2. GAS LIQUID CHROMATOGRAPHY Abroo Arshad (06) Ghulam Fatima (05) Zara Qadeer (03) M.Sc. (Hons). Food and Nutrition Semester – I INSTITUTE OF FOOD SCIENCE AND NUTRTION (IFSN) UNIVERSITY OF SARGODHA, SARGODHA - PAKISTAN 2

3. CONTENTS Introduction History The Distribution Coefficient Instrumentation Properties of Sample to be Analyzed in GLC Advantages and Disadvantages Applications 3

4. INTRODUCTION GLC is a chromatographic technique in which the mobile phase is an inert gas (He, H­ 2 or N 2 ) and the stationary phase is liquid rather than solid. GLC uses a gaseous mobile phase to transport components through either packed columns or hollow capillary columns containing a polymeric liquid stationary phase. 4

5. HISTORY Development of GC as an analytical technique was pioneered by Martin and Synge 1941 They predicted that the mobile phase need not be a liquid but may be a vapor The concept of gas chromatography was envisioned in the early forties but unfortunately little notice was taken of the suggestion 5

6. HISTORY It was left to Martin himself and his co-worker A. T. James to bring the concept to practical reality some years later in 1951 They demonstrated the technique by separating and quantitatively determining the twelve components of a C1-C5 fatty acid mixture The importance of GC was recognized almost immediately by petrochemical laboratories, which faced the challenge of analyzing complex hydrocarbon mixtures 6

7. HISTORY 7

8. THE DISTRIBUTION COEFFICIENT (K c ) The ‘distribution coefficient’ measures the tendency of an analyte to be attracted to the stationary phase Large Kc values lead to longer retention analyte times The value of Kc can be controlled by the chemical nature of the stationary phase and the column temperature K c = [C s ] / [C m ] 8

9. INSTRUMENTATION Gas inlet Pneumatic controls Injector Column Column Oven Detector Data System 9

10. INSTRUMENTATION Gas Inlet Gas is fed from cylinders through supply piping to the instrument Required gases might include: Carrier - (H 2, He, N 2 ) Make-up gas - (H 2, He, N 2 ) Detector Fuel Gas - (H 2 & Air, Ar or Ar & CH 4, N 2 ) depending on the detector 10

11. INSTRUMENTATION Pneumatic Controls The gas supply is regulated to the correct pressure (or flow) and then fed to the required part of the instrument Modern GLC instruments have Electronic Pneumatic pressure controllers – older instruments may have manual pressure control via regulators. 11

12. INSTRUMENTATION Injector Here the sample is volatilized and the resulting gas entrained into the carrier stream entering the GLC column. Many inlet types exist including:  Split / Split less  Programmed Thermal Vaporizing (PTV)  Cool-on-column (COC) etc. 12

13. THE SPLIT / SPLITLESS INJECTOR 13

14. INSTRUMENTATION Column In GC, retention of analyte molecules occurs due to stronger interactions with the stationary phase than the mobile phase. The interaction types can be divided into three broad categories:  Dispersive  Dipole  Hydrogen bonding 14

15. INSTRUMENTATION Columns vary in length and internal diameter depending on the application type and can be either packed or capillary  Packed columns (typical dimension 1.5 m x 4 mm) are packed with a solid support coated with immobilized liquid stationary phase material  Capillary columns (typical dimension 30 m x 0.32 mm x 0.1 mm film thickness) are long hollow silica tubes with the inside wall of the column coated with immobilized liquid stationary phase material of various film thickness. 15

16. THEORETICAL PLATE MODEL OF COLUMN (Efficiency of a Column) Theoretical plate number (N N = 16 (t R /w b ) 2 HETP = L/N 16

17. INSTRUMENTATION Column Oven Temperature in GLC is controlled via a heated oven The injector and detector connections are also contained in the GLC oven In temperature programmed GLC the oven temperature is increased according to the temperature program during the analysis. 17

18. INSTRUMENTATION Detector The detector responds to a physicochemical property of the analyte, amplifies this response and generates an electronic signal for the data system to produce a chromatogram Many different detector types exist and the choice is based mainly on application, analyte chemistry and required sensitivity – also on whether quantitative or qualitative data is required 18

19. INSTRUMENTATION Detector choices include:  Flame Ionization (FID)  Electron Capture (ECD)  Flame Photometric (FPD)  Nitrogen Phosphorous (NPD)  Thermal Conductivity (TCD)  Mass Spectrometer (MS) 19

20. General Features of GC Detectors DetectorsApplicationsSensitivity Linear dynamic range FIDMost organic compounds10-100 pg Excellent. up to 10 7 TCDGeneral, responds to all substances5-100 ngup to 10 7 ECD All substances that have affinity to capture electrons (halides, nitrates, nitriles, peroxides, anhydrides, organometallics) 0.05-1.00 pgup to 10 5 NPDNitrogen and phosphorus compounds0.1-10 pgup to 10 6 FPD Sulphur and phosphorus compounds 10 pg S, 1 pg P up to 10 3 MSNearly all substancesExcellent 20

21. INSTRUMENTATION Data System The data system receives the analogue signal from the detector and digitizes it to form the record of the chromatographic separation known as the ‘Chromatogram’. The data system can also be used to perform various quantitative and qualitative operations on the chromatogram – assisting with sample identification and quantitation. 21

22. PROPERTIES OF SAMPLE Samples analyzed by GLC must be volatile (have a significant vapor pressure below 250 °C) Derivatization to increase volatility is possible but can be cumbersome and introduces possible quantitative errors Most GLC analytes are under 500 Da Molecular Weight for volatility purposes Highly polar analytes may be less volatile than suspected when dissolved in a polar solvent or in the presence of other polar species due to intermolecular forces such as hydrogen bonding. 22

23. ADVANTAGES Fast analysis High efficiency – leading to high resolution Sensitive detectors (ppb) Non-destructive – enabling coupling to Mass Spectrometers (MS) High quantitative accuracy (<1% RSD typical) Requires small samples (<1 mL) Rugged and reliable techniques Well established with extensive literature and applications 23

24. DISADVANTAGES Limited to volatile samples Not suitable for samples that degrade at elevated temperatures (thermally labile) Not suited to preparative chromatography Requires MS detector for analyte structural elucidation (characterization) Most non-MS detectors are destructive 24

25. APPLICATIONS Pharmaceutical In the pharmaceutical industry GLC is used to analyze residual solvents in both raw materials (drug substance) and finished products (drug product). Biopharmaceutical applications include urine drug screens for barbiturates and underivatized drugs and for ethylene oxide in sterilized products such as sutures. 25

26. APPLICATIONS Food/Flavors/Fragrances The food industry uses GLC for a wide variety of applications including quality testing and solvents testing. The Flavors and Fragrances industries use GLC for quality testing and fingerprinting of fragrances for characterization. 26

27. APPLICATIONS Petrochemical GLC applications include natural gas analysis or refineries, gasoline characterization and fraction quantitation, aromatics in benzene, etc. Geochemical applications include mapping of oil reserves and tracing of reservoirs etc. 27

28. APPLICATIONS Chemical/Industrial Chemical / Industrial uses include determination of product content, determination of purity, monitoring production processes, etc. GLCs are used to detect organic acids, alcohols, amines, esters, and solvents. 28

29. APPLICATIONS Environmental Environmental GLC applications include detection of pollutants such as pesticides, fungicides, herbicides, purgeable aromatics, etc. Industrial environmental protection applications include stack and waste emissions as well as water discharges. 29

30. APPLICATIONS Clinical Microbiology Demonstration of microbial metabolites in cultures (studies of short chain fatty acids, lactic and succinic acids are the bases of taxonomy of obligate anaerobes on the genus level) GLC combined with mass spectrometry has been applied to study of sera, fecal specimens and cerebrospinal fluids 30

31. 31