Principle of bioanalytical extraction methods MCQs With Answer

This set of MCQs on the “Principle of Bioanalytical Extraction Methods” is designed for M.Pharm students studying Modern Bio-Analytical Techniques (MPA 202T). The questions focus on core extraction principles used in bioanalysis — liquid–liquid extraction, solid-phase extraction, protein precipitation, supported liquid extraction, ion-pairing, and microextraction techniques. Emphasis is placed on pH/pKa relationships, partition and distribution concepts, sorbent selection, cleanup strategies, matrix effects, and sample preparation troubleshooting. These items will help reinforce theoretical understanding and develop practical decision-making skills for designing and optimizing extraction methods in quantitative bioanalysis.

Q1. What fundamental property does liquid–liquid extraction primarily exploit to separate an analyte between two immiscible phases?

• Partition coefficient (ratio of concentrations in two phases)
• Surface tension differences
• Magnetic susceptibility
• Optical refractive index

Correct Answer: Partition coefficient (ratio of concentrations in two phases)

Q2. For efficient extraction of a weak acidic drug into an organic solvent from aqueous media, which pH condition is most favorable?

• pH much greater than the drug pKa (analyte ionized)
• pH equal to the drug pKa
• pH much less than the drug pKa (analyte unionized)
• pH has no effect on extraction efficiency

Correct Answer: pH much less than the drug pKa (analyte unionized)

Q3. How does the distribution ratio (D) differ from the partition coefficient (P) in bioanalytical extraction?

• D represents only the unionized species’ concentration ratio
• D includes all forms of the analyte in each phase at a given pH
• P accounts for ionized plus unionized species at a given pH
• P varies with pH while D is constant

Correct Answer: D includes all forms of the analyte in each phase at a given pH

Q4. What is the main purpose of protein precipitation as a sample preparation technique in bioanalysis?

• Remove proteins and release protein-bound drug
• Derivatize analytes for GC analysis
• Increase sample viscosity for easier handling
• Convert nonpolar compounds to polar forms

Correct Answer: Remove proteins and release protein-bound drug

Q5. What is the operative principle of solid-phase extraction (SPE) for sample cleanup?

• Selective retention of analyte on a sorbent followed by washing and elution
• Evaporation of solvent to concentrate analyte
• Centrifugal partitioning based on density differences
• Denaturation of matrix components by heat

Correct Answer: Selective retention of analyte on a sorbent followed by washing and elution

Q6. Which sorbent is most commonly used in reverse-phase SPE for nonpolar drug extraction from biological matrices?

• Silica gel without modification
• C18 (octadecyl bonded silica)
• Strong cation exchange resin only
• Alumina (neutral)

Correct Answer: C18 (octadecyl bonded silica)

Q7. What does the term “breakthrough volume” in SPE refer to?

• The volume of eluent required to elute all retained analyte
• The volume of sample that causes analyte to first appear in the column effluent
• The minimum wash volume required to remove impurities
• The gas volume displaced during sorbent conditioning

Correct Answer: The volume of sample that causes analyte to first appear in the column effluent

Q8. What is the mechanism by which ion-pair extraction enhances the extraction of charged analytes into organic solvents?

• Formation of a volatile salt that partitions to the organic phase
• Formation of a neutral ion-pair between analyte and counterion
• Increase of sample temperature to favor partitioning
• Covalent modification of the analyte to increase hydrophilicity

Correct Answer: Formation of a neutral ion-pair between analyte and counterion

Q9. How does supported liquid extraction (SLE) differ from classical liquid–liquid extraction?

• SLE uses energetic mixing to form microemulsions
• In SLE, the aqueous sample is immobilized on an inert support and analyte eluted with organic solvent
• SLE relies on evaporation of solvent under high vacuum
• SLE requires derivatization of analytes prior to extraction

Correct Answer: In SLE, the aqueous sample is immobilized on an inert support and analyte eluted with organic solvent

Q10. What is the primary cause of matrix effects observed in LC–MS bioanalysis after sample extraction?

• Ion suppression or enhancement from co-eluting matrix components
• Excessive column backpressure
• Insufficient chromatographic resolution only
• Decomposition of analyte in the autosampler vial

Correct Answer: Ion suppression or enhancement from co-eluting matrix components

Q11. Which property is most important when selecting an organic solvent for liquid–liquid extraction of a moderately lipophilic drug?

• High miscibility with water to improve phase contact
• Low dielectric constant to increase analyte ionization
• Low miscibility with water and high partition affinity for the analyte
• High vapor pressure to ensure rapid evaporation

Correct Answer: Low miscibility with water and high partition affinity for the analyte

Q12. Why is back-extraction (re-extraction) used after an initial LLE step in bioanalytical workflows?

• To convert analyte into its ionic form for GC analysis
• To change solvent polarity to further purify or concentrate the analyte
• To sterilize the sample for microbiological testing
• To increase the sample pH to extreme values for stability

Correct Answer: To change solvent polarity to further purify or concentrate the analyte

Q13. How does the use of an internal standard (IS) improve quantitation in extraction-based bioanalytical methods?

• It eliminates the need for calibration standards
• It compensates for extraction variability, losses and matrix effects
• It increases the intrinsic signal of the analyte
• It prevents protein binding of the analyte

Correct Answer: It compensates for extraction variability, losses and matrix effects

Q14. What primarily determines elution strength in SPE protocols?

• The pH of the conditioning solvent only
• The polarity of the elution solvent and strength of sorbent–analyte interactions
• The flow rate through the sorbent only
• The temperature of the elution solvent only

Correct Answer: The polarity of the elution solvent and strength of sorbent–analyte interactions

Q15. For extraction of a weak basic drug (pKa 8.5) into an organic solvent, which aqueous pH is most appropriate to maximize recovery?

• pH 5.0
• pH 7.0
• pH 10.0
• pH has no effect on basic drugs

Correct Answer: pH 10.0

Q16. What is the key advantage of solid-phase microextraction (SPME) compared with conventional solvent extraction?

• Requires larger sample volumes for enhanced sensitivity
• Uses a coated fiber to extract analytes directly without solvents
• Always gives quantitative recovery without calibration
• Eliminates the need for chromatographic separation

Correct Answer: Uses a coated fiber to extract analytes directly without solvents

Q17. How is “recovery” defined in the context of bioanalytical extraction methods?

• The ratio of signal intensity in matrix to that in pure solvent
• The percentage of analyte removed from the sample by precipitation
• The percentage of analyte extracted and measured relative to the known amount present
• The change in analyte pKa after extraction

Correct Answer: The percentage of analyte extracted and measured relative to the known amount present

Q18. Why might derivatization be performed before extraction in some bioanalytical methods?

• To reduce the analyte molecular weight
• To increase detectability, volatility or stability of the analyte
• To make the analyte more hydrophilic for aqueous-only methods
• To remove matrix components selectively

Correct Answer: To increase detectability, volatility or stability of the analyte

Q19. After protein precipitation, why is centrifugation routinely performed before analysis?

• To evaporate solvent from the supernatant
• To separate precipitated proteins from the clear supernatant containing the analyte
• To induce chemical modification of analytes
• To neutralize the sample pH

Correct Answer: To separate precipitated proteins from the clear supernatant containing the analyte

Q20. What is the most common cause of persistent emulsions during liquid–liquid extraction of biological samples?

• Excessive temperature during mixing
• Presence of surface-active agents (lipids, phospholipids, detergents)
• Too large a density difference between phases
• Using an organic solvent with very low boiling point

Correct Answer: Presence of surface-active agents (lipids, phospholipids, detergents)

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