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| 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.
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