High performance liquid chromatography (HPLC) – principles and theory MCQs With Answer

High performance liquid chromatography (HPLC) is an essential analytical technique for B. Pharm students, focusing on separation, identification, and quantification of pharmaceutical compounds. This introduction covers core principles and chromatographic theory: mobile phase and stationary phase interactions, retention time, capacity factor (k′), selectivity (α), resolution, column efficiency (N), van Deemter theory, isocratic and gradient elution, detector types, solvent selection, pH effects, and troubleshooting. Instrument components such as pump, injector, column and detector are emphasized along with method development and validation for drug analysis. Keywords: High performance liquid chromatography, HPLC, retention, resolution, column efficiency, mobile phase, detectors, chromatographic theory, B. Pharm, MCQs. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. What is the fundamental separation principle of HPLC?

• Separation based on volatility differences
• Separation based on distribution between mobile and stationary phases
• Separation by gel filtration only
• Separation by electrophoretic mobility

Correct Answer: Separation based on distribution between mobile and stationary phases

Q2. Which HPLC component is responsible for delivering the mobile phase at a constant, controlled flow?

• Detector
• Pump
• Injector
• Column

Correct Answer: Pump

Q3. In reverse-phase HPLC, the stationary phase is typically:

• Polar silica gel
• Ion exchange resin
• Nonpolar bonded phase such as C18
• Porous polymer for size exclusion

Correct Answer: Nonpolar bonded phase such as C18

Q4. Retention time (tR) is defined as:

• The time between gradients changes
• The time from injection to detector response maximum for an analyte
• The time required to equilibrate the column
• The time solvent spends in the pump

Correct Answer: The time from injection to detector response maximum for an analyte

Q5. The capacity factor (k′) is calculated by which expression?

• k′ = t0 / (tR − t0)
• k′ = tR / t0
• k′ = (tR − t0) / t0
• k′ = (t0 − tR) / tR

Correct Answer: k′ = (tR − t0) / t0

Q6. Selectivity (α) in chromatography is defined as:

• The ratio of plate heights of two peaks
• The difference in retention times divided by peak width
• The ratio of capacity factors k′2/k′1 for two solutes
• The slope of the van Deemter curve

Correct Answer: The ratio of capacity factors k′2/k′1 for two solutes

Q7. Which expression represents chromatographic resolution (Rs) between two adjacent peaks?

• Rs = (tR2 − tR1) / (w1 + w2)
• Rs = 2(tR2 − tR1) / (w1 + w2)
• Rs = (t0) / (tR1 + tR2)
• Rs = (k′1 + k′2) / α

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

Q8. The van Deemter equation relates plate height (H) to which variable?

• Detector sensitivity
• Column dead time
• Linear mobile phase velocity
• Sample concentration

Correct Answer: Linear mobile phase velocity

Q9. Reducing stationary phase particle size generally results in:

• Lower column efficiency and lower backpressure
• Higher column efficiency and higher backpressure
• No change in efficiency but decreased retention
• Decreased resolution with reduced pressure

Correct Answer: Higher column efficiency and higher backpressure

Q10. Which elution mode changes mobile phase composition during the run to improve separation over wide polarity ranges?

• Isocratic elution
• Isothermal elution
• Gradient elution
• Step-wise injection

Correct Answer: Gradient elution

Q11. Which detector gives a universal response based on changes in refractive index?

• UV-Vis detector
• Fluorescence detector
• Refractive index (RI) detector
• Electrochemical detector

Correct Answer: Refractive index (RI) detector

Q12. The most commonly used detector in pharmaceutical HPLC due to sensitivity and versatility is:

• Mass spectrometer
• UV-Vis detector
• Conductivity detector
• Refractive index detector

Correct Answer: UV-Vis detector

Q13. Peak tailing in reversed-phase HPLC is often caused by:

• Excessive column temperature
• Active silanol groups on silica interacting with basic analytes
• Using gradient elution
• Too low mobile phase flow rate only

Correct Answer: Active silanol groups on silica interacting with basic analytes

Q14. What is the primary purpose of a guard column?

• To increase detector sensitivity
• To modify mobile phase pH automatically
• To protect the analytical column from particulates and contaminants
• To act as the detector

Correct Answer: To protect the analytical column from particulates and contaminants

Q15. Mobile phase pH most strongly affects the HPLC behavior of:

• Nonpolar neutral molecules
• Ionizable compounds
• High molecular weight polymers only
• All compounds equally regardless of pKa

Correct Answer: Ionizable compounds

Q16. Ion-exchange chromatography separates analytes primarily by:

• Molecular size differences
• Hydrophobic interactions
• Charge-based interactions with the stationary phase
• Volatility of analytes

Correct Answer: Charge-based interactions with the stationary phase

Q17. In normal-phase HPLC, which is true?

• The stationary phase is nonpolar and the mobile phase is polar
• The stationary phase is polar and the mobile phase is nonpolar
• Both phases are highly aqueous
• Only volatile solvents are used

Correct Answer: The stationary phase is polar and the mobile phase is nonpolar

Q18. Size-exclusion chromatography separates molecules based on:

• Hydrophobicity
• Polarity
• Molecular size and shape
• Charge

Correct Answer: Molecular size and shape

Q19. Dead time (t0) in an HPLC run refers to:

• The time required to condition the column before analysis
• The time taken by an unretained compound to pass through the system
• The total run time including re-equilibration
• The detector response delay only

Correct Answer: The time taken by an unretained compound to pass through the system

Q20. The theoretical plate number (N) for a Gaussian peak can be calculated using which relationship?

• N = 5.54 (tR / w1/2)^2
• N = 16 (tR / w)^2
• N = (t0 / tR) × 100
• N = (k′ × α) / Rs

Correct Answer: N = 16 (tR / w)^2

Q21. A tailing factor (Tf) greater than 1 indicates:

• Peak fronting
• Perfect symmetry
• Peak tailing
• No peak observed

Correct Answer: Peak tailing

Q22. A major advantage of gradient elution over isocratic elution is:

• Longer equilibration times always
• Shorter analysis times and better separation for a wide polarity range of analytes
• Elimination of the need for detectors
• Guaranteed better sensitivity for all analytes

Correct Answer: Shorter analysis times and better separation for a wide polarity range of analytes

Q23. Why is degassing of the mobile phase important in HPLC?

• To increase solvent viscosity
• To prevent bubble formation that affects pump performance and detector baselines
• To sterilize the mobile phase
• To change pH of the solvent

Correct Answer: To prevent bubble formation that affects pump performance and detector baselines

Q24. When choosing solvents for UV detection, an important property is:

• High refractive index
• Low UV absorbance (low UV cutoff)
• High viscosity regardless of UV transparency
• Ability to fluoresce strongly

Correct Answer: Low UV absorbance (low UV cutoff)

Q25. Which factor most strongly increases HPLC system backpressure?

• Using longer wavelength detector
• Decreasing stationary phase particle size
• Reducing column temperature by 0.1 °C
• Using a less polar mobile phase

Correct Answer: Decreasing stationary phase particle size

Q26. To reduce interaction of basic analytes with residual silanols and improve peak shape, a common mobile phase additive is:

• Formic acid only
• Triethylamine (TEA) or other amine modifiers
• Sodium chloride
• Calcium carbonate

Correct Answer: Triethylamine (TEA) or other amine modifiers

Q27. A photodiode array (PDA) detector advantage over a single-wavelength UV detector is:

• Higher mass sensitivity
• Ability to record full UV-Vis spectra of peaks and monitor multiple wavelengths simultaneously
• No need for calibration
• Universal response for non-UV active compounds

Correct Answer: Ability to record full UV-Vis spectra of peaks and monitor multiple wavelengths simultaneously

Q28. Preparative HPLC differs from analytical HPLC primarily in:

• Using the same column dimensions but different detectors only
• Larger column dimensions and higher sample load for purification/collection
• Only using isocratic elution
• Exclusively using RI detectors

Correct Answer: Larger column dimensions and higher sample load for purification/collection

Q29. Which ionization technique is commonly used to interface HPLC with mass spectrometry for pharmaceuticals?

• Flame ionization (FI)
• Electrospray ionization (ESI)
• MALDI only
• Thermal ionization

Correct Answer: Electrospray ionization (ESI)

Q30. Which system suitability parameter is most directly used to assess column efficiency?

• Retention time reproducibility
• Theoretical plates (N)
• Gradient slope
• Detector wavelength

Correct Answer: Theoretical plates (N)

G S Sachin

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.

Mail- Sachin@pharmacyfreak.com