474 PHG 5

GC

GC is divided into two types1- Gas – solid chromatography when

the stationary phase is solid2- Gas – liquid chromatography when

the stationary phase is liquid Principle of GC- the sample mixture is introduced as

a liquid volatile at a head of a column filled with the stationary phase

- when the mobile phase is pressed through the column the mixture components distribute themselves according to their partition coefficient between the mobile phase an the stationary phase

- compounds are eluted according to their chemical composition, mole wt, velocity of compound

- detection is carried out by a very sensitive detector and then transferred to recorder to draw the separated peaks

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 the chromatogram

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 be obtained

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

5- very small sample size always required less than 0.1 mg

Retention time is the time required for maximum of the solute peak to reach the detector

Retention volume is the volume of mobile phase required for maximum of the solute peak to reach the detector

Resolution For complete separation of two

chromatographic peaks Resolving power of column relates

to the width of the eluted peaks to the distance between 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. of liquid/ conc. Of solute in total vol. of gas

Instrumentation

1- carrier gas 2- sample inlet system 3- column and oven 4- detector 5-recorder and amplifier

GC

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 overload of the column, leading to lowered resolution of the components

5-10 µl for thermal conductivity detector

1 µl for other high sensitivity detector

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 give multiple 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 pressure is measured by mercury manometer

2 -The mobile phase (carrier gas)

•Commonly used gases includes nitrogen, helium, argon, hydrogen, and carbon dioxideChoice of carrier gas depend on types of detectorCharacters of carrier gas1- very low viscosity low viscosity afford high flow rate hydrogen2- high thermal conductivity for thermal conductivity detector e. g. hydrogen, helium3- diffusabilityGases with high diffusion in the stationary phases such as H2 and helium cause lowering the column efficiency. Nitrogen and other heavy gases are used to give high efficiency

4- ionization Gas as H2 are very high ionization

(requiring least energy to ionization) this is useful when the detectors are of ionization type.

5- compressibility When temp. increase the viscosity of

gas increases and this causes decrease in velocity

6- safety requirement H2 is flammable and He in non

flammable

7- purity of the gas From water so 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 tube before the column

8- chemical inertness

3 -GLC columns Types of columns are used in GCA- packed column 1.5-10 m in length 2-6 mm in diameter Stainless or glass Contains finely divided inert solid support

material e.g. diatomaceous earth or active adsorbent material like alumina

These columns work by adsorption or may be coated with a liquid phase efficiency (30.000 theoretical plates) (15mx0.2mm)

Advantages used for large size of sample without overloading

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

Column performance is measured by1- Column efficiency - is the number of theoretical plates2- measurement of resolution factor for

two closely eluting peaks It is a measure of degree of separation

of adjacent peaks 3- measurement of peak symmetry Peak asymmetry

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

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

1- Alumina: Al2O3 is powerful adsorbent, it can

form hydrogen bond through hydroxyl groups formed on its surface by hydration

2- Carbon black Used for gas solid chromatography 3- Zeolites these are the original

alumino-silicate molecular sieve It is powerful adsorbentAdsorb water and CO2 so it must be the

atmosphere protected form

Al O

H

H X

3- Silica gel OH group is the main site of adsorption

SI O

H

H X

4 – Porous polymer Styrene can polymerise to give

porous beads5- chromosorb-W (white) This is a mixture of sod. carbonate

and diatomites ignited at 900° C to produce iron-sodium-silicate

6- chromosorb –P (pink) Diatomites and clay, heated at 900 °

C afford silicates with excess of iron oxide

II - liquid stationary phase-liquid is directly packed in capillary

column- adsorbed on inert solid support Liquid phase should have the following

characters1- non volatileBleeding is the volatilization of liquid

phase2- high thermal stability3-chemically inert4- having low viscosity

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 at

higher 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

Mechanisms of separation on liquid phase

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

For unknown compounds in volatile oils use two liquid phases for better separation

The compounds of the same boiling point and different chemical structure yield different retention volume

Ability of column to separate these components is called selectivity

- retention volume increase by increase in carbon skeleton

Detectors 1- highly sensitive (10-7μg)

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

6-inert response

7- complete release of the gas

Types of detectors

1- Thermal conductivity type (TCD, Katharometers)

The principle- Heat is conducted away from hot body

at a rate depending on the nature of the surrounding gas

- A metal wire (platinum or tungsten) having high temp. Resistance, it is put in a metal glass or metal tube the wire is heated by a constant electric current

- the temp. difference is established between the other wall and the hot wire , the difference in temp. depends on the thermal conductivity 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 by means of a wheatstone bridge

- To reduce the effects of gas rate to minimum another wire is used as a reference and is exposed only to pure carrier gas

- The signal given by the detector is then magnified by an amplifier

- Gas used hydrogen, helium

2 -Flame ionization detector

Carrier gas leaving the column usually nitrogen is mixed with hydrogen and then burned in the detector at a jet above which a collector electrode is placed

- when hydrogen alone is burned water and small electric current are produced

- however when the organic compounds are burned in the hydrogen flame, ions and CO2 are formed and the current increases according to the sum of the resulting electrons and negative ions

H2 +O2 + RHCO2 +H2O + (ions)- current flow

The lack of response to water and air gases 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

3 -Electron capture detector The ECD is a particular selective

detector that utilizes the ability of electrophilic compounds to absorb free electrons

- an ionisable carrier gas passes into an ionization chamber contains electrodes across which is applied a potential (10 to 50 volt) just sufficient to collect free electrons that are produced as a result of ionization of the carrier gas molecules by the β particles

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

When the electrophilic component enters the chamber it captures the electrons to form stable negative molecular ion or charged particles

AB+ e- (AB)- + or - energy AB+ e- A + B- + or - energy The net result is decrease in electric

current measured by detector which displayed as a peak in the chromatogram

It is used for halogenated compounds, phosphorous compounds

Used for pesticides of the chlorinated compounds

Not used for hydrocarbons (no electrophilicity)

4- Alkali flame-ionization detector It makes use of the fact that the electric

current increases when certain hetero-compounds such as organic-phosphorous , halogen or nitrogen compounds (alkaloids) are burned in a flame which is being seeded with alkali metal salt as rubidium, cesium or potassium

It is used for alkaloids and pesticides 5- Flame photometric detector It is used for compounds which contain

phosphorous or sulpher these compounds when burned in hydrogen flame give chemiluminescent species as HPO and S2 which give characterstic emission 526 and 394 nm wavelength respectively.

Programming of GLC 1- isothermal technique Fixed temp.2- programmed temp techniques - linear and non linear3- programmed pressure In this case the temp is fixed and the

change in the flow rate Advantages low temp can be used , it is suitable

for unstable compounds -pressure is more controlled than

temp.

Temp. programming

Stepwise

Linear

Time

Temp.

0 5` 10` 15` 20` 25` 30`

Isocratic

Gradient

Sample preparation for GC

It must be volatile Non volatile compounds can be

converted into volatile by 1- methylation 2- acetylation 3- silylation

Qualitative and quantitative analysis Qualitative analysis by

determination of retention time Quantitative analysis by measuring

the area under the peak

Applications of GC

1- free fatty acid 2- fatty alcohols 3- fatty acids methyl ester 4- amines 5- hydrocarbons 6- carbohydrates 7- Flavours and fragrances 8- pesticides determination 9- food analysis 10- drug analysis 11- environmental analysis