1. The first step in preparation of affinity chromatography column is _____________
a) ligand attachment to matrix
b) coupling of aromatic amines to matrix
c) activation process
d) precipitation
Explanation: Activation process is the first step in preparation in affinity chromatography column which involves introduction of reactive groups into the chemically inert polymeric matrix material. The second step is attaching the ligand to the matrix covalently. After the activation, the activated matrix may be coupled to primary aliphatic or aromatic amines. Elution is the process of removing the adsorbed material from the column. This is the last step in affinity chromatography.
2. __________ refers to eluting a compound by creating a competition between the bounded enzymes with its suitable complimentary protein in solution.
a) Specific elution
b) Nonspecific elution
c) Filtration
d) Dialysis
Explanation: In affinity chromatography, when the elution is done by creating a competition between the bounded enzymes with its suitable complementary protein in solution, the elution is referred to as specific elution. Nonspecific elution is done when specific ligand fail to elute desired enzyme. Filtration is separation method based on the size. Dialysis is a separation technique in which large molecules are retained in the membrane eluting the smaller molecules.
3. Which among these is not a way of non-specific elution?
a) Solvent change
b) pH change
c) Reversible denaturation
d) Competition to suitable complementary protein
Explanation: Nonspecific elution can be done by using solvent or buffer changes, pH or ionic strength changes, the application of chaotropic reagents, temperature changes, reversible denaturation and destruction of spacer arm. Competition to suitable complementary protein is a type of specific elution.
4. Acetylcholine esterase can be purified by using affinity chromatography with the use of _______ as a ligand.
a) ATP
b) NADP
c) Acrdinium
d) Con A
Explanation: For acetylcholine esterase, acrdinium can be used as ligand. ATP can be used as ligand to purify adenosine kinase. Alcohol dehydrogenase is purified by using NADP as a ligand. Con A and mannose is used to purify acid phosphatase.
5. Acid phosphatase: Mannose:: Alpha amylase: ______________
a) KCl
b) Glycogen
c) Adenosine
d) Methyl-D-mannoside
Explanation: For purifying acid phosphatase, the eluent used is Mannose. In the same way, alpha amylase can be purified by using glycogen as an eluent. Adenosine is used as an eluent for purifying adenosine kinase and methyl-D-mannoside is used as an eluent for acetylcholine esterase.
6. The chromatography in which the sample is forced through a column of the stationary phase by pumping a liquid (mobile phase) at high pressure through the column is referred to as ___________
a) affinity chromatography
b) fast protein liquid chromatography (FPLC)
c) thin layer chromatography (TLC)
d) high performance liquid chromatography (HPLC)
Explanation: The basic principle of HPLC is that the sample is forced through a column of the stationary phase by pumping a liquid at high pressure through the column. In TLC, there is a stationary phase immobilized on a glass plate and an organic solvent that acts as a mobile phase. Affinity chromatography is a separation technique based on the binding affinities between two molecules. FPLC is a technique in which the stationary phase is liquid or solid, in the form of a matrix.
7. The time at which specific analyte elute out is termed as _________
a) Km
b) half-life
c) retention time
d) pKa
Explanation: The time at which specific analyte elute out is referred to as retention time and is the characteristic of that analyte. pKa is the pH at which half of the groups are ionized. Half-life is the time which is required the activity to reduce to half of its original activity. Km is the substrate concentration at which Vmax is reduced to half.
8. _______________ refers to process of isolation and purification of compounds.
a) FPLC
b) Analytical HPLC
c) Preparative HPLC
d) TLC
Explanation: Fast protein liquid chromatography (FPLC) is type of chromatography wherein the stationary phase is a liquid or solid, in the form of matrix. TLC is thin layer chromatography wherein the stationary phase is immobilized on a glass plate and mobile phase is organic solvent. Preparative HPLC refers to process of isolation and purification of compounds. In contrast to analytical HPLC, which involves obtaining the information about the sample compound.
9. Which of the following information cannot be obtained by using analytical HPLC?
a) Isolation of a compound
b) Identification of a compound
c) Quantification of a compound
d) Resolution of a compound
Explanation: Isolation and purification can be obtained by using preparative HPLC. Analytical HPLC is used when information about the compound. Hence identification, quantification and resolution of a compound can be obtained by using analytical HPLC.
10. _____________ can be achieved using HPLC by utilizing the fact that certain compounds have different migration rates given a particular column and mobile phase.
a) Purification of a compound
b) Chemical separations
c) Identification of a compound
d) Quantification of compound
Explanation: Chemical separations can be achieved by using HPLC. Separations can happen because of certain compound have different migration rate given a particular column and mobile phase. The extent of separation is mostly determined by the choice of stationary and mobile phase. The separation of target molecules from other compounds or contaminants is referred to as purification. Identification can be done by using HPLC with a detector. Quantification by using HPLC is determining the unknown concentration in solution.