Principle of chromatography MCQs With Answer

Principle of chromatography MCQs With Answer

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Principle of Chromatography MCQs With Answer for M. Pharm Students

Chromatography is a cornerstone of modern pharmaceutical analytical techniques, enabling the separation, identification, and quantification of complex mixtures based on differential interactions between analytes and two phases. Mastery of its principles—distribution equilibria, adsorption, ion-exchange, size-exclusion, and affinity mechanisms—empowers M. Pharm students to optimize methods, improve resolution, and troubleshoot separations. This curated set of MCQs focuses on the fundamental and advanced principles that govern retention, selectivity, efficiency, and band broadening across TLC, HPLC, GC, and specialized modes. Each question is designed to deepen conceptual understanding, linking theory (plate and rate concepts, Van Deemter behavior) to practical decision-making (pH control, gradient design, stationary phase choice) in real-world pharmaceutical analysis.

Q1. What best describes the fundamental principle underlying chromatographic separation?

  • Differential distribution of solute between stationary and mobile phases leading to different migration rates
  • Selective crystallization of analyte from the mobile phase
  • Differences in sample vapor pressure under isothermal conditions
  • Osmotic pressure differences across the column

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

Q2. In normal-phase liquid chromatography, which species elute first and why?

  • Less polar compounds elute first due to weaker interactions with the polar stationary phase
  • More polar compounds elute first due to stronger solvation in the polar mobile phase
  • Ionic compounds elute first due to ion-exchange with the stationary phase
  • Larger molecules elute first because of size exclusion

Correct Answer: Less polar compounds elute first due to weaker interactions with the polar stationary phase

Q3. In reversed-phase liquid chromatography (RPLC), retention generally increases with which molecular property?

  • Hydrophobicity (nonpolarity) due to stronger partitioning into the nonpolar stationary phase
  • Net charge magnitude due to electrostatic attraction to the stationary phase
  • Molecular size due to pore exclusion
  • Volatility due to vapor-phase partitioning

Correct Answer: Hydrophobicity (nonpolarity) due to stronger partitioning into the nonpolar stationary phase

Q4. The capacity factor k′ = (tR − t0)/t0 primarily reflects what aspect of chromatographic behavior?

  • Extent of analyte retention relative to an unretained marker
  • Detector response linearity
  • Column backpressure normalized to flow rate
  • Sample loading capacity at breakthrough

Correct Answer: Extent of analyte retention relative to an unretained marker

Q5. The selectivity factor (α) in chromatography describes which property of a separation?

  • Relative retention of two analytes and their ability to be separated
  • Absolute efficiency of the column independent of analyte
  • Ratio of mobile-phase viscosity to diffusivity
  • Detector selectivity to the analyte’s chromophore

Correct Answer: Relative retention of two analytes and their ability to be separated

Q6. According to the resolution equation, which change most effectively improves resolution for two closely eluting peaks?

  • A modest increase in selectivity (α) by changing mobile phase or stationary phase chemistry
  • Doubling flow rate while keeping other variables constant
  • Increasing injection volume to enhance signal
  • Using a shorter column of the same particle size

Correct Answer: A modest increase in selectivity (α) by changing mobile phase or stationary phase chemistry

Q7. Ion-exchange chromatography separates analytes primarily based on which interaction?

  • Electrostatic interactions between charged analytes and oppositely charged functional groups on the stationary phase
  • Differential vapor pressures at column temperature
  • Hydrophobic partitioning into alkyl-bonded phases
  • Molecular sieving through well-defined pores

Correct Answer: Electrostatic interactions between charged analytes and oppositely charged functional groups on the stationary phase

Q8. In size-exclusion chromatography (SEC) of globular proteins, which statement is correct?

  • Larger molecules elute earlier because they are excluded from the pores and take a shorter path
  • Smaller molecules elute earlier because they diffuse faster
  • Elution order depends solely on charge
  • Strong adsorption to the stationary phase is desirable

Correct Answer: Larger molecules elute earlier because they are excluded from the pores and take a shorter path

Q9. The key principle behind affinity chromatography is:

  • Specific, reversible binding between a ligand immobilized on the stationary phase and the target analyte
  • Non-specific hydrophobic adsorption of analytes
  • Phase transition of the mobile phase under pressure
  • Permanent covalent attachment of analytes to the matrix

Correct Answer: Specific, reversible binding between a ligand immobilized on the stationary phase and the target analyte

Q10. In thin-layer chromatography (TLC), the retention factor (Rf) is defined as:

  • Distance traveled by solute divided by distance traveled by solvent front
  • Time to reach the detector divided by column length
  • Solvent polarity index divided by analyte polarity
  • Peak width at half height divided by retention time

Correct Answer: Distance traveled by solute divided by distance traveled by solvent front

Q11. According to plate theory, a chromatographic column is modeled as:

  • A series of discrete equilibrium steps where solute re-distributes between phases at each plate
  • A continuous reactor with homogeneous mixing
  • A vacuum distillation unit
  • A purely kinetic system without any equilibrium

Correct Answer: A series of discrete equilibrium steps where solute re-distributes between phases at each plate

Q12. In the Van Deemter equation (H = A + B/u + Cu), which contribution to band broadening decreases as linear velocity (u) increases?

  • Longitudinal diffusion term (B/u), reflecting axial diffusion in the mobile phase
  • Eddy diffusion term (A), reflecting multiple flow paths
  • Mass transfer term (C·u), reflecting finite equilibration rates
  • Extra-column dispersion

Correct Answer: Longitudinal diffusion term (B/u), reflecting axial diffusion in the mobile phase

Q13. The “eddy diffusion” (A-term) component of band broadening arises from:

  • Multiple flow paths through a packed bed causing path-length differences
  • Slow diffusion of solute in the stationary phase film
  • Molecular adsorption–desorption kinetics at the detector
  • Thermal gradients along the column

Correct Answer: Multiple flow paths through a packed bed causing path-length differences

Q14. The distribution constant K = Cs/Cm directly influences which chromatographic property?

  • Retention; higher K means longer retention due to greater time spent in the stationary phase
  • Detector sensitivity; higher K yields stronger signals
  • Column backpressure; higher K reduces pressure
  • Sample solubility limit; higher K increases precipitation

Correct Answer: Retention; higher K means longer retention due to greater time spent in the stationary phase

Q15. In gradient elution of reversed-phase HPLC, the main principle enabling elution of late-retained compounds is:

  • Increasing mobile-phase strength (e.g., higher organic fraction) to reduce their partitioning into the stationary phase
  • Decreasing column temperature to sharpen peaks
  • Increasing ionic strength to enhance ion-exchange
  • Reducing column length to decrease retention time

Correct Answer: Increasing mobile-phase strength (e.g., higher organic fraction) to reduce their partitioning into the stationary phase

Q16. For a weakly acidic analyte in RPLC, adjusting the mobile-phase pH below its pKa typically:

  • Suppresses ionization, increases hydrophobicity, and increases retention
  • Increases ionization, increases polarity, and increases retention
  • Has no effect on retention because pH only matters in ion-exchange
  • Causes size-exclusion effects to dominate

Correct Answer: Suppresses ionization, increases hydrophobicity, and increases retention

Q17. Peak tailing in silica-based reversed-phase columns often results from residual silanol interactions. A principle-based mitigation is:

  • Using end-capped bonded phases or adding mobile-phase modifiers to mask silanol interactions
  • Increasing sample load to saturate the column
  • Lowering detector wavelength
  • Using a vacuum pump to degas post-column

Correct Answer: Using end-capped bonded phases or adding mobile-phase modifiers to mask silanol interactions

Q18. Chiral chromatography achieves enantiomer separation based on which principle?

  • Formation of transient diastereomeric interactions with a chiral stationary phase leading to different retention
  • Differences in molecular mass between enantiomers
  • Differences in UV absorbance spectra
  • Differences in polarity in achiral solvents

Correct Answer: Formation of transient diastereomeric interactions with a chiral stationary phase leading to different retention

Q19. When column capacity is exceeded (overload) in adsorption chromatography, the peak typically shows:

  • Fronting due to nonlinear adsorption isotherms at high concentrations
  • Tailing due to slow mass transfer at infinite dilution
  • Perfect Gaussian symmetry independent of load
  • No change in shape but reduced height

Correct Answer: Fronting due to nonlinear adsorption isotherms at high concentrations

Q20. In displacement chromatography, separation is driven by which mechanism?

  • Competitive displacement of sample components by a higher-affinity displacer, creating contiguous, self-sharpening zones
  • Continuous dilution of the sample band by the mobile phase
  • Partitioning solely based on volatility differences
  • Temperature programming of the column oven

Correct Answer: Competitive displacement of sample components by a higher-affinity displacer, creating contiguous, self-sharpening zones

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