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HPLC in Chromatography –


HPLC – High Performance Liquid Chromatography


1. Introduction

High Performance Liquid Chromatography (HPLC) is an advanced analytical technique used for the separation, identification, and quantification of components present in a mixture. It is based on the differential distribution of analytes between a stationary phase and a liquid mobile phase under high pressure.
HPLC is widely used in biochemistry, biotechnology, pharmaceuticals, food analysis, environmental studies, and clinical diagnostics.


2. Principle of HPLC

The principle of HPLC is based on partition, adsorption, ion-exchange, or size-exclusion mechanisms, depending on the type of column used.
A liquid mobile phase is pumped at high pressure through a column packed with fine stationary phase particles
Sample components interact differently with the stationary phase
Components with stronger interaction elute slower
Components with weaker interaction elute faster
Separated components are detected and recorded as chromatographic peaks


3. Instrumentation of HPLC
The major components of an HPLC system are:
3.1 Solvent Reservoir
Contains mobile phase solvents (water, methanol, acetonitrile, buffers)
Must be HPLC grade
Degassed to remove air bubbles
3.2 Pump
Delivers mobile phase at constant flow rate
Operates at high pressure (up to 6000 psi)
Types:
Reciprocating pump
Syringe pump
Constant pressure pump
3.3 Injector
Introduces sample into the mobile phase
Types:
Manual injector (Rheodyne)
Auto-sampler
Typical sample volume: 5–20 µL
3.4 Column
Heart of the HPLC system
Packed with small particles (3–10 µm)
Common columns:
C18 (ODS)
C8
Silica
Column length: 10–30 cm
3.5 Detector
Detects eluted components
Converts concentration into electrical signals
Common detectors:
UV–Visible detector
Photodiode Array (PDA)
Fluorescence detector
Refractive Index detector
Mass spectrometry (LC-MS)


3.6 Data System

Records chromatograms
Measures peak area, height, and retention time
Used for quantitative analysis
4. Mobile Phase
Liquid that carries the sample through the column
Must be:
Pure
Degassed
Compatible with detector
Common solvents:
Water
Methanol
Acetonitrile
Buffer solutions
Elution Types
Isocratic elution – constant solvent composition
Gradient elution – solvent composition changes during separation
5. Types of HPLC
5.1 Normal Phase HPLC
Polar stationary phase (silica)
Non-polar mobile phase
Polar compounds elute last
5.2 Reverse Phase HPLC (RP-HPLC)
Non-polar stationary phase (C18)
Polar mobile phase
Most widely used type
5.3 Ion Exchange HPLC
Separation based on charge
Used for proteins, amino acids
5.4 Size Exclusion HPLC
Separation based on molecular size
Large molecules elute first
5.5 Affinity HPLC
Highly specific interactions
Used for biomolecules
6. Factors Affecting HPLC Separation
Mobile phase composition
Flow rate
Column temperature
Particle size
pH of mobile phase
Sample concentration
7. Applications of HPLC
7.1 Pharmaceutical Industry
Drug analysis and quality control
Determination of drug purity
Stability testing
7.2 Biotechnology & Biochemistry
Protein and peptide separation
Nucleic acid analysis
Metabolite profiling
7.3 Clinical Applications
Detection of hormones
Analysis of blood and urine samples
7.4 Food Industry
Detection of additives
Vitamin analysis
Pesticide residue analysis
7.5 Environmental Analysis
Detection of pollutants
Water and soil analysis
8. Advantages of HPLC
High resolution
High sensitivity
Rapid analysis
Accurate quantification
Suitable for non-volatile compounds
9. Limitations of HPLC
High cost of equipment
Requires skilled operation
Uses large volumes of organic solvents
Maintenance is expensive
10. Conclusion
HPLC is a powerful and versatile analytical technique with wide applications in research, industry, and diagnostics. Due to its high accuracy, reproducibility, and sensitivity, it has become an essential tool in modern analytical laboratories.

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