Sample preparation for HPLC MCQs With Answer

Introduction: Sample preparation is a critical step before high-performance liquid chromatography (HPLC) analysis, directly influencing accuracy, precision, and instrument performance. This blog provides focused multiple-choice questions on sample preparation techniques commonly required for HPLC in pharmaceutical analysis, including filtration, centrifugation, protein precipitation, liquid–liquid extraction, solid-phase extraction (SPE), dilution/reconstitution strategies, solvent compatibility, and sample stability. The questions emphasize practical decision-making—selecting membrane types, pore sizes, sorbents, pH adjustments, and methods to minimize matrix effects and carryover—so M.Pharm students gain applied understanding for method development and routine bioanalytical workflows. Answers are provided to reinforce learning and support exam preparation.

Q1. Which membrane pore size is most appropriate to remove sub-2 µm particulates for UHPLC sample injection?

• 0.45 µm
• 0.2 µm
• 1.2 µm
• 5 µm

Correct Answer: 0.2 µm

Q2. For filtering samples containing high organic content (acetonitrile or methanol), which membrane material is most chemically resistant?

• Polyvinylidene fluoride (PVDF)
• Polytetrafluoroethylene (PTFE)
• Nylon
• Cellulose acetate

Correct Answer: Polytetrafluoroethylene (PTFE)

Q3. What is the primary purpose of using solid-phase extraction (SPE) in HPLC sample preparation?

• Selective extraction and concentration of analytes from complex matrices
• Simple particle filtration before injection
• Adjusting the mobile phase pH inside the column
• Direct derivatization of analytes

Correct Answer: Selective extraction and concentration of analytes from complex matrices

Q4. Which reagent is most commonly used for protein precipitation in plasma samples prior to HPLC analysis?

• Acetonitrile
• n-Hexane
• Sodium chloride
• Diethyl ether

Correct Answer: Acetonitrile

Q5. A sample requires removal of precipitated proteins after adding organic solvent; which centrifugation condition is typically appropriate?

• 500 x g for 1 minute
• 1,000 x g for 5 minutes
• 3,000 x g for 2 minutes
• 10,000 x g for 10 minutes

Correct Answer: 10,000 x g for 10 minutes

Q6. For concentrating analytes from extracts without heat degradation, which evaporation method is preferred?

• Heating on a hotplate to dryness
• Evaporation under a gentle nitrogen stream at ambient temperature
• Open-air evaporation at room temperature
• Microwave-assisted evaporation

Correct Answer: Evaporation under a gentle nitrogen stream at ambient temperature

Q7. Which extraction solvent is most appropriate for liquid–liquid extraction (LLE) of highly nonpolar analytes from aqueous matrices?

• Hexane
• Water
• Methanol
• Acetonitrile

Correct Answer: Hexane

Q8. To extract a weakly basic drug (pKa ~8) into an organic solvent, at which aqueous pH should the sample be adjusted to maximize recovery?

• pH 2
• pH 4
• pH 7
• pH 10

Correct Answer: pH 10

Q9. Which SPE sorbent provides a balanced retention for a wide polarity range (polar to mid-polar) and is commonly used in bioanalysis?

• C18 (octadecyl silica)
• Hydrophilic-lipophilic balance (HLB)
• Strong cation exchange (SCX)
• Normal phase silica

Correct Answer: Hydrophilic-lipophilic balance (HLB)

Q10. In LC–MS sample prep, what does the term “matrix effect” commonly refer to?

• Ion suppression or enhancement caused by coeluting matrix components
• Change in sample pH over time
• Loss of analyte due to evaporation
• Mechanical clogging of the column by particulates

Correct Answer: Ion suppression or enhancement caused by coeluting matrix components

Q11. Which sample vial material is often preferred to minimize adsorption of basic or peptide analytes?

• Non-silanized glass
• Silanized glass
• Polypropylene
• Aluminum foil

Correct Answer: Polypropylene

Q12. When is chemical derivatization recommended before HPLC analysis?

• When the analyte is volatile enough for GC
• When the analyte lacks a chromophore or fluorophore required for detection
• When the analyte is highly polar and water soluble
• When the sample has particulate matter

Correct Answer: When the analyte lacks a chromophore or fluorophore required for detection

Q13. What is the most effective practice to minimize autosampler carryover between injections?

• Use a larger injection volume
• Perform thorough needle and injection port washes with a strong wash solvent between injections
• Use water as a wash for all analytes
• Decrease column temperature between runs

Correct Answer: Perform thorough needle and injection port washes with a strong wash solvent between injections

Q14. To improve sample stability and reduce degradation during long autosampler sequences, what is the recommended autosampler condition?

• Keep samples at ambient laboratory temperature
• Maintain a refrigerated autosampler (e.g., 4 °C)
• Store samples in direct sunlight
• Shake samples continuously at room temperature

Correct Answer: Maintain a refrigerated autosampler (e.g., 4 °C)

Q15. Which filter membrane should generally be avoided for protein-containing samples due to high non-specific protein binding?

• Cellulose acetate
• Polytetrafluoroethylene (PTFE)
• Polyvinylidene fluoride (PVDF)
• Polypropylene

Correct Answer: Polyvinylidene fluoride (PVDF)

Q16. In liquid–liquid extraction, adding sodium chloride (salting out) primarily serves to:

• Decrease ionic strength to solubilize analytes
• Increase partitioning of analytes into the organic phase by reducing analyte solubility in water
• Neutralize the pH of the aqueous phase
• Act as an organic solvent modifier

Correct Answer: Increase partitioning of analytes into the organic phase by reducing analyte solubility in water

Q17. When reconstituting a dried extract prior to injection, which solvent choice minimizes peak distortion and poor chromatography?

• Poorly miscible solvent with the initial mobile phase
• Solvent identical or similar in composition to the initial mobile phase
• Pure water regardless of initial mobile phase
• Strong acid regardless of analyte chemistry

Correct Answer: Solvent identical or similar in composition to the initial mobile phase

Q18. What is the correct sequence of SPE cartridge preparation before sample loading?

• Equilibrate with water, then condition with organic solvent
• Condition with organic solvent, then equilibrate with aqueous buffer
• Load sample directly without conditioning
• Dry the cartridge, then condition with strong acid

Correct Answer: Condition with organic solvent, then equilibrate with aqueous buffer

Q19. Why is degassing (removal of dissolved gases) of samples and mobile phases important for HPLC systems?

• To increase sample viscosity
• To prevent bubble formation that causes baseline noise and pump cavitation
• To sterilize the mobile phase
• To neutralize analyte pH

Correct Answer: To prevent bubble formation that causes baseline noise and pump cavitation

Q20. If an unknown sample concentration is above the upper limit of quantitation (ULOQ), what is the appropriate action before reporting results?

• Report the value as exceeding ULOQ without further action
• Dilute the sample into the calibration range and reanalyze validating dilution integrity
• Inject a smaller volume without dilution
• Discard the sample

Correct Answer: Dilute the sample into the calibration range and reanalyze validating dilution integrity

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