Gas Chromatography

Mobile phase: Inert gas such as N2 or He. Mobile phase: Inert gas such as N2 or He. Stationary phase: May be solid (GSC) or Stationary phase: May be solid (GSC) or Non volatile liquid at operating temperature over inert support (GLC). Non volatile liquid at operating temperature over inert support (GLC). Mixture: Mixture components must be: Mixture: Mixture components must be: 1- Volatile: Volatile material usually have molecular weight not exceed 400 – 500 Dalton. 1- Volatile: Volatile material usually have molecular weight not exceed 400 – 500 Dalton. 2- Thermo stable. 2- Thermo stable.

Instruments: Instruments: 1- Gas Tank. 1- Gas Tank. 2- Flow meter. 2- Flow meter. 3- Injector system for sample application. 3- Injector system for sample application. 4- Column and oven. 4- Column and oven. 5- Detector. 5- Detector. 6- Amplifier and Recorder. 6- Amplifier and Recorder.

stationary phase: stationary phase: GSC: GSC: 1- Silica gel. 1- Silica gel. 2- Charcoal (Activated). 2- Charcoal (Activated). 3- Molecular sieves (synthetic inorganic materials). 3- Molecular sieves (synthetic inorganic materials).

GLC: GLC: Liquid or low – melting solid coating an inert support. e.g. of supports: 1- Chromosorb. 1- Chromosorb. 2- Celite. 2- Celite. 3- Glass beeds. 3- Glass beeds. 4- Teflon 4- Teflon e.g. of liquids or low-melting solids: Each liquid have a maximum operating Temperature. Each liquid have a maximum operating Temperature.  Silicon gum rubber (higher than 250 ˚C).  Paraffin oil (250 ˚C).  Polyethylene glycol (up to 250 ˚C).

Column: Column: 1- Packed Columns: 2-6 mm id and 1-3 meters in length. 2- Capillary Column: 0.2 – 0.5 mm id and meters in length. Some columns may reach up to 300 meters.

Types of capillary columns: 1-Wall Coated Open tubular (WCOT): Used in GLC, the stationary liquid coats the column wall. 2-Support Coated Open tubular (SCOT): The inert support is fused to the column wall by special machine then the stationary liquid coat the support. It is used in GLC. 3-Porous layer Open tubular (PLOT): The stationary solid is bonded to the wall of the column. Not commercially produced. 4-Micro-Packed Columns: The columns are coiled and packed with the desired support with stationary liquid or solid stationary phase under pressure and vibrations.

Capillary Columns give mush better resolution but their sample capacity is much lower than the packed columns (about 1/10 or less).

Operating Temperature: Operating Temperature: Operating Temperature can be looked at as the polarity of mobile phase in case of adsorption chromatography. Very low temp: Tailing of peaks. Very low temp: Tailing of peaks. Very high temp: No separation of peaks. Very high temp: No separation of peaks. Temperature programming: Temperature programming: Allows starting the process at low temperature and gradually increasing the temperature till reach the maximum required temperature. Column bleeding: Column bleeding: Elution of the liquid stationary phase itself if very high temperature is used (exceed maximum operating temperature).

Conditioning of Columns: Conditioning of Columns: Means heating the column before use at a temperature C above the normal working temperature. However, that must not exceed the maximum operating temperature. The mobile phase is allowed to flow during the conditioning but the column outlet must be disconnected from the detector. Conditioning aims to clean the column from any impurities.

Detectors: Detectors: They are used to indicate the presence and measure the amount of the components in the column effluent. Examples of the commonly used detectors are: Examples of the commonly used detectors are: 1- Thermal Conductivity Detector (TCD). 1- Thermal Conductivity Detector (TCD). 2- Flame Ionization Detector (FID). 2- Flame Ionization Detector (FID). 3- Electron Capture Detector (ECD). 3- Electron Capture Detector (ECD).

Sample Introduction Sample Introduction Sample introduction will affect the separation, accuracy and precision of the results. The method of sample application will depends mainly on the nature of the sample. Gaseous samples: Gaseous samples: They are introduced using special valves. They are introduced using special valves. Liquid Samples: Liquid Samples: Include liquids, liquids or solids in suitable solvents. They are introduced by injection via septum seal.

Capillary column have low capacity so we have to apply very small amount of samples. This can be achieved by: Capillary column have low capacity so we have to apply very small amount of samples. This can be achieved by: 1- Dilution of sample with solvent: 1- Dilution of sample with solvent: This will lead to problem due to large amount of solvents that may affect separation and quantitation. This will lead to problem due to large amount of solvents that may affect separation and quantitation. 2- Injection of very small amount of more concentrated solution: 2- Injection of very small amount of more concentrated solution: That leads to increase the error in measurements. That leads to increase the error in measurements. 3- Using modified injectors that allow splitting of samples: 3- Using modified injectors that allow splitting of samples: Only part of the injected sample go to the column and the rest of the sample go to vent. The split ratio to give good results is usually 1: 100. Only part of the injected sample go to the column and the rest of the sample go to vent. The split ratio to give good results is usually 1: 100.

Head Space Sampling: Head Space Sampling: The sample is contained in sealed vial and maintained at constant temperature for about 20 min. so equilibrium will be achieved between gas and liquid states. The vapour phase is then allowed to enter the column.

Derivatization of non volatile samples: Non volatile samples containing acidic hydrogen can be converted into more volatile more thermally stable derivatives as: 1- Silylation: 1- Silylation: This is the most widely used method. Alkylsilyl group will replace the acidic hydrogen. pyridine pyridine R-OH+Cl-SiMe 3 R-O-SiMe 3 + HCl R-OH+Cl-SiMe 3 R-O-SiMe 3 + HCl (TMS) (TMS) Trimethylchlorosilane Trimethylchlorosilane

2- Acylation: 2- Acylation: COCF 3 COCF 3 R-OH + O R-O-COCF 3 R-OH + O R-O-COCF 3 COCF 3 COCF 3 (TFAA) (TFAA) Trifluoroaceticanhydride Trifluoroaceticanhydride 3- Alkylation: 3- Alkylation: RCOOH + BF 3 /MeOH RCOOMe RCOOH + BF 3 /MeOH RCOOMe Boron trifluoride Boron trifluoride

Peak symmetry: Good system must result in a symmetric peaks with narrow base. Asymmetry ratio (As) can be calculated: b As = As = a Where a and b should by measured at10% height of the peak. As for good peak will be

The peak shape also can be predicted by calculation of the Tailing factor (T): W T= T= F 2F Where W and F must be measured at 5% height of the peak. Ideal peak will give Tailing factor = 1. Increasing the value of T means Tailed peak.

Resolution: Resolution: It is the measure of the efficiency of the separation of two components in a mixture. Resolution "R" can be calculated:

1.18(V Rb -V Ra ) 1.18(V Rb -V Ra ) R = R = W ha + W hb W ha + W hb V Ra distance along the base line between the point of Injection and the maxima of a. W ha is the peak width measured at half peak height. Increase in R means better separation and absence of overlapping between peaks.

Identification of peaks Retention time (t R ): Retention time (t R ): It is the time between sample injection and the maximum detection of a substance under specified conditions (temperature, gas flow, Column type…………….etc).

Relative Retention time (α): Since tR is vary due to many factor so it is hard to use it as a method for identification. To overcome this problem we camper t R of the substance with t S (Retention time of a standard substance). t R - t a t R - t a α = α = t S - t a t S - t a t a : Retention time of air peak. The standard is mixed with the substance and injected together so conditions are the same.

Retention Index (I): The identification based on the comparison of the retention time of the substance not only with one standard but with series of hydrocarbons (n-paraffins C n H 2 n+ 2 ). “I” can be calculated by equation or from graphs or by the aid of computer programs. Peak Identification by GC-MS: The peaks elapsed from the GC are subjected to mass spectrometer to determine their molecular weight and fragmentation pattern for the identification purpose.