HILIC approaches in HPLC MCQs With Answer

Introduction: Hydrophilic Interaction Liquid Chromatography (HILIC) has become an essential technique in the M. Pharm curriculum for analyzing polar and highly hydrophilic drug molecules, metabolites, and formulation excipients that are poorly retained by reversed-phase HPLC. This blog-style MCQ set focuses on HILIC principles, stationary phases (silica, zwitterionic, amide, diol), retention mechanisms (partitioning into a water-enriched layer, adsorption, electrostatic interactions), mobile phase design (high organic content, buffer type, salt concentration, pH), method development strategies and MS compatibility. The questions are tailored to deepen conceptual understanding and practical troubleshooting for M.Pharm students preparing for advanced instrumental analysis exams.

Q1. What is the primary retention mechanism in classical HILIC?

• Hydrophobic partitioning into the stationary phase bulk
• Partitioning into a water-enriched layer on the stationary phase surface
• Ion exchange on a strongly acidic resin
• Size exclusion through pore size differences

Correct Answer: Partitioning into a water-enriched layer on the stationary phase surface

Q2. Which stationary phase is most characteristic of mixed-mode HILIC due to both polar and ionic interactions?

• Octadecyl (C18) bonded silica
• Zwitterionic (e.g., sulfobetaine) stationary phase
• Pure polystyrene-divinylbenzene
• Graphitized carbon black

Correct Answer: Zwitterionic (e.g., sulfobetaine) stationary phase

Q3. In HILIC mobile phases, why is acetonitrile commonly used as the organic modifier?

• It forms strong hydrogen bonds with analytes, improving retention
• It is miscible with water, provides high elution strength for nonpolar compounds, and gives low viscosity with good MS compatibility
• It increases the polarity of the mobile phase to mimic reversed-phase behavior
• It suppresses electrostatic interactions by forming ion pairs

Correct Answer: It is miscible with water, provides high elution strength for nonpolar compounds, and gives low viscosity with good MS compatibility

Q4. How does increasing the water content in a HILIC mobile phase typically affect retention of polar neutral analytes?

• Retention increases due to stronger partitioning into the stationary phase
• Retention decreases because the water layer becomes thicker and reduces partitioning from mobile phase
• Retention is unaffected; only ionizable analytes are influenced
• Retention becomes unpredictable and independent of water content

Correct Answer: Retention decreases because the water layer becomes thicker and reduces partitioning from mobile phase

Q5. Which buffer is most commonly recommended for HILIC when coupling to electrospray MS for small molecules?

• Sodium phosphate at high concentration
• Ammonium acetate or ammonium formate at low millimolar concentration
• Potassium chloride at high ionic strength
• Tris-HCl at pH 9.0

Correct Answer: Ammonium acetate or ammonium formate at low millimolar concentration

Q6. Which of the following best explains poor peak shapes (tailing) for basic analytes on bare silica HILIC columns?

• Excessive organic content in the mobile phase
• Ion-exchange interactions between residual silanols and protonated basic analytes
• Formation of micelles in the mobile phase
• Too high column temperature causing analyte decomposition

Correct Answer: Ion-exchange interactions between residual silanols and protonated basic analytes

Q7. For ionizable analytes in HILIC, how does increasing buffer ionic strength generally influence retention?

• It always increases retention by strengthening partitioning
• It decreases electrostatic interactions and can reduce retention of charged analytes
• It has no effect because retention is only driven by hydrophobicity
• It causes immediate column collapse and loss of retention

Correct Answer: It decreases electrostatic interactions and can reduce retention of charged analytes

Q8. What is the role of a water-rich layer at the stationary phase surface in HILIC?

• It acts as a stationary aqueous phase into which polar analytes partition from the organic-rich mobile phase
• It repels polar analytes, causing faster elution
• It serves purely as a rinse to clean the stationary phase
• It prevents any ionic interactions between analyte and stationary phase

Correct Answer: It acts as a stationary aqueous phase into which polar analytes partition from the organic-rich mobile phase

Q9. Which adjustment is most effective to improve retention of very polar neutral compounds in HILIC?

• Reduce acetonitrile content in the mobile phase drastically to <10%
• Increase the organic content (acetonitrile) to strengthen partitioning into the water layer
• Use only water as the mobile phase
• Replace HILIC column with a C8 reversed-phase column

Correct Answer: Increase the organic content (acetonitrile) to strengthen partitioning into the water layer

Q10. Which column conditioning practice is particularly important when switching between reversed-phase and HILIC modes on the same silica-based column?

• Flushing with pure methanol followed by aqueous buffer
• Equilibrating with high acetonitrile content mobile phase until stable baseline and retention are achieved
• Running at high flow rates for short times to speed equilibration
• Storing column dry between runs without equilibration

Correct Answer: Equilibrating with high acetonitrile content mobile phase until stable baseline and retention are achieved

Q11. Which statement describes the effect of pH on retention of weak bases in HILIC?

• Lowering mobile phase pH (more acidic) generally reduces retention of weak bases by fully deprotonating them
• Lowering mobile phase pH generally increases retention of weak bases via stronger electrostatic attraction to negatively charged sites
• pH has no effect on ionizable compounds in HILIC
• Raising pH always improves peak shape for bases because silanol interactions are eliminated

Correct Answer: Lowering mobile phase pH generally increases retention of weak bases via stronger electrostatic attraction to negatively charged sites

Q12. Which of the following mobile phase additives is most likely to suppress ionization and reduce MS sensitivity when used at high concentration in HILIC-MS?

• 10 mM ammonium formate
• 100 mM ammonium acetate
• 0.1% formic acid
• 5 mM ammonium bicarbonate

Correct Answer: 100 mM ammonium acetate

Q13. When is gradient elution preferred over isocratic elution in HILIC method development?

• When analytes have very similar polarities and retention times
• When sample contains a wide polarity range from highly polar to moderately polar compounds requiring different retention strengths
• When column equilibration time must be minimized
• When using nonvolatile buffers incompatible with MS

Correct Answer: When sample contains a wide polarity range from highly polar to moderately polar compounds requiring different retention strengths

Q14. What is a common cause of retention time drift in HILIC methods during repeated injections?

• Insufficient column equilibration and varying water layer thickness between runs
• Using high-purity acetonitrile only
• Excessive column backpressure stabilizing retention
• Analyte degradation by ultraviolet light in the detector

Correct Answer: Insufficient column equilibration and varying water layer thickness between runs

Q15. Which sample solvent practice is best when injecting aqueous samples onto a HILIC column with 90% acetonitrile mobile phase?

• Inject the aqueous sample directly without modification
• Dilute the sample with acetonitrile to reduce mismatch and avoid breakthrough/peak distortion
• Add strong acid to the sample to promote retention
• Use methanol as a diluent to improve retention consistency

Correct Answer: Dilute the sample with acetonitrile to reduce mismatch and avoid breakthrough/peak distortion

Q16. Which effect does increasing column temperature typically have on HILIC separations?

• It always increases retention for all analytes
• It can decrease mobile phase viscosity and change partitioning equilibrium, often reducing retention and sometimes improving peak shape
• It increases ion exchange interactions selectively for acidic analytes only
• It eliminates the need for buffer in the mobile phase

Correct Answer: It can decrease mobile phase viscosity and change partitioning equilibrium, often reducing retention and sometimes improving peak shape

Q17. Which analyte class is most suitable for HILIC analysis compared to reversed-phase HPLC?

• Highly lipophilic APIs with logP > 5
• Very polar small molecules, sugars, nucleotides, and highly polar metabolites
• Large neutral polymers only
• Nonpolar volatile organic solvents

Correct Answer: Very polar small molecules, sugars, nucleotides, and highly polar metabolites

Q18. How does using a zwitterionic stationary phase influence selectivity compared with neutral amide or diol HILIC phases?

• Zwitterionic phases provide only hydrophobic selectivity similar to C18
• Zwitterionic phases can provide both electrostatic and polar interactions, offering different selectivity for charged analytes
• Zwitterionic phases eliminate all water layer formation
• Zwitterionic phases are incompatible with organic modifiers

Correct Answer: Zwitterionic phases can provide both electrostatic and polar interactions, offering different selectivity for charged analytes

Q19. Which troubleshooting step is recommended when observing irreproducible retention due to variability in salt concentration from buffer preparation?

• Use nonvolatile salts at higher concentrations to stabilize retention
• Prepare buffer by weight or use commercially prepared buffered solvents with accurate ionic strength and use consistent lot-to-lot practice
• Eliminate buffer entirely and use pure acetonitrile
• Change detector wavelength to avoid variability

Correct Answer: Prepare buffer by weight or use commercially prepared buffered solvents with accurate ionic strength and use consistent lot-to-lot practice

Q20. In HILIC-MS method transfer between instruments, which parameter is most critical to match to preserve retention and selectivity?

• Column oven color
• Organic solvent percent (acetonitrile/water ratio), buffer type and concentration, and pH
• Detector brand
• Autosampler injection needle length

Correct Answer: Organic solvent percent (acetonitrile/water ratio), buffer type and concentration, and pH

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