing this value by 10. You recall the phosphate was originally in a 100 mlvolumetric flask, but when the absorbance was measured, the phosphate was contained in a 10 ml volumetric flask. Specific items that belong in this section are listed below. Again, you will have done virtually all this work for your pre-lab and you should be able to adapt it for your report. Make generous use of sub-headings to let the reader know the content of each subsection. A table showing the data for the standards. The table should identify each standard and include the concentrations, the measured absorbances, and the absorbances corrected for reagent blank. A plot for standards showing absorbance corrected for reagent blank vs. molarity of phosphate. Comment on the shape of the plot. The plot should be a best-fit straight line. The results of least squares fit to the data for standards and in particular a calculation of the molar absorptivity coefficient. A table showing the data for samples. The table should identify each sample and include the absorbances, and the absorbances corrected for procedural blank. A calculation of the molarity of phosphate in the two samples of seawater as measured, i.e., after concentrating the solution. Use the results of the least squares fit and the absorbances corrected for procedural blank for this purpose. A calculation of the original concentration of phosphate in your seawater sample. 2/14/07 9

10 Grading of Lab Report Please type your lab report and submit a stapled hardcopy to your instructor at the beginning of the next lab period. Please write your name on every page. Your lab report will generally be graded based on the following: 1. Abstract: report best estimate of phosphate in mol/l and P-phosphate in mg P/L 2. Introduction 3. Sample calculation for concentrations of the standards 4. Table entries of concentration, absorbance and corrected absorbance of the standards 5. Table entries for absorbance and corrected absorbance of the seawater 6. Plot of corrected absorbance vs concentration of standards 7. Regression analysis output 8. Best fit straight line plotted 9. Molar absorptivity calculation 10. Average molar absorptivity reported 11. Calculation of molarity of phosphate in prepared seawater samples 12. Calculation of phosphorus, mg P/L in prepared seawater samples 13. Calculation of molarity of phosphate in original seawater 14. Calculation of phosphorus, mg P/L in original seawater Please make sure all the above information is included in your lab report. For ease of grading, include a grading grid on your cover page. Please type and paginate your lab report and submit a stapled hardcopy to your instructor at the beginning of the next lab period. Please write your name on every page. Your lab report will generally be graded based on the above information. Please make sure all the above is included in your lab report. Make sure your answers have the correct number of significant segues and the correct required units. Presentation, your understanding, discussion and explanation and accuracy are taken into account when reports are graded. You may receive bonus points or lose points when appropriate. 2/14/07 10

11 Grading Grid Prepare a grading grid on the cover sheet of your report similar to the following table:. Name Sec No. Spectrophotometric Det of Phosphate 1 Abstract 2. Sample Calculation for Concentration of the Standards 3. Table Showing the Data for Standards 4. Plot of Corrected Absorbance vs. Concentration of Standards 5. Regression Analysis Output 6. Best Fit Straight Line Plotted 7. Molar Absorptivity Calculation 8. Average Molar Absorptivity Reported 9. Calculation of Molarity of Phosphate in Prepared Seawater Samples 10 Calculation of Phoshorus, mg P/L in Prepared seawater samples 11. Calculation of Molarity of Phosphater in Original Seawater 12. Calculation of Phosphorus mg/l in Original Seawater 13. Answer to Questions etc. TOTAL Max Points Please make sure all the above is included in your lab report. Make sure your answers have the correct number of significant figures and the correct required units. Presentation, your understanding, discussion, and explanation and accuracy are taken into account when reports are graded. You may receive bonus points or lose points when appropriate. 2/14/07 11

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