Megan Roadley

Biol 211, Cell Biology, Fall 2007

The Spectrophotometric Analysis of Blue Dye

Purpose of the Experiment

The purpose of this experiment is to determine the absorption spectrum of blue dye, to create a standard curve for blue dye concentration, and to use the standard curve to determine the concentration of blue dye is a series of unknowns. In the process, students gain a working understanding of pipetting and spectrophotometry


Materials


Procedure. Exercise 1B., Part A. Construction of the Absorption Spectrum.

Perform the following procedure at each of 13 wavelengths of light:

1. Set the wavelength on the spectrophotometer.

2. Calibrate the spectrophotometer with the blank by inserting the blank into the cuvette holder, closing the lid, then pressing the 100% T button. (wait for a stable reading of 100, repeat if necessary.)

3. Separately, place each of the three cuvettes containing the stock dye solution (S1, S2, and S3) into the cuvette holder, close the lid, then wait for a stable %T reading.

4. Record the reading in the data table.

Procedure, Exercise 1B., Part B. Construction of the Standard Curve.

1.Set the spectrophotometer to the Amax wavelength.

2. Perform the following procedure to each of the 15 cuvettes containing one of the dilutions of stock blue pigment solution.

a. Calibrate the spectrophotometer with the blank.

b. Place one of the cuvettes containing a dilution of the stock blue pigment solution into the cuvette holder of the spectrophotmeter, close the lid and measure the %T.

Record the data in the data table.

Procedure, Exercise 1B., Part C. Determination of the Unknowns.

1. Set the wavelength of your spectrophotometer to the Amax wavelength.

2. Perform the following procedure on each of the 6 cuvettes containing the samples of the unknowns:

a. Zero the spectrophotometer with the blank.

b. Place the cuvette into the cuvette holder, close the lid, then measure the transmittance. Record the data in the data table to the right.

Results

Table 1. Absorption Spectrum Transmittance Data
Wavelength (nm) S1 (%T) S2 (%T) S3 (%T) Mean %T SE
400 68 68 68 68 0
425 84 84 84 84 0
450 98 99 99 99 0.058
475 98 99 99 99 0.058
500 96 95 96 96 0.059
525 87 87 87 87 0
550 68 69 69 69 0.07
575 35 35 35 35 0
600 15 16 16 16 0.144
625 2 2 2 2 0
650 16 16 16 16 0
675 78 79 78 78 0.065
700 98 98 98 98 0
Table 2. Absorption Spectrum Absorbance Data
Wavelength (nm) S1 (A) S2 (A) S3 (A) Mean A SE
400 0.167 0.167 0.167 0.167 0
425 0.076 0.076 0.076 0.076 0
450 0.009 0.004 0.004 0.006 0.031
475 0.009 0.004 0.004 0.006 0.031
500 0.018 0.022 0.018 0.019 0.014
525 0.06 0.06 0.06 0.06 0
550 0.167 0.161 0.161 0.163 0.007
575 0.456 0.456 0.456 0.456 0
600 0.824 0.796 0.796 0.805 0.015
625 1.699 1.699 1.699 1.699 0
650 0.796 0.796 0.796 0.796 0
675 0.108 0.102 0.108 0.106 0.009
700 0.009 0.009 0.009 0.009 0
































































Table 3. Standard Curve. %T vs. Conc.
Concentration (%) 1 2 3 Mean %T SE
1.25 95 95 96 95 0.059
3.125 87 87 88 87 0.062
6.25 77 77 77 77 0
12.5 59 59 59 59 0
25 35 34 34 34 0.099
Table 4. Standard Curve. A vs. Conc.
Concentration (%) 1 2 3 Mean A SE
1.25 0.022 0.022 0.018 0.021 0.013
3.125 0.06 0.06 0.056 0.059 0.008
6.25 0.114 0.114 0.114 0.114
12.5 0.229 0.229 0.229 0.229 0
25 0.456 0.469 0.469 0.465 0.009









































































































Table 5. Determination of Unknowns
Unknown (%) %T 1 %T 2 %T 3 Abs 1 Abs 2 Abs 3
A 13 12 12 0.886 0.921 0.921
B 35 35 35 0.456 0.456 0.456






















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