STUDY PROBLEMS AND CALCULATIONS: UV/VIS SPECTROSCOPY

1. Which chemical groups are responsible for the absorption of ultra-violet radiation in
proteins? Are proteins able to absorb visible light?
2. Which chemical groups absorb UV light in nucleic acids? What is LAMDAmax of this
absorption?
3. Alternating double and single bonds are referred to as a conjugated system. How does
such a structure influence UV/Vis absorption?
4. List chemical groups that form chromophores in proteins and nucleic acids and match
them to the appropriate wavelength: a) 190–205 nm, b) 260 nm, c) 280 nm. Is it
possible for a protein to absorb visible light?
5. How can you check if a solution of nucleic acids is free of protein contaminants?
6. The A280 of a 1 g/L solution of bovine serum albumin is equal to 0.66. What is the
absorbance of 0.25% BSA solution? (the cuvette thickness is 1 cm)
7. 0.3 ml of a patient’s serum was diluted with 2.7 ml 0.9% NaCl. The biuret reaction was
carried out and the absorbance was equal to 0.4. The absorbance of a 0.5 mg/ml standard
solution was 0.25. Calculate the protein concentration in the patient’s serum.
8. A serum sample was diluted 50X and protein content was determined by the biuret
method. The absorbance was equal to 0.3. If the absorbance of 0.25 mg/ml of standard
solution was 0.15, calculate the protein concentration in the serum.
9. The absorptivity (k) at 280 nm (A 280) of a 1 g/L solution of the enzyme trypsin in a 1 cm
cuvette is 1.43. What is the concentration of a solution of trypsin that has an A 280 of 1.5 in
a 0.5 cm cuvette?
10. 50 μl of phenylalanine solution was diluted with 0.95 ml of phosphate buffer. This diluted
sample was used to determine amino-acid concentration with the ninhydrin method. The
absorbance measured at 630 nm was equal to 0.28. For a calibrator of 6 mg/ml
concentration the absorbance value was 0.82. Calculate the concentration of the
phenylalanine solution.