Preparative HPLC principles MCQs With Answer

Preparative HPLC principles MCQs With Answer

Introduction: Preparative HPLC is a cornerstone technique in M.Pharm programs for isolating and purifying drug candidates, impurities, and synthetic intermediates at milligram-to-kilogram scale. This quiz set focuses on the core principles needed to design, scale, and optimize preparative separations: column selection and packing, loadability, solvent choice, gradient scaling, mass-transfer phenomena, detection and fraction collection, and strategies to maximize recovery and purity. Questions are designed to deepen understanding beyond basics—covering scale-up equations, kinetic limitations, solvent compatibility and practical troubleshooting—so students can translate analytical HPLC knowledge into efficient preparative workflows in research and industrial settings.

Q1. What is the primary objective of preparative HPLC?

• To obtain high-resolution chromatograms for method validation
• To quantify trace impurities with high sensitivity
• To isolate and purify substantial quantities of a target compound for further use
• To perform routine quality control assays in small sample volumes

Correct Answer: To isolate and purify substantial quantities of a target compound for further use

Q2. Which scaling principle is most appropriate when moving from an analytical column to a preparative column?

• Keep flow rate identical to the analytical method
• Maintain constant linear velocity and scale flow proportionally to column cross-sectional area
• Increase flow rate proportionally to column length only
• Keep injection volume constant while increasing column diameter

Correct Answer: Maintain constant linear velocity and scale flow proportionally to column cross-sectional area

Q3. What primarily determines the maximum sample mass that can be loaded onto a preparative HPLC column without unacceptable loss of resolution?

• The sample injection solvent viscosity
• The stationary phase binding capacity and acceptable resolution loss
• The detector sensitivity only
• The pump flow rate stability

Correct Answer: The stationary phase binding capacity and acceptable resolution loss

Q4. Which chromatographic symptom is most characteristic of mass overload in preparative HPLC?

• Baseline noise increase without peak shape change
• Peak fronting and loss of resolution between adjacent peaks
• Shorter retention times with unchanged peak shape
• Improved peak symmetry and resolution

Correct Answer: Peak fronting and loss of resolution between adjacent peaks

Q5. Why are slightly larger particle sizes often chosen for preparative HPLC columns compared to analytical columns?

• Larger particles provide higher theoretical plates for better resolution
• Larger particles reduce backpressure and allow higher flow and load, sacrificing some efficiency
• Larger particles increase detector sensitivity
• Larger particles prevent any peak broadening regardless of flow

Correct Answer: Larger particles reduce backpressure and allow higher flow and load, sacrificing some efficiency

Q6. Which method is commonly used to trigger fraction collection in preparative HPLC to achieve good purity and recovery?

• Collecting fixed-volume fractions irrespective of detector signal
• Detector-triggered collection with threshold settings and timed windows as backup
• Manual collection based solely on column retention time without detection
• Collecting only the first and last fractions of every run

Correct Answer: Detector-triggered collection with threshold settings and timed windows as backup

Q7. When selecting a mobile phase for preparative HPLC, which factor is most important to facilitate downstream processing?

• The mobile phase must have the highest possible UV absorbance
• The solvent should be highly nonpolar regardless of analyte solubility
• The solvent must dissolve the analyte and be sufficiently volatile or removable for isolation
• The mobile phase composition should be identical to analytical runs at all times

Correct Answer: The solvent must dissolve the analyte and be sufficiently volatile or removable for isolation

Q8. What is a critical consideration when scaling gradient elution from analytical to preparative HPLC?

• Gradient %B/min can be kept identical without change
• The gradient slope must be preserved in terms of column volumes and system dwell volume accounted for
• Use the same dwell volume as analytical instruments to ensure exact gradients
• Gradient scaling is unnecessary because fraction collection masks differences

Correct Answer: The gradient slope must be preserved in terms of column volumes and system dwell volume accounted for

Q9. What happens if the injection solvent is significantly stronger than the starting mobile phase in preparative HPLC?

• The peaks sharpen and resolution improves
• Peaks may exhibit severe broadening or fronting and resolution decreases
• The column lifetime increases due to better wetting
• Detector baseline remains perfectly flat

Correct Answer: Peaks may exhibit severe broadening or fronting and resolution decreases

Q10. Which term in the Van Deemter equation is directly related to mass transfer resistance between mobile and stationary phases?

• A term (eddy diffusion)
• B term (longitudinal diffusion)
• C term (mass transfer resistance)
• D term (detector noise)

Correct Answer: C term (mass transfer resistance)

Q11. In preparative method development, what is the “critical pair”?

• The pair of solvents used to elute the sample
• The two detectors used for simultaneous detection
• The pair of analytes (target and closest impurity) whose separation determines method acceptability
• The first and last fractions collected during a run

Correct Answer: The pair of analytes (target and closest impurity) whose separation determines method acceptability

Q12. Which stationary phase mode is most commonly employed for preparative purification of small drug-like molecules?

• Ion-exchange exclusively
• Normal phase (bare silica) for all compounds
• Reversed-phase (C18/C8) because of broad applicability and robustness
• Affinity chromatography for nonpolar small molecules

Correct Answer: Reversed-phase (C18/C8) because of broad applicability and robustness

Q13. Why is pH control important during preparative reversed-phase HPLC of ionizable compounds?

• Because pH has no effect on retention or selectivity for ionizable compounds
• Because pH influences ionization state, which alters retention, selectivity and peak shape
• pH only affects detector sensitivity, not chromatography
• pH control is only relevant for normal-phase chromatography

Correct Answer: Because pH influences ionization state, which alters retention, selectivity and peak shape

Q14. For preparative separations requiring extreme pH stability, which column packing material is preferred?

• Silica bonded phases exclusively
• Polymeric (e.g., polystyrene-divinylbenzene) supports due to broader pH stability
• Unbonded silica only for better retention
• Cellulose acetate supports for all small molecules

Correct Answer: Polymeric (e.g., polystyrene-divinylbenzene) supports due to broader pH stability

Q15. Which detection technique is most commonly used to trigger fraction collection in preparative HPLC for drug-like compounds?

• Mass spectrometry is always used as the primary trigger
• Evaporative light scattering detector (ELSD) is the universal trigger
• UV-visible absorption detection is most common; ELSD or MS used when compounds lack chromophores
• Fluorescence detection is used for all preparative purifications

Correct Answer: UV-visible absorption detection is most common; ELSD or MS used when compounds lack chromophores

Q16. Which factor most commonly causes lower than expected mass recovery after preparative HPLC?

• Excessive detector sensitivity
• Nonspecific irreversible adsorption to the stationary phase or container surfaces
• Too high mobile phase volatility
• Using too short a column

Correct Answer: Nonspecific irreversible adsorption to the stationary phase or container surfaces

Q17. When is gradient elution preferred over isocratic elution in preparative HPLC?

• For simple mixtures where all components elute close together
• When a wide polarity range must be separated and to shorten run time for strongly retained components
• Gradient is never preferred for preparative work due to reproducibility issues
• Only when the detector cannot detect late-eluting peaks

Correct Answer: When a wide polarity range must be separated and to shorten run time for strongly retained components

Q18. How does injecting highly concentrated crude samples repeatedly affect preparative column lifetime?

• It improves column efficiency by packing analyte into the stationary phase
• It can foul the stationary phase with particulates or lipophilic contaminants and shorten column life
• It has no effect if mobile phase is compatible
• It always increases theoretical plates due to overloading

Correct Answer: It can foul the stationary phase with particulates or lipophilic contaminants and shorten column life

Q19. What is the principal effect of increasing column temperature in preparative reversed-phase HPLC?

• Increases mobile phase viscosity and backpressure
• Improves mass transfer, reduces viscosity and backpressure, and can change selectivity
• Eliminates the need for gradient elution
• Always causes irreversible degradation of analytes

Correct Answer: Improves mass transfer, reduces viscosity and backpressure, and can change selectivity

Q20. When scaling injection from analytical to preparative HPLC, how should injected mass generally be adjusted to maintain similar chromatographic behavior?

• Keep injected mass identical regardless of column size
• Scale injected mass proportionally to column bed volume (column volume) while considering acceptable load per unit volume
• Decrease injected mass as column diameter increases
• Double the injected mass for every twofold increase in column length only

Correct Answer: Scale injected mass proportionally to column bed volume (column volume) while considering acceptable load per unit volume

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