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David Feldman 9/15/14

AS 020.315 Biochemistry Lab Dr. Horner

Laboratory 2 Report

Introduction

Different compounds, because of their molecular and sub-molecular structures, will interact in
different ways with the visible light and ultraviolet spectrums by absorbing differing
wavelengths at varying amounts. In order to conduct accurate Biochemical reactions within a
laboratory setting, it is often necessary to determine the concentration of an unknown solution;
we may use Spectrophotometry, which is based upon the light and UV absorbance properties, to
do just this. Using a spectrophotometer we obtain different absorbances of a single concentration
of a compound at varying UV wavelengths, we determine an unknown concentration of a
compound based upon it's UV absorbance, and we determine the concentration of an unknown
solution using the standard curve method and the Biuret assay.

Materials and Methods

Materials and Methods are exactly as described in Dr. Horner's "Laboratory 2:
Spectrophotometry" from the lab manual for the 020.315 Fall 2014 course. All 3 experiments
listed in this chapter were done in succession. As described, we work with varying
concentrations of Bovine Serum Albumin (BSA), and use a Genesys 10 UV-Visible Light
Spectrophotometer, and a Genesys 20 Visible Light Spectrophotometer to conduct experiments.

Results

1. Below is a graph representing the absorbance of 1 mg/ml BSA versus wavelength as
measured using the Genesys 10 UV-Visible Light Spectrophotometer. A blank sample was run
prior to each measurement to ensure accuracy.


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2. The calculation of the 1 mg/ml extinction coefficient of BSA at the absorbance maximum
(280 nm) is demonstrated below.

3. The calculation of the 1 mg/ml extinction coefficient of BSA at the absorbance maximum
(280 nm) is demonstrated below.

4. Below is a graph representing the absorbance of 10 mg/ml BSA versus wavelength as
measured using the Genesys 20 Visible Light Spectrophotometer. A blank sample was run prior
to each measurement to ensure accuracy -- hence no "net" calculation is needed. Values for 0 and
.3 mg of BSA are mean experimental values.


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5. The concentration of the unknown BSA solution in mgs/ml is calculated as follows:

a. From the UV absorbance of the diluted unknown BSA solution, the dilution factor, and the
mg/ml :

2.41 mg/ml = c


b. From the A540 of the unknown BSA solution samples in the Biuret Assay and the standard
curve method:

The first sample (.400 ml) yielded and absorbance of .265 which translated to 1.68 mg.
1.68/.4 = 4.2

The second sample (.200 ml) yielded and absorbance of .157 which translated to .87 mg.
.87/.2 = 4.35

The third sample (.100 ml) yielded and absorbance of .116 which translated to .51 mg.
.51/.1 = 5.1

The average concentration determined by this method is

4.5 mg/ml = c

6. From the absorbances of the five separate chemical blanks, we determine the mean of the
readings and the standard deviation.


std dev
x x
n
i
i
i n
. .

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2
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The readings for the chemical blanks were: A540 = 0.063, 0.054, 0.054, 0.053, and 0.059


mean = .056
std. dev. = .0043



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7. From the absorbances of the five separate samples with 0.30 mg of BSA we determine the
mean of the readings and the standard deviation.

The readings for the .3 mg of BSA samples were: A540 = 0.098, 0.089, 0.113, 0.099, and 0.098.

mean = .099
std. dev. = .0086

8.
Standard Error for Blanks: 0.0019
Standard Error for .3mg: 0.0038

Multiplying these standard errors by 2.776 and then adding and subtracting them from the means
as calculated above we get the 95% confidence ranges for:

Blanks: A = .05 - .06

.3 mg: A = .09 - .11

From this we can indeed say with 95% certainty that our methods can distinguish between 0 and
.3 mg of protein. This is because there is no overlap between the ranges for the 95% confidence
limits.


Conclusion

Through this lab we were indeed able to use a spectrophotometer to obtain different absorbances
of a single concentration of a compound at varying UV wavelengths, an unknown concentration
of a compound, and the concentration of an unknown solution using the standard curve method
and the Biuret assay. Nonetheless, it was apparent that results were not perfect from this
experiment -- the Biuret assay yielded significantly different results from the A280. While it
would be nice to easily assign blame for this discrepancy (and my personal hunch is that the
A280 is the more accurate of the two) it is uncertain where mistakes were made. Should accurate
results be desired a repeat of both experiments should be done.