Top 22 HPLC Interview Questions and Answers

Top 22 HPLC Interview Questions and Answers

 

1. What is HPLC?

Answer: High Performance Liquid Chromatography (HPLC) is an analytical technique used to separate, identify, and quantify components in a mixture.

2. What are the main components of an HPLC system?

Answer: Pump, injector, column, detector, and data system.

3. What types of HPLC are there?

Answer: Normal-phase, reversed-phase, ion-exchange, and size-exclusion chromatography.

4. What is the difference between normal-phase and reversed-phase HPLC?

Answer: Normal-phase uses a polar stationary phase and non-polar mobile phase. Reversed-phase uses a non-polar stationary phase (e.g., C18 column) and polar mobile phase (e.g., water, methanol).

5. What is a C18 column?

Answer: A column packed with octadecylsilane bonded silica particles, commonly used in reversed-phase HPLC.

6. What are common detectors used in HPLC?

Answer: UV-Visible, PDA (Photodiode Array), Fluorescence, and RI (Refractive Index) detectors.

7. What is retention time (Rt)?

Answer: The time taken by a compound to travel from the injector to the detector in the column.

8. What factors affect retention time?

Answer: Mobile phase composition, flow rate, column temperature, and column type.

9. What is resolution in HPLC?

Answer: A measure of how well two peaks are separated. Good resolution is essential for accurate quantification.

10. How can you improve resolution in HPLC?

Answer: Adjust mobile phase composition, change flow rate, use a different column, or modify temperature.

11. What is tailing in HPLC?

Answer: When a peak is asymmetrical, extending to the right side. Caused by column overloading or poor sample solubility.

12. What is fronting?

Answer: When a peak leans toward the left. Often due to column overloading or poor injection technique.

13. What is the mobile phase?

Answer: The solvent(s) that carry the sample through the column. Can be isocratic (constant composition) or gradient (changing composition).

14. What is a gradient method in HPLC?

Answer: A technique where the mobile phase composition changes during the run, often used to separate complex mixtures.

15. What is system suitability in HPLC?

Answer: Tests run before analysis to ensure the system is working correctly (e.g., resolution, theoretical plates, tailing factor).

16. What are theoretical plates?

Answer: A measure of column efficiency. More plates = better separation.

17. What is the tailing factor?

Answer: A measure of peak symmetry. Ideal value is close to 1.0.

18. How do you prepare an HPLC sample?

Answer: Filter the sample (usually with a 0.22 or 0.45 µm syringe filter), dilute appropriately, and degas if needed.

19. What is the purpose of degassing the mobile phase?

Answer: To remove dissolved gases that can form bubbles and cause baseline noise or detector issues.

20. What causes baseline drift or noise?

Answer: Temperature fluctuations, air bubbles, dirty detector flow cell, or unstable mobile phase.

21. What is peak area and peak height?

Answer: Peak area is used for quantitation; peak height is less common but can be used if peaks are well-separated.

22. How do you validate an HPLC method?

Answer: By evaluating parameters like accuracy, precision, specificity, linearity, range, LOD, and LOQ.