PART-1

"HPLC Interview Guide: Frequently Asked Questions

& Practical Insights"

Prepared by – Amit U Vernekar Purushan |

Senior Executive, Quality Control
>>>>>>>
 "HPLC Interview Guide: Frequently Asked Questions
& Practical Insights"
1) What is High-Performance Liquid Chromatography (HPLC),
and how does it differ from other chromatography methods?

Solution: HPLC is a high-pressure liquid chromatography

technique used to separate, identify, and quantify
components in a mixture. Unlike gas chromatography (GC),
which uses a gas mobile phase, HPLC uses a liquid mobile
phase under high pressure to pass through a stationary
phase.

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https://www.thermofisher.com/blog/analyteguru/tired-of-hplc-
downtime-time-for-a-technology-boost/

Prepared by – Amit U Vernekar Purushan | Senior Executive, Quality

Control >>>>>>>
 "HPLC Interview Guide: Frequently Asked Questions
& Practical Insights"

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

Solution:

1.Reservoir: Stores the mobile phase (solvent).

2. Pump: Delivers the mobile phase at high pressure and
consistent flow.
3. Injector: Introduces the sample into the system.
4. Column: Packed with stationary phase for separation.
5. Detector: Identifies and quantifies the analytes.
6. Data System: Processes chromatographic data and
generates reports.

Figure 1: Block Diagram of HPLC System“

Picture Courtesy: [https://microbenotes.com/high-performance-
liquid-chromatography-hplc/]"

Prepared by – Amit U Vernekar Purushan | Senior Executive, Quality

Control >>>>>>>
 "HPLC Interview Guide: Frequently Asked Questions
& Practical Insights"
3) Who discovered HPLC?

Solution:
The principle of chromatography was first introduced by
Mikhail Tsvet (1903) for separating plant pigments. The
modern concept of HPLC was developed in the 1960s by
Csaba Horváth and S. R. Lipsky, who introduced high-pressure
liquid chromatography for efficient separation in analytical
chemistry.

4)What are the commonly used types of HPLC columns, and

how do they differ in application?
Solution: Commonly used HPLC columns include:
•C18 (Octadecylsilane): Non-polar; used for hydrophobic
compounds.
•C8 (Octylsilane): Moderately non-polar; faster elution than
C18.
•Phenyl: Moderately polar; used for aromatic and π-π
interacting compounds.
•Silica: Highly polar; used for normal-phase chromatography.
•HILIC: Highly polar; retains polar compounds.
•SEC (Size Exclusion Chromatography): Separates based on
molecular size.

Prepared by – Amit U Vernekar Purushan | Senior Executive, Quality

Control >>>>>>>
 "HPLC Interview Guide: Frequently Asked Questions
& Practical Insights"
5)What is the principle of HPLC?
Solution Part-1:
HPLC operates based on the differential interaction of analytes
between a stationary phase (column) and a mobile phase
(solvent system). Separation occurs due to differences in
properties like polarity, hydrophobicity, charge, and molecular
size.
•Retention Mechanism: Compounds with stronger interactions
(higher affinity) for the stationary phase will elute later, while
those more soluble in the mobile phase elute faster.

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Performance-Liquid-Chromatography-(HPLC)

Prepared by – Amit U Vernekar Purushan | Senior Executive, Quality

Control >>>>>>>
 "HPLC Interview Guide: Frequently Asked Questions
& Practical Insights"
Solution Part-2:

Eddy Diffusion Theory in HPLC

Eddy diffusion, also called the multipath effect, describes how analyte
molecules travel through multiple paths within the packed stationary
phase, causing peak broadening in the chromatogram.
Key Factors in Eddy Diffusion:
•The stationary phase is made of small particles packed tightly in the
column.
•When the mobile phase pushes analytes through the column, molecules
follow different paths due to the uneven packing.
•This variation in flow paths causes some molecules to reach the detector
slightly earlier or later, broadening the peaks.
H=L/N
Whereas H is the height equivalent to the plate, also represented by HETP
(height equivalent to theoretical plate)
Here is the derivation of the Van Deemter equation where the equation
relates to a height equivalent to a theoretical plate (HETP) of a
chromatographic column to several flows and is associated with several
kinetic parameters that cause peak broadening.
HETP = A + B/u (CS + Cm). u
Where
A = Eddy diffusion parameter
B = diffusion coefficient of eluting particles in the longitudinal direction
C = Resistance to mass transfer coefficient of the analyte between the
stationary and mobile phase
u = speed

Prepared by – Amit U Vernekar Purushan | Senior Executive, Quality

Control >>>>>>>
 "HPLC Interview Guide: Frequently Asked Questions
& Practical Insights"
6)What are the main types of HPLC?
Solutions:

•Normal Phase HPLC (NP-HPLC) – Polar stationary phase, non-

polar mobile phase
•Reverse Phase HPLC (RP-HPLC) – Non-polar stationary phase,
polar mobile phase
•Ion Exchange HPLC (IEX-HPLC) – Separation based on ionic charge
•Size Exclusion HPLC (SEC-HPLC) – Separation based on molecular
size
7)What is retention time in HPLC?
Solutions : The time taken for an analyte to pass from injection
to detection, measured in minutes.

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Prepared by – Amit U Vernekar Purushan | Senior Executive, Quality

Control >>>>>>>
 "HPLC Interview Guide: Frequently Asked Questions
& Practical Insights"
8): What types of detectors are used in HPLC?
Solutions:
•UV-Vis Detector – Most common, detects based on absorbance
•PDA Detector – Detects full UV spectrum
•Fluorescence Detector – High sensitivity for fluorescent
compounds
•Refractive Index Detector (RID) – Used for non-UV absorbing
compounds

9)What is the difference between isocratic and gradient elution?

Solutions:

•Isocratic Elution – Mobile phase composition remains constant

•Gradient Elution – Mobile phase composition changes over time,
improving separation of complex samples

Prepared by – Amit U Vernekar Purushan | Senior Executive, Quality

Control >>>>>>>
 "HPLC Interview Guide: Frequently Asked Questions
& Practical Insights"
10)What causes high back pressure in HPLC?
Solutions:
Clogged column or frit
Mobile phase contamination
Particulate matter in the system

11)Why Do Peaks Tail or Split in HPLC

Poor Column Packing
Strong Sample-Solvent Interactions
Overloading of the Column
Column Deterioration (Column Goes Off)

Prepared by – Amit U Vernekar Purushan | Senior Executive, Quality

Control >>>>>>>
 Thank you !!
Looking Forward to your thoughts
and Discussions.

“Be unreasonable,follow your

Passion”-Sudha Murty
Let’s Connect
Email Id :[email protected]

Prepared by – Amit U Vernekar Purushan | Senior

Executive, Quality Control >>>>>>>