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A

Seminar
On

GLC: Principle and Application

DELIVERED BY: UNDER GUIDANCE BY:


Deepak Kr. Verma Dr. P. S. Kendurkar (Prof. & Head)
M,Sc,(Agri. Biochemistry) Dr, Madhu Vajpeyi (Assoc. Prof.)
ID No.7483/09
E-mail:rajadkv@rediffmail.com

Department Of Agricultural Biochemistry


CHANDRA SHEKHAR AAZAD UNIVERSITY OF
AGRICULTURE & TECHNOLOGY
KANPUR-
KANPUR-208002
GLC: Principle & Application

INTRODUCTION

Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504


GLC: Principle & Application
Chromatography
Chroma = a color
Greek word Made up by &
Graphing = to write the words.
Russian Botanist,
Botanist,
Michael Semonavich Tswett (L.T. 1872-
1872-1919)
In 1906
Tswett had told to the chromatography that it is the procedure
where mixture of different colored pigments (such as chlorophyll &
xanthophylls) is separated from each other, means originally the
separated colored substance from the extract of the leaf. By this
method Tswett showed that the chlorophyll is the made up of two
pigments and it becomes more clear when the petroleum ether was
pure in the packed column.
Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504
GLC: Principle & Application

DEFINITION
Of
CHROMATOGRAPHY

Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504


GLC: Principle & Application

Chromatography = Biochemical Techniques = Component of a Mixture


Through their different migration.
Separated
The technique in which the components of the mixture
are caused to migrate at different rates through on apparatus
which involve in the equilibrium of the compounds between a
stationary and a mobile phase.
A process by which the component of a mixture are
separated by different movement through two phase system in
which the movement is effected by a mobile phase through a
stationary phase.
Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504
GLC: Principle & Application

The technique of separation of substances according to


their partition coefficient or distribution coefficient (Kd)
below (i.e. their relation solubility) in two impressible
phase.
Here,
(Kd) = Concentration of phase A
Concentration of phase B

Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504


GLC: Principle & Application

MODES
Of
CHROMATOGRAPHY
Chromatography separation
may be achieved by
two basic techniques,
they are
1-Column chromatography
2-Thin-layer or Planer chromatography

Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504


GLC: Principle & Application

PRINCIPLE
OF
CHROMATOGRAPHY

Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504


GLC: Principle & Application

1:-Different rates of migration of the component


of a mixture on the solvent system.
2:-Different rates of adsorption of the
components in the stationary phase.

Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504


GLC: Principle & Application

Types
Of
CHROMATOGRAPHY
In all the chromatography techniques the phenomena
of the adsorption or partition is involved. In the
adsorption, the binding of compounds to the surface of
solid phase (stationary phase) takes place, where as in the
partition the relatively solubility of the compound in two
phase, result in the partition of the compounds in two
phase.
Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504
GLC: Principle & Application

1:-Adsorption chromatography (Compt. b/w S &L/G)


1:--Adsorption Column Chromatography (Compt. b/w S&G)
2:--Thin Layer Chromatography (Compt. b/w S&L)
3:--Ion-exchange Chromatography (Compt. b/w S&L)
2:-Partition chromatography (Compt. b/w L &L/G)
1:-Paper Chromatography (Compt. b/w L&L)
2:--Thin Layer Chromatography (Compt. b/w L&L)
3:--Gel Filtration (Compt. b/w L&L)
4:--Gas Liquid Chromatography
Or (Compt. b/w L&G)
Vapor Phase Chromatography

Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504


GLC: Principle & Application

Gas-solid Chromatography (GSC)


Or
Gas liquid chromatography (GLC)

Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504


GLC: Principle & Application

1-In GSC and GLC, the separation is carried out in a tubular


column made up of a glass, metal or telfon.
2-In GSC, the adsorbent are the first, first finely divided into
the fine size graded powder and then fill in the column
while that in GLC, the column wall is either coated with
non-
non-volatile liquid.
3-GSC is based upon the selectivity adsorption on a solid while
that in GLC is based upon the partition between the gas
and immobile (stationary phase) liquid phase.
4-The basic component of the GSC and GLC type of
chromatography are the same and shown in the figure.
Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504
GLC: Principle & Application
G.L.C.
A gas liquid chromatography is the analysis of the organic
metabolites has been used widely for the analysis of the fat, fatty
acids, pesticides and several volatile substances. Extensive studies
have been made possible on the organic volatile in food and
fermented beverage where flavor and aroma are of almost
importance.
In general consideration of GLC techniques, the column is
packed with a pure inert solid coated with a thin layer of a
nonvolatile liquid as the stationary phase. The components of the
mixture are separated by being partitioned b/w this phase and the
mobile phase.

Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504


GLC: Principle & Application

GLC exploits differences in the partition coefficient


between a stationary liquid phase and a mobile gas phase of
volatilized analyst as they are carried out through the column
by the mobile gas phase means the technique is based upon
the partitioning compounds between a liquid and a gas
phase.
Therefore, it’s used to confine the analyst that are
volatile but thermally stable, ’The partition coefficient are inversely
proportional to the volatility of the analyst’.
The temperature of the column is raised to 50-300 ºC to
facilitate analyst volatilization. There is optimum flow arte of
the mobile phase for maximum column efficiency (minimum
plate height, H)
Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504
GLC: Principle & Application

The stationary phase is consists of…


►A high boiling points liquid materials
such as silicon, grease or wax
The materials is either coated onto the
internal wall of the column or supported on
inert granular solid and packed into the
column.

Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504


GLC: Principle & Application

1-A column house in an oven that can be


temperature programmed
2-A Sample Inlet Points
3- A Carrier Gas Supply & Control
4- A Detector
5- An Amplifier
6- Data Record System

Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504


GLC: Principle & Application

Filters/Traps Data system


H

RESET

Regulators Syringe/Sampler

Inlets

Detectors  gas
system
Gas Carrier
Hydrogen
Air

Column
 inlet
 column
 detector
 data
system

Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504


GLC: Principle & Application

There are two type of column used in the GLC, they are …
1- Packed Conventional Column
2- Capillary (open tubular) Column

Packed Conventional Column- These consist of coil glass or stainless


steel column 1-2 mm long and 2-4 mm internal diameter. They are
packed with stationary phase coated onto on inert silica support.
Capillary (open tubular) Column-These are made by high quality
fazed quartz and or 10-100 m and 0.1-1.0 mm diameter.
This type of column are further divided in two type, they are
1-Wall-coated Open Tubular (WCOT)
2-Support-coated Open Tubular (SCOT)
also called as Porous Layer Open Tubular (PLOT)
The operating temperature for all type of column must be
compatible with stationary phase chosen for use column temperature is
controlled to ±0.1°C.

Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504


GLC: Principle & Application

DETECTOR
Used
In
GLC
Several types of detectors are in common used in the
conjunction with gas-liquid chromatography system…

Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504


GLC: Principle & Application

1- Flame Ionization Detector (FID)


2- Nitrogen Phosphorus Detector
(NPD)
3- Electron Capture Detector (NPD)
4- Flame Photometric Detector (ECD)
5- Rapid Scanning Furriers
Transforms Infrared Detector
6- Mass Spectrometer Detector (MSD)
Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504
GLC: Principle & Application

APPLICATION
Of
CHROMATOGRAPHY

Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504


GLC: Principle & Application

1-GLC is very useful for the analysis of complex mixture.


2-GLC is widely used for the qualitative and quantitative analysis
of the large number of a low polarity compounds be4couse it has
high sensitivity, and speed o0f resolutions.
3-Analytically, very powerful technique when coupled to mass
spectrometry
4-Use for the separation of volatile substances
5-In the clinical or medical chemistry, it is used for the separation
of plasma, lipids, blood, alcohol, urinary steroid etc

Deepak Kr. Verma, 2010 Techniques in Biochemistry ABC-504


Bibliography
Lehninger, Principle Of Biochemistry by Nelson David L. & Cox Micheal M,
Macmillan, Worth Publications
Principles and Techniques of Biochemistry & Molecular Biology
by Keith Wilson & John Walker
Edition Sixth 2005, Cambridge University Press
Practical Biochemistry, Principles and Techniques by Keith Wilson & John
Walker
Edition Fifth 2000, Cambridge University Press
Modern Practices of Gas Chromatography by Grob R.L.
Edition 3rd 1995, Wiley Intersciences, New York
A Biologist Guide to Principles & Techniques of Practical Biochemistry
by Wilson K., Gouleling KH (Editors)
Edition 3rd 1986, Edword Arnold (Publisher’s) Ltd. London
Fundamental of Biochemistry by Jain J.L., Jain Sanjay, Jain Nitin
Edition 2008, S.Chand & Company Ltd
Text Books of Plant Physiology and Biochemistry by Verma S.K.
Edition 2005, S.Chand & Company Ltd
Biochemistry by Fatima, Narayan & Meyya R.P.
Edition Fourth 2002, Saras Publications
Web Links
www.biochem.orizona.edu
www.biocore.com