This set of Enzyme Technology Multiple Choice Questions & Answers (MCQs) focuses on “Enzymes Purification – High Performance Liquid Chromatography”.
1. 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.
2. The time at which specific analyte elute out is termed as _________
c) retention time
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.
3. _______________ refers to process of isolation and purification of compounds.
b) Analytical HPLC
c) Preparative HPLC
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.
4. 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.
5. _____________ 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.
6. __________________ refers to the process of separating or extracting the target compound from other compounds or contaminants.
a) Quantification of a compound
b) Identification of a compound
c) Purification of a compound
d) Chemical separations
Explanation: The process of separating or extracting the target compound from other compounds or contaminants is referred to as purification. Chromatography produces characteristic peaks for a specific compound. Hence the target compounds, contaminants and other compounds must have different peaks which means they must have different migration values so that the desired product may be eluted, and not the undesired ones.
7. The process of determining the unknown concentration of a compound in a known solution by using HPLC is referred to as _______________
a) quantification of a compound
b) purification of a compound
c) chemical separations
d) identification of a compound
Explanation: Quantification by using HPLC refers to the process of determining the unknown concentration of a compound in a known solution. It involves injection of series of known concentration of the standard solution onto HPLC. This is detected as series of peaks that is correlated to concentration of compound injected. By comparing the peak of unknown concentration these known peaks, the concentration can be quantified.
8. To alter the retention time, which of the following parameters need not be manipulated?
a) Choice of column
b) Choice of mobile phase
c) Choice in flow rate
d) Sample type
Explanation: Identification of a compound by using HPLC is done by using a detector. The detector identifies in the form of peaks. The identifying peak should have a reasonable retention time and should be well separated from other extraneous peaks at detection level. To alter the retention time, the parameters which can be manipulated are choice of column, mobile phase and flow rate. Sample type does not matter.
9. The gradient separates the analyte mixture as a function of affinity of the analyte for the mobile phase relative to stationary phase.
Explanation: If the analytes have stronger interactions with mobile phase as compared to stationary phase, the analytes will elute faster and have shorter retention time. Whereas the analytes with stronger interactions with stationary phase as compared to mobile phase, will elute the analytes at later point of time and have longer retention time. Hence the above statement is true.
10. Which block diagram is represented in the following diagram?
c) Affinity chromatography
Explanation: The block diagram represented above is of HPLC. The sample is forced through a column of stationary phase by pumping piqued at high pressure through the column. The sample to be analyzed in introduced in low amount over mobile and is retained by specific chemical or physical interactions with stationary phase as it passes over the entire length of the column. The use of pressure increases linear velocity giving less time to the compounds to diffuse within the column which leads to improved resolution in the resulting chromatograph.
11. In HPLC, the compounds are eluted using a constant mobile phase composition and is referred to as _____________
a) nonspecific elution
b) isocratic elution
c) specific elution
d) gradient elution
Explanation: In isocratic elution, the compounds are eluted using a constant mobile phase composition. As compared to gradient elution in which the compounds are eluted using increasing strength of the organic solvent. Specific and nonspecific elution occurs during affinity chromatography. As compared gradient and isocratic elution which is taking place during HPLC.
12. In _________ elution, the compounds are eluted by increasing the strength of the organic solvent during HPLC.
Explanation: Specific and nonspecific elution occurs during affinity chromatography. Isocratic elution is one which the elution is done using a constant mobile phase. The process of eluting compounds during HPLC by increasing the strength of the organic solvent is referred to as gradient HPLC. The sample is injected on weaker mobile phase and the strength is increased in linear fashion by raising the organic solvent fraction which results in the elution of retained compounds.
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