Principle and application of HPLC MCQs With Answer

Principle and application of HPLC MCQs With Answer helps M. Pharm students consolidate core concepts of modern pharmaceutical analysis. High-Performance Liquid Chromatography (HPLC) is central to assay, impurity profiling, dissolution testing, bioanalysis, and stability studies. Mastery of its principle, instrumentation, method development, and troubleshooting is essential for robust, regulatory-compliant methods. This quiz focuses on reversed-phase and normal-phase modes, column chemistry, mobile phase design, gradient strategy, detector selection, and system suitability. It also probes understanding of critical parameters such as capacity factor, resolution, plate theory, van Deemter behavior, and dwell volume. Applications across small molecules and biomolecules, plus practical concerns like pH control, sample solvent effects, degassing, and guard columns, are covered to reinforce both theory and practice.

Q1. The fundamental principle of HPLC separation is best described as:

• Differential distribution of analytes between a stationary phase and a mobile phase under high pressure
• Separation based solely on molecular size exclusion through porous beads
• Electromigration of ions under an applied electric field
• Thermal desorption of analytes from a heated column

Correct Answer: Differential distribution of analytes between a stationary phase and a mobile phase under high pressure

Q2. In reversed-phase HPLC, which change will most likely decrease the retention time of a neutral hydrophobic analyte?

• Increase the percentage of organic modifier (e.g., acetonitrile) in the mobile phase
• Decrease column temperature
• Use a longer C18 column
• Lower the flow rate

Correct Answer: Increase the percentage of organic modifier (e.g., acetonitrile) in the mobile phase

Q3. Which detector is generally unsuitable for gradient elution in HPLC due to baseline drift with changing mobile phase composition?

• UV-Visible absorbance detector
• Photodiode array detector
• Refractive index detector
• Fluorescence detector

Correct Answer: Refractive index detector

Q4. In the van Deemter equation (H = A + B/u + C·u), the B term primarily represents:

• Eddy diffusion due to multiple flow paths
• Longitudinal diffusion of analyte molecules along the column axis
• Mass transfer resistance between phases
• Viscous heating at high flow rates

Correct Answer: Longitudinal diffusion of analyte molecules along the column axis

Q5. If tR = 6.0 min and t0 (dead time) = 1.0 min, the capacity factor (k’) is:

• 0.17
• 5.0
• 6.0
• 1.2

Correct Answer: 5.0

Q6. Two peaks elute at 5.20 min and 6.00 min with baseline widths of 0.40 min and 0.50 min, respectively. The chromatographic resolution (Rs) is approximately:

• 1.0
• 1.6
• 2.0
• 2.4

Correct Answer: 1.6

Q7. Reducing fully porous particle size from 5 μm to 2.5 μm (same column dimensions and flow per linear velocity) will generally:

• Quarter the backpressure and lower efficiency
• Double the backpressure and improve efficiency
• Increase backpressure fourfold and improve efficiency
• Not change backpressure or efficiency

Correct Answer: Increase backpressure fourfold and improve efficiency

Q8. Which combination best describes normal-phase HPLC conditions?

• Polar silica stationary phase with nonpolar mobile phase (e.g., hexane/isopropanol)
• Nonpolar C18 stationary phase with water/acetonitrile mobile phase
• Ion-exchange stationary phase with buffered aqueous mobile phase
• Size-exclusion stationary phase with isocratic aqueous mobile phase

Correct Answer: Polar silica stationary phase with nonpolar mobile phase (e.g., hexane/isopropanol)

Q9. For a basic drug (pKa ~9) in reversed-phase HPLC, which strategy typically increases retention while reducing tailing?

• Mobile phase pH 3 with ion-pair reagent addition
• Mobile phase pH 11 without buffer
• Mobile phase pH 7 with non-endcapped C18
• Mobile phase pH 2 without ion-pair reagent

Correct Answer: Mobile phase pH 3 with ion-pair reagent addition

Q10. Dwell volume (gradient delay volume) in an HPLC system is:

• The volume of the column packed bed
• The volume from mixer to column head that determines how soon a programmed gradient reaches the column
• The volume of the detector flow cell
• The swept volume between injector and detector

Correct Answer: The volume from mixer to column head that determines how soon a programmed gradient reaches the column

Q11. When analyzing a sample containing analytes spanning a wide polarity range, the preferred elution mode is:

• Isocratic elution at constant high organic
• Isocratic elution at constant low organic
• Gradient elution increasing organic content over time
• Stepwise temperature programming

Correct Answer: Gradient elution increasing organic content over time

Q12. Injecting a sample prepared in a much stronger solvent than the initial mobile phase typically causes:

• Symmetric peaks with improved efficiency
• Peak fronting or splitting for early eluters
• Increased retention for late eluters
• No effect if using a UV detector

Correct Answer: Peak fronting or splitting for early eluters

Q13. A typical pharmacopoeial system suitability limit for peak tailing factor (T) in assay methods is:

• T ≤ 1.0
• T ≤ 1.5
• T ≤ 2.0
• T ≤ 3.0

Correct Answer: T ≤ 2.0

Q14. A stability-indicating HPLC method is one that:

• Measures assay without separating degradants
• Separates and quantifies the drug in the presence of its degradation products and impurities
• Uses only isocratic elution for robustness
• Requires fluorescence detection for sensitivity

Correct Answer: Separates and quantifies the drug in the presence of its degradation products and impurities

Q15. At equal volume fraction in reversed-phase HPLC, which organic modifier generally provides stronger elution strength (shorter retention) for small molecules?

• Methanol
• Acetonitrile
• Tetrahydrofuran is weaker than methanol
• Water

Correct Answer: Acetonitrile

Q16. The primary purpose of a guard column in HPLC is to:

• Increase theoretical plates dramatically
• Protect the analytical column from particulates and strongly retained contaminants
• Reduce detector noise
• Decrease system dwell volume

Correct Answer: Protect the analytical column from particulates and strongly retained contaminants

Q17. Which is an effective degassing method to minimize pump cavitation and baseline noise?

• Helium sparging or in-line vacuum degassing
• Increasing column temperature
• Using a longer injection loop
• Switching to a larger detector cell

Correct Answer: Helium sparging or in-line vacuum degassing

Q18. For enantiomeric separation by HPLC in pharmaceuticals, a common approach is to:

• Use a C18 column with high pH mobile phase only
• Use a chiral stationary phase or derivatize enantiomers to diastereomers
• Use size-exclusion chromatography
• Use refractive index detection in gradient mode

Correct Answer: Use a chiral stationary phase or derivatize enantiomers to diastereomers

Q19. Compared with conventional HPLC, UHPLC typically uses:

• Particles ≥ 10 μm and pressures ≤ 200 bar
• Sub-2 μm particles and system pressures up to ~1000 bar
• Only polymeric columns at ambient pressure
• Open tubular columns without packing

Correct Answer: Sub-2 μm particles and system pressures up to ~1000 bar

Q20. For quantifying trace-level drugs in plasma with high selectivity and sensitivity, the preferred HPLC detection is:

• UV at 254 nm with isocratic elution
• Photodiode array with large flow cell
• Fluorescence with no sample cleanup
• Tandem mass spectrometry (LC–MS/MS) after appropriate sample prep (e.g., SPE)

Correct Answer: Tandem mass spectrometry (LC–MS/MS) after appropriate sample prep (e.g., SPE)

Download