Gel chromatography – principle, theory and instrumentation MCQs With Answer

Gel chromatography, also called size-exclusion chromatography (SEC) or gel filtration, separates molecules by size using porous stationary phases. This introduction explains the principle and theory—pore size, exclusion limit, partition coefficient (Kav), elution volume, void and total column volumes—and describes instrumentation such as columns, gels (Sephadex, agarose, polyacrylamide), pumps, detectors and fraction collectors. Practical considerations for B. Pharm students include column packing, calibration with standards, sample loading, flow rate effects and data interpretation for molecular weight estimation and purification. Emphasis is on applications in drug analysis, biomolecule purification and quality control. ‘Now let’s test your knowledge with 30 MCQs on this topic.’

Q1. Which fundamental property does gel chromatography primarily separate molecules by?

• Molecular size or hydrodynamic volume
• Net electrical charge
• Hydrophobicity
• Specific binding affinity

Correct Answer: Molecular size or hydrodynamic volume

Q2. Gel chromatography is also commonly known as:

• Ion-exchange chromatography
• Size-exclusion chromatography (SEC) or gel filtration
• Affinity chromatography
• Reverse-phase chromatography

Correct Answer: Size-exclusion chromatography (SEC) or gel filtration

Q3. What is the exclusion limit of a gel?

• The molecular weight below which molecules are completely retained
• The minimum pore diameter in micrometers
• The molecular weight above which molecules are completely excluded from pores and elute in the void volume
• The maximum sample volume that can be applied to the column

Correct Answer: The molecular weight above which molecules are completely excluded from pores and elute in the void volume

Q4. The partition coefficient Kav is defined by which expression?

• Kav = Vo / Vt
• Kav = (Ve – Vo) / (Vt – Vo)
• Kav = Ve / Vo
• Kav = (Vt – Ve) / Vo

Correct Answer: Kav = (Ve – Vo) / (Vt – Vo)

Q5. In gel chromatography, which molecules elute first?

• The smallest molecules that enter most pores
• Neutral molecules only
• The largest molecules that are excluded from pores
• Molecules with highest affinity for the stationary phase

Correct Answer: The largest molecules that are excluded from pores

Q6. Which stationary phase material is a cross-linked dextran commonly used in gel filtration?

• Silica gel
• Sephadex
• Polystyrene reversed-phase beads
• Activated carbon

Correct Answer: Sephadex

Q7. Which detector is commonly used for protein detection after gel chromatography?

• Flame ionization detector (FID)
• Ultraviolet (UV) absorbance detector
• Electrochemical detector
• Mass spectrometer as a routine pack-in detector

Correct Answer: Ultraviolet (UV) absorbance detector

Q8. What is the void volume (Vo) of a gel filtration column?

• The volume inside the gel beads available to solute molecules
• The total column volume including bead pores and interstitial space
• The interstitial column volume outside the beads through which excluded molecules elute first
• The sample injection volume

Correct Answer: The interstitial column volume outside the beads through which excluded molecules elute first

Q9. How does increasing flow rate generally affect resolution in gel chromatography?

• Resolution increases linearly with flow rate
• Resolution usually decreases at high flow rates due to reduced equilibration
• Flow rate has no effect on resolution
• Resolution improves for very small molecules only

Correct Answer: Resolution usually decreases at high flow rates due to reduced equilibration

Q10. Which parameter is most important when calibrating a gel filtration column for molecular weight estimation?

• Retention time of salts only
• Elution volumes of standard molecules of known molecular weight and shape
• Column back pressure measurements
• pH profile of the mobile phase across the run

Correct Answer: Elution volumes of standard molecules of known molecular weight and shape

Q11. The total column volume (Vt) in SEC equals:

• Void volume plus solvent excluded volume only
• Void volume (Vo) plus internal pore volume (Vi)
• Volume of the sample injected
• Only the volume of mobile phase used for elution

Correct Answer: Void volume (Vo) plus internal pore volume (Vi)

Q12. Which factor does NOT influence the fractionation range of a gel?

• Degree of cross-linking of the gel matrix
• Pore size distribution of the beads
• Column temperature within a practical range
• Detector wavelength selection

Correct Answer: Detector wavelength selection

Q13. Which gel material is most appropriate for high-molecular-weight proteins and nucleic acids?

• Low-crosslinked agarose (Sepharose)
• Highly cross-linked polystyrene divinylbenzene
• Silica with small pores
• Activated alumina

Correct Answer: Low-crosslinked agarose (Sepharose)

Q14. Which practice helps minimize secondary (non-size) interactions in SEC?

• Use a mobile phase with appropriate ionic strength and pH to mask charges
• Use a hydrophobic organic solvent as mobile phase
• Increase sample concentration greatly
• Use very small sample injection volumes only

Correct Answer: Use a mobile phase with appropriate ionic strength and pH to mask charges

Q15. What is the effect of sample volume relative to column volume on resolution?

• Larger sample volumes improve resolution
• Smaller sample volumes (typically ≤5% of column volume) maintain better resolution
• Sample volume has no effect on resolution in SEC
• Resolution is only determined by detector sensitivity

Correct Answer: Smaller sample volumes (typically ≤5% of column volume) maintain better resolution

Q16. What is a common method to prepare and pack SEC columns?

• Dry packing the beads without solvent
• Slurry packing the gel into the column under low pressure or gravity
• Heating the beads inside the column to melt them into place
• Adding beads incrementally while running at high flow rates

Correct Answer: Slurry packing the gel into the column under low pressure or gravity

Q17. For accurate molecular weight estimation by SEC, which shape assumption is commonly made about standards and samples?

• All molecules are perfectly rod-like
• All molecules have similar hydrodynamic shapes (often globular) to the standards
• All molecules are flat sheets
• Shape does not matter for SEC calibration

Correct Answer: All molecules have similar hydrodynamic shapes (often globular) to the standards

Q18. Which of the following is an application of gel chromatography in pharmaceutical analysis?

• Estimating degradation products by charge heterogeneity
• Buffer exchange and desalting of protein formulations
• Analyzing volatile organic impurities by headspace
• Measuring tablet friability

Correct Answer: Buffer exchange and desalting of protein formulations

Q19. What does a Kav value close to zero indicate for a given molecule?

• The molecule is retained deeply within bead pores
• The molecule elutes near the void volume and is largely excluded from pores
• The molecule is strongly adsorbed to the stationary phase
• The molecule is degraded during the run

Correct Answer: The molecule elutes near the void volume and is largely excluded from pores

Q20. Which mobile phase characteristic is most important for preserving native structure of proteins during SEC?

• High organic solvent content (>50% acetonitrile)
• Use of denaturing detergents
• Aqueous buffer with suitable pH and ionic strength
• Strong acids to protonate all groups

Correct Answer: Aqueous buffer with suitable pH and ionic strength

Q21. Which statement about gel permeation chromatography (GPC) used for polymers is correct?

• GPC separates polymers primarily by their chemical functionality
• GPC is SEC for synthetic polymers and separates by hydrodynamic volume/molecular weight
• GPC requires ion-exchange resins to work
• GPC cannot provide molecular weight distribution data

Correct Answer: GPC is SEC for synthetic polymers and separates by hydrodynamic volume/molecular weight

Q22. Which parameter best describes column efficiency in SEC?

• Number of theoretical plates (N) or peak width metrics
• Detector noise level only
• Pump horsepower
• Sample color

Correct Answer: Number of theoretical plates (N) or peak width metrics

Q23. How are very small molecules like salts typically eluted in gel filtration?

• They elute in the void volume
• They are retained longest because they adsorb to beads
• They enter most pores and elute near the total column volume (Vt)
• They are permanently trapped inside the beads

Correct Answer: They enter most pores and elute near the total column volume (Vt)

Q24. Which operational precaution reduces shear stress on fragile biomolecules during SEC?

• Using very high pressure pumps and high flow rates
• Using low to moderate flow rates and gentle packing
• Heating the column to high temperatures
• Adding harsh chaotropes to the mobile phase

Correct Answer: Using low to moderate flow rates and gentle packing

Q25. What causes band broadening in gel chromatography?

• Perfectly monodisperse bead sizes
• Multiple diffusion paths, eddy diffusion and longitudinal diffusion
• Using only ultrapure water as mobile phase
• Eluting only very large molecules

Correct Answer: Multiple diffusion paths, eddy diffusion and longitudinal diffusion

Q26. Which detector provides absolute molecular weight information when coupled with SEC?

• UV detector alone
• Differential refractive index alone
• Multi-angle light scattering (MALS) coupled with concentration detector
• Flame ionization detector

Correct Answer: Multi-angle light scattering (MALS) coupled with concentration detector

Q27. During column cleaning and storage of agarose-based SEC columns, which condition should be avoided?

• Storing in recommended buffer with preservative
• Drying the gel completely and storing dry for long periods
• Using gentle antimicrobial agents at recommended concentrations
• Keeping at moderate temperature within manufacturer’s limits

Correct Answer: Drying the gel completely and storing dry for long periods

Q28. Which of the following best describes the fractionation range of a gel?

• The pH range over which the gel is stable
• The molecular weight range over which the gel can separate molecules effectively
• The temperature range for column operation
• The number of columns that can be connected in series

Correct Answer: The molecular weight range over which the gel can separate molecules effectively

Q29. When connecting two SEC columns in series to increase fractionation range, what is a likely consequence?

• Decreased overall resolution always
• Increased total column volume and potential improvement in separation of broader size ranges
• Immediate clogging regardless of sample
• No change in elution profile

Correct Answer: Increased total column volume and potential improvement in separation of broader size ranges

Q30. Which statement is TRUE regarding isocratic elution in SEC?

• Gradient elution is required for SEC to separate by size
• SEC typically uses isocratic elution because separation is based on size, not solvent strength
• Mobile phase composition is continuously changed to elute large molecules first
• Isocratic elution causes irreversible binding of analytes to the gel

Correct Answer: SEC typically uses isocratic elution because separation is based on size, not solvent strength

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