HPLC troubleshooting techniques MCQs With Answer

Introduction: HPLC troubleshooting techniques MCQs With Answer is designed for M.Pharm students preparing for Advanced Instrumental Analysis. This concise quiz set focuses on practical problems encountered in liquid chromatography — from baseline instability, pressure fluctuations and peak distortion to detector and column-related faults. Each question targets diagnostic reasoning and corrective actions used in routine laboratory practice, helping you link instrumental symptoms to root causes. By working through these 20 scenario-based MCQs with clear answers, you will strengthen method development, validation and routine maintenance skills essential for reliable HPLC results in pharmaceutical analysis.

Q1. What is the most likely cause of sudden, sharp spikes in the HPLC baseline?

• Mobile phase microbial contamination
• Air bubbles in the mobile phase or detector flow cell
• Column packing degradation
• High salt concentration in sample

Correct Answer: Air bubbles in the mobile phase or detector flow cell

Q2. If retention times gradually increase over repeated injections, which is the most probable cause?

• Autosampler carryover
• Column contamination or buildup of strongly retained material
• Detector lamp intensity drop
• Mobile phase pH perfectly controlled

Correct Answer: Column contamination or buildup of strongly retained material

Q3. A progressive rise in system backpressure during a run typically indicates:

• Air trapped in the detector cell
• Clogged filter or frit, or particulate buildup in the column
• Incorrect injection volume
• Degraded detector lamp

Correct Answer: Clogged filter or frit, or particulate buildup in the column

Q4. What troubleshooting step should you perform first when you observe noisy UV detector baseline but stable flow and pressure?

• Replace the column
• Check and clean the detector flow cell and lamp alignment
• Change the mobile phase composition drastically
• Increase injection volume

Correct Answer: Check and clean the detector flow cell and lamp alignment

Q5. Which problem is most likely if peaks show fronting (leading edge tailing opposite) in an HPLC chromatogram?

• Overloading of the column by too large sample mass
• Presence of strong ionic interactions with stationary phase
• Mobile phase degassing failure
• Incorrect detector wavelength

Correct Answer: Overloading of the column by too large sample mass

Q6. If two peaks that used to be baseline resolved start to co-elute (reduced resolution), which actions are appropriate to diagnose the issue?

• Check column temperature, mobile phase composition and pump flow rate
• Only replace the detector lamp
• Increase injection speed of the autosampler
• Use a different vial cap

Correct Answer: Check column temperature, mobile phase composition and pump flow rate

Q7. Which cause is most likely when a chromatogram shows ghost peaks (recurrent small peaks) even with blank injections?

• Insufficient column equilibration and carryover from previous runs
• Normal detector noise
• Ambient room temperature fluctuations
• Using ultra-pure water in mobile phase

Correct Answer: Insufficient column equilibration and carryover from previous runs

Q8. What is the primary remedy for a system that shows irregular pressure pulsations linked to the pump?

• Replace the sample vials
• Service or replace pump check valves and pistons, and degas solvents
• Switch detector from UV to PDA
• Increase column temperature to maximum

Correct Answer: Service or replace pump check valves and pistons, and degas solvents

Q9. Which factor most commonly causes broad, low-efficiency peaks in HPLC?

• Dead volume in the system (extra-column broadening)
• Using a guard column
• Too low detector sensitivity
• Excessive degassing

Correct Answer: Dead volume in the system (extra-column broadening)

Q10. When a reversed-phase method shows peaks eluting earlier than expected, the best initial check is:

• Confirm mobile phase composition was prepared correctly (e.g., organic solvent fraction)
• Replace the autosampler syringe
• Switch to a new batch of standard without verification
• Reduce detector gain

Correct Answer: Confirm mobile phase composition was prepared correctly (e.g., organic solvent fraction)

Q11. Persistent high baseline drift in gradient HPLC is most likely caused by:

• Incomplete mixing or incompatible solvents causing refractive index changes
• Using a freshly sonicated mobile phase
• Correct column temperature control
• Blank injections between samples

Correct Answer: Incomplete mixing or incompatible solvents causing refractive index changes

Q12. Sudden loss of detector signal while injection and system appear normal suggests:

• Detector lamp failure or quenched lamp emission
• Column contamination
• Mobile phase pH change
• Ambient humidity increase

Correct Answer: Detector lamp failure or quenched lamp emission

Q13. Which troubleshooting approach reduces ionic contamination that causes peak tailing for basic analytes?

• Use a mobile phase with a low concentration of an appropriate volatile buffer or add ion-pair reagent
• Use unfiltered solvents
• Increase sample injection temperature
• Shorten column length

Correct Answer: Use a mobile phase with a low concentration of an appropriate volatile buffer or add ion-pair reagent

Q14. If the autosampler shows variable injection volumes producing inconsistent peak areas, what is the most likely fault?

• Autosampler syringe wear or air in the syringe line; check and service syringe and seals
• Column temperature fluctuation
• Mobile phase pH drift
• Using a different detector wavelength

Correct Answer: Autosampler syringe wear or air in the syringe line; check and service syringe and seals

Q15. What is the recommended action when an HPLC method shows significant carryover of a strongly retained analyte?

• Implement an effective needle/loop wash with strong solvent and adjust wash volume
• Reduce column temperature to low
• Use smaller injection vials
• Decrease detector sensitivity

Correct Answer: Implement an effective needle/loop wash with strong solvent and adjust wash volume

Q16. A baseline that slowly drifts upward over the course of many runs on an isocratic method may indicate:

• Accumulation of strongly retained contaminants on the column or detector cell fouling
• Perfectly clean system
• Incorrect autosampler programming only
• Stable mobile phase composition

Correct Answer: Accumulation of strongly retained contaminants on the column or detector cell fouling

Q17. Which adjustment improves resolution between closely eluting peaks without changing column chemistry?

• Modify mobile phase composition, reduce flow rate, or adjust column temperature
• Always inject larger sample volumes
• Remove the guard column
• Use unfiltered solvents

Correct Answer: Modify mobile phase composition, reduce flow rate, or adjust column temperature

Q18. When using gradient elution, reproducibility problems in retention times between runs are often caused by:

• Inadequate gradient delay volume calibration or poor gradient formation (mixing)
• Using freshly prepared standards
• Stable column temperature
• Constant detector lamp output

Correct Answer: Inadequate gradient delay volume calibration or poor gradient formation (mixing)

Q19. Which preventive maintenance step helps avoid particulate-related pressure spikes and column blockage?

• Install and regularly replace inlet filters and guard cartridges, and filter/degass mobile phases
• Never filter the mobile phase to save time
• Only use glass syringes for injections
• Keep column at room temperature regardless of method

Correct Answer: Install and regularly replace inlet filters and guard cartridges, and filter/degass mobile phases

Q20. A chromatogram shows asymmetric peak tailing specific to one analyte; the most focused troubleshooting step is:

• Investigate secondary interactions (active sites) on the stationary phase or silica deactivation and consider using a guard column or column replacement
• Switch off detector between runs
• Reduce sample concentration to zero
• Only change injection vials brand

Correct Answer: Investigate secondary interactions (active sites) on the stationary phase or silica deactivation and consider using a guard column or column replacement

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