Selectivity in HPLC MCQs With Answer

Selectivity in HPLC MCQs With Answer

Introduction: Understanding selectivity is central to optimizing high-performance liquid chromatography (HPLC) methods in pharmaceutical analysis. This question set focuses on thermodynamic and mechanistic aspects that determine how analytes discriminate between mobile and stationary phases — the selectivity factor (α), its relation to resolution, and how changes in stationary phase chemistry, mobile phase composition, pH, temperature, ion-pairing and mixed-mode interactions alter selectivity. These carefully curated MCQs are designed for M.Pharm students preparing for Advanced Instrumental Analysis (MPA 201T). They probe both conceptual theory and practical method-development strategies to deepen your ability to predict and control separations in drug analysis.

Q1. What is the standard definition of the selectivity factor (α) between two chromatographic peaks?

• The difference in retention times (tR2 − tR1)
• The ratio of retention factors k2/k1
• The product of retention factors k1 × k2
• The reciprocal of resolution (1/RS)

Correct Answer: The ratio of retention factors k2/k1

Q2. Which expression correctly shows the dependence of chromatographic resolution (RS) on selectivity (α), efficiency (N) and retention (k)?

• RS = (N/4) × (α − 1) × k
• RS = (√N/4) × [(α − 1)/α] × [k/(1 + k)]
• RS = (√N) × α × k/(1 + k)
• RS = (√N/2) × (1/α) × (1 + k)

Correct Answer: RS = (√N/4) × [(α − 1)/α] × [k/(1 + k)]

Q3. Which single change is usually most effective to alter selectivity in reversed‑phase HPLC?

• Changing detector wavelength
• Changing the stationary phase chemistry (different bonded phase)
• Changing flow rate
• Changing injection volume

Correct Answer: Changing the stationary phase chemistry (different bonded phase)

Q4. How does temperature most commonly affect selectivity in HPLC separations?

• Temperature never affects selectivity; it only affects viscosity
• Temperature changes the thermodynamic partitioning, so selectivity can increase or decrease
• Increasing temperature always increases selectivity
• Temperature only affects detector response, not chromatographic selectivity

Correct Answer: Temperature changes the thermodynamic partitioning, so selectivity can increase or decrease

Q5. A selectivity factor (α) close to 1.0 indicates which situation?

• Excellent baseline separation
• Co-elution or very poor separation between the two analytes
• One analyte is retained infinitely longer than the other
• That column efficiency is extremely high

Correct Answer: Co-elution or very poor separation between the two analytes

Q6. If k1 = 2.0 and k2 = 4.0, what is the selectivity factor (α)?

• 0.5
• 2.0
• 6.0
• 1.0

Correct Answer: 2.0

Q7. To change selectivity for two ionizable acidic compounds in RP‑HPLC, which mobile phase adjustment is most directly useful?

• Adjust the mobile phase pH to change the ionization state relative to their pKa values
• Only increase the flow rate
• Change the detector from UV to MS
• Decrease injection volume

Correct Answer: Adjust the mobile phase pH to change the ionization state relative to their pKa values

Q8. In hydrophilic interaction liquid chromatography (HILIC), selectivity is primarily governed by which interaction type?

• Hydrophobic partitioning into the alkyl chain
• Polar interactions and aqueous layer partitioning at the stationary phase
• Ion-exchange on nonpolar surfaces
• Covalent binding to the stationary phase

Correct Answer: Polar interactions and aqueous layer partitioning at the stationary phase

Q9. How does increasing the carbon load (higher bonded phase density) of an RP stationary phase typically affect selectivity?

• It decreases hydrophobic retention and reduces selectivity
• It increases hydrophobic retention and can change relative selectivity for nonpolar analytes
• It converts the column into a normal-phase column
• It only affects plate count, not selectivity

Correct Answer: It increases hydrophobic retention and can change relative selectivity for nonpolar analytes

Q10. In ion-exchange chromatography, which factor primarily determines the intrinsic selectivity among ions?

• The type of ion-exchange functional group and resin chemistry
• The UV detection wavelength
• The particle size only
• The injection volume

Correct Answer: The type of ion-exchange functional group and resin chemistry

Q11. How do ion-pairing reagents change selectivity for ionic analytes in reversed-phase HPLC?

• They oxidize analytes and reduce retention
• They form ion-pair complexes that increase apparent hydrophobicity and retention of ionic analytes
• They only change detector sensitivity, not retention
• They remove the stationary phase coating

Correct Answer: They form ion-pair complexes that increase apparent hydrophobicity and retention of ionic analytes

Q12. Which interaction is NOT a principal contributor to enantioselective recognition on chiral stationary phases?

• Steric (shape) complementarity
• Hydrogen bonding and electrostatic interactions
• Covalent bond formation between analyte and stationary phase
• π-π and van der Waals interactions

Correct Answer: Covalent bond formation between analyte and stationary phase

Q13. Is the selectivity factor (α) dependent on column efficiency (N)?

• Yes — α increases with N
• No — α is a thermodynamic ratio (k2/k1) and is independent of N
• Yes — only at high flow rates
• Yes — but only when detector sensitivity changes

Correct Answer: No — α is a thermodynamic ratio (k2/k1) and is independent of N

Q14. Which change will typically NOT significantly alter selectivity for an isocratic reversed‑phase separation?

• Changing mobile phase organic composition
• Changing stationary phase chemistry
• Changing flow rate
• Changing column temperature

Correct Answer: Changing flow rate

Q15. What is a common cause of peak order reversal (two peaks swapping elution order) when method conditions are changed?

• A small change in detector slit width
• A change in mobile phase pH that differentially alters ionization of analytes
• Reducing injection volume
• Using a longer tubing connector

Correct Answer: A change in mobile phase pH that differentially alters ionization of analytes

Q16. Which chromatographic metric is most sensitive to relatively small changes in selectivity (α) between two nearby peaks?

• Retention time of the first peak
• Resolution (RS) between the two peaks
• Detector noise level
• Column physical length only

Correct Answer: Resolution (RS) between the two peaks

Q17. Mixed-mode stationary phases can enhance selectivity by combining interaction types. Which pair correctly describes a common mixed-mode RP–ion exchange mechanism?

• Hydrophobic partitioning + ionic (electrostatic) interactions
• Hydrophobic partitioning + covalent bonding
• Normal-phase adsorption + size exclusion
• Chiral covalent binding + detector ionization

Correct Answer: Hydrophobic partitioning + ionic (electrostatic) interactions

Q18. How does reducing particle size (e.g., 5 μm → 1.7 μm) primarily influence selectivity in HPLC?

• It dramatically changes the thermodynamic selectivity α
• It has minimal direct effect on α but greatly increases efficiency and peak capacity
• It converts reversed-phase retention to normal-phase retention
• It removes silanol activity completely

Correct Answer: It has minimal direct effect on α but greatly increases efficiency and peak capacity

Q19. Which plot is most useful to evaluate how selectivity between two analytes changes with temperature?

• van’t Hoff plot of ln k vs 1/T
• Retention time vs injection volume
• Peak area vs detector wavelength
• Flow rate vs column length

Correct Answer: van’t Hoff plot of ln k vs 1/T

Q20. Given k1 = 1.5, k2 = 1.8 and N = 10,000, estimate the resolution (RS) using RS ≈ (√N/4)·[(α − 1)/α]·[k/(1 + k)] where k ≈ (k1 + k2)/2. Which is the closest value?

• 0.8
• 1.2
• 2.6
• 4.0

Correct Answer: 2.6

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