Top HPLC Interview Questions and Answers for Freshers and Experienced Candidates

High-Performance Liquid Chromatography (HPLC) is a vital analytical tool in the pharmaceutical industry, helping to identify, separate, and quantify compounds. Here’s a detailed guide covering frequently asked interview questions on HPLC to help freshers and experienced ones

1. What is HPLC?

• Answer: HPLC, or High-Performance Liquid Chromatography, is a powerful technique used to separate, identify, and quantify components within a mixture. It’s known for delivering high resolution and accuracy, making it a preferred method in pharmaceutical quality control and research.

2. Explain the Principle of HPLC.

• Answer: HPLC separates components based on interactions between the stationary and mobile phases. Techniques include adsorption, partition, ion exchange, and size exclusion, all contributing to effective separation based on how each component interacts with the column’s materials.

3. What Phases Are Used in HPLC?

• Answer: HPLC involves two key phases: the mobile phase (liquid that carries analytes through the system) and the stationary phase (a solid or gel packed in the column to interact with the sample compounds and aid separation).

4. How Does the Mobile Phase Move Through the System?

• Answer: Stainless steel capillaries are typically used to transport the mobile phase throughout the HPLC system, ensuring efficient flow between modules without compromising pressure or stability.

5. What Is the Internal Diameter of HPLC Capillaries?

• Answer: The internal diameter usually ranges from 0.5 to 1 mm between the pump and injector, and around 0.2 mm beyond the injector, ensuring optimal flow rates for accurate results.

6. Why Is HPLC Sometimes Referred to as High-Pressure Liquid Chromatography?

• Answer: The term “high-pressure” is used because pumps are employed to push the mobile phase through the column under high pressure. HPLC is also called liquid chromatography due to the liquid nature of the mobile phase.

7. What Are Chromatographic Conditions in HPLC?

• Answer: Chromatographic conditions refer to the method parameters, such as mobile phase composition, column type, wavelength, flow rate, injection volume, and run time, that need to be optimized for effective HPLC analysis.

8. What Is a Chromatogram in HPLC?

• Answer: A chromatogram is a graphical plot that shows the detector response over time, helping to visualize the separation of components as they exit the column.

9. Define Retention Time in HPLC.

• Answer: Retention time is the time taken by a particular analyte to travel through the column and reach the detector. It’s a key identifier for compounds in the mixture.

10. What Is the Separation Mechanism in HPLC?

• Answer: The mechanism relies on each compound’s unique interactions with the stationary phase. These interactions cause different movement rates, resulting in the separation of individual components.

11. What Are the Main Modes of HPLC?

• Answer: The primary modes include normal phase (polar stationary phase, non-polar mobile phase), reverse phase (non-polar stationary phase, polar mobile phase), ion exchange, size exclusion, and affinity chromatography. Each mode caters to specific types of compounds and separations.

12. What’s the Difference Between Isocratic and Gradient Elution?

• Answer: In isocratic elution, the mobile phase composition remains constant, while gradient elution gradually changes the solvent mix, making it ideal for separating complex samples.

13. What Is the USP General Chapter for Chromatography?

• Answer: Chromatography guidelines and practices in the U.S. Pharmacopeia (USP) are detailed in chapter <621>, which offers standardized procedures for chromatographic analysis.

14. What Is a Frit in HPLC?

• Answer: A frit is a small, inert filter placed before columns to prevent particles from entering the HPLC system, which could compromise results or damage the equipment.

15. What Causes Baseline Drift or Noise?

• Answer: Temperature fluctuations, mobile phase mixing issues, contamination, or air bubbles in the detector cell can all cause baseline drift or noise, impacting detection accuracy.

16. What Are the Key Components of an HPLC System?

• Answer: The essential components include the solvent delivery pump, injector, column oven, and detector. Each part is crucial for efficient separation, flow control, and detection.

17. What Types of Detectors Are Used in HPLC?

• Answer: Common detectors include PDA (Photodiode Array), fluorescence, electrochemical, UV-VIS, conductivity, and refractive index detectors. Each is selected based on the properties of the analytes.

18. Why Is Degassing Important for the Mobile Phase?

• Answer: Degassing removes dissolved gases that can form bubbles under pressure, potentially affecting the flow and causing inconsistencies in peak shapes and baseline stability.

19. Why Is Caffeine Commonly Used in HPLC Calibration?

• Answer: Caffeine is popular for calibration due to its availability, stability, and multiple UV absorbance peaks, which aid in accurate wavelength calibration across a range of detection limits.

20. What Is a Gradient System in HPLC?

• Answer: A gradient system involves varying the mobile phase composition during a run, allowing for improved resolution and sensitivity, particularly for complex mixtures with diverse compounds.