Chromatography is a fundamental separation technique in pharmaceutical analysis, combining theory and practical classification to resolve complex mixtures. This introduction covers chromatographic mechanisms—adsorption, partition, ion-exchange, size-exclusion and affinity—along with common systems such as TLC, HPLC, and GC. Key concepts like stationary phase, mobile phase, retention time, Rf, resolution, theoretical plates, van Deemter equation, and detector types are explained to build strong laboratory and analytical skills for B. Pharm students. Understanding classification by mechanism and application helps in method selection, optimization and validation for drug analysis, impurity profiling and quality control. Now let’s test your knowledge with 30 MCQs on this topic.
Q1. Which principle best describes partition chromatography?
- Separation by differential adsorption onto a solid surface
- Separation by differential solubility between two immiscible phases
- Separation by molecular size through pores
- Separation by ionic interactions with charged sites
Correct Answer: Separation by differential solubility between two immiscible phases
Q2. Which stationary phase is most commonly used in reversed-phase HPLC?
- Silica gel with polar surface
- Polystyrene divinylbenzene
- C18-bonded silica
- Cellulose powder
Correct Answer: C18-bonded silica
Q3. In thin layer chromatography (TLC), what does Rf represent?
- Retention factor defined as ratio of retention time to dead time
- Resolution factor between two peaks
- Ratio of distance traveled by solute to distance traveled by solvent front
- Recovery fraction after extraction
Correct Answer: Ratio of distance traveled by solute to distance traveled by solvent front
Q4. Which chromatographic mode separates molecules primarily by molecular size?
- Affinity chromatography
- Ion-exchange chromatography
- Size-exclusion chromatography
- Partition chromatography
Correct Answer: Size-exclusion chromatography
Q5. The van Deemter equation relates plate height (H) to which experimental variable?
- Detector sensitivity
- Column temperature
- Linear velocity of the mobile phase
- Solvent polarity
Correct Answer: Linear velocity of the mobile phase
Q6. In gas chromatography, which detector is most commonly used for organic compounds and provides near-universal response to hydrocarbons?
- Flame ionization detector (FID)
- Electron capture detector (ECD)
- Refractive index detector (RID)
- Mass spectrometer (MS)
Correct Answer: Flame ionization detector (FID)
Q7. What is the main advantage of gradient elution in HPLC over isocratic elution?
- Simpler mobile phase preparation
- Better separation of compounds with a wide polarity range
- Lower solvent consumption for all analyses
- Higher column backpressure
Correct Answer: Better separation of compounds with a wide polarity range
Q8. Which parameter describes the capacity of an analyte to interact with the stationary phase relative to the mobile phase?
- Resolution (Rs)
- Retention factor (k’)
- Selectivity (α)
- Theoretical plates (N)
Correct Answer: Retention factor (k’)
Q9. Which chromatographic technique employs specific biological interactions such as antigen–antibody binding?
- Ion-exchange chromatography
- Affinity chromatography
- Paper chromatography
- Chiral chromatography
Correct Answer: Affinity chromatography
Q10. In column chromatography, what does a higher number of theoretical plates (N) indicate?
- Lower column efficiency
- Higher column efficiency and better separation power
- Faster analysis time regardless of conditions
- Greater solvent consumption
Correct Answer: Higher column efficiency and better separation power
Q11. Which detector is most suitable for gradient HPLC when analytes lack strong chromophores?
- UV-Visible detector
- Fluorescence detector
- Evaporative light scattering detector (ELSD)
- Conductivity detector
Correct Answer: Evaporative light scattering detector (ELSD)
Q12. What is the primary role of a guard column in HPLC?
- To increase retention time of analytes
- To protect the analytical column from particulates and strongly retained contaminants
- To act as a detector
- To change mobile phase polarity
Correct Answer: To protect the analytical column from particulates and strongly retained contaminants
Q13. In ion-exchange chromatography, what determines whether a cation or anion exchanger is used?
- Molecular weight of analyte
- Polarity of the mobile phase
- Net charge of the analyte at working pH
- Viscosity of the mobile phase
Correct Answer: Net charge of the analyte at working pH
Q14. Which term describes the time an unretained solute takes to elute from a chromatographic column?
- Retention time (tr)
- Dead time or void time (t0)
- Peak width at baseline
- Retention factor (k’)
Correct Answer: Dead time or void time (t0)
Q15. What is the main difference between normal-phase and reversed-phase liquid chromatography?
- Normal-phase uses nonpolar stationary phase; reversed-phase uses polar stationary phase
- Normal-phase uses a polar stationary phase and nonpolar mobile phase; reversed-phase uses nonpolar stationary phase and polar mobile phase
- Normal-phase uses gas mobile phase; reversed-phase uses liquid mobile phase
- There is no difference; terms are interchangeable
Correct Answer: Normal-phase uses a polar stationary phase and nonpolar mobile phase; reversed-phase uses nonpolar stationary phase and polar mobile phase
Q16. Which factor does NOT directly influence chromatographic resolution (Rs)?
- Selectivity (α)
- Retention factor (k’)
- Number of theoretical plates (N)
- Detector wavelength
Correct Answer: Detector wavelength
Q17. For GC analysis, why are many pharmaceutical compounds derivatized before injection?
- To increase volatility and thermal stability
- To decrease molecular weight
- To make compounds nonpolar only
- To remove impurities completely
Correct Answer: To increase volatility and thermal stability
Q18. Which chromatographic technique is best suited for chiral separation of enantiomers in drug analysis?
- Size-exclusion chromatography
- Chiral stationary phase HPLC
- Ion-exchange chromatography
- Paper chromatography
Correct Answer: Chiral stationary phase HPLC
Q19. In the context of HPLC, what does selectivity factor (α) describe?
- The number of theoretical plates per meter
- Ratio of retention factors (k’) of two analytes indicating how well they are chemically distinguished
- The efficiency of the detector
- Flow rate of mobile phase
Correct Answer: Ratio of retention factors (k’) of two analytes indicating how well they are chemically distinguished
Q20. What is the primary purpose of sample pre-treatment before chromatographic analysis?
- To color the sample for better detection
- To concentrate analytes, remove matrix interferences and protect the column
- To change the detector type
- To volatilize nonvolatile compounds
Correct Answer: To concentrate analytes, remove matrix interferences and protect the column
Q21. Which statement about theoretical plate height (H) is true?
- H increases when column efficiency increases
- H is inversely proportional to the number of theoretical plates (N)
- H equals column length times number of plates
- H is unrelated to peak broadening
Correct Answer: H is inversely proportional to the number of theoretical plates (N)
Q22. In HPLC, what effect does increasing column temperature generally have?
- Increases mobile phase viscosity and slows flow
- Increases retention times for all analytes
- Can reduce viscosity, improve mass transfer, and often decrease retention times
- Removes the need for a detector
Correct Answer: Can reduce viscosity, improve mass transfer, and often decrease retention times
Q23. Which mobile phase component would you choose for reversed-phase HPLC of a highly polar analyte to increase retention?
- Increase organic modifier percentage (e.g., acetonitrile)
- Use pure nonpolar solvent like hexane
- Decrease organic modifier percentage and increase aqueous portion
- Use a stronger ion-pairing reagent only
Correct Answer: Decrease organic modifier percentage and increase aqueous portion
Q24. What is the main mechanism of separation in adsorption chromatography?
- Partition of solute between two liquid phases
- Adsorption of solute onto the surface of the stationary solid phase
- Filtration based on particle size
- Enzymatic transformation of analytes
Correct Answer: Adsorption of solute onto the surface of the stationary solid phase
Q25. Which parameter describes how well two adjacent peaks are separated in chromatography?
- Retention factor (k’)
- Selectivity (α)
- Resolution (Rs)
- Plate height (H)
Correct Answer: Resolution (Rs)
Q26. Which of the following is a limitation of size-exclusion chromatography?
- It cannot be used for separating molecules by size.
- It provides strong interactions with analytes causing irreversible binding.
- Limited resolution for molecules of very similar size and potential column exclusion for very large molecules.
- It requires derivatization of all samples.
Correct Answer: Limited resolution for molecules of very similar size and potential column exclusion for very large molecules.
Q27. Which chromatography variant is most suitable for desalting protein samples with minimal loss?
- Reverse-phase HPLC with C18
- Ion-exchange with strong anion exchanger
- Size-exclusion chromatography (gel filtration)
- Affinity chromatography with non-specific ligand
Correct Answer: Size-exclusion chromatography (gel filtration)
Q28. In gradient HPLC method development, why is equilibration time important between runs?
- To allow detector cooling
- To ensure column re-equilibration to initial mobile phase composition, maintaining retention reproducibility
- To change column dimensions
- To dry the column packing
Correct Answer: To ensure column re-equilibration to initial mobile phase composition, maintaining retention reproducibility
Q29. Which property of silica makes it a common stationary phase support in HPLC?
- Insolubility, high surface area and availability of silanol groups for bonding
- Strong basicity and ionic exchange capacity
- Biological activity for affinity interactions
- Nonporous hydrophobic surface only
Correct Answer: Insolubility, high surface area and availability of silanol groups for bonding
Q30. For quantitative chromatographic analysis, which practice is essential to ensure accuracy and precision?
- Ignoring sample matrix effects
- Using consistent injection volumes, validated methods, calibration with standards and appropriate controls
- Changing detector settings between each injection
- Never using internal standards
Correct Answer: Using consistent injection volumes, validated methods, calibration with standards and appropriate controls
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