Size exclusion chromatography MCQs With Answer

Introduction:

This quiz set on Size Exclusion Chromatography (SEC) is designed for M.Pharm students studying Advanced Instrumental Analysis (MPA 201T). It covers theory, column properties, calibration, detectors, and practical troubleshooting with emphasis on separation by molecular size/hydrodynamic volume rather than chemical interaction. Questions include calculations (Vo, Ve, Kav), choice of stationary phases, detector selection (RI, UV, MALS), universal calibration concepts, and common artifacts such as adsorption, shear degradation and aggregation. These MCQs will help you consolidate conceptual understanding and prepare for exams and lab applications where selecting conditions and interpreting SEC data for polymers, proteins and pharmaceutical macromolecules are critical.

Q1. What is the primary basis for separation in size exclusion chromatography (SEC)?

• Partitioning based on polarity differences
• Adsorption to the stationary phase
• Differences in molecular hydrodynamic volume
• Affinity interactions between analyte and ligand

Correct Answer: Differences in molecular hydrodynamic volume

Q2. In SEC, a molecule that is larger than the largest pore size of the packing will elute at which volume?

• Elution volume (Ve)
• Void volume (Vo)
• Total column volume (Vt)
• Partial inclusion volume

Correct Answer: Void volume (Vo)

Q3. The partition coefficient Kav used in SEC is defined by which of the following equations?

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

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

Q4. Which detector provides absolute molar mass determination in SEC without standards?

• Refractive index (RI) detector
• Ultraviolet (UV) detector
• Multi-angle light scattering (MALS) detector
• Evaporative light scattering (ELSD) detector

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

Q5. Universal calibration for polymer SEC relates elution to which combined parameter?

• Hydrodynamic radius only
• Intrinsic viscosity multiplied by molecular weight (η · M)
• Refractive index increment (dn/dc) alone
• UV absorbance at 254 nm

Correct Answer: Intrinsic viscosity multiplied by molecular weight (η · M)

Q6. Which stationary phase material is commonly used for protein gel filtration columns?

• Polystyrene-divinylbenzene beads
• Silica gel with ion exchange groups
• Agarose or cross-linked dextran
• C18 reversed-phase silica

Correct Answer: Agarose or cross-linked dextran

Q7. Which of the following best describes the fractionation range of a SEC column?

• The pH range over which the packing is stable
• The solvent compatibility window for the column
• The range of molecular sizes resolved between Vo and Vt
• The temperature range for isocratic operation

Correct Answer: The range of molecular sizes resolved between Vo and Vt

Q8. How does increasing sample load (mass applied) typically affect SEC resolution?

• Improves resolution due to better signal
• Reduces resolution due to band broadening and overloading
• Has no effect on resolution
• Causes earlier elution of all species equally

Correct Answer: Reduces resolution due to band broadening and overloading

Q9. Which mobile phase practice is essential in SEC to minimize secondary interactions for protein samples?

• Use highly acidic solvents to denature proteins
• Add low concentrations of salt or buffer at physiological pH
• Use strong organic modifiers like acetonitrile
• Operate with a gradient elution

Correct Answer: Add low concentrations of salt or buffer at physiological pH

Q10. What effect does increasing column temperature generally have on SEC separations for polymers?

• Decreases diffusion and broadens peaks dramatically
• Increases solvent viscosity and slows elution
• Reduces solvent viscosity and can improve mass transport, sometimes improving resolution
• Makes SEC equivalent to reversed-phase chromatography

Correct Answer: Reduces solvent viscosity and can improve mass transport, sometimes improving resolution

Q11. Which statement correctly distinguishes GPC from gel filtration?

• GPC refers to biomolecules while gel filtration is for synthetic polymers
• They are identical terms used interchangeably, but GPC often refers to polymers and gel filtration to biomolecules
• GPC uses affinity ligands, gel filtration uses size only
• Gel filtration always uses organic solvents, GPC is aqueous

Correct Answer: They are identical terms used interchangeably, but GPC often refers to polymers and gel filtration to biomolecules

Q12. When calibrating an SEC column for molecular weight determination using narrow standards, what plot is typically used?

• Log(Molecular weight) vs elution volume (Ve)
• Elution time vs detector baseline
• UV absorbance vs concentration
• Void volume vs total column volume

Correct Answer: Log(Molecular weight) vs elution volume (Ve)

Q13. Which phenomenon causes high-molecular-weight polymers to appear at slightly different positions than expected due to shear during SEC?

• Adsorptive retention
• Shear degradation of polymer chains
• Ion-exchange coupling
• Column overpacking

Correct Answer: Shear degradation of polymer chains

Q14. Secondary interactions in SEC (e.g., ionic or hydrophobic) typically result in which chromatographic behavior?

• Ideal size-based separation with sharper peaks
• Shifted elution volumes and peak tailing or extra retention
• Complete exclusion of all analytes at Vo
• Lower backpressure and improved flow

Correct Answer: Shifted elution volumes and peak tailing or extra retention

Q15. Which detector is most sensitive to changes in solvent composition and baseline drift in SEC?

• Refractive index (RI) detector
• Multi-angle light scattering (MALS) detector
• UV absorbance detector
• Conductivity detector

Correct Answer: Refractive index (RI) detector

Q16. For a SEC column, total column volume (Vt) is composed of which two volumes?

• Dead volume and retention volume
• Void (interstitial) volume and pore volume
• Mobile phase volume and stationary phase volume
• Detector cell volume and injection loop volume

Correct Answer: Void (interstitial) volume and pore volume

Q17. Which packing feature primarily determines the fractionation range of a SEC column?

• Particle color
• Pore size distribution of the gel beads
• Length of tubing from pump to detector
• Detector sensitivity

Correct Answer: Pore size distribution of the gel beads

Q18. Why is SEC typically run under isocratic conditions rather than gradient elution?

• Because gradient pumps are incompatible with SEC columns
• Because size-based equilibrium within pores requires constant mobile phase composition to maintain retention characteristics
• Because detectors cannot handle changing mobile phases
• Because gradients increase sample adsorption irreversibly

Correct Answer: Because size-based equilibrium within pores requires constant mobile phase composition to maintain retention characteristics

Q19. Which practical step reduces nonspecific adsorption of proteins to SEC column packing?

• Using deionized water with no buffer
• Including low concentrations of salt and a non-ionic stabilizer like 0.02% sodium azide
• Lowering ionic strength to zero
• Increasing flow rate to maximum

Correct Answer: Including low concentrations of salt and a non-ionic stabilizer like 0.02% sodium azide

Q20. In an SEC experiment, two similarly sized analytes are not resolved. Which adjustment is most likely to improve resolution?

• Increase detector wavelength
• Reduce column length
• Use a column with narrower pore size distribution or increase column length to increase theoretical plates
• Switch to gradient elution

Correct Answer: Use a column with narrower pore size distribution or increase column length to increase theoretical plates

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