Gas Liquid Chromotography by Deepak Kr. Verma

ASeminar

On

Delivered By:Deepak Kr. VermaM,Sc,(Agri. Biochemistry)ID No.7483/09E-mail:[email protected]

Under Guidance By:Dr. P. S. Kendurkar (Prof. & Head)

Dr, Madhu Vajpeyi (Assoc. Prof.)

Chandra Shekhar Aazad University Of Agriculture & Technology

Kanpur-208002

Department Of Agricultural Biochemistry

GLC: Principle and Application

INTRODUCTIONINTRODUCTION

Deepak Kr. Verma, 2010

GLC: Principle & Application

Techniques in Biochemistry ABC-504

Chromatography Chroma = a color Greek word Made up by & Graphing = to write the words.

Russian Botanist,Michael Semonavich Tswett (L.T. 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.




DEFINITIONDEFINITIONOfOf

CHROMATOGRACHROMATOGRAPHYPHY




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.




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




MODESMODES

OfOfCHROMATOGRACHROMATOGRA

PHYPHYChromatography separationmay be achieved by

two basic techniques, they are

1-Column chromatography2-Thin-layer or Planer chromatography




PRINCIPLEPRINCIPLE

OFOFCHROMATOGRACHROMATOGRA

PHYPHY




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.




TypesTypesOfOf


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.




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




Gas-solid Chromatography (GSC)

Or Gas liquid chromatography (GLC)




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-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.




G.L.C.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.




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)




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.




1-A column house in an oven that can be temperature programmed2-A Sample Inlet Points3- A Carrier Gas Supply & Control4- A Detector5- An Amplifier 6- Data Record System




Filters/Traps

Air

Hyd

rog

en

Gas C

arrier

Column

gas system

inlet column detector data

system

Data system

Syringe/Sampler

Inlets

Detectors

Regulators

H

RESET




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.




DETECTORDETECTORUsedUsed

InInGLCGLC

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




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 Detector6- Mass Spectrometer Detector

(MSD)




APPLICATIONAPPLICATIONOfOf





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 substances5-In the clinical or medical chemistry, it is

used for the separation of plasma, lipids, blood, alcohol, urinary steroid etc




Lehninger, Principle Of Biochemistry by Nelson David L. & Cox Micheal M,Macmillan, Worth PublicationsPrinciples and Techniques of Biochemistry & Molecular Biologyby Keith Wilson & John WalkerEdition Sixth 2005, Cambridge University PressPractical Biochemistry, Principles and Techniques by Keith Wilson & John WalkerEdition Fifth 2000, Cambridge University PressModern Practices of Gas Chromatography by Grob R.L.Edition 3rd 1995, Wiley Intersciences, New YorkA Biologist Guide to Principles & Techniques of Practical Biochemistryby Wilson K., Gouleling KH (Editors)Edition 3rd 1986, Edword Arnold (Publisher’s) Ltd. LondonFundamental of Biochemistry by Jain J.L., Jain Sanjay, Jain NitinEdition 2008, S.Chand & Company LtdText Books of Plant Physiology and Biochemistry by Verma S.K.Edition 2005, S.Chand & Company LtdBiochemistry by Fatima, Narayan & Meyya R.P.Edition Fourth 2002, Saras Publications

Bibliography

Web Linkswww.biochem.orizona.edu

www.biocore.com

Gas Liquid Chromotography by Deepak Kr. Verma

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