Applications of gel filtration chromatography in pharmaceuticals MCQs With Answer

Introduction: Gel filtration chromatography, also known as size-exclusion chromatography (SEC), is a vital technique in pharmaceutical analysis for separating molecules by size. B. Pharm students should understand how porous gel beads (e.g., Sephadex, Sephacryl) fractionate proteins, peptides, polymers, and drug aggregates without altering biological activity. Key applications include desalting, buffer exchange, removal of aggregates, molecular weight estimation, and quality control of biopharmaceuticals. Important concepts are void volume, elution volume, fractionation range, and the partition coefficient (Kav). Mastery of operating parameters—column dimensions, flow rate, sample volume, and pore size—improves resolution and recovery. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. What is the primary principle behind gel filtration chromatography?

• Separation based on charge interactions with the matrix
• Separation based on molecular size and exclusion from pores
• Separation based on hydrophobic interactions
• Separation based on affinity between ligand and target

Correct Answer: Separation based on molecular size and exclusion from pores

Q2. Gel filtration chromatography is also commonly referred to as:

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

Correct Answer: Size-exclusion chromatography (SEC)

Q3. Which of the following materials is typically used as the stationary phase in gel filtration columns?

• Silica gel with C18 groups
• Porous polysaccharide beads like Sephadex or Sephacryl
• Strong anion exchange resins
• Hydrophobic polymer membranes

Correct Answer: Porous polysaccharide beads like Sephadex or Sephacryl

Q4. What does the void volume (V0) of a gel filtration column represent?

• The volume of pores accessible to all solutes
• The volume outside the beads where very large molecules elute
• The total packed bed volume including beads and interstitial spaces
• The volume at which the smallest molecules are retained

Correct Answer: The volume outside the beads where very large molecules elute

Q5. How is the total column volume (Vt) defined in gel filtration chromatography?

• Volume of mobile phase applied during the run
• Volume of stationary phase only
• Sum of void volume and internal pore volume of beads
• Volume occupied by sample injection only

Correct Answer: Sum of void volume and internal pore volume of beads

Q6. The elution volume (Ve) is best described as:

• The volume of buffer needed to equilibrate the column
• The volume at which a particular analyte exits the column
• The volume of the column bed excluding beads
• The volume of sample loaded onto the column

Correct Answer: The volume at which a particular analyte exits the column

Q7. Which formula correctly defines the partition coefficient (Kav) used in SEC?

• Kav = (Vt – Ve) / (Ve – V0)
• Kav = (Ve – V0) / (Vt – V0)
• Kav = Ve / V0
• Kav = V0 / Vt

Correct Answer: Kav = (Ve – V0) / (Vt – V0)

Q8. Which factor primarily improves resolution in gel filtration chromatography?

• Reducing column length
• Increasing sample volume dramatically
• Using smaller bead diameter and longer column
• Running at extremely high flow rates

Correct Answer: Using smaller bead diameter and longer column

Q9. What is the typical effect of injecting a large sample volume on SEC separation?

• Improves resolution by spreading peaks
• Decreases resolution due to band broadening
• Has no effect on separation
• Always increases retention times

Correct Answer: Decreases resolution due to band broadening

Q10. Gel filtration chromatography is commonly used in pharmaceuticals for:

• Ionizing small molecules to improve solubility
• Desalting and buffer exchange of protein samples
• Covalently attaching drugs to solid supports
• Measuring pKa values of drugs

Correct Answer: Desalting and buffer exchange of protein samples

Q11. Which application uses SEC to separate monomers from aggregates in biopharmaceuticals?

• Determination of isoelectric point
• Aggregate analysis for product quality and stability
• Enzymatic activity assay
• Solid phase synthesis monitoring

Correct Answer: Aggregate analysis for product quality and stability

Q12. The fractionation range of a gel filtration medium refers to:

• The pH range in which the gel is stable
• The molecular weight range over which the medium can separate molecules effectively
• The temperature range for column operation
• The ionic strength limits for buffer use

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

Q13. The exclusion limit of a gel matrix means:

• The smallest molecule that can enter the pores
• The largest molecule that cannot enter the pores and elutes in void volume
• The ionic strength above which the gel degrades
• The minimal flow rate to avoid packing collapse

Correct Answer: The largest molecule that cannot enter the pores and elutes in void volume

Q14. Which detector is commonly used with SEC for protein quantification in pharmaceuticals?

• Flame ionization detector (FID)
• UV absorbance detector at 280 nm
• Electrochemical detector
• Gas detector

Correct Answer: UV absorbance detector at 280 nm

Q15. How does increasing the flow rate generally affect resolution in gel filtration?

• Resolution improves linearly with flow rate
• Resolution decreases because of reduced analyte–pore equilibration time
• Resolution remains unchanged
• Resolution increases due to reduced diffusion

Correct Answer: Resolution decreases because of reduced analyte–pore equilibration time

Q16. Which statement best describes gel permeation chromatography (GPC) compared to SEC?

• GPC is a form of SEC applied mainly to synthetic polymers often using organic solvents
• GPC separates based on charge while SEC separates by size
• GPC is used only for desalting proteins
• GPC uses ion exchange resins rather than porous beads

Correct Answer: GPC is a form of SEC applied mainly to synthetic polymers often using organic solvents

Q17. Which of these is a common calibration standard for sizing proteins in SEC?

• Glucose polymers only for RP-HPLC
• Known molecular weight proteins such as albumin, ferritin, and thyroglobulin
• C18 peptides
• Monosaccharides measured by FID

Correct Answer: Known molecular weight proteins such as albumin, ferritin, and thyroglobulin

Q18. Spin columns used for rapid desalting employ gel filtration in which mode?

• High-pressure HPLC mode
• Gravity or centrifuge-driven batch column mode for small volumes
• Gas-phase chromatography
• Electrophoretic flow mode

Correct Answer: Gravity or centrifuge-driven batch column mode for small volumes

Q19. Which parameter must be known to calculate Kav for a solute?

• Sample injection concentration only
• Void volume (V0), total column volume (Vt), and elution volume (Ve)
• Mobile phase pH only
• Detector wavelength only

Correct Answer: Void volume (V0), total column volume (Vt), and elution volume (Ve)

Q20. Why is gel filtration preferred for gentle purification of proteins in pharmaceuticals?

• It uses strong denaturing agents that improve purity
• It separates without strong ionic or hydrophobic interactions, preserving native structure
• It always yields higher resolution than affinity chromatography
• It covalently modifies proteins for better detection

Correct Answer: It separates without strong ionic or hydrophobic interactions, preserving native structure

Q21. Which practice helps minimize band broadening in SEC?

• Using very wide sample injection volumes relative to column volume
• Optimizing column packing, using appropriate flow rates, and minimizing extra-column volume
• Running at extremely low temperatures only
• Using highly viscous solvents to slow diffusion

Correct Answer: Optimizing column packing, using appropriate flow rates, and minimizing extra-column volume

Q22. In pharmaceutical QC, SEC can detect which important attribute of therapeutic proteins?

• Primary amino acid sequence
• Presence and amount of high-molecular-weight aggregates
• Exact site-specific glycosylation patterns
• Metal ion contamination at trace levels

Correct Answer: Presence and amount of high-molecular-weight aggregates

Q23. Which of the following is an advantage of SEC over dialysis for desalting?

• SEC requires much longer processing times than dialysis
• SEC provides faster buffer exchange with higher sample recovery and fractions collected immediately
• Dialysis is compatible with high throughput small-volume sample runs
• SEC always achieves lower salt concentrations than dialysis

Correct Answer: SEC provides faster buffer exchange with higher sample recovery and fractions collected immediately

Q24. Cross-linking density of gel beads affects SEC by:

• Determining the bead color only
• Altering pore size distribution and thus the fractionation range
• Changing the pH of mobile phase
• Preventing solvent flow through the column

Correct Answer: Altering pore size distribution and thus the fractionation range

Q25. Which technique combined with SEC provides accurate molar mass and size information for biopharmaceuticals?

• Flame photometry
• Multi-angle light scattering (MALS) coupled to SEC
• Gas chromatography
• Thin layer chromatography

Correct Answer: Multi-angle light scattering (MALS) coupled to SEC

Q26. Why must sample concentration be controlled in SEC injections?

• High concentration always improves resolution
• Too concentrated samples can lead to column overloading and distorted elution profiles
• Concentration has no impact on SEC performance
• Low concentration causes columns to collapse

Correct Answer: Too concentrated samples can lead to column overloading and distorted elution profiles

Q27. Which of the following is NOT a common cause of band broadening in gel filtration?

• Longitudinal diffusion
• Eddy diffusion from poor packing
• Mass transfer resistance between mobile and stationary phases
• Use of appropriately sized beads and optimal flow rates

Correct Answer: Use of appropriately sized beads and optimal flow rates

Q28. For molecular weight estimation using SEC, a calibration curve is constructed by plotting:

• Log(molecular weight) versus Kav values for standards
• Retention time versus buffer pH
• Peak area versus detector wavelength
• Flow rate versus column temperature

Correct Answer: Log(molecular weight) versus Kav values for standards

Q29. Which statement about cleaning and regenerating SEC columns in pharmaceutical labs is correct?

• Columns are disposable and never regenerated
• Regular flushing with appropriate buffers and mild detergents or salts helps maintain performance
• Strong acids must always be used to clean any column
• Columns should be dried at high temperature between runs

Correct Answer: Regular flushing with appropriate buffers and mild detergents or salts helps maintain performance

Q30. Compared to ultrafiltration, one advantage of gel filtration for buffer exchange in small sample volumes is:

• SEC causes more protein denaturation than ultrafiltration
• SEC provides simultaneous size-based separation and is less likely to concentrate aggregates
• Ultrafiltration is always faster than SEC for small volumes
• SEC cannot remove small ions from protein solutions

Correct Answer: SEC provides simultaneous size-based separation and is less likely to concentrate aggregates

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