HPLC Interview Questions & Answers

HPLC

Md. Ahasan Alam

BSc & MSc in Applied Chemistry & Chemical
Engineering.
Executive, ACI Consumer Brands
+8801703504343
[email protected]
1. What is chromatography?
Chromatography is a laboratory technique used
to separate, identify, and analyze the components
of a mixture based on how they interact with
mobile & stationary phases.

2. How many types of chromatography are there?

Chromatography can be classified based on the
type of mobile phase, stationary phase & support
medium.
i) Liquid chromatography
ii) Gas chromatography Md. Ahasan Alam
BSc & MSc in Applied Chemistry &
iii) Supercritical fluid chromatography Chemical Engineering.
Executive, ACI Consumer Brands
+8801703504343
[email protected]
3. What is HPLC?
HPLC (High-Performance Liquid Chromatography / High-Pressure Liquid
Chromatography ) is an advanced analytical technique for separating, identifying, and
quantifying components in a liquid mixture. Due to its precision, sensitivity, and
efficiency, it is widely used in industries such as pharmaceuticals, food testing, and
environmental analysis.

4. Why is it called High-pressure liquid chromatography?

High pressure is applied by using pump to force the mobile phase through the
chromatographic column.

Md. Ahasan Alam

BSc & MSc in Applied Chemistry &
Chemical Engineering.
Executive, ACI Consumer Brands
+8801703504343
[email protected]
5. What is the principle of HPLC?
The principle of HPLC is based on separating components in a mixture due to their
different interactions with a stationary phase and a mobile phase. Components with a
stronger affinity for the stationary phase move more slowly, while those with a stronger
affinity for the mobile phase move faster, resulting in separation.

6. What is the normal phase?

Stationary Phase: Polar (silica gel).
Mobile Phase: Non-polar solvents (hexane, chloroform). Md. Ahasan Alam
BSc & MSc in Applied Chemistry &
Chemical Engineering.
Executive, ACI Consumer Brands
+8801703504343
[email protected]
7. What is the reverse phase chromatography?
Stationary Phase: Non-polar solvents (C18 column coated with alkyl
chains).
Mobile Phase: Polar (water, methanol, acetonitrile).

9. What are the components of HPLC?

There are 6 components in HPLC:
1) Reservoir
2) Pump
3) Injector
4) Column
5) Detector Md. Ahasan Alam
BSc & MSc in Applied Chemistry &
6) Data System Chemical Engineering.
Executive, ACI Consumer Brands
+8801703504343
[email protected]
10. What is column?
An HPLC column is a critical component of the
HPLC system where the actual separation of
sample components takes place. It contains the
stationary phase, which interacts with the
components of the sample to separate them based
on their physical and chemical properties.
11. What are the common types of detectors used in HPLC?
➢ UV detector
➢ PDA detector
➢ Fluorescence detector
➢ Conductivity detector
➢ Refractive index detector Md. Ahasan Alam

➢ Light scattering detector BSc & MSc in Applied Chemistry &

Chemical Engineering.
Executive, ACI Consumer Brands
+8801703504343
[email protected]
12. What is gradient run?
A gradient run in HPLC refers to a method in which the composition of the mobile phase
is gradually changed during the separation process. This contrasts with an isocratic run,
where the mobile phase composition remains constant throughout.

13. What is isocratic run?

An isocratic run in HPLC refers to a separation method where the composition of the
mobile phase remains constant throughout the entire analysis.

14. What is linear gradient?

A linear gradient in HPLC is a method where the composition of the mobile phase
changes uniformly over time.
Md. Ahasan Alam
BSc & MSc in Applied Chemistry &
Chemical Engineering.
Executive, ACI Consumer Brands
+8801703504343
[email protected]
15. What is linear gradient?
A linear gradient in HPLC refers to a method where the mobile phase composition
changes at a constant rate over time.

16. Tell about retention time.

Retention time (tᵣ) in chromatography is the amount of time it takes for a specific
compound in a sample to pass through the chromatography system and be
detected by the detector.

17. Which material is commonly used in the stationary phase?

Silica gel

Md. Ahasan Alam

BSc & MSc in Applied Chemistry &
Chemical Engineering.
Executive, ACI Consumer Brands
+8801703504343
[email protected]
18. Why is silica gel used in the stationary phase?
It is an inert material & does not react with the mobile phase.

19. What is calibration?

Calibration is the process of making sure a measuring instrument is accurate.

20. Which standard do we use for HPLC calibration?

Caffein standard used for HPLC calibration.

Md. Ahasan Alam

BSc & MSc in Applied Chemistry &
Chemical Engineering.
Executive, ACI Consumer Brands
+8801703504343
[email protected]
20. Why is the caffeine standard used for calibration?
✓ It is very stable
✓ It is durable
✓ Readily available in market
✓ It shows 2 maxima and 1 minima at 205, 273 nm respectively.
21. What are the parameters in HPLC calibration?
✓ Lamp intensity
✓ Pump flow rate accuracy
✓ Gradient composition
✓ Column oven temperature & thermostat
✓ Injection volume accuracy
✓ Detector linearity
✓ Detector wavelength accuracy Md. Ahasan Alam
BSc & MSc in Applied Chemistry &
✓ Carryover test Chemical Engineering.
Executive, ACI Consumer Brands
+8801703504343
[email protected]
22. Difference between C8 & C18 column?
➢ C8 has 8 carbon atoms chain & C18 has 18 carbon atom chain.
➢ C18 is more hydrophobic than C8 due to that C18 has a longer retention time and
C8 has a short retention time.

23. What is the baseline in HPLC?

The baseline is a detector response to the mobile phase.

Md. Ahasan Alam

BSc & MSc in Applied Chemistry &
Chemical Engineering.
Executive, ACI Consumer Brands
+8801703504343
[email protected]
Thank you