BIOL 244 Absorbance Spectrum and Standard Curve Worksheets As the concentration of a substance increases, its absorbance will also increase. The exact rela

BIOL 244 Absorbance Spectrum and Standard Curve Worksheets As the concentration of a substance increases, its absorbance will also increase. The exact relationship between absorbance and concentration for a particular substance is determined by creating a standard curve. To create a standard curve, first measure the absorbances of a series of samples with known concentrations using a spectrophotometer (this data is provided for you below). Then, graph each data point with concentration on the x-axis and absorbance on the y-axis. Create a line of best-fit. Now you can use interpolation to estimate the concentration of an unknown sample by measuring its absorbance (absorbance values also provided below). This is an absorbance spectrum of two concentrations of the
compound methylene blue: line (a) is a concentration of 100mg/ml and
line (b) is a concentration of 40mg/ml.
1. Do you see a pattern in the absorbance spectra? What is the
wavelength of maximum absorbance?
2. What effect does concentration have on the a) wavelength of
maximum absorption, and b) the amount of absorbamce?
This is the absorbance spectrum of three different concentrations of
phenol red.
1. Do you see a pattern in the absorbance spectra? What is the
wavelength of maximum absorbance? Are they different from
methylene blue?
2. What effect does concentration have on the a) wavelength of
maximum absorption, and b) the amount of absorbance?
Biology 244 Standard Curve Activity
As the concentration of a substance increases, its absorbance will also increase. The exact relationship
between absorbance and concentration for a particular substance is determined by creating a standard
curve. To create a standard curve, first measure the absorbances of a series of samples with known
concentrations using a spectrophotometer (this data is provided for you below). Then, graph each data
point with concentration on the x-axis and absorbance on the y-axis. Create a line of best-fit. Now you
can use interpolation to estimate the concentration of an unknown sample by measuring its absorbance
(absorbance values also provided below).
1. Examine the following absorbance values for known concentrations of bovine serum albumin:
Concentration (g/ml) 1.5
1.0
0.75
0.5
0.25
0.125
Absorbance
4.2
3.7
2.6
1.9
0.8
5.0
0.025
0.2
Use EXCEL to create a standard curve with the data. Cut and paste your figure in the space below.
Include all labels including axes, the equation for the line and the squared correlation coefficient
(r2).
2. Here are duplicate absorbances for unknowns #1 and #2 run in the same assay as in question 1:
Unknown #1: 3.1
Unknown #1: 3.7
Unknown #2: 0.9
Unknown #2 1.2
Estimate the concentration of protein in Unknown #1: __________________
Estimate the concentration of protein in Unknown #2:___________________

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