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 1

CONTENTS Introduction History The Distribution Coefficient Instrumentation Properties of Sample to be Analyzed in GLC Advantages and Disadvantages Applications 2

 Gas chromatography is a technique used for separation of volatile substances, or substances that can be made volatile, from one another in a gaseous mixture at high temperatures without decomposition 3 GAS CHROMATOGRAPHY

 Gas solid chromatography (GSC) The stationary phase, in this case, is a solid like silica or alumina. It is the affinity of solutes towards adsorption onto the stationary phase which determines, in part, the retention time.  Gas Liquid chromatography (GLC) A liquid phase immobilizes on surface of an inert solid. TYPES OF GAS CHROMATOGRAPHY

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

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 HISTORY…

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GAS LIQUID CHROMATOGRAPHY GLC is a chromatographic technique in which the stationary phase is a liquid while the mobile phase is a suitable inert carrier gas. GLC is the most widely used technique for separation of volatile species GLC uses a gaseous mobile phase to transport components through either packed columns or hollow capillary columns containing a polymeric liquid stationary phase. 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 ] 9

10 A carrier gas should have the following properties: 1.Highly pure (> 99.9%) 2.Inert so that no reaction with stationary phase can take place 3.A higher density carrier gas is preferred. 4.Compatible with the detector 5.A cheap and available carrier gas is an advantage. CARRIER GAS

INSTRUMENTATION Gas inlet Pneumatic controls Injector Column Column Oven Detector Data System 11

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 12 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. 13 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. 14 INSTRUMENTATION…

THE SPLIT / SPLITLESS INJECTOR 15

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 16 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. 17 INSTRUMENTATION…

18 General properties of a good liquid stationary phase are:  Very low volatility liquids that have good absolute and differential solubility for analyte are required for successful separations  Thermal stability and chemically inert  Nonvolatile liquids to assure minimum bleeding of the stationary phase STATIONARY PHASE

THEORETICAL PLATE MODEL OF COLUMN (Efficiency of a Column) Theoretical plate number (N N = 16 (t R /w b ) 2 HETP = L/N 19

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. 20 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 21 INSTRUMENTATION…

Detector choices include:  Flame Ionization (FID)  Electron Capture (ECD)  Flame Photometric (FPD)  Nitrogen Phosphorous (NPD)  Thermal Conductivity (TCD)  Mass Spectrometer (MS) 22 INSTRUMENTATION…

Hydrogen Air Capillary tube (column) Platinum jet Collector Sintered disk Teflon insulating ring Flame Gas outlet Coaxial cable to Analog to Digital converter Ions Why do we need hydrogen? FLAME IONIZATION DETECTOR

GENERAL FEATURES OF GC DETECTORS DetectorsApplicationsSensitivity Linear dynamic range FIDMost organic compounds 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) pgup to 10 5 NPDNitrogen and phosphorus compounds pgup to 10 6 FPD Sulphur and phosphorus compounds 10 pg S, 1 pg P up to 10 3 MSNearly all substancesExcellent 24

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. 25 INSTRUMENTATION…

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. 26 PROPERTIES OF SAMPLE

27 Three temperature zones should be adjusted before a GC separation can be done:  Temperature of Injector  Temperature of Column  Temperature of Detector TEMPERATURE ZONES

28  The injector temperature should be such that fast evaporation of all sample components is achieved. The temperature of the injector is always more than that of the column.  In general, the column temperature is selected to compromise between the length of the analysis and the level of separation  The detector temperature should be kept at some level so as to prevent any solute condensation in the vicinity of the detector body. TEMPERATURE ZONES

ADVANTAGES Fast analysis High efficiency – leading to high resolution Sensitive detectors (ppb) Non-destructive – enabling coupling to Mass Spectrometers (MS) High quantitative accuracy Requires small samples (<1 mL) Reliable techniques Well established with extensive literature and applications 29

Limited to volatile samples Not suitable for samples that degrade at elevated temperatures (thermally labile) Requires MS detector for analyte structural elucidation (characterization) Most non-MS detectors are destructive 30 DISADVANTAGES

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. 31 APPLICATIONS

Food Industry 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. 32 APPLICATIONS…

Petrochemical GLC applications include natural gas analysis or refineries, gasoline characterization and fraction quantitation, aromatics in benzene, etc. 33 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. 34 APPLICATIONS…

Environmental Environmental GLC applications include detection of pollutants such as pesticides, fungicides, herbicides, purgeable aromatics, etc. 35 APPLICATIONS…

Clinical Microbiology Demonstration of microbial metabolites in cultures GLC combined with mass spectrometry has been applied to study of fecal specimens and cerebrospinal fluids 36 APPLICATIONS…

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