Adil Quraish

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

1. Media bottle containing stock solution of blue pigment
2. Media bottle of deionized water
3. Media bottle of unknown A
4. Media bottle of unknown B
5. A supply of graduated transfer pipets
6. A pipetting device to be used with the graduated transfer pipets
7. Automatic pipettors and pipet tips
8. A supply of test tubes and requisite test tube racks
9. Genesys 20D spectrophotometer.

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	67	66	67	67	0.071
425	80	80	79	80	0.065
450	98	99	98	98	0.058
475	99	99	99	99	0
500	95	96	96	96	0.059
525	87	87	87	87	0
550	66	66	66	66	0
575	32	33	33	33	0.101
600	5	5	5	5	0
625	1	1	1	1	0
650	11	11	11	11	0
675	79	79	79	79	0
700	98	98	98	98	0

**Table 2. Absorption Spectrum Absorbance Data**
Wavelength (nm)	S1 (A)	S2 (A)	S3 (A)	Mean A	SE
400	0.174	0.18	0.174	0.176	0.007
425	0.097	0.097	0.102	0.099	0.008
450	0.009	0.004	0.009	0.007	0.028
475	0.004	0.004	0.004	0.004	0
500	0.022	0.018	0.018	0.019	0.014
525	0.06	0.06	0.06	0.06	0
550	0.18	0.18	0.18	0.18	0
575	0.495	0.481	0.481	0.486	0.009
600	1.301	1.301	1.301	1.301	0
625	2	2	2	2	0
650	0.959	0.959	0.959	0.959	0
675	0.102	0.102	0.102	0.102	0
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	93	94	94	94	0.06
3.125	86	86	87	86	0.062
6.25	75	75	75	75	0
12.5	57	57	57	57	0
25	29	32	32	31	0.254

**Table 4. Standard Curve. A vs. Conc.**
Concentration (%)	1	2	3	Mean A	SE
1.25	0.032	0.027	0.027	0.029	0.014
3.125	0.066	0.066	0.06	0.064	0.011
6.25	0.125	0.125	0.125	0.125	0
12.5	0.244	0.244	0.244	0.244	0
25	0.538	0.495	0.495	0.509	0.028

**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	54.65	54.65	54.65	54.65
A	19.15	19.15	19.15	19.15