Biol 211, Cell Biology, Fall 2008

Exercise #1: Pipetting & Spectrophotometry

Investigator: Casey Snyder

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 66 66 67 66 0.071
425 81 81 82 81 0.064
450 99 99 99 99 0
475 97 97 97 97 0
500 95 95 95 95 0
525 86 86 86 86 0
550 66 66 66 66 0
575 33 33 33 33 0
600 14 13 14 14 0.154
625 1 1 1 1 0
650 10 10 10 10 0
675 80 97 97 91 0.841
700 98 98 98 98 0
































Table 2. Absorption Spectrum Absorbance Data
Wavelength (nm) S1 (A) S2 (A) S3 (A) Mean A SE
400 0.18 0.18 0.174 0.178 0.007
425 0.092 0.092 0.086 0.09 0.009
450 0.004 0.004 0.004 0.004 0
475 0.013 0.013 0.013 0.013 0
500 0.022 0.022 0.022 0.022 0
525 0.066 0.066 0.066 0.066 0
550 0.18 0.18 0.18 0.18 0
575 0.481 0.481 0.481 0.481 0
600 0.854 0.886 0.854 0.865 0.016
625 2 2 2 2 0
650 1 1 1 1 0
675 0.097 0.013 0.013 0.041 0.196
700 0.009 0.009 0.009 0.009 0

































Conclusion: The absorption maximum (Amax) for blue pigment is 625 nm


Table 3. Standard Curve. %T vs. Conc.
Concentration (%) 1 2 3 Mean %T SE
1.25 94 95 94 94 0.06
3.125 87 87 87 87 0
6.25 75 75 75 75 0
12.5 57 54 57 56 0.189
25 34 34 33 34 0.099

















Table 4. Standard Curve. A vs. Conc.
Concentration (%) 1 2 3 Mean A SE
1.25 0.027 0.022 0.027 0.025 0.015
3.125 0.06 0.06 0.06 0.06 0
6.25 0.125 0.125 0.125 0.125 0
12.5 0.244 0.268 0.244 0.252 0.023
25 0.469 0.469 0.481 0.473 0.008




















The standard curve equation (approximate) is A = 0.02C + 0

where A is absorbance and C is concentration of blue pigment (% of solution)


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 42 41 41 0.377 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 18.85 19.35 19.35 19.1833333333333