1. Which chromatography technique consist of a stationary phase immobilized on a glass plate and an organic solvent acting as a mobile phase?
a) Ion exchange chromatography
b) Affinity chromatography
c) Thin layer chromatography (TLC)
d) Gel exclusion chromatography
Explanation: In TLC, the stationary phase is immobilized on a glass plate and organic solvent act as the mobile phase. The sample dissolved in the solvent is placed on the stationary phase. The solvent mixture moves up by the capillary action and meet the sample mixture. Different compounds travel at different rates due to their solubility and interactions with the stationary phase absorbents. Gel exclusion, affinity and ion-exchange chromatography are used for purification of enzymes based on the property of the enzyme.
2. If the solvent phase in TLC was 80:20 mixture of hexane and ethyl acetate, the polar compounds will not move as much as non-polar compounds.
a) true
b) false
Explanation: When the solvent is 80:20 mixture of hexane and ethyl acetate, the solvent will be mostly nonpolar. This means that the non-polar compounds will move further up the plate. In contrast the polar compounds will not have moved as much as the non-polar compounds. Hence the statement is true.
3. When the solvent phase in TLC is 20:80 mixture of hexane and ethyl acetate, the polar compounds will not move as much as non-polar compounds.
a) true
b) false
Explanation: In TLC, when the mixture 20:80 mixture of hexane and ethyl acetate, the solvent will become mostly polar. This means polar compounds will move further up in the plate as compared to non-polar compounds. Hence the above statement is false.
4. Pick odd one out.
a) Silica gel
b) Alumina
c) Cellophane
d) Cellulose
Explanation: The odd one out is cellophane which is a tubing kind of bag used in dialysis as a permeable membrane. Silica gel, Alumina and cellulose are the adsorbents used in TLC. Some other are acetylated cellulose, kieselghur etc.
5. What is the coloring agent used to detect laccase?
a) ABTS
b) Acetylated cellulose
c) Kieselghur
d) Hexane and Ethyl acetate mixture
Explanation: The coloring agent used to detect laccase is ABTS. Acetylated cellulose and kieselghur are the adsorbents used in stationary phase of TLC. Hexane and ethyl acetate mixture is the solvent used as the mobile phase in TLC.
6. On spraying the coloring agent, the enzyme is detected as a __________
a) square
b) triangle
c) line
d) spot
Explanation: After performing the TLC, the plate is sprayed with coloring agent and the protein or enzyme is detected as spot. The coloring agent used is ABTS which can detect laccase enzyme.
7. The ratio of distance travelled by the sample over the total distance covered by the solvent is referred to as ___________
a) solubility
b) miachelis menton constant
c) retention factor
d) volumetric throughput
Explanation: Retention factors refers to the ratio of distance travelled by the sample to the distance travelled by the solvent. Volumetric throughput comes into picture when dealing with filtration. This is proportional to pressure and filter area and inversely proportional to filter cake thickness and dynamic viscosity. Solubility comes into picture when dealing with ammonium sulphate precipitation and is given by Log S = Kintercept – Salt T.
Miachelis Menton constant deals with enzyme kinetics is given by Km which is nothing but the substrate concentration when Vmax is reduced to half.
8. _______________ is a liquid column chromatographic method of separating solute molecules according to differences in molecular size.
a) Thin layer chromatography (TLC)
b) Ion exchange chromatography
c) Gel filtration
d) Affinity chromatography
Explanation: The liquid chromatographic method of separating solute molecules according to differences in molecular size is referred to as gel filtration. TLC is a type of chromatography wherein the stationary phase is immobilized on a glass or plastic plate and organic solvent acts as the mobile phase. Ion-exchange and affinity chromatography are used in enzyme preparation according to the property of the enzyme.
9. Which of the following technique separates the sample by percolating it through a bed of porous, uncharged particles?
a) Gel filtration
b) Filtration
c) Dialysis
d) Ultrafiltration
Explanation: Gel filtration separates the sample by percolating it through a bed of porous, uncharged particles. Filtration simply separates based on the size across a permeable membrane. Ultrafiltration is a type crossflow microfiltration technique where in the sample flow is tangential to the membrane to avoid the building of the cake. Dialysis is a process which allows the passage of low molecular weight compounds retaining the high molecular weight compounds inside the membrane.
10. Which of the following is not a step involved in gel filtration?
a) Gel preparation
b) Precipitation
c) Sample application
d) Elution of the sample
Explanation: Precipitation is the step involved in purification of enzyme or protein. Gel filtration is a technique used to purify enzyme or protein. The steps involved are as follows:
(i) Gel preparation by using sponge like porous material which is equilibrated an applied in the column.
(ii) Sample application to the column which consists of different molecules.
(iii) To elute the sample, same solvent is applied to the column. Large molecules which cannot penetrate the gel, pass the column faster and get eluted first and smaller molecules they get penetrate the gel and take a longer time elute and thus, separating the sample of interest.