Supercritical fluid chromatography and ion-exclusion chromatography MCQs With Answer

This set of multiple-choice questions focuses on Supercritical Fluid Chromatography (SFC) and Ion-Exclusion Chromatography (IEC), two specialized separation techniques critical for advanced pharmaceutical analysis. The questions are designed for M.Pharm students preparing for MPC 201T Advanced Spectral Analysis and emphasize mechanistic understanding, method development, and practical troubleshooting. Expect items covering thermodynamic principles, mobile phase selection, stationary phase chemistry, detector compatibility, retention behavior, and common applications such as chiral separations and organic acid analysis. Practice with these MCQs will deepen conceptual clarity and prepare you for laboratory method optimization, interpretation of chromatographic behaviour under varying pressure/temperature, and real-world pharmaceutical separations.

Q1. Which property of carbon dioxide makes it the most commonly used supercritical fluid in SFC for pharmaceutical separations?

• High polarity similar to water
• Low critical temperature and moderate critical pressure
• Intrinsic UV absorbance suitable for detection
• Strong hydrogen bonding ability

Correct Answer: Low critical temperature and moderate critical pressure

Q2. In SFC, adding a polar organic modifier (e.g., methanol) to supercritical CO2 primarily affects separation by:

• Increasing mobile phase viscosity drastically
• Decreasing the critical temperature of CO2
• Increasing mobile phase polarity and solvating polar analytes
• Converting stationary phase chemistry to ion exchange

Correct Answer: Increasing mobile phase polarity and solvating polar analytes

Q3. Which of the following retained analytes is most suitable for separation by ion-exclusion chromatography?

• Neutral nonpolar hydrocarbons
• Strongly basic amines with pKa > 10
• Weak organic acids like acetic and formic acid
• Large hydrophobic peptides

Correct Answer: Weak organic acids like acetic and formic acid

Q4. What is the primary retention mechanism in ion-exclusion chromatography for weak acids?

• Partitioning into a hydrophobic stationary phase
• Size-exclusion based on molecular weight
• Donnan exclusion and electrostatic repulsion from a charged stationary phase
• Hydrogen-bonding interactions with silica

Correct Answer: Donnan exclusion and electrostatic repulsion from a charged stationary phase

Q5. Which column packing is typically used in ion-exclusion chromatography for separation of inorganic anions and organic acids?

• Silica gel with C18 bonded phase
• Polystyrene-divinylbenzene with sulfonic acid groups (strong cation exchanger)
• Polymeric sulfonated styrene-divinylbenzene weakly acidic resin
• Alumina neutral support

Correct Answer: Polystyrene-divinylbenzene with sulfonic acid groups (strong cation exchanger)

Q6. Which parameter in SFC most directly controls mobile-phase density and hence chromatographic selectivity?

• Mobile-phase pH
• Column length
• Backpressure and temperature
• Detector wavelength

Correct Answer: Backpressure and temperature

Q7. Why is a backpressure regulator (BPR) essential in SFC systems?

• To reduce detector noise by stabilizing UV lamp output
• To maintain the supercritical state of CO2 by controlling system pressure
• To remove dissolved gases from the mobile phase
• To inject samples via loop injection

Correct Answer: To maintain the supercritical state of CO2 by controlling system pressure

Q8. In ion-exclusion chromatography, increasing the ionic strength of the eluent typically results in:

• Longer retention times for excluded ions due to increased Donnan potential
• Shorter retention times for weak electrolytes by reducing electrostatic exclusion
• Conversion of the stationary phase to reversed-phase behavior
• No change since IEC is independent of eluent composition

Correct Answer: Shorter retention times for weak electrolytes by reducing electrostatic exclusion

Q9. Which detection technique is most commonly paired with IEC for analysis of low-molecular-weight inorganic anions and organic acids?

• Mass spectrometry without suppression
• Conductivity detection, often with chemical suppression
• Fluorescence detection with pre-column derivatization
• Evaporative light scattering detection (ELSD)

Correct Answer: Conductivity detection, often with chemical suppression

Q10. When developing an SFC method for a chiral pharmaceutical compound, which stationary phase type is commonly used?

• Octadecyl silica (C18)
• Silica gel with amino modification
• Polysaccharide-based chiral stationary phases (e.g., cellulose or amylose derivatives)
• Strong anion-exchange resin

Correct Answer: Polysaccharide-based chiral stationary phases (e.g., cellulose or amylose derivatives)

Q11. Which effect explains why retention of analytes in SFC often decreases with increasing temperature at constant pressure?

• Decrease in detector sensitivity with temperature
• Decrease in mobile phase density reducing solvating power of supercritical CO2
• Increase in stationary phase hydrophobicity
• Formation of microbubbles that enhance retention

Correct Answer: Decrease in mobile phase density reducing solvating power of supercritical CO2

Q12. In ion-exclusion chromatography, separation of neutral species and ionized species in the same sample is possible because:

• Neutral species are strongly retained by ion-exchange sites
• Ionized species are excluded from the porous gel phase and elute earlier
• Neutral species cannot enter the column packing and elute in the void
• Ion-exclusion columns selectively react chemically with neutrals only

Correct Answer: Ionized species are excluded from the porous gel phase and elute earlier

Q13. Which modifier additive is commonly used in SFC to improve peak shape for basic pharmaceutical analytes?

• Trifluoroacetic acid (TFA) as an acidic modifier
• Sodium chloride to increase ionic strength
• Hexane to reduce polarity
• EDTA to chelate metal ions

Correct Answer: Trifluoroacetic acid (TFA) as an acidic modifier

Q14. For IEC of weak organic acids, adjusting the sample pH to be much lower than the pKa results in:

• Increased ionization and stronger exclusion
• Conversion to the neutral form leading to increased retention in the gel pores
• Degradation of the stationary phase
• No effect because IEC responds only to ionic strength

Correct Answer: Conversion to the neutral form leading to increased retention in the gel pores

Q15. Which statement best describes the role of co-solvent percentage in SFC gradient elution?

• Increasing co-solvent percentage decreases elution strength and prolongs retention
• Co-solvent percentage has no effect in SFC since CO2 is the primary solvent
• Increasing co-solvent percentage increases eluting power and can reduce retention and change selectivity
• Only temperature changes, not co-solvent, affect SFC selectivity

Correct Answer: Increasing co-solvent percentage increases eluting power and can reduce retention and change selectivity

Q16. Which chromatographic parameter in IEC often shows minimal temperature dependence compared to SFC?

• Mobile phase density
• Donnan potential created by fixed charge on the resin
• Critical pressure of the mobile phase
• Compressibility of the eluent

Correct Answer: Donnan potential created by fixed charge on the resin

Q17. Which compatibility advantage does SFC generally offer over reversed-phase HPLC for certain pharmaceutical analytes?

• Universal solubility of all drug classes in supercritical CO2 without modifiers
• Lower operating pressures than HPLC
• Faster separations for moderately nonpolar and chiral compounds with reduced organic solvent consumption
• No need for temperature control

Correct Answer: Faster separations for moderately nonpolar and chiral compounds with reduced organic solvent consumption

Q18. In ion-exclusion chromatography, what is the expected elution order for a mixture of strong acid (fully ionized), weak acid (partially ionized), and neutral compound under acidic eluent conditions?

• Neutral > Weak acid > Strong acid
• Strong acid > Weak acid > Neutral
• Weak acid > Strong acid > Neutral
• Neutral > Strong acid > Weak acid

Correct Answer: Neutral > Weak acid > Strong acid

Q19. Which of the following is a common challenge when coupling SFC to mass spectrometry, and its typical mitigation?

• Excessive nonvolatile salts — mitigate by adding more salt
• CO2 gas expansion causing spray instability — mitigate with an efficient make-up solvent and proper BPR/ESI interface design
• High water content in mobile phase damaging MS — mitigate by increasing water to 100%
• Too low pressure at MS source — mitigate by removing the backpressure regulator

Correct Answer: CO2 gas expansion causing spray instability — mitigate with an efficient make-up solvent and proper BPR/ESI interface design

Q20. When validating an IEC method for quantifying organic acids in a drug matrix, which validation parameter is particularly important due to potential matrix ionic effects?

• System suitability based only on column temperature
• Specificity and robustness with respect to eluent ionic strength and pH
• Detector linearity at a single ionic strength only
• Mass balance of neutral species

Correct Answer: Specificity and robustness with respect to eluent ionic strength and pH

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