Aliza Sial

Biol 211, Cell Biology, Fall 2008

Exercise #1: Pipetting & Spectrophotometry

Purpose of the Experiment

The scientific objective of this experiment is to determine the concentration of blue pigment in two unknowns.


Materials


Preparation

1. Turn on the spectrophotometer and allow to warm up.

2. Gather, label, and fill 24 test tubes with volumes of blue dye and water according to the components list.

3. Prepare the blanks (deionized water).

Procedure. Part 1. Construction of the Absorption Spectrum.

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

1. Set the wavelength on the spectrophotometer to one of 13 wavelengths specified.

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. Part 2. 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 5 known 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. Part 3. Determination of the Concentration of Blue Pigment in Two Unknowns.

1. Set the 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.

Results

Table 1. Absorption Spectrum Transmittance Data
Wavelength (nm) S1 (%T) S2 (%T) S3 (%T) Mean %T SE
400 65 65 66 65 0.072
425 82 82 82 82 0
450 97 98 99 98 0.082
475 99 98 99 99 0.058
500 95 95 95 95 0
525 85 86 86 86 0.062
550 68 67 67 67 0.071
575 33 32 33 33 0.101
600 14 14 14 14 0
625 1 1 1 1 0
650 12 12 12 12 0
675 77 77 78 77 0.066
700 98 97 97 97 0.059


































Table 2. Absorption Spectrum Absorbance Data
Wavelength (nm) S1 (A) S2 (A) S3 (A) Mean A SE
400 0.187 0.187 0.18 0.185 0.008
425 0.086 0.086 0.086 0.086 0
450 0.013 0.009 0.004 0.009 0.039
475 0.004 0.009 0.004 0.006 0.031
500 0.022 0.022 0.022 0.022 0
525 0.071 0.066 0.066 0.068 0.009
550 0.167 0.174 0.174 0.172 0.008
575 0.481 0.495 0.481 0.486 0.009
600 0.854 0.854 0.854 0.854 0
625 2 2 2 2 0
650 0.921 0.921 0.921 0.921 0
675 0.114 0.114 0.108 0.112 0.008
700 0.009 0.013 0.013 0.012 0.017

























Table 3. Standard Curve. %T vs. Conc.
Concentration (%) 1 2 3 Mean %T SE
1.25 95 94 94 94 0.06
3.125 88 87 88 88 0.062
6.25 76 77 69 74 0.414
12.5 54 55 55 55 0.078
25 32 32 32 32 0






















Table 4. Standard Curve. A vs. Conc.
Concentration (%) 1 2 3 Mean A SE
1.25 0.022 0.027 0.027 0.025 0.015
3.125 0.056 0.06 0.056 0.057 0.008
6.25 0.119 0.114 0.161 0.131 0.058
12.5 0.268 0.26 0.26 0.263 0.007
25 0.495 0.495 0.495 0.495 0
















Table 5. Determination of Unknowns
Unknown (%) %T 1 %T 2 %T 3 Abs 1 Abs 2 Abs 3
A 8 8 8 1.097 1.097 1.097
B 41 41 41 0.387 0.387 0.387
















Table 6. Concentraion of Blue Pigment in Unknowns
Unknown (%) C 1 C 2 C 3 C mean
A 54.85 54.85 54.85 54.85
A 19.35 19.35 19.35 19.35