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STUDY PROBLEMS AND CALCULATIONS: UV/VIS SPECTROSCOPY

1. Describe the general principle of colorimetric assays.


2. Which chemical groups are responsible for the absorption of ultra-violet radiation
in proteins? Are proteins able to absorb visible light?
3. Which chemical groups absorb UV light in nucleic acids? What is max of this
absorption?
4. Alternating double and single bonds are referred to as a conjugated system. How
does such a structure influence UV/Vis absorption?
5. Why are some solutions coloured? (give chemical explanations of this feature).
6. Define molar absorptivity (molar extinction coefficient).
7. How can you determine the purity of a protein solution using absorption
spectroscopy?
8. List chemical groups that form chromophores in proteins and nucleic acids and
match them to the appropriate wavelength: a) 190205 nm, b) 260 nm, c) 280
nm. Is it possible for a protein to absorb visible light?
9. How can you check if a solution of nucleic acids is free of protein contaminants?
10. Describe the principle of the biuret protein assay.
11. 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)
12. 0.3 ml of a patients 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
patients serum.
13. A serum sample was diluted 50 and protein content was determined by the biuret
method. The absorbance was equal to 0.3. Using the calibration curve on page 8-9,
calculate the protein concentration in the serum.
14. The absorptivity (k) at 280 nm (A280) 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
A280 of 1.5 in a 0.5 cm cuvette?
15. 50 l of phenylalanine solution was diluted with 0.95 ml of phosphate buffer. This
diluted sample was used to determine amino-acid concentration with the
ninhydrine 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.
16. Glucose concentration was determined in a serum sample with the phenol/H2SO4
method. The sample was diluted 20-fold before the assay. The absorbance
measured at =750 nm was equal to 0.7. Using the appropriate calibration curve,
calculate the result of the assay. Remember sample dilution!
STUDY PROBLEMS AND CALCULATIONS: CHROMATOGRAPHY

1. What type of chromatography should be used for the following separations:


a) Serum albumin (protein, Mw=60,000 Da, pI=4.5) from immunoglobulin G (protein,
Mw=95,000 Da, pI=7.8)
b) Transferrin (protein) from isoleucine (amino acid)
c) -carotene from chlorophyll (plant dyes)
2. Which chromatographic techniques exploit difference in protein charge for
separation?
3. Which chromatographic techniques exploit differences in molecular size of
compounds for separation?
4. If an amino acid has an Rf value of 0.45, how far will it move on a TLC plate in which
the solvent front moved 15.2 cm?
5. A peptide hydrolysate was analysed in thin-layer chromatography. Three spots were
obtained in the chromatogram, located at 2.9 cm, 6.8 cm, and 14.4 cm from the start
line. The solvent front covered 18 cm. What was the amino-acid content of the
peptide? The amino-acid standards moved as follows:
Amino acid Phe Leu Arg Asp Ile Gly Ser Lys
Rf 0.88 0.72 0.28 0.16 0.80 0.38 0.56 0.24

6. Which chromatographic methods enable the determination of amino-acid content


in proteins?
7. How do you separate the following mixture of proteins:
Protein A: pI=6.5, Mw=20,000 Da; Protein B: pI=7.8, Mw=20,000 Da;
Protein C: pI=6.5, Mw=83,000 Da
8. The following mixture of amino acids was separated by ion-exchange
chromatography: Asp (pI=2.77), Cys (pI=4.60), and Phe (pI=5.48). The column was
equilibrated with the buffer of pH=2 and eluted with an increasing pH gradient.
What type of ion-exchange resin was used? In which order were the amino acids
eluted?
9. Choose the conditions for separation of the following mixture of proteins: protein
A: pI=4.0, protein B: pI=5.5, protein C: pI=7.0. Use anion exchange chromatography.
10. You have to isolate the enzyme trypsin using affinity chromatography. What ligand
should be immobilised to agarose beads? Choose all possibilities.
a) Chymotrypsin (proteolytic enzyme), b) soybean trypsin inhibitor, c) casein
(protein, trypsin substrate), d) goat anti-trypsin antibodies.