Pharmaceutical applications of HPLC MCQs With Answer

Introduction: This quiz collection focuses on pharmaceutical applications of High-Performance Liquid Chromatography (HPLC) tailored for M.Pharm students studying Advanced Instrumental Analysis (MPA 201T). The questions cover core principles, column and detector choices, mobile phase selection, method development strategies, validation parameters, stability-indicating assays, sample preparation for biological matrices, troubleshooting, and regulatory expectations. Designed to deepen conceptual understanding and practical decision-making, the MCQs emphasize real-world analytical challenges encountered in drug development, quality control, and stability studies. Each question includes plausible alternatives and a clear correct answer to help reinforce learning, prepare for exams, and support routine method development in pharmaceutical laboratories.

Q1. What is the primary chromatographic retention mechanism exploited in reversed-phase HPLC?

• Adsorption of analytes onto polar stationary phase
• Partitioning between a nonpolar stationary phase and a polar mobile phase
• Size-based exclusion through porous beads
• Ionic exchange between charged analytes and stationary phase

Correct Answer: Partitioning between a nonpolar stationary phase and a polar mobile phase

Q2. Which advantage makes reversed‑phase HPLC the most widely used mode in pharmaceutical analysis?

• Best for extremely volatile analytes
• Excellent reproducibility and compatibility with aqueous mobile phases for a broad range of drug molecules
• Requires no organic solvents
• Provides size-based separations independent of polarity

Correct Answer: Excellent reproducibility and compatibility with aqueous mobile phases for a broad range of drug molecules

Q3. How does decreasing column particle size influence chromatographic performance?

• Reduces efficiency but lowers backpressure
• Increases efficiency (more theoretical plates) and raises column backpressure
• Has no effect on efficiency but increases selectivity
• Only affects detector sensitivity, not separation

Correct Answer: Increases efficiency (more theoretical plates) and raises column backpressure

Q4. In the Van Deemter equation, which term becomes most significant at high mobile phase velocities?

• A term (eddy diffusion)
• B term (longitudinal diffusion)
• C term (mass transfer resistance)
• D term (extra-column dispersion)

Correct Answer: C term (mass transfer resistance)

Q5. For quantifying non‑UV absorbing, semi‑volatile drugs in a formulation, which detector is most suitable?

• UV‑Vis diode array detector (DAD)
• Refractive index detector (RID)
• Charged aerosol detector (CAD)
• Fluorescence detector (FLD)

Correct Answer: Charged aerosol detector (CAD)

Q6. What is the principal benefit of gradient elution when analyzing complex pharmaceutical mixtures?

• Always eliminates the need for sample preparation
• Improves separation of analytes with widely different polarities and shortens run time
• Makes method validation unnecessary
• Reduces column backpressure compared to isocratic runs

Correct Answer: Improves separation of analytes with widely different polarities and shortens run time

Q7. Which parameter is NOT typically considered a system suitability requirement for an HPLC assay?

• Resolution between critical peak pairs
• Theoretical plate count (efficiency)
• Tailing factor for analyte peak
• Limit of detection (LOD)

Correct Answer: Limit of detection (LOD)

Q8. Why is precise control of mobile phase pH critical in pharmaceutical HPLC?

• It only affects column temperature
• It alters detector lamp intensity
• It controls ionization state of analytes, affecting retention and selectivity
• pH has no significant effect if organic modifier is present

Correct Answer: It controls ionization state of analytes, affecting retention and selectivity

Q9. Which mobile phase buffer is most appropriate when coupling HPLC to a mass spectrometer for drug analysis?

• Phosphate buffer (non‑volatile)
• Tris buffer
• Ammonium formate or ammonium acetate (volatile)
• Sodium sulfate buffer

Correct Answer: Ammonium formate or ammonium acetate (volatile)

Q10. For trace-level drug quantification in plasma where matrix cleanup is needed, which sample preparation method is preferred?

• Direct injection of diluted plasma
• Protein precipitation only
• Solid‑phase extraction (SPE)
• Simple filtration through a syringe filter

Correct Answer: Solid‑phase extraction (SPE)

Q11. What is the main objective of forced degradation studies in method development for pharmaceuticals?

• To determine the drug’s taste profile
• To develop a stability‑indicating method and identify degradation pathways and products
• To optimize tablet coating aesthetics
• To replace routine stability studies

Correct Answer: To develop a stability‑indicating method and identify degradation pathways and products

Q12. Which validation parameter assesses whether detector response is directly proportional to analyte concentration over a specified range?

• Precision
• Specificity
• Linearity
• Ruggedness

Correct Answer: Linearity

Q13. Why is column equilibration important before running analytical samples in HPLC?

• Equilibration sterilizes the column
• It prevents detector saturation only
• It ensures reproducible retention times and a stable baseline by establishing steady-state mobile phase–stationary phase conditions
• Equilibration reduces mobile phase viscosity permanently

Correct Answer: It ensures reproducible retention times and a stable baseline by establishing steady-state mobile phase–stationary phase conditions

Q14. What is a common cause of pronounced peak tailing for basic pharmaceutical compounds on silica‑based reversed‑phase columns?

• Excess mobile phase organic modifier
• Interaction with residual silanol groups on the silica support
• Too low column temperature only
• Detector noise

Correct Answer: Interaction with residual silanol groups on the silica support

Q15. How does UHPLC (ultra‑high performance liquid chromatography) mainly differ from conventional HPLC?

• UHPLC requires lower system pressure than HPLC
• UHPLC uses larger particles for better selectivity
• UHPLC uses sub‑2 μm particles and higher pressures to achieve faster separations with higher efficiency
• UHPLC cannot be used for pharmaceutical analysis

Correct Answer: UHPLC uses sub‑2 μm particles and higher pressures to achieve faster separations with higher efficiency

Q16. What is the purpose of ion‑pair chromatography in drug analysis?

• To remove ions from the mobile phase
• To increase retention of ionic analytes on reversed‑phase columns by forming neutral ion pairs
• To separate enantiomers using chiral selectors
• To perform size exclusion separations

Correct Answer: To increase retention of ionic analytes on reversed‑phase columns by forming neutral ion pairs

Q17. A stability‑indicating assay is best described as:

• An assay that measures only degradation products
• An assay that quantifies the active pharmaceutical ingredient (API) without separation from degradation products
• An assay that accurately and selectively measures the API in the presence of degradation products, impurities, and excipients
• An assay used solely for potency testing during clinical trials

Correct Answer: An assay that accurately and selectively measures the API in the presence of degradation products, impurities, and excipients

Q18. When is an isocratic HPLC method preferred over a gradient method in routine QC of pharmaceuticals?

• When analytes have widely varying polarities and require strong elution strength
• When instrument backpressure must be maximized
• When analytes have similar retention behavior and sufficient resolution can be achieved with a constant mobile phase composition
• When MS detection is mandatory

Correct Answer: When analytes have similar retention behavior and sufficient resolution can be achieved with a constant mobile phase composition

Q19. Which common cause leads to sample carryover in HPLC and how can it be prevented?

• Excess mobile phase degassing; prevented by increasing column temperature
• Inadequate needle wash between injections; prevented by optimizing wash solvents and wash volume
• Using volatile buffers; prevented by switching to phosphate buffers
• Low detector sensitivity; prevented by increasing injection volume

Correct Answer: Inadequate needle wash between injections; prevented by optimizing wash solvents and wash volume

Q20. If heteroscedasticity (variance increasing with concentration) is observed in calibration data, which weighting strategy is commonly recommended for linear regression in pharmaceutical assays?

• No weighting (ordinary least squares)
• Weighting by concentration (x)
• Weighting by 1/x
• Weighting by x^2

Correct Answer: Weighting by 1/x

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