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GC

GC is divided into two types

1- Gas solid chromatography when thestationary phase is solid

2- Gas liquid chromatography when thestationary phase is liquid

Principle of GC

- the sample mixture is introduced as a liquidvolatile at a head of a column filled with thestationary phase

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- when the mobile phase is pressed throughthe column the mixture componentsdistribute themselves according to theirpartition coefficient between the mobilephase an the stationary phase

- compounds are eluted according to theirchemical composition, mole wt, velocity of

compound - detection is carried out by a very sensitive

detector and then transferred to recorder todraw the separated peaks

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Component which prefer the mobile phase

will appear first in the chromatogram while

that dissolve in the stationary phase better

than mobile gas will appear later on thechromatogram

Advantages of GC

1- capable of resolving more than 150 mixed

compounds in one experiment

2- time of analysis of any sample takes less

than five minutes

3- qualitative and quantitative analysis could

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4- can be used for analysis of most of thenatural products in the volatile form

5- very small sample size always required

less than 0.1 mg

Retention time is the time required formaximum of the solute peak to reach the

detector Retention volume is the volume of mobile

phase required for maximum of the solutepeak to reach the detector

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Resolution

For complete separation of twochromatographic peaks

Resolving power of column relates to thewidth of the eluted peaks to the distancebetween maxima

Partition coefficient

Is conc. Of solute in unit vol. of liquid/ conc.Of solute in unit vol. of gas

Capacity : conc. Of solute in total vol. ofliquid/ conc. Of solute in total vol. of gas

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Instrumentation

1- carrier gas

2- sample inlet system

3- column and oven

4- detector

5-recorder and amplifier

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GC

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1- Sample introduction

system

Sample is liquid or solid dissolved in ether

or chloroform is introduced by a micro-

syringe (10ul-100 ul)

Too much sample usually causes overloadof the column, leading to lowered

resolution of the components

5-10 l for thermal conductivity detector 1 l for other high sensitivity detector

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The sample is injected within 0.1 second

longer time e.g. one second causes several

feeding (multiple feeding) to the column

with the same sample which will givemultiple appearance of the same peak

Inlet temperature

The injection port has heater this heater to

gives 5 -10 over the temperature of the

column

The flow rate of the gas is 10 -100 ml gas

/min it is measured by flow meter while the

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4- ionization

Gas as H2 are very high ionization(requiring least energy to ionization) this isuseful when the detectors are of ionizationtype.

5- compressibility

When temp. increase the viscosity of gas

increases and this causes decrease invelocity

6- safety requirement

H2 is flammable and He in non flammable

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7- purity of the gas

From waterso dried by molecular sieve

tube in front of the gas and just before the

column From dust and other impurities using filter

before the column

From oxygen by using copper oxide tubebefore the column

8- chemical inertness

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3- GLC columns

Types of columns are used in GC

A- packed column

1.5-10 m in length

2-6 mm in diameter

Stainless or glass

Contains finely divided inert solid support materiale.g. diatomaceous earth or active adsorbent materiallike alumina

These columns work by adsorption or may be coatedwith a liquid phase efficiency (30.000 theoreticalplates) (15mx0.2mm)

Advantages used for large size of sample withoutoverloading

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B- capillary column (Golay column)

The liquid stationary phase is applied to

the inner wall of capillary instead of being

adsorbed on a support

200.000 plates

0.2-1 mm diameter

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Column performance is measured by

1- Column efficiency

- is the number of theoretical plates

2- measurement of resolution factor for two closely

eluting peaks

It is a measure of degree of separation of

adjacent peaks3- measurement of peak symmetry

Peak asymmetry

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Factors affecting the column performance

1- particle size

2- high affinity of solute in carrier gas

3- reduction in sample size

4- velocity of the carrier gas

5- column diameter

6- column temperature

7- column length

8- type of stationary phase

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4- The stationary phase

1- Solid stationary phase Characters:

-Chemically inert - porous- High thermal conductivity

- Good mechanical resistance

-No tendency for solute

Types: charcoal, silica gel, alumina,molecular sieves, porous polymer,chromosorb-w, chromosorb-p

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1- Alumina:

Al2O3 is powerful adsorbent, it can form

hydrogen bond through hydroxyl groups

formed on its surface by hydration2- Carbon black

Used for gas solid chromatography

3- Zeolites these are the original alumino-silicate molecular sieve

It is powerful adsorbent

Adsorb water and CO2

so it must be the

atmosphere protected form

Al O

H

H X

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3- Silica gel OH group is the main site of

adsorption

SI O

H

H X

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4Porous polymer

Styrene can polymerise to give porous

beads

5- chromosorb-W (white)

This is a mixture of sod. carbonate and

diatomites ignited at 900 C to produce

iron-sodium-silicate6- chromosorbP (pink)

Diatomites and clay, heated at 900 C afford

silicates with excess of iron oxide

http://en.wikipedia.org/wiki/File:Styrene.svghttp://en.wikipedia.org/wiki/File:Styrene.svg

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liquid stationary phase-II

-liquid is directly packed in capillary column

- adsorbed on inert solid support

Liquid phase should have the following

characters1- non volatile

Bleeding is the volatilization of liquid phase

2- high thermal stability3-chemically inert

4- having low viscosity

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Temperature choice and

control

The control of temp. Is very important as

high temp. Cause bleeding

It affects column separation

The column operate more efficiently athigher temp. There is much less tailing, few

poorly shaped peaks because of volatilities

of the components At high temperature the bleeding or

volatilization of liquid stationary phase will

destroy the column

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Mechanisms of separation on

liquid phase

Generally, polar components are held for longtime on polar liquid phase (like dissolves like)

For unknown compounds in volatile oils usetwo liquid phases for better separation

The compounds of the same boiling point anddifferent chemical structure yield differentretention volume

Ability of column to separate thesecomponents is called selectivity

- retention volume increase by increase incarbon skeleton

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Detectors

1- highly sensitive (10-7g)2- fast responsibility to any change in eluted

components

3- linearity to concentration

The intensity of the response or the reading

should bear some linear qualitative relationship

to the concentration of a component of the

effluent

4- simplicity

5- stability

Withstand the large temp. Range during the

operation

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Types of detectors

1- Thermal conductivity type (TCD,

Katharometers)

The principle

- Heat is conducted away from hot body at arate depending on the nature of the

surrounding gas

- A metal wire (platinum or tungsten) havinghigh temp. Resistance, it is put in a metal

glass or metal tube the wire is heated by a

constant electric current

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- the temp. difference is established betweenthe other wall and the hot wire , thedifference in temp. depends on the thermalconductivity of the gas

- if the gas conduct much heat from the wire

the temp. of the wire will lower conductivity

- This temp. Difference is measured bymeans of a wheatstone bridge

- To reduce the effects of gas rate tominimum another wire is used as a

reference and is exposed only to purecarrier gas

- The signal given by the detector is thenmagnified by an amplifier

- Gas used h dro en helium

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The lack of response to water and airgases is useful in

- There will be no need to remove them

from the gas-The detector is sensitive to organic

compounds while insensitive to water ,

insecticide and inorganic compounds

including gases as He, N2, CO2, NH2

alkaloids peptides amino acids

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3- Electron capture detector

The ECD is a particular selective detector thatutilizes the ability of electrophilic compoundsto absorb free electrons

- an ionisable carrier gas passes into an

ionization chamber contains electrodes acrosswhich is applied a potential (10 to 50 volt) justsufficient to collect free electrons that areproduced as a result of ionization of thecarrier gas molecules by the particles

The migration of electrons to the anodeproduces a steady current of about 10-8 to 10-9A

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Sample preparation for GC

It must be volatile

Non volatile compounds can be converted

into volatile by

1- methylation

2- acetylation

3- silylation

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