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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 unknownconcentration in a known solution.

Procedure:

Step 1) Determine a Reference Wavelength

a. Prepare a blank dH2O and a sample of 0.1M solutionb. Insert them into Genesys 10 Spectrophotometerc. Press [TEST]; scroll to Scanning & press [ENTER]d. Select stored tests; scroll to Scan(PAR); select Load Teste. Ensure it will scan from 400-700 nmf. Set to read the blank; select Collect Baselineg. Select Measure Sample; select Edit Graph: Math: Peaks: & Valleysh. Print graph; select tabular & print tablei. 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 solutiona) Vs = Volume of stock solution required to reach target molarityb) Vs = (0.002 L (0.0075 M))/0.100 M = 1.5 mLc) Vs = (0.002 L (0.0050 M))/0.100 M = 1.0 mLd) Vs = (0.002 L (0.0025 M))/0.100 M = 500 Le) 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 1-5determined above

d. Select Measure Standards; print absorbance, slope, and interceptStep 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 0.187Based 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 naturalseawater- 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. IfA=lc then either a

change in l (pathlength) orc (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.