Resolution in HPLC MCQs With Answer

Resolution in HPLC MCQs With Answer

This quiz set focuses on chromatographic resolution in high-performance liquid chromatography (HPLC), an essential concept for achieving reliable separations in pharmaceutical analysis. It covers fundamental definitions, mathematical expressions, practical measurement methods, and the influence of instrumental and operational factors such as column efficiency, selectivity, retention, particle size, flow rate, temperature and extra-column broadening. The questions are designed for M. Pharm students preparing for Advanced Instrumental Analysis (MPA 201T) and system suitability evaluations, with emphasis on applying equations and understanding how to improve or interpret resolution in real laboratory situations.

Q1. What is the standard definition of chromatographic resolution (Rs) between two adjacent peaks?

• The time difference between two peaks
• Two times the retention difference divided by the sum of baseline widths of the peaks
• The ratio of retention times of two peaks
• The sum of peak heights divided by baseline noise

Correct Answer: Two times the retention difference divided by the sum of baseline widths of the peaks

Q2. Which algebraic expression correctly represents resolution (Rs) using retention times (tR1, tR2) and baseline widths (w1, w2)?

• Rs = (tR2 – tR1) / (w1 + w2)
• Rs = 2 (tR2 – tR1) / (w1 + w2)
• Rs = (tR2 – tR1) / (w1 – w2)
• Rs = 4 (tR2 – tR1) / (w1 + w2)

Correct Answer: Rs = 2 (tR2 – tR1) / (w1 + w2)

Q3. Using the standard resolution formula, calculate Rs if tR1 = 5.0 min, tR2 = 6.2 min, w1 = 0.4 min and w2 = 0.5 min.

• 1.33
• 2.67
• 0.89
• 3.50

Correct Answer: 2.67

Q4. Which of the following formulae expresses Rs in terms of plate number (N), selectivity (α = k2/k1) and retention factor (k2)?

• Rs = (sqrt(N)/4) * (α – 1) / α * k2 / (1 + k2)
• Rs = (N/4) * (α – 1) * k2
• Rs = (sqrt(N)/2) * α / (1 + k2)
• Rs = (4/sqrt(N)) * (α – 1) / k2

Correct Answer: Rs = (sqrt(N)/4) * (α – 1) / α * k2 / (1 + k2)

Q5. Which parameter has the largest direct effect on increasing theoretical plate number (N) for a silica packed column?

• Increasing particle diameter
• Decreasing column length
• Decreasing particle diameter
• Using a wider internal diameter with same length

Correct Answer: Decreasing particle diameter

Q6. If column length is doubled, leaving all else unchanged, how does resolution (approximately) change assuming resolution is mainly related to plate number?

• It doubles
• It increases by a factor of √2 (about 1.41)
• It remains the same
• It increases by a factor of 4

Correct Answer: It increases by a factor of √2 (about 1.41)

Q7. Which van Deemter term is mainly responsible for loss of resolution at high linear velocities due to mass transfer resistance?

• A term (eddy diffusion)
• B term (longitudinal diffusion)
• C term (mass transfer)
• D term (detector dispersion)

Correct Answer: C term (mass transfer)

Q8. Which experimental change will most likely reduce the column plate height (H) and thus improve efficiency?

• Increasing particle size
• Decreasing particle size
• Adding longer extra-column tubing
• Lowering detector sensitivity

Correct Answer: Decreasing particle size

Q9. The baseline peak width (wb) and width at half-height (w0.5) are related. Which factor relates wb to w0.5 for a Gaussian peak?

• wb = 0.5 × w0.5
• wb = 1.699 × w0.5
• wb = 2.3548 × w0.5
• wb = 4 × w0.5

Correct Answer: wb = 1.699 × w0.5

Q10. Which of the following changes will primarily alter selectivity (α) between two solutes in reversed-phase HPLC?

• Changing flow rate
• Changing stationary phase chemistry (e.g., C8 → C18)
• Decreasing extra-column dead volume
• Increasing detector lamp intensity

Correct Answer: Changing stationary phase chemistry (e.g., C8 → C18)

Q11. For two closely eluting peaks with significant tailing, what system behavior most directly causes reduced resolution?

• Lowered detector wavelength
• Increased peak width due to tailing
• Shorter column length
• Higher mobile phase viscosity only

Correct Answer: Increased peak width due to tailing

Q12. Which components contribute to extra-column broadening that degrades resolution in HPLC?

• Injector, tubing, detector cell
• Only column packing imperfections
• Mobile phase composition only
• Only sample solubility

Correct Answer: Injector, tubing, detector cell

Q13. Typical acceptance criterion for baseline resolution for two neighboring pharmacopoeial peaks is approximately:

• Rs ≥ 0.5
• Rs ≥ 1.0
• Rs ≥ 1.5
• Rs ≥ 2.5

Correct Answer: Rs ≥ 1.5

Q14. How does increasing linear flow velocity generally affect chromatographic resolution, assuming selectivity remains unchanged?

• Resolution increases due to higher retention
• Resolution decreases because efficiency (N) falls due to mass transfer
• Resolution remains constant
• Resolution increases because peaks become taller

Correct Answer: Resolution decreases because efficiency (N) falls due to mass transfer

Q15. In the Rs expression Rs = (sqrt(N)/4) * (α – 1)/α * k2/(1 + k2), which variable reflects the compound-specific tendency to stay on the stationary phase?

• N (plate number)
• α (selectivity)
• k2 (retention factor)
• 4 (constant)

Correct Answer: k2 (retention factor)

Q16. Which practical approach is most effective to increase resolution when two peaks have very similar selectivity (α ≈ 1)?

• Shortening column length
• Optimizing mobile phase composition or stationary phase to change α
• Only increasing detector gain
• Lowering mobile phase pH to extreme values without rationale

Correct Answer: Optimizing mobile phase composition or stationary phase to change α

Q17. What is the main effect of increasing column temperature on HPLC separations relevant to resolution?

• Increases mobile phase viscosity and increases backpressure
• Generally decreases retention times and can change selectivity and efficiency
• Has no effect on diffusion coefficients
• Makes peaks always wider and lowers resolution

Correct Answer: Generally decreases retention times and can change selectivity and efficiency

Q18. When using gradient elution, why is measurement of resolution more complex than in isocratic runs?

• Detector response is unstable during gradients
• Retention factors change with time during the gradient, altering effective selectivity and peak shapes
• You cannot measure retention time in gradients
• Columns cannot be equilibrated after gradients

Correct Answer: Retention factors change with time during the gradient, altering effective selectivity and peak shapes

Q19. Which action is most likely to improve resolution without significantly increasing run time for two partially resolved peaks?

• Increase column length by 100% (double length)
• Switch to a stationary phase with a slightly different chemistry to increase α
• Reduce particle size by a large factor and double flow rate
• Increase injection volume drastically

Correct Answer: Switch to a stationary phase with a slightly different chemistry to increase α

Q20. Which statement best describes the interplay of selectivity (α), efficiency (N) and retention (k) for achieving good resolution?

• Only increasing N will always guarantee adequate resolution irrespective of α and k
• Selectivity (α) is the most powerful parameter; small changes in α often produce larger gains in Rs than comparable changes in N or k
• Retention (k) is irrelevant for resolution
• Decreasing N improves resolution if k is very high

Correct Answer: Selectivity (α) is the most powerful parameter; small changes in α often produce larger gains in Rs than comparable changes in N or k

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