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
