gas chromatography

Gas chromatography:

Gas chromatography K.Jyothsna M.Pharmacy Pharmaceutical analysis . by 1 1

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CONTENTS : INTRODUCTION PRINCIPLE ADVANTAGES INSRUMENTATION DERIVATISATION PARAMETERS USED IN GC 2

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INTRODUCTION : CHROMATOGRAPHY-- Chromatography is a separation of mixture into individual components using a stationary phase and a mobile phase. There are various advanced chromatographic techniques, widely used for the estimation of multicomponent drugs in their formulations like: High Performance Liquid Chromatography High Performance Thin Layer Chromatography Gas Chromatography 3

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Gas chromatography In Gas chromatography, the components of a vapouraised sample are fractionated as a consequence of a partition between a mobile gaseous phase and a stationary phase held in a column. According to the nature of stationary phase, Gas chromatography may be (a). Gas solid chromatography [GSC] (b). Gas liquid chromatography [GLC] Carrier gas column detector Basic chromatographic arrangement 4

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SCHEMATIC DIAGRAM 5

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PRINCIPLE : In GLC, the main principle is partition. Gas is used as mobile phase, liquid which is coated on to a solid support is used as stationary phase. The mixture of components to be separated is converted to vopour and mixed with gaseous mobile phase. The component which is more soluble in stationary phase travels slower and eluted later. The components which is less soluble in stationary phase travels faster and eluted out first. No two components has the same partition co-efficient for a fixed combination of stationary phase , mobile phase. Hence the components are separated according their partition coefficients. 6

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THE CHROMATOGRAPHIC PROCESS - PARTITIONING (gas or liquid) MOBILE PHASE STATIONARY PHASE Sample out Sample in (solid or heavy liquid coated onto a solid or support system) 7

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ADVANTAGES: Both qualitative & quantitative analysis are possible. Time of analysis is short. Instrument is simple & high sensitive. the method is applicable to about 60% of the organic compounds. Separation, which are very difficult or virtually impossible by other technique, can be simple & straight forward with GC. The successful separation of pesticides is a matter of selecting the proper column & conditions. CRITERIA FOR COMPOUNDS TO BE ANALYSED BY GC: Volatility: Thermo stability: 8

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Hardware and Columns 9

INSTRUMENTATION::

INSTRUMENTATION : Mobile Phase : He, Ar, N 2 , H 2 Flow regulators & flow meters: Injection Port: Rubber septum barrier (usually maintained at a higher temperature than the boiling point of the least volatile component in the sample mixture) Column: (fused silica with a thin coating of stationary phase on the inner surface) Oven: Thermostat controlled forced air oven Detector: Data System: recorders & integrators 10

CARRIER GAS::

CARRIER GAS : Carrier gas should be: Inert, high purity, easily available. Low cost, due to large quantities are used Allow the detector or respond in an adequate manner less risk of explosion or hazards, should give best column performance consistent with the required speed & analysis. 11

CHOICE OF CARRIER GAS::

CHOICE OF CARRIER GAS : Advantages Disadvantages Hydrogen Cheap Gives the most time efficient separation Still very efficient at high gas velocities i.e.. 60 cm/ sec Can form an explosive mixture with air Some industries in some countries have regulated AGAINST the use of hydrogen is a reductive gas Helium Very inert, will not react with analytes Gives a very time efficient separation Non flammable Expensive A non-replenishable resource Nitrogen Cheap Very inert, will not react with analytes Non flammable Very slow velocity to achieve good efficiency Narrow range for maximum efficiency 12

FLOW REGULATORS::

FLOW REGULATORS : To regulate pressure& control the gas flow through the seperation column. They are two types : 1. Rotometer 2. soap bubble flow rate ROTOMETER : It is like a burette with a float held on to a spring SOAP BUBBLE METER : It has a glass tube with inlet tube at bottom through gas comes in. In this soap film is formed in path of gas. The time required for the soapfilm to move between two graduates on the burette is then measured & converted to flow rate. 13

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Introduced as a plug of vapor with suitable size Slow injection or oversized samples cause band spreading and poor resolution Micro syringes Injection ports microsyringe Gastightsyringe INJECTION SYSTEM : 14

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Split/Splitless Injector Splitless Injection , (where the split vent is closed) attempts to transfer all of the sample to the column and is used for trace analysis. Split Mode , only a small portion (maybe 1-10% of the sample moves into the column, and the rest is sent to waste. This is used when the analytes are in high concentration and would overload the column. Sample is injected through the septum with a syringe. 15

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COLUMNS : Columns in GC are two types 1) packed column 2) capillary column Packed column (capillary column) Open tubular column 16

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Packed column : Glass or metals 2-3 m long, 2-4 mm I'd. Densely packed with packing materials or solid support coated with thin layer of stationary liquid phase Diatomaceous earth Size: 60-80 mesh (250-170 m) or 80-100 mesh (170-149 m) Better resolution – efficient mass transfer between gas and SP Tubing – fused silica, glass, copper, stainless steel Open tubular column 17

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Liquid phase Solid support coated with liquid phase Porous Adsorbent Porous Layer Open Tubular (PLOT) Wall-coated Open Tubular (WCOT) Support-coated Open Tubular (SCOT) Types of open tubular column : 18

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Type of Column FSOT WCOT SCOT Packed Length (m) 10-100 10-100 10-100 1-6 ID (mm) 0.1-0.3, 0.53* 0.25-0.75 0.5 2-4 Efficiency (Plate/m) 2000-4000 1000-4000 600-1200 500-1000 Sample size (ng) 10-75 10-1000 10-1000 10-10 6 Relative pressure Low Low Low High Relative speed Fast Fast Fast Slow Flexible Yes No No No Chemical inertness Best Poor CHARACTERISTICS 19

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HO-CH 2 -CH 2 -(O-CH 2 -CH 2 ) n -OH polyethylene glycol 20

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The oven Inside here Column 21

Temperature Control:

Temperature Control Isothermal Gradient Instrumentation - Oven 22

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TEMPARATURE 23

Instrumentation - Detectors:

Instrumentation - Detectors Non-Destructive Thermal Conductivity (TCD) Electron Capture (ECD) [551.1] Photo Ionization (PID) [502.2] 24

Instrumentation - Detectors:

Instrumentation - Detectors Destructive Mass Spectral (CI/EI) [625] Flame Ionization (FID) [604] Nitrogen-Phosphorus (NPD) [8141A] Flame Photometric (FPD) [8141A] Electrolytic Conductivity (Hall/ELCD) [502.2] 25

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ELECTRON CAPTURE DETECTOR 26

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The TCD is a nondestructive, concentration sensing detector. A heated filament is cooled by the flow of carrier gas. When the carrier gas is contaminated by sample , the cooling effect of the gas changes. The difference in cooling is used to generate the detector signal. Flow Flow Flow THERMAL CONDUCTIVITY DETECTOR 27

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FLAME IONIZATION DETECTOR 28

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DERIVATISATION: Precolumn derivatisation: To improve the separation of sample by Column.(more volatile&thermostable,l.tailing) Ex: carboxylic acids, sugars, phenols, alcohols, etc can be converted to less polar compounds by using reagents like BSA reagent( Bis trimetinyl Silyl Acetamide reagent). They can also be converted to acetyl derivative or triflouro acetyl derivative. Post column derivatisation: To improve the response shown by detector . The components may not be detected by detector unless derivatisation is done. (online detection technique, where the flow rate is neither stopped nor altered) The components may be converted in such away that their ionisation or affinity towards electrons is increased. Pretreatment of solid support: solid support is to hold the stationary phase liquid as a thin film. 29

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PARAMETERS USED IN GC: Retention time: Difference in time between the point of injection and appearance of peak maxima . Retention volume: The volume of carrier gas required to elute 50%of the component from the column . Vr = retention time * flow rate Separation factor: The ratio of partition coefficient of the two components to be separated . Air peak Peak width Base line Point of injection Retention time Point of injection Air peak Peak 1 peak2 Base line Retention time Rt1 Retention time Rt2 Less separation factor Point of injection Air peak Retention time Rt1 Retention time Rt2 Peak 1 peak2 Base line separation factor More separation factor 30

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Resolution: It is a measure of the extent of separation of two components and the baseline separation achieved . Theoretical plate : it is an imaginary or hypothetical unit of a column where distribution of solute between stationary phase and mobile phase has attained equilibrium. (functional unit plate) Asymmetry factor: Air peak Retention time Rt1 Peak 1 peak2 Base line Retention time Rt2 Point of injection w1 w2 More resolution Air peak Point of injection Retention time Rt2 Retention time Rt1 peak2 Peak 1 Base line Less resolution Point of injection Point of injection Tailing Fronting 31

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THANK YOU 34