Introduction: Instrumentation of HPLC and its pharmaceutical applications MCQs With Answer provides B.Pharm students with a practical, focused review of high-performance liquid chromatography (HPLC) instrumentation, detectors, columns, mobile phases, and method validation. This introduction covers essential keywords such as HPLC, chromatography, stationary phase, mobile phase, pumps, detectors (UV, PDA, RID, MS), system suitability, assay, impurity profiling, dissolution testing, stability-indicating methods, and regulatory compliance. Emphasis is placed on instrument components, method optimization, troubleshooting, and real-world pharmaceutical applications to prepare students for lab work and exams. Now let’s test your knowledge with 30 MCQs on this topic.
Q1. What is the primary function of the pump in an HPLC system?
- To separate analytes based on polarity
- To deliver a constant, precise flow of mobile phase
- To detect analytes by absorption
- To load samples onto the column
Correct Answer: To deliver a constant, precise flow of mobile phase
Q2. Which detector is most commonly used for routine pharmaceutical UV-visible detection?
- Refractive index detector (RID)
- Evaporative light scattering detector (ELSD)
- PDA (photodiode array) / UV detector
- Mass spectrometer (MS)
Correct Answer: PDA (photodiode array) / UV detector
Q3. What does column particle size primarily influence?
- Detector sensitivity
- Mobile phase pH
- Chromatographic efficiency and backpressure
- Sample solubility
Correct Answer: Chromatographic efficiency and backpressure
Q4. Which mobile phase factor most strongly affects retention of ionizable drugs?
- Buffer pH
- Column length
- Detector wavelength
- Sample injection volume
Correct Answer: Buffer pH
Q5. In reversed-phase HPLC, the stationary phase is typically:
- Hydrophilic silica without modification
- Polar bonded phase like amino
- Nonpolar bonded phase such as C18
- Ion exchange resin
Correct Answer: Nonpolar bonded phase such as C18
Q6. Which parameter is NOT part of typical system suitability tests?
- Number of theoretical plates (N)
- Tailoring factor (incorrect term)
- Tailing factor
- Resolution between peaks
Correct Answer: Tailoring factor (incorrect term)
Q7. What is the main advantage of gradient elution over isocratic elution?
- Simpler mobile phase preparation
- Better separation of analytes with wide polarity range
- Lower initial instrument cost
- Eliminates need for column equilibration
Correct Answer: Better separation of analytes with wide polarity range
Q8. Which detector is most suitable for non-UV absorbing compounds like sugars?
- PDA detector
- Refractive index detector (RID)
- UV detector at 254 nm
- Electrochemical detector
Correct Answer: Refractive index detector (RID)
Q9. What does the term ‘dead volume’ (extra-column volume) refer to?
- Volume of solvent in the mobile phase reservoir
- Unavoidable volume in injector, tubing, and detector that broadens peaks
- Volume inside the stationary phase pores
- Volume of the column packing
Correct Answer: Unavoidable volume in injector, tubing, and detector that broadens peaks
Q10. Which mobile phase additive is commonly used to control pH and improve peak shape for basic drugs?
- Sodium chloride
- Triethylamine (TEA) or phosphate buffer
- Acetonitrile without buffer
- Sugar alcohols
Correct Answer: Triethylamine (TEA) or phosphate buffer
Q11. What is the role of the injector/sampler in HPLC?
- To detect analytes as they elute
- To introduce a precise, reproducible volume of sample onto the column
- To change the mobile phase composition
- To control column temperature
Correct Answer: To introduce a precise, reproducible volume of sample onto the column
Q12. Which parameter indicates peak symmetry and is important for quantitation?
- Retention factor (k’)
- Tailing factor
- Alpha (selectivity)
- Backpressure
Correct Answer: Tailing factor
Q13. In HPLC method validation, LOD refers to:
- Limit of destruction
- Lowest operating detector
- Limit of detection, smallest amount reliably distinguished from noise
- Limit of dilution
Correct Answer: Limit of detection, smallest amount reliably distinguished from noise
Q14. Which hyphenated technique is most powerful for structural identification of impurities?
- HPLC-UV
- HPLC-MS (mass spectrometry)
- HPLC with RID
- HPLC with refractive detection
Correct Answer: HPLC-MS (mass spectrometry)
Q15. What effect does increasing column temperature generally have?
- Increases solvent viscosity and backpressure
- Decreases retention and can improve peak shape
- Precipitates buffer salts in the detector
- Eliminates need for mobile phase filtration
Correct Answer: Decreases retention and can improve peak shape
Q16. Which factor most directly increases column backpressure?
- Decreasing mobile phase flow rate
- Using larger particle size packing
- Using smaller particle size packing or higher viscosity mobile phase
- Shortening column length
Correct Answer: Using smaller particle size packing or higher viscosity mobile phase
Q17. What is the capacity factor (k’) used to describe?
- Detector linearity range
- Relative retention of an analyte versus dead time
- Buffer ionic strength
- Column frit porosity
Correct Answer: Relative retention of an analyte versus dead time
Q18. For chiral separations in pharmaceuticals, which stationary phase is typically used?
- Achiral C18 only
- Chiral stationary phases (e.g., polysaccharide-based)
- Strong cation exchanger only
- Plain silica with no modification
Correct Answer: Chiral stationary phases (e.g., polysaccharide-based)
Q19. Which troubleshooting step is appropriate if all peaks show degraded resolution and high tailing?
- Increase detector wavelength
- Check column for contamination or replace/clean column
- Decrease injection volume to zero
- Remove the mobile phase buffer
Correct Answer: Check column for contamination or replace/clean column
Q20. Why is degassing the mobile phase important?
- To increase analyte solubility dramatically
- To prevent bubble formation that causes baseline noise and pump cavitation
- To sterilize the solvent
- To change mobile phase pH
Correct Answer: To prevent bubble formation that causes baseline noise and pump cavitation
Q21. Which HPLC mode is preferred for separating highly polar, water-soluble drugs?
- Normal phase with nonpolar solvent
- Reversed-phase or hydrophilic interaction liquid chromatography (HILIC)
- Gas chromatography
- Thin-layer chromatography only
Correct Answer: Reversed-phase or hydrophilic interaction liquid chromatography (HILIC)
Q22. What is ‘resolution’ (Rs) a measure of in chromatography?
- Pump flow stability
- Degree of separation between two adjacent peaks
- Detector lamp intensity
- Column pore size distribution
Correct Answer: Degree of separation between two adjacent peaks
Q23. Which solvent is commonly used as an organic modifier in reversed-phase HPLC?
- Hexane
- Acetonitrile or methanol
- Water-free ethanol only
- Glycerin
Correct Answer: Acetonitrile or methanol
Q24. What is the primary reason to filter mobile phase and samples before injection?
- To change analyte polarity
- To remove particulates that can clog column and affect reproducibility
- To sterilize samples for consumption
- To increase detector response
Correct Answer: To remove particulates that can clog column and affect reproducibility
Q25. In pharmaceutical analysis, a stability-indicating HPLC method must:
- Detect only the parent drug, ignoring degradation products
- Separate and quantify the drug from its degradation products
- Be based solely on UV detection at a single wavelength
- Use only isocratic elution
Correct Answer: Separate and quantify the drug from its degradation products
Q26. What is gradient delay volume (gradient dwell volume) important for?
- Determining detector wavelength
- Predicting the time before gradient composition reaches the column, affecting retention reproducibility
- Calculating buffer concentration
- Choosing column particle size
Correct Answer: Predicting the time before gradient composition reaches the column, affecting retention reproducibility
Q27. Which maintenance practice prolongs column life?
- Using unfiltered crude samples directly
- Flushing with strong solvent after use and storing in appropriate solvent
- Leaving the column connected to the system indefinitely without flow
- Operating at extreme pH outside column specifications
Correct Answer: Flushing with strong solvent after use and storing in appropriate solvent
Q28. What advantage does UHPLC offer compared to conventional HPLC?
- Lower system backpressure
- Higher efficiency, faster separations using sub-2 μm particles and higher pressures
- No need for method validation
- Works only with RID detectors
Correct Answer: Higher efficiency, faster separations using sub-2 μm particles and higher pressures
Q29. During method transfer between labs, which parameter is most critical to match for comparable retention?
- Detector brand only
- Column chemistry and dimensions, mobile phase composition, flow rate, and temperature
- Color of the mobile phase reservoir
- Type of sample vial cap
Correct Answer: Column chemistry and dimensions, mobile phase composition, flow rate, and temperature
Q30. Which guideline is commonly referenced for HPLC method validation in pharmaceuticals?
- ICH Q2(R1) — Validation of analytical procedures
- OECD GLP-1
- CLSI blood culture guideline
- WHO vaccine storage manual
Correct Answer: ICH Q2(R1) — Validation of analytical procedures
I am a Registered Pharmacist under the Pharmacy Act, 1948, and the founder of PharmacyFreak.com. I hold a Bachelor of Pharmacy degree from Rungta College of Pharmaceutical Science and Research. With a strong academic foundation and practical knowledge, I am committed to providing accurate, easy-to-understand content to support pharmacy students and professionals. My aim is to make complex pharmaceutical concepts accessible and useful for real-world application.
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