Experiment 3.

2 Dye Binding Method

Sample Concentration (g/ml) 10.00 8.00 BSA 6.00 4.00 2.00 Unknown 157 Unknown 6 Table 2: Absorption readings from Spectrophotometer at wavelength of 595nm Absorbance 0.433 0.281 0.187 0.135 0.060 0.395 0.185

3.2.3 Questions
1. Equation of Best Fit Line: y = 0.038x

For Unknown 157, Protein content in diluted sample = = Protein content in undiluted sample =

0.3950.038
10.39g/ml 10.39g/ml 100 (3 significant figures)

= =

1039g/ml 1.04mg/ml (3 significant figures)

For Unknown 6, Protein content in diluted sample = = Protein content in undiluted sample =

0.1850.038
4.87 g/ml 4.87 g/ml 100 (3 significant figures)

= = 2.

487 g/ml 0.487mg/ml (3 significant figures)

Method 1 Method 2

Protein Content(mg/ml) Unknown 157 Unknown 6 1.760 0.709 1.039 0.487

Table 3: Comparison of Protein content of Unknown samples between experiment methods

Method 1 - The protein content values are calculated using E1% values of 6.67. Method 2 -The protein content values are obtained from the best fit line interpolated from graph 2. From the above table, it is shown that method 1 gave a higher value for both unknown 157 and unknown 6 as compared to method 2. It is because the first method depends on the ability of the unknown solution to absorb light of the particular wavelength, which means it would include any impurities found present that absorbed the light. Therefore, any absorbance measured would be inaccurate if the unknown solutions are not pure. However, in method 2, a dye that will bind specifically to the proteins alone is used. Besides that , the maximum absorptions is raised to 595nm .At this higher wavelength ,the effect of light absorptions by the impurities would be very much lessened as the impurities in the unknown solution would be very small and would not absorb he light with such high wavelength. Thus, any absorption measured would be from the dye bound to the proteins in the solution and would give a more accurate result. In conclusion method 2 would be a better method as it gave a more accurate result. 3. According to Beer-Lambert Law: A= C L

Where,

A=Absorbance E=Molar absorption coefficient / Extinction coefficient C=Molar Concentration (mol dm-3) L=Path length (cm)

Absorbance of BSA : A=0.667

Given: Molecular Weight of BSA Concentration of BSA = = = = Molarities of BSA = = = 66000 1.0mg/ml 1000mg dm-3 1g dm-3 1g dm-3 molecular weight 1g dm-3 66000

166000mol dm-3

Path length:

1 cm

Hence,

= = =

ACL 0.667(166000)1
44022 mol-1dm3 cm-1