Revised phase chiral method development MCQs With Answer

Revised phase chiral method development MCQs With Answer

This question set is designed for M.Pharm students to strengthen practical and theoretical understanding of chiral method development in reversed-phase chromatography. The focus is on advanced instrumental considerations: chiral stationary phases, mobile phase composition and modifiers, retention and selectivity optimization, detection challenges, and method validation for enantiomeric separations. Each MCQ tests decision-making skills used in real method development — column selection, buffer choice, solvent effects, temperature control, and troubleshooting for peak shape and resolution. Use these questions for self-assessment or classroom discussion to master key parameters that drive robust, reproducible enantioselective separations in pharmaceutical analysis.

Q1. In reversed‑phase chiral separations, which primary interaction is most commonly exploited by polysaccharide‑based chiral stationary phases to achieve enantioselectivity?

• Strong ionic exchange between analyte and CSP
• Inclusion complexation within a cavity
• Hydrogen bonding and multiple secondary interactions (π–π, dipole)
• Size exclusion based segregation

Correct Answer: Hydrogen bonding and multiple secondary interactions (π–π, dipole)

Q2. When screening mobile phases for reversed‑phase chiral method development of a basic drug, which mobile phase modifier typically improves peak shape and reduces tailing?

• Trifluoroacetic acid (TFA)
• Sodium chloride
• Tetrabutylammonium hydroxide
• Acetonitrile without buffer

Correct Answer: Trifluoroacetic acid (TFA)

Q3. Which chromatographic parameter directly quantifies the ability of a method to separate two enantiomers?

• Capacity factor (k’)
• Resolution (Rs)
• Theoretical plate number (N)
• Retention time (tR) of the first peak

Correct Answer: Resolution (Rs)

Q4. In method development, increasing column temperature often affects enantioselectivity by:

• Always increasing resolution due to faster kinetics
• Decreasing retention and commonly reducing selectivity due to weakened secondary interactions
• Causing irreversible denaturation of chiral selectors only
• Changing detection wavelength requirements

Correct Answer: Decreasing retention and commonly reducing selectivity due to weakened secondary interactions

Q5. Which approach is most appropriate when no enantioresolution is observed on multiple CSPs in reversed‑phase mode?

• Derivatize the analyte to introduce a chromophore
• Switch to polar organic or normal phase chiral mode or try alternative CSP chemistries
• Increase injection volume dramatically
• Decrease detector sensitivity

Correct Answer: Switch to polar organic or normal phase chiral mode or try alternative CSP chemistries

Q6. For ionizable analytes, why is buffer pH critical in reversed‑phase chiral separations?

• It affects mobile phase viscosity only
• It controls analyte ionization state which alters interaction strength with the CSP and retention/selectivity
• It changes the column length
• It only influences detector baseline noise

Correct Answer: It controls analyte ionization state which alters interaction strength with the CSP and retention/selectivity

Q7. Which of the following is a reliable strategy to increase enantioselectivity (α) without significantly increasing analysis time?

• Use a shorter column with smaller particle size at higher flow rate
• Change organic modifier composition (e.g., MeOH to ACN) or add small amounts of polar additives
• Increase injection volume tenfold
• Replace buffer with pure water

Correct Answer: Change organic modifier composition (e.g., MeOH to ACN) or add small amounts of polar additives

Q8. What is the principal reason to include an achiral reversed‑phase column in early-stage method development alongside CSP screening?

• To confirm method reproducibility under nonselective conditions
• To check for coeluting achiral impurities and matrix effects that affect peak purity and assay specificity
• To increase run time for stress testing
• To intentionally reduce sensitivity

Correct Answer: To check for coeluting achiral impurities and matrix effects that affect peak purity and assay specificity

Q9. Which detector technique provides additional orthogonal confirmation of enantiomer identity beyond UV when developing chiral methods?

• Refractive index (RI) detection
• Mass spectrometry (MS) and chiroptical detection (e.g., circular dichroism, ECD)
• Evaporative light scattering (ELSD)
• Turbidity measurement

Correct Answer: Mass spectrometry (MS) and chiroptical detection (e.g., circular dichroism, ECD)

Q10. In reversed‑phase chiral chromatography, the term “enantioselectivity (α)” is defined as:

• The ratio of plate numbers of two peaks
• The ratio of capacity factors k’2/k’1 for the two enantiomers
• The sum of the retention times
• The difference between peak widths at baseline

Correct Answer: The ratio of capacity factors k’2/k’1 for the two enantiomers

Q11. A major advantage of using polar organic mode on polysaccharide CSPs compared with aqueous reversed‑phase is:

• Polar organic mode always eliminates sample preparation
• It often increases enantioselectivity for polar analytes and improves solubility of hydrophobic modifiers
• It prevents any degradation of the CSP
• It removes the need for buffers entirely for ionizable analytes

Correct Answer: It often increases enantioselectivity for polar analytes and improves solubility of hydrophobic modifiers

Q12. Which experimental design strategy reduces the number of experiments needed when optimizing mobile phase composition, pH, and temperature for chiral separation?

• One‑factor‑at‑a‑time (OFAT)
• Design of experiments (DoE) approach such as factorial or response surface methods
• Random trial and error
• Increasing column length sequentially until resolved

Correct Answer: Design of experiments (DoE) approach such as factorial or response surface methods

Q13. If an enantiomer peak exhibits broadening and tailing on a CSP, the first troubleshooting step should be:

• Change detector wavelength
• Check sample solvent compatibility with the mobile phase and reduce solvent mismatch
• Switch to a longer column immediately
• Increase column temperature by 50 °C

Correct Answer: Check sample solvent compatibility with the mobile phase and reduce solvent mismatch

Q14. Which statement describes the role of chiral recognition by macrocyclic glycopeptide CSPs (e.g., vancomycin, teicoplanin)?

• They rely solely on hydrophobic interactions
• They provide multiple interaction sites including hydrogen bonding, ionic interactions, steric fit and inclusion for broad chiral recognition
• They function only in gas chromatography
• They are limited to separating sugars only

Correct Answer: They provide multiple interaction sites including hydrogen bonding, ionic interactions, steric fit and inclusion for broad chiral recognition

Q15. During method validation for an enantiomeric purity assay, which parameter is most critical to demonstrate the method’s ability to distinguish and quantify a minor enantiomer in presence of a major enantiomer?

• Linearity over a single concentration
• Limit of detection (LOD) only
• Specificity and limit of quantitation (LOQ) for the minor enantiomer, plus accuracy and precision at low levels
• Detector lamp intensity

Correct Answer: Specificity and limit of quantitation (LOQ) for the minor enantiomer, plus accuracy and precision at low levels

Q16. Which mobile phase additive can enhance enantioselectivity for basic compounds by forming transient ion pairs on some CSPs?

• Nonionic surfactants like Tween 20
• Triethylamine (TEA) or alkylamines as basic modifiers
• Sodium sulfate at high concentration
• Glycerol as a viscosity modifier

Correct Answer: Triethylamine (TEA) or alkylamines as basic modifiers

Q17. When transferring a chiral method between columns with the same chemistry but different lengths and particle sizes, which scaling parameter is most important to preserve resolution?

• Detector type
• Gradient profile, linear velocity (flow adjusted for particle size), and column efficiency (N)
• Injection solvent color
• Ambient room lighting

Correct Answer: Gradient profile, linear velocity (flow adjusted for particle size), and column efficiency (N)

Q18. Which scenario indicates that enantioseparation is dominated by inclusion complexation (e.g., cyclodextrin derivatives) rather than surface interactions?

• Resolution improves with increased column diameter
• Significant dependence of retention/selectivity on the size and polarity of guest substituents and temperature-dependent binding constants suggesting cavity binding
• Complete independence from mobile phase composition
• Only ionic strength changes affect selectivity

Correct Answer: Significant dependence of retention/selectivity on the size and polarity of guest substituents and temperature-dependent binding constants suggesting cavity binding

Q19. Which practice improves robustness of a reversed‑phase chiral assay intended for routine QC use?

• Using a very narrow operational window for pH and temperature
• Optimizing for maximum resolution with minimal changes in mobile phase composition, using stable buffers, and validating system suitability criteria
• Never performing system suitability tests
• Avoiding documentation of column lot changes

Correct Answer: Optimizing for maximum resolution with minimal changes in mobile phase composition, using stable buffers, and validating system suitability criteria

Q20. In LC–MS analysis of chiral separations, which mobile phase consideration is crucial to maintain MS sensitivity while achieving chiral selectivity?

• Use nonvolatile salts at high concentration to maximize buffering power
• Prefer volatile buffers (ammonium acetate/formate) and volatile modifiers while balancing pH and organic content to preserve both MS response and chiral recognition
• Always use TFA at 1% for best MS signal
• Eliminate organic modifier to improve electrospray stability

Correct Answer: Prefer volatile buffers (ammonium acetate/formate) and volatile modifiers while balancing pH and organic content to preserve both MS response and chiral recognition

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