Practical aspects of preparative HPLC MCQs With Answer

Introduction: This collection of practical multiple-choice questions on preparative High-Performance Liquid Chromatography (HPLC) is designed specifically for M.Pharm students studying Advanced Instrumental Analysis (MPA 201T). The quiz emphasizes real-world operational considerations — column selection, sample loading, solvent choice, scale-up principles, fraction collection, detection methods, column care and validation metrics. Questions focus on troubleshooting and decision-making during preparative runs, helping students bridge theory with laboratory practice. Answers are provided to reinforce learning and self-assessment. Use these MCQs to sharpen practical skills required for efficient, reproducible purification of pharmaceutical compounds by preparative HPLC.

Q1. In preparative HPLC, which factor most directly limits the maximum sample mass that can be loaded onto a reversed-phase column without severe loss of resolution?

• Detector sensitivity
• Mobile phase viscosity
• Column loading capacity relative to stationary phase surface area
• Temperature stability of the detector

Correct Answer: Column loading capacity relative to stationary phase surface area

Q2. When scaling up from analytical to preparative HPLC using constant linear velocity scaling, which parameter must be adjusted proportionally to maintain chromatographic selectivity?

• Column length only
• Flow rate based on column cross-sectional area
• Detector wavelength
• Sample solvent identity

Correct Answer: Flow rate based on column cross-sectional area

Q3. What is the primary practical reason to prefer larger particle sizes (e.g., 5–10 µm) in preparative columns versus sub-2 µm particles often used analytically?

• Improved resolution for complex mixtures
• Lower backpressure enabling higher flow rates and larger columns
• Better UV transparency of the stationary phase
• Compatibility with gradient elution

Correct Answer: Lower backpressure enabling higher flow rates and larger columns

Q4. Which mobile phase modification is commonly used in preparative reversed-phase HPLC to improve peak shape for basic compounds and aid in mass spectrometry compatibility?

• Adding sodium hydroxide to raise pH to 9–10
• Adding trifluoroacetic acid (TFA) at high concentration
• Adding 0.1% formic acid or ammonium formate buffer
• Using pure water without additives

Correct Answer: Adding 0.1% formic acid or ammonium formate buffer

Q5. During preparative gradient runs, why is dwell volume (system void volume) more critical than in analytical HPLC?

• Dwell volume governs detector sensitivity for UV detection
• Large dwell volume changes the effective gradient shape and retention times at preparative scale
• Dwell volume affects column packing quality
• Dwell volume causes solvent evaporation in the injector

Correct Answer: Large dwell volume changes the effective gradient shape and retention times at preparative scale

Q6. What is the most appropriate action if sample solvent is stronger (higher organic content) than the initial mobile phase in a preparative reversed-phase injection, causing peak broadening and splitting?

• Increase column temperature to improve solvent mixing
• Pre-dilute or exchange sample into a weaker solvent similar to initial mobile phase
• Increase detector gain to compensate for peak height
• Shorten the column to reduce peak broadening

Correct Answer: Pre-dilute or exchange sample into a weaker solvent similar to initial mobile phase

Q7. Which detection method is most suitable for preparative HPLC when target compounds lack strong UV chromophores and collection of non-volatile solvents is required?

• UV-Vis detector at 254 nm
• Evaporative light scattering detector (ELSD)
• Diode-array detection with 4 nm slit
• Fluorescence detection without derivatization

Correct Answer: Evaporative light scattering detector (ELSD)

Q8. In preparative HPLC, what is the practical purpose of performing breakthrough curve experiments before routine fractionation?

• To calibrate the UV detector wavelength
• To determine the maximum sample load that avoids sample loss on the column and ensures acceptable recovery
• To measure column temperature gradients
• To check solvent miscibility with detector cell

Correct Answer: To determine the maximum sample load that avoids sample loss on the column and ensures acceptable recovery

Q9. For preparative separations of stereoisomers, which stationary phase choice is most directly compatible with chiral resolution without derivatization?

• Reversed-phase C18 silica
• Chiral stationary phase (e.g., polysaccharide-based)
• Normal phase bare silica only
• Ion-exchange resin

Correct Answer: Chiral stationary phase (e.g., polysaccharide-based)

Q10. When collecting fractions in preparative HPLC, which practice best ensures high purity and reproducible recovery for closely eluting peaks?

• Collecting broad fractions that include both peaks
• Using real-time UV threshold with time-based windows and visually confirming chromatograms for every run
• Collecting at fixed time intervals irrespective of detector signal
• Stopping fraction collection between injections to save solvent

Correct Answer: Using real-time UV threshold with time-based windows and visually confirming chromatograms for every run

Q11. Which cleaning procedure is most appropriate for removing strongly retained lipophilic contaminants from a preparative C18 column without damaging bonded phase?

• Flush with 100% water at high flow rate
• Flush sequentially with high-organic solvents (acetonitrile or isopropanol) followed by a mild aqueous-organic mixture
• Use strong base (5 M NaOH) at elevated temperature
• Backflush with pure hexane

Correct Answer: Flush sequentially with high-organic solvents (acetonitrile or isopropanol) followed by a mild aqueous-organic mixture

Q12. Which parameter calculated from preparative HPLC runs is most useful to assess process efficiency when scaling purification between batches?

• Retention factor (k’) only
• Percent recovery (mass of purified compound divided by mass loaded) and purity
• Detector lamp life
• Injector loop dead volume

Correct Answer: Percent recovery (mass of purified compound divided by mass loaded) and purity

Q13. In preparative HPLC, what is the effect of increasing column diameter while keeping the same stationary phase and linear velocity, assuming perfect scaling?

• Decreased column capacity and lower recovery
• Increased throughput (more mass per injection) with similar resolution and retention times
• Increased backpressure drastically
• Significant change in selectivity of separations

Correct Answer: Increased throughput (more mass per injection) with similar resolution and retention times

Q14. Which statement best describes why isocratic elution is sometimes preferred over gradient elution for preparative HPLC despite lower resolution for complex mixtures?

• Isocratic elution gives narrower peaks for all compounds
• Isocratic elution simplifies solvent recycling and fraction composition because mobile phase composition is constant
• Isocratic elution eliminates the need for detectors
• Isocratic elution always reduces solvent consumption

Correct Answer: Isocratic elution simplifies solvent recycling and fraction composition because mobile phase composition is constant

Q15. When performing fraction concentration after preparative HPLC, which approach minimizes loss of volatile analyte and contamination from non-volatile salts?

• Evaporate to dryness at high heat without inert gas
• Use rotary evaporation or vacuum centrifugation at reduced temperature, and if non-volatile salts are present, perform a desalting step prior to concentration
• Freeze-dry aqueous-organic fractions directly without desalting
• Neutralize fractions to pH 12 to precipitate salts

Correct Answer: Use rotary evaporation or vacuum centrifugation at reduced temperature, and if non-volatile salts are present, perform a desalting step prior to concentration

Q16. Which column parameter is most critical to inspect when troubleshooting unusually high backpressure in a preparative HPLC system?

• Detector wavelength alignment
• Integrity of frits and presence of particulate blockage
• Outlet tubing color
• Sample injection volume accuracy

Correct Answer: Integrity of frits and presence of particulate blockage

Q17. For preparative purification of an ionizable drug where pH affects retention, what is the practical strategy to improve resolution and peak shape?

• Operate at extreme pH (pH 1 or pH 12) regardless of column stability
• Choose a mobile phase pH where the analyte is predominantly in one ionization state and use compatible buffers at preparative concentrations
• Always use neutral pH water without buffer
• Use salt gradients instead of organic modifiers

Correct Answer: Choose a mobile phase pH where the analyte is predominantly in one ionization state and use compatible buffers at preparative concentrations

Q18. Which safety and sustainability practice is particularly important in preparative HPLC labs due to large solvent volumes?

• Discard all waste solvents down the sink because they are organic
• Implement solvent recycling, proper labeled waste segregation, and use of low-toxicity solvents where possible
• Always use the highest purity solvents regardless of cost
• Store all used fractions at room temperature in open containers

Correct Answer: Implement solvent recycling, proper labeled waste segregation, and use of low-toxicity solvents where possible

Q19. During preparative method validation, which parameter indicates how much impurity co-elutes with the target peak and is therefore critical for acceptance criteria?

• System suitability retentive time only
• Purity as determined by peak purity assessment (e.g., PDA spectral homogeneity) and orthogonal analysis
• Injector loop volume
• Detector lamp hours

Correct Answer: Purity as determined by peak purity assessment (e.g., PDA spectral homogeneity) and orthogonal analysis

Q20. If a preparative C18 column shows progressive loss of retention and resolution over multiple runs, the most likely practical causes to investigate are:

• Detector lamp intensity fluctuations and injector temperature
• Stationary phase degradation due to high pH, irreversible adsorption of sample matrix, or bleed of bonded phase
• Incorrect data processing parameters only
• Using fresh solvents for each run

Correct Answer: Stationary phase degradation due to high pH, irreversible adsorption of sample matrix, or bleed of bonded phase

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