HPLC method development MCQs With Answer

Introduction: HPLC method development MCQs With Answer is a focused quiz set designed for M.Pharm students studying Advanced Instrumental Analysis (MPA 201T). These multiple-choice questions cover core and advanced concepts in reversed-phase and other HPLC modes, addressing mobile phase selection, stationary phases, retention mechanisms, buffer choice, gradient vs isocratic strategies, system suitability, method validation and stability-indicating approaches. Each question is crafted to deepen understanding and support practical decision-making during method development and transfer. Use this quiz to test, revise and reinforce the theoretical knowledge and practical reasoning needed to design robust, reproducible HPLC methods in pharmaceutical analysis.

Q1. What is the primary objective of HPLC method development in pharmaceutical analysis?

• Achieve the fastest possible run time regardless of resolution
• Achieve adequate separation (selectivity) and accurate quantitation of analytes
• Use the most expensive column to ensure quality
• Minimize solvent consumption at the expense of peak shape

Correct Answer: Achieve adequate separation (selectivity) and accurate quantitation of analytes

Q2. How is the chromatographic retention factor (k’) defined?

• k’ = t0 / tR
• k’ = tR / t0
• k’ = (tR – t0) / t0
• k’ = tR – t0

Correct Answer: k’ = (tR – t0) / t0

Q3. Which parameter is most commonly used to describe column efficiency?

• Resolution (Rs)
• Theoretical plate number (N)
• Retention factor (k’)
• Tailing factor (Tf)

Correct Answer: Theoretical plate number (N)

Q4. According to the resolution equation, which factor generally provides the biggest improvement in resolution per unit change?

• Column length
• Retention factor (k’)
• Selectivity (alpha)
• Mobile phase flow rate

Correct Answer: Selectivity (alpha)

Q5. What is the commonly recommended rule for mobile phase pH selection for ionizable analytes?

• Set pH equal to analyte pKa
• Set pH at least 2 units away from the analyte pKa to favor a single ionization state
• Always use pH 7 for all analyses
• Use pH within 0.5 units of pKa for better sensitivity

Correct Answer: Set pH at least 2 units away from the analyte pKa to favor a single ionization state

Q6. Which buffer choice is most compatible with LC–MS detection during method development?

• Sodium phosphate buffer (non-volatile)
• Ammonium acetate or ammonium formate (volatile)
• Potassium dihydrogen phosphate (non-volatile)
• High concentration sodium chloride

Correct Answer: Ammonium acetate or ammonium formate (volatile)

Q7. When is gradient elution preferred over isocratic elution in HPLC method development?

• When all analytes elute at nearly the same retention time
• When analytes span a wide range of polarities and retention times
• When sample throughput is very low
• When using only aqueous mobile phases

Correct Answer: When analytes span a wide range of polarities and retention times

Q8. What is the primary function of a guard column in an HPLC system?

• To act as the primary analytical column
• To protect the analytical column by trapping particulates and strongly retained contaminants
• To increase system backpressure deliberately
• To change selectivity before the main column

Correct Answer: To protect the analytical column by trapping particulates and strongly retained contaminants

Q9. How does increasing column temperature typically influence retention and peak shape in reversed-phase HPLC?

• Increases retention times and causes peak broadening
• Decreases retention times and often improves peak shape and efficiency
• Has no effect on retention or efficiency
• Always causes peak tailing regardless of analyte

Correct Answer: Decreases retention times and often improves peak shape and efficiency

Q10. Which metric is used to quantify peak tailing?

• Retention factor (k’)
• Symmetry factor or tailing factor (Tf)
• Theoretical plates (N)
• Capacity factor

Correct Answer: Symmetry factor or tailing factor (Tf)

Q11. Which additive is commonly used as an ion-pairing reagent to retain strongly basic compounds on reversed-phase columns?

• Triethylamine (TEA) with perchlorate
• Sodium octane sulfonate (anionic ion-pair reagent)
• Ammonium bicarbonate
• Acetic acid only

Correct Answer: Sodium octane sulfonate (anionic ion-pair reagent)

Q12. What is the effect of increased extra-column volume on chromatographic performance?

• Improves resolution by increasing retention
• Reduces band broadening and increases efficiency
• Causes additional band broadening and reduces apparent column efficiency
• Has no measurable effect on narrow peaks

Correct Answer: Causes additional band broadening and reduces apparent column efficiency

Q13. Which of the following is NOT typically assessed as part of system suitability before sample analysis?

• Resolution between critical peaks
• Theoretical plates (column efficiency)
• Limit of detection (LOD)
• Peak tailing factor

Correct Answer: Limit of detection (LOD)

Q14. To improve peak shape for a basic analyte that shows tailing, which mobile phase additive is often used?

• Triethylamine (TEA) or another basic modifier to block silanol interactions
• Strong oxidizing agent
• High concentration phosphate buffer for MS detection
• Sodium chloride to increase ionic strength dramatically

Correct Answer: Triethylamine (TEA) or another basic modifier to block silanol interactions

Q15. When transferring a method from a 4.6 mm ID column to a 2.1 mm ID column, how should you scale the flow rate to maintain similar linear velocity?

• Keep the same flow rate
• Scale flow rate by the square of the ratio of diameters (area ratio)
• Double the flow rate because the column is shorter
• Reduce flow by 10% regardless of diameter

Correct Answer: Scale flow rate by the square of the ratio of diameters (area ratio)

Q16. Which detector is most negatively affected by nonvolatile buffers such as sodium phosphate in the mobile phase?

• UV–Visible detector
• PDA detector
• Evaporative light scattering detector (ELSD)
• Mass spectrometer (MS)

Correct Answer: Mass spectrometer (MS)

Q17. Which experimental strategy significantly reduces the number of experiments needed to optimize multiple HPLC factors simultaneously?

• One-factor-at-a-time (OFAT) optimization
• Design of Experiments (DoE) or factorial design
• Random trial-and-error
• Only changing column temperature

Correct Answer: Design of Experiments (DoE) or factorial design

Q18. In an isocratic method a late-eluting impurity greatly increases run time. What is an effective method development solution?

• Reduce column temperature to retain late peak further
• Switch to gradient elution or increase organic content toward a stronger mobile phase
• Decrease injection volume to zero
• Replace detector with a less sensitive detector

Correct Answer: Switch to gradient elution or increase organic content toward a stronger mobile phase

Q19. What chromatographic phenomenon is most likely responsible when a peak exhibits pronounced fronting?

• Strong secondary interactions with silanol groups
• Column overloading (sample too concentrated)
• Incorrect detector wavelength
• Use of volatile buffer in MS

Correct Answer: Column overloading (sample too concentrated)

Q20. Which step is essential to demonstrate that an HPLC method is stability-indicating?

• Use of a guard column during routine analysis
• Perform forced-degradation studies that show degradation products are separated from the API peak
• Only verify system suitability once
• Use a single-point calibration curve

Correct Answer: Perform forced-degradation studies that show degradation products are separated from the API peak

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