Pre-lab due at the beginning of the laboratory period. EXPERIMENT: SPECTROPHOTOM

Question

Pre-lab due at the beginning of the laboratory period. EXPERIMENT: SPECTROPHOTOMETRIC ANALYSIS OF PROTEINS 1. Which of the 20 essential amino acids are likely to be useful during spectrophotmetric determination of protein concentration? What is it about these amino acids that makes them useful for spectrophotometry? 2. Which of the methods described in the manual is specific for polypeptides with two or more peptide bonds? What is the reason for the complex formed? 3. What is the Beer-Lambert law and why is it useful? How would you determine the molar absorptivity of a substance using the law? How would you determine the concentration of an unknown solution from the law? 4. What is the concentration (mg/mL) of the protein stock solution you will make? (You will find information in the Reagents section of your lab.) How will you dilute this to obtain a 40 mg/mL solution for standards? Show calculations for preparing standard solutions with concentrations of 0.4, 1.2, 6.0, and 12.0 mg/mL from the 40 mg/mL solution.

Explanation / Answer

1. Tyrosine, tryptophan and phenylalanine are used in spectrophotometric determination of protein concentration, because these three amino acids have aromatic groups which gives a peak at 280nm, while others give at 190nm.

2.Biuret or Gornall method is used for detection of polypeptides with two or more peptide bonds.

The reason behind the complex formation is that the copper ions of CuSO4 used reacts with the CO-NH(peptide bond ) of proteins and forms a purple color complex.

3.Beer lambert's law - it states that the quantity of light absorbed (A) by the substance is directly proportional to the concentration (c)of substance and the path length (l) of light through the solution.

A = e l c

where A= absorbance

e= molar absorptivity constant

l= path length and,

c= concentration

This law is useful in determining the unknown concentration of a substance if the amount of light absorbed by the substance is known.

To determine the molar absorptivity-

A = elc

e= A/lc *Absorbance, path length and concentration should be known

Molar absorptivity can be determined by dividing Absorbance by path length and concentration.

To determine concentation of unknown solution-

A= elc

c= A/el * Absorbance , path length and molar absorptivity should be known

Unknown concentration of the solution can be determined by dividing Absorbance with path length and molar absorptivity constant

4. Protein stock solution that was made in lab was 400mg /10 ml.

Therefore, it corresponds to 40mg/ml solution , that is to be made

We will dilute the solution 10 times to go from 400mg/10 ml to 40mg/ml, so we will add another 100 ml to the stock to dilute it .

Now , we have to go from 40mg/ml to 0.4, 1.2, 6 and 12 mg/ml

For doing this , there is a formula C1V1 = C2V2 , where C is concentration and V is volume

a) For 40mg/ml = 4mg/ml

40*1 = 4*V2

V2 = 40*1/4 = 10

Taking 4 mg in 10 ml will make the solution, 4mg/10ml

Similarly for 1.2mg/ml , take 33.3ml volume for dilution, i.e 1.2mg/33.3ml

For 6mg/ml , Take 6.66ml for dilution , i.e 6mg/6.66ml

Foe 12mg/ml , take 3.33 ml for dilution, i.e 12mg/3.33ml

So , in this way the above solutions can be diluted from 40mg/ml to thr respective above calculated ones.

Hope it helps, Good luck !!!!

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