Hello, I\'m studying for my bio lab practical and was given some practice proble

Question

Hello, I'm studying for my bio lab practical and was given some practice problems. These are the ones I am unsure about. Any explanations would be much appreciated. Will rate.

1. Why is 595nm used as the wavelength to measure the absorbance of Coomassie Blue? What does the wavelength tell us about Coomassie blue, or any other substance?

2. The pKa for R group of glutamic acid is 4.3 (The pKa values of amino group and carboxyl group are 9 and 2.5 each. At pH 2, is this amino acid posivitely charged or negatively charged and why?

Explanation / Answer

1). When light falls on a substance, three things can happen. They are

- Absorbance

- Reflectance and

- Some do both and show neutral effect.

The absorbance value of a substance depends on the wavelength at which it absorbs that light.

The wavelength is determined by the color which it observed. In the case of coomasive blue, it imparts blue color to the sample, and this blue color shows observance at 560nm - 650 nm.

In this way different wavelengths are parted depending on the color. They are

Wavelength(nm)           colour observed

400                                  yelow-green

435                                   yellow

495                                   purple

560                                   blue

650                                  greenish blue

800                                  bluish green

2) The pKa values of amino group and carboxyl group are 9 and 2.5 each

Therefore the pI value is

pI = pK1 + pK2 / 2

   = 9+ 2.5 / 2

   = 5.75

As the pH value is 2 and it is less than the pI value, amino acid is positively charged.

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