Chromatography is a cornerstone technique in phytochemical analysis, enabling separation, identification, and quantification of plant-derived compounds. B.Pharm students must understand core concepts such as stationary and mobile phases, partition and adsorption mechanisms, Rf values, retention time, and detector selection (UV, PDA, MS). Practical topics include TLC screening, HPLC method development, GC for volatile oils, sample preparation, column selection, mobile phase optimization, gradient versus isocratic elution, and method validation parameters like sensitivity, precision, and accuracy. These skills are vital for herbal drug standardization, quality control, and pharmacognosy research. Now let’s test your knowledge with 30 MCQs on this topic.
Q1. What is the fundamental basis of chromatographic separation?
- Separation based on differential solubility in a single solvent
- Separation based on differential partitioning between stationary and mobile phases
- Separation based on complete chemical reaction with the stationary phase
- Separation based solely on molecular weight
Correct Answer: Separation based on differential partitioning between stationary and mobile phases
Q2. In thin-layer chromatography (TLC), the Rf value is defined as:
- The ratio of analyte spot height to plate height
- The ratio of distance traveled by solvent front to distance traveled by analyte
- The ratio of distance traveled by analyte to distance traveled by solvent front
- The difference between analyte and solvent front distances
Correct Answer: The ratio of distance traveled by analyte to distance traveled by solvent front
Q3. Retention time in HPLC refers to:
- The time taken for the column to equilibrate
- The time between injection and detector response maximum for a compound
- The time solvent spends in the reservoir
- The time required to change mobile phase composition
Correct Answer: The time between injection and detector response maximum for a compound
Q4. Which of the following describes reversed-phase HPLC?
- Nonpolar stationary phase and polar mobile phase
- Polar stationary phase and nonpolar mobile phase
- Stationary phase is gas and mobile phase is liquid
- Stationary phase is ionic membrane
Correct Answer: Nonpolar stationary phase and polar mobile phase
Q5. Which detector is most commonly used for routine quantification of UV-absorbing phytochemicals in HPLC?
- Flame ionization detector (FID)
- Refractive index detector (RID)
- Ultraviolet-visible (UV-Vis) detector / PDA
- Fluorescence detector only
Correct Answer: Ultraviolet-visible (UV-Vis) detector / PDA
Q6. One major advantage of HPLC over TLC in phytochemical analysis is:
- HPLC provides only qualitative, not quantitative, data
- HPLC allows precise quantification and higher sensitivity
- TLC has better reproducibility than HPLC
- TLC consumes more mobile phase than HPLC
Correct Answer: HPLC allows precise quantification and higher sensitivity
Q7. The Van Deemter equation is used to describe:
- The chemical stability of stationary phases
- The relationship between linear velocity and plate height (band broadening)
- The solubility of analytes in mobile phase
- The pH dependence of ionizable analytes
Correct Answer: The relationship between linear velocity and plate height (band broadening)
Q8. In Van Deemter theory, which change typically decreases plate height and increases column efficiency?
- Increasing particle size of stationary phase packing
- Decreasing column temperature drastically
- Using smaller particle size packing
- Increasing detector dead volume
Correct Answer: Using smaller particle size packing
Q9. Which detector is considered universal and commonly used for gas chromatography of essential oils?
- Photodiode array (PDA)
- Flame ionization detector (FID)
- UV-Vis detector
- Conductivity detector
Correct Answer: Flame ionization detector (FID)
Q10. Which chromatographic mechanism is primarily responsible for separation on silica gel TLC?
- Size exclusion
- Ion exchange
- Adsorption
- Affinity binding
Correct Answer: Adsorption
Q11. What is the retention factor (k’) formula used in liquid chromatography?
- k’ = t0 / (tR – t0)
- k’ = (tR – t0) / t0
- k’ = tR × t0
- k’ = tR – t0
Correct Answer: k’ = (tR – t0) / t0
Q12. Which mobile phase modification is commonly used to control ionization of acidic or basic phytochemicals in reversed-phase HPLC?
- Addition of nonpolar solvents only
- Use of buffers to control pH
- Use of detergents to denature analytes
- Increasing column length without changing mobile phase
Correct Answer: Use of buffers to control pH
Q13. In gradient elution, the main advantage over isocratic elution is:
- Better separation for a narrow polarity range only
- Faster elution and improved separation for complex mixtures
- Elimination of need for detectors
- Lower method development complexity always
Correct Answer: Faster elution and improved separation for complex mixtures
Q14. Which reagent is classically used to visualize alkaloids on a TLC plate?
- Dragendorff’s reagent
- Ninhydrin
- Vanillin-sulfuric acid for terpenes
- Potassium permanganate for sugars
Correct Answer: Dragendorff’s reagent
Q15. Size exclusion chromatography separates analytes primarily based on:
- Polarity differences
- Charge interactions with stationary phase
- Molecular size and hydrodynamic volume
- Affinity to ligands on the stationary phase
Correct Answer: Molecular size and hydrodynamic volume
Q16. Ion-exchange chromatography is most suitable for separation of which phytochemical property?
- Hydrophobicity
- Volatility
- Charge
- Optical activity
Correct Answer: Charge
Q17. Which detector provides spectral information across multiple wavelengths simultaneously in HPLC?
- Refractive index detector (RID)
- Electrochemical detector (ECD)
- Photodiode array (PDA) detector
- Flame ionization detector (FID)
Correct Answer: Photodiode array (PDA) detector
Q18. Which interface is most commonly used to couple liquid chromatography with mass spectrometry for polar phytochemicals?
- Electron ionization (EI)
- Matrix-assisted laser desorption (MALDI)
- Electrospray ionization (ESI)
- Atomic absorption interface
Correct Answer: Electrospray ionization (ESI)
Q19. A peak tailing factor greater than 1 indicates:
- Perfectly symmetrical peak
- Peak fronting
- Peak tailing (asymmetry)
- No peak detected
Correct Answer: Peak tailing (asymmetry)
Q20. Which phytochemical class is typically analyzed by gas chromatography after suitable derivatization?
- Non-volatile glycosides without derivatization
- Volatile essential oils and small terpenes
- Large polar polysaccharides
- Peptide alkaloids that are non-volatile
Correct Answer: Volatile essential oils and small terpenes
Q21. For quantitative HPLC analysis, which sample preparation step is most critical to protect column life and detector performance?
- Freeze-drying the sample only
- Filtration through a 0.45 μm or 0.22 μm membrane
- Boiling the sample to concentrate analytes
- Adding salt crystals before injection
Correct Answer: Filtration through a 0.45 μm or 0.22 μm membrane
Q22. Which parameter is NOT part of method validation in chromatographic analysis?
- Accuracy and precision
- Linearity and range
- Resolution of unrelated instruments
- Limit of detection and limit of quantification
Correct Answer: Resolution of unrelated instruments
Q23. Which statement correctly describes preparative chromatography?
- It is used only for analytical purposes and low sample amounts
- It is employed to purify and isolate significant quantities of a compound
- It does not require method validation
- It always uses GC instead of LC
Correct Answer: It is employed to purify and isolate significant quantities of a compound
Q24. In reversed-phase HPLC for weak acids, increasing mobile phase pH above pKa will generally:
- Increase retention of weak acids
- Decrease ionization of weak acids
- Increase ionization and generally decrease retention of weak acids
- Have no effect on retention
Correct Answer: Increase ionization and generally decrease retention of weak acids
Q25. Which chromatographic factor most directly improves resolution between two closely eluting peaks?
- Reducing column efficiency (fewer plates)
- Increasing selectivity (alpha) and/or efficiency (N)
- Using a detector with lower sensitivity
- Shortening column length dramatically
Correct Answer: Increasing selectivity (alpha) and/or efficiency (N)
Q26. Which visualization method is commonly used to detect flavonoids on TLC plates after chromatography?
- Observation under UV light at 254/365 nm and use of NP/PEG spray
- Flame ionization of the TLC plate
- Measuring retention time directly on the plate
- Direct mass spectrometry without any spray
Correct Answer: Observation under UV light at 254/365 nm and use of NP/PEG spray
Q27. Which of the following best describes limit of detection (LOD) in chromatographic methods?
- The highest concentration that can be accurately measured
- The lowest concentration that can be reliably distinguished from background noise
- The concentration at which the detector saturates
- The retention time of the first peak
Correct Answer: The lowest concentration that can be reliably distinguished from background noise
Q28. Which chromatographic technique is appropriate for analysis of high-molecular-weight polysaccharides?
- Gas chromatography without derivatization
- Reverse-phase HPLC on C18 columns
- Size exclusion chromatography (SEC/GPC)
- Thin-layer chromatography on silica with nonpolar solvent
Correct Answer: Size exclusion chromatography (SEC/GPC)
Q29. Which factor most commonly causes poor reproducibility and retention time shifts in HPLC?
- Consistent mobile phase composition and pH
- Variations in mobile phase composition, pH, or column temperature
- Using fresh, high-purity solvents every run
- Properly equilibrated column and system
Correct Answer: Variations in mobile phase composition, pH, or column temperature
Q30. When developing an HPLC method for a phytochemical herbal extract, which initial strategy is recommended?
- Start with a very long column and random mobile phases
- Perform sample cleanup, choose column chemistry based on analyte polarity, and screen mobile phases systematically
- Inject crude extract without filtration or consideration of solvent compatibility
- Always use GC methods first regardless of analyte volatility
Correct Answer: Perform sample cleanup, choose column chemistry based on analyte polarity, and screen mobile phases systematically
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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