Bergeron

Laboratory Exercise 1: Spectrophotometry

Joshua Bergeron 9-5-2013

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Introduction: This lab exercised the use of a spectrophotometer in the determination of the unknown concentration of a known solution. The Genesys 10 spectrophotometer has the capability of determining a reference wavelength and generating a standard curve based on an analysis of known concentrations of a solution. This exercise is relevant to any accurate determination of the concentration of a solution. The methodology in this lab is an efficient and accurate method for determining an unknown concentration in a known solution. Procedure: Step 1) Determine a Reference Wavelength a. b. c. d. e. f. g. h. i. Prepare a blank dH2O and a sample of 0.1M solution Insert them into Genesys 10 Spectrophotometer Press [TEST]; scroll to Scanning & press [ENTER] Select stored tests; scroll to Scan(PAR); select Load Test Ensure it will scan from 400-700 nm Set to read the blank; select Collect Baseline Select Measure Sample; select Edit Graph: Math: Peaks: & Valleys Print graph; select tabular & print table The reference wavelength corresponds to the largest absorbance value

Step 2) Determine a Standard Curve a. Prepare 5 samples from 0.1 M to 0.005 M of known solution a) Vs = Volume of stock solution required to reach target molarity b) Vs = (0.002 L (0.0075 M))/0.100 M = 1.5 mL c) Vs = (0.002 L (0.0050 M))/0.100 M = 1.0 mL d) Vs = (0.002 L (0.0025 M))/0.100 M = 500 L e) Vs = (0.002 L (0.0005 M))/0.100 M = 1oo L b. Press [TEST]; scroll to Standard Curve; press [ENTER] a) Set appropriate test parameters c. Select Run Standards & key in the concentrations of samples 1-5 determined above d. Select Measure Standards; print absorbance, slope, and intercept Step 3) Determine the unknown concentration from the known solution a. press [ESC] to exit b. Set the reference wavelength and measure the absorbance of the sample c. Compare the absorbance to the standard curve in order to estimate the unknown concentration

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Results: Wavelength 400 produced the highest absorbance value of 0.289 and was used as the Reference Wavelength. A standard curve with a standard deviation of 0.012 was produced with a slope of 3.38 The unknown sample was measure at 400nm and yielded a measured absorbance of 0.187 Based on the standard curve results, the unknown concentration is estimated to be 0.050 M See Attached Lab Results Discussion: My initial experiment resulted in errors within my standard curve. After repeating the experiment, I realized I had failed to fill the blank cuvette with a blank sample. This prevented a proper determination of absorbance within my standard curve. I find this procedure to be highly efficient and accurate. Once the error in my method was corrected, I was able to follow the procedure and determine the unknown concentration. Questions: 1) What was the point of blanking the spectrophotometer with dH2O? dH2O is free of anything that would alter absorbance. Its important to use a blank that only lacks the element you are trying to measure. 2) If you wanted to measure the absorbance of a sample of natural seawater, what would you suggest using as your BLANK? What potential sources of error would be inherent in the method you suggest? Be specific. I would attempt to produce a blank that only lacked the element I was trying to measure. If it was a chlorophyll determination, I would either filter the seawater or attempt to create artificial seawater. The difficulty arises in the recreation of natural seawater- even artificial seawater doesnt accurately represent the content of any one random natural seawater sample. Your method for producing an appropriate blank depends entirely on which element within natural seawater you are measuring the absorbance of. 3) Take a closer look at Equation 1 as it relates to Beers Law. Which would have a more significant impact on absorbance: molecular abundance (concentration) or the distance through which the light beam must pass (path length)? Explain your rationale. If A=lc then either a change in l (pathlength) or c (concentration) would effect absorbance. They represent an equal ratio in the equation. 4) When you were calculating the concentration of your unknown (using your standard curve), why were you able to assume that the y-intercept was zero? The standard curve used for the concentration determination was graphed from the origin. This allows you to assume that the y-intercept is always zero.