Chromatographic Methods

Basic of HPLC & GC

Dr. Hitesh Pawar

DBT-ICT Centre for Energy Biosciences
Institute of Chemical Technology, Mumbai

Applied Chemistry Chromatographic Methods (HPLC and GC) Institute of Chemical Technology Mumbai
Applied Chemistry Chromatographic Methods (HPLC and GC) Institute of Chemical Technology Mumbai
 High Performance Liquid Chromatography
(HPLC) System

Applied Chemistry Chromatographic Methods (HPLC and GC) Institute of Chemical Technology Mumbai
 Why HPLC….?
✓ Smaller particle sizes [<10 microns] are required to improve separation power.
✓ However, smaller particles have greater resistance to flow, so higher pressures are needed to
create the desired solvent flow rate.
✓ Pumps and columns designed to withstand high pressure are necessary.
✓ When moderate to high pressure is used to flow the solvent through the chromatographic column, the
technique is called HPLC.
✓ The acronym HPLC, coined by the late Prof. Csaba Horváth for his 1970 Pittcon paper, originally
indicated the fact that high pressure was used to generate the flow required for liquid
chromatography in packed columns.

Thus, HPLC offers a powerful analytical technique for identification, quantification and
HPLC purification of mixtures with high performance and high speed as compared to traditional
column chromatography because of forcibly pumped mobile phase
Applied Chemistry Chromatographic Methods (HPLC and GC) Institute of Chemical Technology Mumbai
 Principle of HPLC
▪ The separation principle of HPLC is based on the distribution of the analyte (sample) between a mobile phase
(eluent) and a stationary phase (packing material of the column).
• Depending on the chemical structure of the analyte, the molecules are retarded while passing the stationary
phase.
• The specific intermolecular interactions between the molecules of a sample and the packing material define their
time “on-column”. Hence, different constituents of a sample are eluted at different times. Thereby, the separation of
the sample ingredients is achieved.
• The Principle of HPLC is based on Van Deemter Equation which relates the efficiency of chromatographic column
to the particle size of column, molecular diffusion and thickness of stationary
H = A + B/u + C*ʋ
Where, H or (HETP)= Height equivalent of theoretical plate
A= eddy diffusion term
B= molecular diffusion term
C= Rate of mass transfer coefficient
ʋ = Represents flow rate
Separation of components of compound into pure individual components takes place between two phases
▪ Stationary Phase - The substance on which adsorption of analyte takes place
▪ Mobile Phase - Solvent which carries the analyte
Applied Chemistry Chromatographic Methods (HPLC and GC) Institute of Chemical Technology Mumbai
 Types of HPLC….?
1.

Based on Mode of Separation.

2.

Based on Principle of Separation.

3.

Based on Elution Technique.

4.

Based on Scale of Operation.

5.

Based on Type of Analysis.

Applied Chemistry Chromatographic Methods (HPLC and GC) Institute of Chemical Technology Mumbai
 Based on Mode of Separation
a) Normal Phase HPLC: This method separates analytes on the basis of polarity. NP-HPLC uses polar stationary
phase and non-polar mobile phase.
- Therefore, the stationary phase is usually Silica and typical mobile phases are hexane, methylene chloride,
chloroform, diethyl ether, and mixtures of these.
b) Reverse Phase HPLC :The stationary phase is non polar (hydrophobic) in nature, such as C18, C8, Cyano
columns used.
- While the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile

Normal Phase Reverse Phase HPLC

• Non-polar compounds elute faster than polar • Polar compounds elute faster than non-polar
compounds compounds
• Polarity of mobile phase increases, elution time • Polarity of mobile phase increases, elution time
increases decreases
• Cannot be reused/reproducible • Can be reused/reproducible
• Mobile phase are non-polar such as isopropyl • Mobile phase are polar compounds such as water,
alcohol (IPA), hexane, dichloromethane, chloroform, methanol, acetonitrile
ethyl ether
Applied Chemistry Chromatographic Methods (HPLC and GC) Institute of Chemical Technology Mumbai
 Based on Principle of Separation
(i) Adsorption chromatography:
• In which stationary phase is an adsorbent.
• The compounds separated based on their affinity
towards stationary phase.
• More affinity-slow elution
• Less affinity-fast elution
(ii) Ion-Exchange HPLC:
• Ion-exchange chromatography separates molecules based
on their respective charged groups.
• Ion-exchange chromatography retains analyte molecules
on the column based on coulombic (ionic) interactions.
• Essentially, molecules undergo electrostatic interactions
with opposite charges on the stationary phase matrix.
Applied Chemistry Chromatographic Methods (HPLC and GC) Institute of Chemical Technology Mumbai
 iii) Partition chromatography:
• A process of separation of solutes utilizing the partition
of the solutes between two liquid phases
• Namely the original solvent and the film of solvent on
the adsorption column.

iv) Size-Exclusion HPLC:

• In which the stationary phase is a gel having a closely
controlled pore size.
• Molecules are separated based on molecular size, shape
smaller molecules being temporarily retained in the pores
and mainly Agarose , Dextran used as stationary phase

Applied Chemistry Chromatographic Methods (HPLC and GC) Institute of Chemical Technology Mumbai
 Based on Elution Technique
During the chromatographic experiment, a pump can deliver a constant mobile phase
composition(isocratic) or an increasing mobile phase composition (gradient).

i) Isocratic Elution ii ) Gradient Elution

• Delivers constant mobile phase composition • Delivers variable mobile phase composition

• Solvent must be pre-mixed • In this mobile phase is programmed to change

• Lowest cost pump in composition during elution time

• Best for simple preparation. • Best for complex preparations.

Applied Chemistry Chromatographic Methods (HPLC and GC) Institute of Chemical Technology Mumbai
 Based on Scale of Operation

i) Analytical Chromatography : No recovery of individual components of

substance

ii) Preparative Chromatography : Individual components of substance can be

recovered, used for small scale experiments, process development and
optimization by packing column with resin.

iii)Process Chromatography : used for commercial scale compounds

purification and separation on large scale.

Applied Chemistry Chromatographic Methods (HPLC and GC) Institute of Chemical Technology Mumbai
 Based on Type of Analysis

i) Qualitative Analysis:

Determine the quality of sample

ii) Quantitative Analysis:

Determine the quantity(concentration) of sample.

Applied Chemistry Chromatographic Methods (HPLC and GC) Institute of Chemical Technology Mumbai
 Basic Instrumentation
I. Solvent Reservoir, Mixing system & Degassing system
II. High Pressure Pump
III. Sample Injector.
IV. Column
V. Detectors
VI. Data Recording System.

Components of the HPLC

A - Mobile Phase Reservoir
B - Pump
C - Sample Injector
D - Column (Stationary Phase)
E - Detector
F - Data Analyser
G - Waste Container
Applied Chemistry Chromatographic Methods (HPLC and GC) Institute of Chemical Technology Mumbai
 Applications of HPLC
▪ Analysis of drugs
▪ Analysis of synthetic polymers
▪ Analysis of pollutants in environmental analytics
▪ Determination of drugs in biological matrices
▪ Isolation of valuable products
▪ Product purity and quality control of industrial products and fine chemicals
▪ Separation and purification of biopolymers such as enzymes or nucleic acids
▪ Water purification
▪ Pre-concentration of trace components
▪ Ligand-exchange chromatography
▪ Ion-exchange chromatography of proteins
▪ High-pH anion-exchange chromatography of carbohydrates and oligosaccharides

Applied Chemistry Chromatographic Methods (HPLC and GC) Institute of Chemical Technology Mumbai
 Gas Chromatography (GC)
Gas chromatography (GC): is a common analytical technique used to separate and analyze volatile and semi-
volatile compounds in a mixture. GC is a popular analytical technique as it combines exceptional resolving power
with speed and sensitivity. It is widely applied in many industries, including environmental, petroleum, chemical,
food and beverage, and pharmaceutical.

▪ Like other forms of chromatography, GC involves a stationary phase and a mobile phase.
▪ In GC, the mobile phase is an inert gas, usually helium or nitrogen, and the stationary phase is either a solid
adsorbent, termed gas-solid chromatography (GSC), or a liquid adsorbed onto an inert support, termed gas-liquid
chromatography (GLC, or just GC).

Applied Chemistry Chromatographic Methods (HPLC and GC) Institute of Chemical Technology Mumbai