According to Beer’s Law, A=Ebc, under ideal conditions, a substance’s concentration and its absorbance are directly proportional: a high-concentration solution absorbs more light, and solution of lower concentration absorbs less light. Since concentration and absorbance are proportional, Beer’s Law makes it possible to determine an unknown concentration of phosphate after determining the absorbance.
The overall goal of this lab was to make a calibration curve with a plot of absorbance vs. concentration and be able to determine the phosphate concentrations in samples of cola, surface water, and other aqueous solutions of interest.
The procedure of this lab was obtained from the student’s laboratory course website or manual.
Table 1: Absorbance vs. Concentration
|Concentration||Absorbance (at 690 nm)|
|2.00 x 10-5 M||.906|
|5.00 x 10-5 M||.969|
|1.00 x 10-4 M||1.05|
|2.00 x 10-4 M||1.41|
|5.00 x 10-4 M||2.37|
Graph 1: Absorbance vs. Concentration Calibration Curve
Table 2: Dilution of Phosphate Stock Solution
|Concentration||mL Phosphate diluted in 100 mL flask|
|2.00 x 10-5 M||2|
|5.00 x 10-5 M||5|
|1.00 x 10-4 M||10|
|2.00 x 10-4 M||20|
|5.00 x 10-4 M||50|
Table 3: Absorbance of Solutions at 690 nm
|Solution||Absorbance (at 690 nm)|
|Unknown Water Sample||0.361|
100 mL of 1.00 x 10-3 phosphate solution was used to prepare five standard solutions with known phosphate concentrations. 5.00 mL of each phosphate solution were added to separate small beakers, and then 1.00 mL of ammonium molybdate solution and 0.40 mL of aminoaphtholsulfonic acid reagent were added to each beaker. After 5 minutes, the absorbances at 690 nm were measured using a spectrometer.
A calibration curve displaying Absorbance vs. Concentration was created using Excel by using the increasing concentrations of the five standard solutions for the x values, and their corresponding absorbances for the y values. In Part 2, a small amount of Cola was heated in a beaker covered with a watch glass to reduce evaporation.
Once cooled, a sample of the soda was diluted to 50-fold with ultra-pure water by combining 2 mL soda and 100 mL of ultra pure water, and then 5.00 mL of that diluted soda pop was delivered to a large test tube. 1.00 mL of ammonium molybdate reagent and 0.40 mL of aminonapthosulfonic acid were also added to the test tube, and after 5 minutes, the absorbance was measured.
The calibration curve created in Part 1 was used to solve for the phosphate concentration in soda pop. In Part 3, the same procedure was used as in Part 2 to determine the phosphate concentration in an unknown solution. However, the solution wasn’t boiled in Part 3 since there wasn’t any carbonation in the unknown. It also was not diluted because it was a water sample.
When only the molybdate binds with phosphate, it turns the solution blue, indicating the presence of PO43-. The linear relationship between absorbance and concentration displays that absorbance depends on the concentration. Beer’s Law, A=Ebc, helped to develop the linear equation, since absorbance was equal to y, Eb was equal to m, and the concentration, c, was equal to the slope, x, in the equation y=mx+b.
To calibrate the spectrometer, a solution containing 5.00 mL of water, 1.00 mL of ammonium molybdate reagent, and 0.4 mL of aminonapthosulfonic acid was used as a blank. Since the species of interest was the phosphate, everything but the phosphate was used in the blank, and subtracted from the measured absorbance of cuvette containing a phosphate solution.
The results were not quite as expected, since the data was askew due to a great amount of experimental error in Part 1 of the lab. This error occurred from not adding the correct amount of solutions to each beaker, throwing off the absorption rate and then the calibration curve. The absorbances of each of the five solutions being wrong also affected the linear equation obtained in Part 1, which made R2 not be as close to the expected value of 1.
The linear equation being not a perfect straight line also affected the determination of concentration of phosphate in the Cola in Part 2. The impact of this experimental error in Part 1 affected the rest of the lab, not allowing for perfect results.
In this experiment, a calibration curve was created by plotting absorbance vs. concentration in Excel. The calibration curve was constructed by measuring the absorbance rate of phosphate in five standard solutions.
The linear equation derived from the calibration curve was then manipulated and used to determine the concentration of phosphate in soda pop, and in an unknown water solution. The concentration of phosphate was experimentally determined to be 0.006834 M in Cola, and 1.41 x 10-4 M in an unknown water sample.