GAS – LIQUID CHROMATOGRAPHY

1. GAS – LIQUID CHROMATOGRAPHY BY M. GLORY HEPSIBAH M.PHARM (PH. ANALYSIS) BHARAT COLLEGE OF PHARMACY

2. Contents • Introduction • History • Principle • Advantages of GLC • Physical Components • Instrumentation of GLC • Method to Carry out GLC • Derivatisation of Sample • Different Parameters involved in GLC • Applications of GLC • Conclusion

3. INTRODUCTION : GLC is one of the newest & largely developed technique between a gaseous mobile phase & a Liquid phase immobilized on the surface of an inert solid. In all forms mobile phase is liquid but in GLC, mobile phase is gas such as He and Stationary phase is high Boiling point liquid absorbed onto a solid.

4. History 1903 - Mikhail semenovich Tswelt 1941 – Martin – Liquid – liquid chromatogram 1944 – Martin – Paper chromatography 1947 – Fritz Prior – gas – solid chromatography 1950 - Laid the foundation for GC and Later developed GLC.

5. Principle: The Components of vapourized sample are fractionated as a consequence of being partitioned between a gaseous mobile phase & a liquid stationary phase which is non – volatile liquid. Retention Volume – Retention time X Flow rate.

6. Advantages of GLC Strong separating power Sensitivity is quite high Gives good precision & accuracy Speed of analysis is fact Cost of low & life is long

7. Physical Components Auto Sample Inlets

8. FLOW SCHEME OF GLC

9. Auto samples: Automatic insertion provides better reproducibility and time optimization Classified as : a) Liquid b) Static – head Space by syringe technology c) Dynamic – head space by transfer – line technology d) SPME

10. Gas Chromatograph with a head Space sampler

11. Inlets : Column Inlet provides the means to introduce a sample into a continuous flow of carrier gas. Types of inlets are S/SL (Split/ Split less) injectors On column inlet PVT injectors Gas source inlet or Gas switching Valve P/T (Purge & Trap) System SPME (Solid Phase micro- extraction)

12. Instrumentation of GLC Carrier gas supply Flow regulators & Flow meters Injection devices Columns Temperature control devices Detectors Recorder & Integrators

13. Schematic Diagram of GLC

14. Carrier Gas Choice of Carrier Gas depends on, their efficiency of chromatographic separation different gases used are H2, He, N2& Ar As carrier gas is compressible, gases are stored under high pressure in cylinders

15. Requirements of Carrier Gas Inertness Suitable to the detectors used High Purity Easily available & less risk of explosion Cheap Should give best column performance consistent with the required speed of analysis

16. Flow regulators & flow meters Flow regulators – to deliver the gas with uniform pressure or flow rate. Flow meters – to measure flow rate of carrier gas. Two types of flow meters are a) Rota meter b) Soap bubble meter

17. Soap bubble meter Rota meter

18. SAMPLE INJECTION Sample injection devices

19. Sample injectors used are depends upon their nature i.e., Solid, Liquid & gases. Solid : dissolved in a suitable solvent & then injected through a septum. Liquids – injected through loop or septum devices Gases – injected through valve devices.

20. Columns: Columns are generally made up of glass or stainless steel. Classified as : Based on its use Analytical column b) Preparative column Based on its nature: Packed columns Open tubular or capillary or Golay columns Scot (support coated open tubular columns)

21. A gas chromatography oven, open to show a capillary column

22. Temperature Control Devices Pre – Heaters Thermo – statically controlled devices

23. Two types of Operations : Isothermal Programming Linear Programming

24. Detectors : Heart of an apparatus Detect the difference between a pure carrier gas and an eluted component. Different detectors used are : Kathorometer or thermal conductivity detector (TCD) Flame Ionization Detector Argon Ionization detector Electron capture detector

25. Thermal Conductivity Detector (TCD) or Kathorometer Thermal Conductivity of some gases are : Gases: H2 He N2 Methane Hexane T.C’s : 32.7 33.9 5.2 6.5 3.0

26. Thermal Conductivity Detector (TCD) or Kathorometer

27. Flame ionization detector The whole detectors is enclosed in its own oven which is hotter thatn the column temperature that slops any thing condensing in the detectors

28. Argon Ionization Detectors Argon irradiation Argon + e` collision met stable state of Ar Collision of Substances • In current Detector ionization

29. Electron Capture Detector It is highly sensitive It can even detect nanogram quantities

30. Recorders & Integrators : Recorder : To Record responses obtained after amplification from detectors. Integrators : Improved version of recorders with some data processing capabilities.

31. Method of carrying out GLC Injection of sample How the column works Packing material Column temperature How separation works on the column

32. Derivatisation: Treatment of Sample to improve the process of separation by column or detection by detectors. It is of two types : Pre – column derivatisation Post – Column derivatisation Pre treatment of solid support is also required.

33. Different parameters in GLC Retention time Retention volume Separation Factor Resolution Plate theory HETP Fronting

34. Retention time (Rt) It is the difference in time between the point of injection and appearance of peak maxima Retention Volume (Vl) It is the volume of carrier gas required to elute 50% of the component from the column Vt = Rt x Flow rate.

35. Separation factors : It is ratio of partition coefficient of the two components to be separated S = Kb/Ka = (Tb – To) / (Ta – To) Where, To = Retention time of unretained substance Ka,Kb = Partition coefficients of b & a Ta, Tb = Retention times of b & a

36. Resolution Resolution is a measure of extent of separation of two components & the Base line separation achieved. Rs = 2(Rt1- Rt2)/W1+W2

37. Plate Theory : (functional unit of column) It is an imaginary or hypothetical unit of a column where distribution of solute between stationary phase & mobile phase has attained equilibrium.

38. HETP (Height Equivalent to a Theoritical Plate) If HETP is Less, column efficiency is more. HETP = Length of the Column/ No of theoretical Plates According to Van = Deemter equation HETP = A+B/U + Cu A = Eddy Diffusion B = Longitudinal diffusion C = Effect of Mass transfer U = flow rate Fronting : It is due to saturation of Stationary Phase & Can be avoided by using less quantity of sample.

39. Applications • Qualitative analysis • Checking the purity of the compound • Quantitative Analysis • Detection of steroidal drugs • Monitoring Hazardous Pollutants • Analysis of Commercial Drug Preparations, drug samples, Blood urine, etc. • Analysis of foods • Separation & identification of lipids, proteins flavors, Carbohydrates, colorants etc.

40. Conclusion Gas – Liquid Chromatography is basically a separation process in which the compound of a vaporized sample are separated fractionated as a consequence of partition between a mobile gaseous phase and stationary phase held in column. Hence the components are separated according to their partition coefficients.

41. Thank You