Chiral analysis of pharmaceuticals by HPLC MCQs With Answer

Introduction:
This quiz set on Chiral analysis of pharmaceuticals by HPLC is designed for M.Pharm students studying Advanced Instrumental Analysis (MPA 201T). It covers theory and practical aspects of chiral separation using HPLC: chiral stationary phases, mobile-phase strategies (normal-, reversed- and polar-organic modes), chiral mobile-phase additives, indirect derivatization methods, detection techniques, and critical chromatographic parameters (selectivity, resolution, plate number). Questions include method development, mechanism of chiral recognition, sample preparation issues such as racemization, and validation metrics like enantiomeric excess. These MCQs aim to deepen understanding and prepare students for practical lab work and exams in chiral pharmaceutical analysis.

Q1. What best describes chiral HPLC used in pharmaceutical analysis?

• A chromatographic technique that uses achiral stationary phases to separate structural isomers
• A method that separates enantiomers by introducing a chiral selector in either stationary phase or mobile phase to form diastereomeric interactions
• A capillary electrophoresis technique with chiral buffering agents
• An HPLC method that uses only temperature gradients to resolve stereoisomers

Correct Answer: A method that separates enantiomers by introducing a chiral selector in either stationary phase or mobile phase to form diastereomeric interactions

Q2. Which type of chiral stationary phase (CSP) is most widely used for a broad range of pharmaceuticals?

• Ion-exchange resins
• Polysaccharide-based CSPs (e.g., cellulose or amylose derivatives)
• Silica monoliths without chiral modification
• Reverse-phase C18 without chiral selector

Correct Answer: Polysaccharide-based CSPs (e.g., cellulose or amylose derivatives)

Q3. What is the primary mechanism responsible for chiral discrimination on many CSPs?

• Solely size exclusion of the larger enantiomer
• Formation of covalent bonds with one enantiomer only
• Combination of stereoselective interactions such as hydrogen bonding, steric fit, π-π stacking and inclusion within chiral cavities
• Complete ionization of one enantiomer in the mobile phase

Correct Answer: Combination of stereoselective interactions such as hydrogen bonding, steric fit, π-π stacking and inclusion within chiral cavities

Q4. Which mobile-phase composition is typical for normal-phase chiral separations on polysaccharide CSPs?

• Water/acetonitrile with 0.1% formic acid
• Hexane/isopropanol (or hexane/ethanol) with small polar modifiers
• PBS buffer at pH 7.4
• Methanol/water gradient with 0.01% triethylamine

Correct Answer: Hexane/isopropanol (or hexane/ethanol) with small polar modifiers

Q5. What is a chiral mobile-phase additive (CMPA) strategy?

• Using high salt concentrations to denature the CSP
• Adding cyclodextrins or chiral ionic liquids to the mobile phase to form transient diastereomeric complexes with enantiomers
• Employing only achiral organic solvents to improve resolution
• Using temperature ramps as the sole means of selectivity control

Correct Answer: Adding cyclodextrins or chiral ionic liquids to the mobile phase to form transient diastereomeric complexes with enantiomers

Q6. What defines the indirect method for chiral HPLC analysis?

• Using chiral stationary phases exclusively
• Converting enantiomers into diastereomeric derivatives using a chiral derivatizing agent and separating them on an achiral column
• Performing HPLC without any chiral reagents and relying on temperature alone
• Using only mass spectrometric detection without separation

Correct Answer: Converting enantiomers into diastereomeric derivatives using a chiral derivatizing agent and separating them on an achiral column

Q7. Which chiral derivatizing reagent is commonly used for amino acid enantiomer analysis?

• Dansyl chloride
• Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide, FDAA)
• Trimethylsilyl chloride
• 2,4-Dinitrophenylhydrazine (DNPH)

Correct Answer: Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide, FDAA)

Q8. Which detection technique directly provides stereochemical information useful in chiral HPLC?

• UV detection at 254 nm
• Mass spectrometry without prior chiral separation
• Circular dichroism (CD) detection coupled to HPLC
• Refractive index detection

Correct Answer: Circular dichroism (CD) detection coupled to HPLC

Q9. How is enantiomeric excess (ee) calculated?

• ee = (concentration of racemate) / (total concentration) × 100
• ee = (major enantiomer − minor enantiomer) / (major enantiomer + minor enantiomer) × 100
• ee = (area of minor peak) / (area of major peak) × 100
• ee = 100 − percent purity

Correct Answer: ee = (major enantiomer − minor enantiomer) / (major enantiomer + minor enantiomer) × 100

Q10. Which formula correctly represents chromatographic resolution (Rs) between two peaks?

• Rs = (tR1 + tR2) / (w1 − w2)
• Rs = 2 × (tR2 − tR1) / (w1 + w2)
• Rs = (k2 − k1) / N
• Rs = α / (k’ average)

Correct Answer: Rs = 2 × (tR2 − tR1) / (w1 + w2)

Q11. How does column temperature generally influence chiral separations and method development?

• Temperature has no effect on enantioselectivity
• Changing temperature affects both retention and selectivity; van’t Hoff plots can reveal enthalpic vs entropic contributions to chiral recognition
• Higher temperature always improves resolution for chiral separations
• Temperature only affects detector response, not chromatographic behavior

Correct Answer: Changing temperature affects both retention and selectivity; van’t Hoff plots can reveal enthalpic vs entropic contributions to chiral recognition

Q12. Which chromatographic parameter most directly reflects column efficiency in HPLC?

• Resolution (Rs)
• Selectivity factor (α)
• The number of theoretical plates (N)
• Retention factor (k’)

Correct Answer: The number of theoretical plates (N)

Q13. Why is derivatization used in indirect chiral HPLC methods?

• To increase column backpressure intentionally
• To convert enantiomers into diastereomers that can be separated on achiral columns and to improve detectability
• To remove chirality from the analyte
• To eliminate the need for detection

Correct Answer: To convert enantiomers into diastereomers that can be separated on achiral columns and to improve detectability

Q14. Ligand-exchange chiral chromatography commonly uses which combination for resolving amino-acid-like chiral drugs?

• Sodium chloride and silica gel
• Copper(II) ions complexed with chiral amino acid ligands to form diastereomeric complexes
• Ammonium acetate and C18 stationary phase
• Gold nanoparticles as a chiral stationary phase

Correct Answer: Copper(II) ions complexed with chiral amino acid ligands to form diastereomeric complexes

Q15. Which CSP type is appropriate for large, polar chiral drugs and biological molecules?

• Pirkle-type small molecule CSPs only
• Protein-based CSPs such as α1-acid glycoprotein (AGP) or ovomucoid
• Unmodified silica gel
• Nonporous polymer beads without chiral functionality

Correct Answer: Protein-based CSPs such as α1-acid glycoprotein (AGP) or ovomucoid

Q16. The selectivity factor (α) in chiral HPLC is defined as which ratio?

• α = N2 / N1 where N is plate number
• α = t0 / tR
• α = k’2 / k’1 where k’ is the capacity factor of the second (more retained) enantiomer divided by the first
• α = (w1 + w2) / (tR2 − tR1)

Correct Answer: α = k’2 / k’1 where k’ is the capacity factor of the second (more retained) enantiomer divided by the first

Q17. What is the best practice to avoid racemization during sample preparation for chiral HPLC?

• Use strong acids and prolonged heating to ensure complete dissolution
• Store and process samples at low temperature, use mild pH conditions, and minimize exposure to heat and strong catalysts
• Always derivatize at pH 12
• Expose samples to air and light to accelerate equilibration

Correct Answer: Store and process samples at low temperature, use mild pH conditions, and minimize exposure to heat and strong catalysts

Q18. Which mobile-phase additive is commonly used to reduce tailing of basic chiral drugs on silica-based CSPs?

• Perchloric acid
• Triethylamine or other basic amines to block residual silanol interactions
• High concentrations of sodium hydroxide
• Strong oxidizing agents

Correct Answer: Triethylamine or other basic amines to block residual silanol interactions

Q19. Which analytical parameter specifically quantifies chiral purity of a pharmaceutical sample?

• Total organic carbon (TOC)
• Enantiomeric excess (ee) or enantiomeric purity expressed as % ee
• Melting point
• Isotopic ratio

Correct Answer: Enantiomeric excess (ee) or enantiomeric purity expressed as % ee

Q20. What is an advantage of polar organic mode (e.g., methanol or acetonitrile) for chiral HPLC separations on some CSPs?

• It always provides better resolution than normal-phase for all analytes
• Improved mass-spectrometry compatibility and faster column re-equilibration for polar analytes compared to nonpolar normal-phase solvents
• It eliminates the need for a chiral selector
• It prevents any hydrogen-bonding interactions between analyte and CSP

Correct Answer: Improved mass-spectrometry compatibility and faster column re-equilibration for polar analytes compared to nonpolar normal-phase solvents

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