Principle and application of column chromatography MCQs With Answer

Principle and application of column chromatography MCQs With Answer is designed to help M. Pharm students master both the theoretical foundations and practical decision-making involved in column-based separations. Column chromatography underpins many modern pharmaceutical analytical techniques—from rapid flash separations to preparative purification of APIs and natural products. This quiz blends core principles (adsorption/partition equilibria, normal- vs reverse-phase, selectivity, plate theory, van Deemter behavior) with hands-on considerations (solvent choice, modifiers, sample loading, gradient design, scale-up). Each question is curated to reflect real-world lab choices, interpretation of chromatographic parameters, and method optimization strategies essential for pharmaceutical R&D and QC.

Q1. What is the fundamental principle governing separation in column chromatography?

• Differential distribution of solute between stationary and mobile phases leading to different migration rates
• Only diffusion-driven mass transfer in a single phase
• Electrophoretic mobility differences under an electric field
• Crystallization based on temperature gradients

Correct Answer: Differential distribution of solute between stationary and mobile phases leading to different migration rates

Q2. In normal-phase column chromatography, which statement is most accurate?

• Polar stationary phase with nonpolar mobile phase
• Nonpolar stationary phase with aqueous mobile phase
• Both stationary and mobile phases are strongly aqueous
• Nonpolar stationary phase with nonpolar mobile phase

Correct Answer: Polar stationary phase with nonpolar mobile phase

Q3. On silica gel, which solvent will generally elute compounds most strongly in an eluotropic series?

• Hexane
• Toluene
• Ethyl acetate
• Methanol

Correct Answer: Methanol

Q4. How does decreasing stationary-phase particle size typically affect a packed column separation?

• Increases efficiency and resolution but raises backpressure requirements
• Decreases efficiency and reduces backpressure
• No change in efficiency; only reduces analysis time
• Increases peak tailing without affecting efficiency

Correct Answer: Increases efficiency and resolution but raises backpressure requirements

Q5. In reverse-phase column chromatography (e.g., C18), which statement is correct regarding gradient elution?

• Increasing the percentage of organic modifier (e.g., acetonitrile) decreases retention time of hydrophobic analytes
• Increasing water content decreases retention time of hydrophobic analytes
• Increasing organic modifier always increases retention for all analytes
• Retention is unaffected by solvent strength in reverse-phase

Correct Answer: Increasing the percentage of organic modifier (e.g., acetonitrile) decreases retention time of hydrophobic analytes

Q6. Which stationary phase most strongly retains basic drugs in normal-phase chromatography?

• Acidic silica gel
• Neutral alumina
• C18-bonded silica
• Polystyrene-divinylbenzene resin

Correct Answer: Acidic silica gel

Q7. What is a common chromatographic consequence of overloading a silica gel column?

• Band broadening with fronting/streaking and poor resolution
• Sharper peaks and improved resolution
• No effect on band shape; only faster elution
• Complete baseline separation of all components

Correct Answer: Band broadening with fronting/streaking and poor resolution

Q8. In preparative open-column or flash chromatography, how are collected fractions most commonly monitored in a lab setting?

• Collect fractions and analyze by TLC
• By mass spectrometry in real time for every fraction
• By conductivity measurements only
• By differential scanning calorimetry

Correct Answer: Collect fractions and analyze by TLC

Q9. What is the primary retention mechanism in ion-exchange column chromatography?

• Electrostatic interactions between oppositely charged analytes and stationary phase
• Size-based exclusion from pores
• Partition into a hydrophobic bonded layer
• Hydrogen-bonding with neutral ligands

Correct Answer: Electrostatic interactions between oppositely charged analytes and stationary phase

Q10. In size-exclusion (gel filtration) column chromatography, which statement is true?

• Larger molecules elute first because they are excluded from the pores
• Smaller molecules elute first because they diffuse faster
• Elution order depends only on charge
• All analytes have identical retention regardless of size

Correct Answer: Larger molecules elute first because they are excluded from the pores

Q11. For silica-based reverse-phase columns, what is the typical mobile-phase pH range recommended to avoid silica dissolution?

• Approximately pH 2–8
• Approximately pH 8–12
• Approximately pH 0–2
• Approximately pH 10–14

Correct Answer: Approximately pH 2–8

Q12. Which adjustment most effectively increases resolution of two closely eluting peaks without excessively increasing analysis time?

• Changing mobile-phase composition to alter selectivity (α)
• Doubling the column length
• Decreasing flow rate far below the optimum
• Increasing injection volume

Correct Answer: Changing mobile-phase composition to alter selectivity (α)

Q13. What is a key advantage of flash chromatography over gravity-packed column chromatography?

• Use of pressurized gas allows smaller particles and faster, higher-resolution separations
• Eliminates the need for solvents
• Works only for proteins and not small molecules
• Requires no fraction collection

Correct Answer: Use of pressurized gas allows smaller particles and faster, higher-resolution separations

Q14. To reduce tailing or irreversible adsorption of basic amines on silica, which mobile-phase modifier is commonly added?

• 0.1–1% triethylamine
• 10% hydrochloric acid
• Sodium dodecyl sulfate
• Ammonium sulfate salt at high ionic strength

Correct Answer: 0.1–1% triethylamine

Q15. In liquid chromatography, the capacity factor k’ is defined as:

• (tR − t0) / t0
• t0 / (tR − t0)
• tR / t0
• tR − t0

Correct Answer: (tR − t0) / t0

Q16. According to the Van Deemter equation, which statement best describes the effect of increasing linear velocity?

• It decreases longitudinal diffusion (B/u term) but increases mass transfer resistance (C·u term)
• It increases the A term (eddy diffusion) only
• It decreases both B and C terms simultaneously to zero
• It has no effect on plate height

Correct Answer: It decreases longitudinal diffusion (B/u term) but increases mass transfer resistance (C·u term)

Q17. Which compound would likely show the longest retention on a C18 column using water/acetonitrile at pH 3 (assuming comparable concentrations)?

• Naphthalene
• Toluene
• Phenol
• Aniline

Correct Answer: Naphthalene

Q18. What is the primary purpose of using a guard column in LC systems?

• To protect the analytical column from particulates and strongly retained contaminants
• To increase the system backpressure
• To serve as the main separation column
• To eliminate the need for filtration of samples

Correct Answer: To protect the analytical column from particulates and strongly retained contaminants

Q19. When scaling a method from an analytical column to a preparative column, how should flow rate be adjusted to maintain similar linear velocity?

• F2 = F1 × (d2/d1)^2
• F2 = F1 × (d2/d1)
• F2 = F1 × (d1/d2)^2
• Flow rate should remain unchanged regardless of diameter

Correct Answer: F2 = F1 × (d2/d1)^2

Q20. In which scenario is normal-phase column chromatography generally preferred over reverse-phase?

• Separation of very nonpolar compounds (e.g., lipid classes) using nonpolar eluents
• Analysis of highly polar peptides in aqueous buffers
• Ionizable drug assays requiring pH control in water
• Routine separations requiring MS-compatible aqueous mobile phases

Correct Answer: Separation of very nonpolar compounds (e.g., lipid classes) using nonpolar eluents

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