474 PHG 5

GC
GC is divided into two types 1- Gas solid chromatography when the stationary phase is solid 2- 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

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 microsyringe (10ul-100 ul) Too much sample usually causes overload of the column, leading to lowered resolution of the components 5-10 l for thermal conductivity detector 1 l 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

2- The mobile phase (carrier gas)

Commonly used gases includes nitrogen, helium, argon, hydrogen, and carbon dioxide Choice of carrier gas depend on types of detector Characters of carrier gas 1- very low viscosity low viscosity afford high flow rate hydrogen 2- high thermal conductivity for thermal conductivity detector e. g. hydrogen, helium 3- diffusability Gases with high diffusion in the stationary phases such as H2 and helium cause lowering the column efficiency. Nitrogen and other heavy

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 GC A- 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 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 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 H O Al 2- Carbon black H Used for gas solid chromatography 3- Zeolites these are the original aluminosilicate molecular sieve It is powerful adsorbent Adsorb water and CO2 so it must be the atmosphere protected form

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

H SI O H X

4 Porous 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-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 characters 1- non volatile Bleeding is the volatilization of liquid phase 2- high thermal stability 3-chemically inert 4- 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 (10-7g) 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

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

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 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+ eA + 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

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 linear 3- 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
Temp.
Linear Gradient Stepwise

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

Time

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