Top 10 interview questions about HPLC apparatus.
1. What is HPLC and why is it used?
HPLC (High-Performance Liquid Chromatography) is an analytical technique used to separate, identify, and quantify components in a liquid sample.
It’s used in pharmaceuticals, food testing, environmental monitoring, and research due to its high accuracy, speed, and sensitivity.
2. What are the main components of an HPLC system?
Solvent reservoir: Holds the mobile phase.
Pump: Moves the mobile phase through the system.
Injector: Introduces the sample.
Column: Separates the sample components.
Detector: Identifies and measures the analytes.
Data system: Records and displays results.
3. What types of detectors are used in HPLC?
UV-Vis Detector: Measures light absorbance.
Fluorescence Detector: Detects compounds that fluoresce.
Refractive Index Detector: Universal detector for non-UV absorbing compounds.
Mass Spectrometer (MS): Provides molecular weight and structure info.
Electrochemical Detector: Detects electroactive substances.
4. What is the function of the HPLC column?
The column contains the stationary phase where the sample components are separated based on their interaction with the stationary and mobile phases.
5. What is the difference between isocratic and gradient elution?
Isocratic elution: The mobile phase composition stays the same throughout the run.
Gradient elution: The mobile phase composition changes gradually to help separate complex mixtures more effectively.
6.What factors affect retention time in HPLC?
Mobile Phase Composition: Changes in polarity, pH, or buffer can alter analyte interactions.
Flow Rate: Faster flow = shorter retention time.
Column Temperature: Higher temps usually reduce retention time
Column Type and Packing Material: Pore size, particle size, and chemistry influence separation.
Analyte Properties: Polarity, size, and ionization state determine how it interacts with the stationary phase.
7.What causes peak tailing or fronting in an HPLC chromatogram?
Peak Tailing: Usually caused by strong or secondary interactions between the analyte and active sites on the column or tubing (e.g., metal sites, contamination).
Peak Fronting: Often due to column overloading (too much sample) or injector issues
Both can result from: Damaged columns, poor mobile phase, or system contamination.
8.How do you choose a mobile phase for HPLC?
Based on Column Type: Use polar solvents for normal phase, and water-organic mixtures (e.g., water + acetonitrile/methanol) for reverse phase.
Analyte Solubility: Must dissolve well in the mobile phase.
pH & Buffering: Important for ionizable compounds.
UV Transparency: For UV detection, solvents should not absorb at detection wavelengths.
Polarity Match: Adjust to control analyte retention (less polar → longer retention in reverse phase).
9.What maintenance is required for an HPLC system?
Regularly Replace Filters: In-line and solvent filters.
Flush System: With clean solvents to avoid blockages or contamination
Check and Replace Pump Seals & Check Valves: To prevent leaks or pressure issues.
Column Care: Flush with appropriate solvents after use, avoid drying.
Calibrate Detector and Check Baseline Noise: For accurate quantification.
10.What is the difference between normal phase and reverse phase HPLC?
Normal Phase HPLC (NP-HPLC):
Stationary Phase: Polar (e.g., silica)
Mobile Phase: Non-polar or less polar (e.g., hexane, chloroform)
Elution Order:
Non-polar compounds elute firs
Polar compounds elute last, because they interact more strongly with the polar stationary phase
Best for: Separating polar compounds or isomers
Example:
If you’re separating alcohols, ketones, or other polar molecules, NP-HPLC may help you distinguish between them based on polarity.
Reverse Phase HPLC (RP-HPLC):
Stationary Phase: Non-polar (e.g., C18 – octadecylsilane)
Mobile Phase: Polar (e.g., water mixed with methanol or acetonitrile
Elution Order:
Polar compounds elute first
Non-polar compounds elute last, as they stick to the non-polar stationary phase longer
Best for: Separating non-polar or moderately polar compounds
Example:
RP-HPLC is the most common type, ideal for biomolecules (like peptides, proteins, and drugs) and organic compounds.
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