K V GOPINATH Pharm Php,cPht Tirumala Tirupati Devasthanams TIRUPATI e-mail:gopinath.karnam@gmail.comIntroduction SSS SSS SSS * Gas chromatography — It is a process of separating component(s) from the given crude drug by using a gaseous mobile phase. * It involves a sample being vaporized and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid, * Two major types + Gas-solid chromatography (stationary phase: solid) * Gas-liquid chromatography (stationary phase: immobilized liquid)of Gas Chromatography has strong separation power and even complex solved into constituents * The sensitivity of the method is quite high * Itgives good precision and accuracy * The analysis is completed in a short time * The cost of instrument is relatively low and its life is generally long * The technique is relatively suitable for routine analysisof Gas chromatography ; (common), N2, H2, Argon * Sample injection port - micro syringe * Columns 2-50 m coiled stainless steel/glass/Teflon * Detectors -Flame ionization (FID) -Thermal conductivity (TCD) -Electron capture (ECD) -Nitrogen-phosphorus -Flame photometric (FPD) -Photo-ionization (PID)schematic diagram of a gas chromatograph ‘Soap-bubble meter Two-stage pressure ‘regulator Column oven* Commonly used gases include nitrogen, helium, argon, and carbon dioxide, * The choice of carrier gas is often dependant upon the type of detector which is used. * The cartier gas system also contains a molecular sieve to remove water and other impurities. - P inlet 10-50 psig -F=25-150 mL/min packed column -F=1-25 mL/min open tubular columnSample injection- Direct Injection 1)Direct injection :into heated port (>T oven) using micro syringe - (i) 1-20 uL Sucker packed column chamber -(ii) 10° uL. capillary columnSample injection- rotary sample valve with sample loop * Split injection: routine method = 0.1-1.% sample to column - remainder to waste * Split less injection: all sample to column - best for quantitative analysis - only for trace analysis, low [sample] On-column injection: -for samples that decompose above boiling Point ( no heated injection port) -column at low temperature to condense sample in narrow band -heating of column starts chromatographyGas Chromatography - Columns * There are two general types of column, packed and capillary (also known as open tubular). * Packed columns contain a finely divided, inert, solid support material ( diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm. * Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of two types; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT). - Wall-coated columns consist of a capillary tube whose walls are coated with liquid stationary phase. In support-coated columns, the inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth, onto which the stationary phase has been adsorbed. - SCOT columns are generally less efficient than WCOT columns, Both types of capillary column are more efficient than packed columns.Gas Chromatography — ~ Common Stationary phases ‘TABLE 27-2 Some Common Stationary Phases for Gas-Liquid Chromatography Polydimettiyl siloxane Polyiphenylmethy Idimetiy!} siloxane (104% phenyl) Poly(phenylmethy!) siloxane (30% phenyl Poly(triftuoropropyldimethy!) siloxane Polyethylene glycot Poly(dicyanoaliyldimethy siloxane ‘OV-1, SE30 OV.3, SES2 ov? ov20 ‘Carbowax 20M ov.218 ‘Common Applications ‘Geneal-purpose nonpolar phase; hydrocarbons: polynuclear aromatics; drugs; steroids: PCBs Fatty acd methyl esters; alkaloids; drugs halogenated compounds Drugs: steroids; pesticides; glycols ‘Chlorinated aromatics; nitroaromatics; alky|-substtuted benzenes Free acids; sloohols; ethers; essential iycols Polyunsaturated faty acids; rosin acids; free acids; alcoholsG C- DETECTORS * There are many detectors which can be used in gas chromatography. Different detectors will give different types of selectivity. * Detectors can be grouped into concentration dependant detectors and mass flow dependant detectors. * The signal from a concentration dependant detector is related to the concentration of solute in the detector, and does not usually destroy the sample Dilution of with make-up gas will lower the detectors response. * Mass flow dependant detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector. The response of a mass flow dependant detector is unaffected by make-up gasStable and reproducible Linear response Wide dynamic range Fast response Simple (reliable) Nondestructive Uniform response to all analytesFlame Ionization Detector (FID) Flame Removable Foaization ‘elector detector oe bolder ngulttor Collector — assembly Hesie fame Grounded ie Inside oven wall 4 Exit end of colomn * It operates by the principle that by change in conductivity of the flame as the compound is burnt. The change in conductivity of the flame does not arise by simple ionization of the compound , it is partial or complete stripping of the compound to give charged hydrogen- deficient polymers or aggregates of carbon of low ionization potential. * Rugged; Sensitive (10%g/s) : Wide dynamic range (10) Signal depends on # C atoms in organic analyte - mass sensitive; not concentration sensitive; Weakly sensitive to carbonyl, amine, alcohol, ‘amine groups; Not sensitive to non-combustibles ~ 14,0, CO, SO,, Nox; DestructiveThermal Conductivity Detector (TCD) * Itis based upon the alteration of the thermal conductivity of the carrier gas. in the presence of an organic compound. The platinum wires are heated electrically and assume equilibrium conditions of temperature and resistance when carrier gas alone passes over them. They are mounted in a whetstone bridge arrangement and when a compound emerges, the thermal conductivity of the gas surrounding wire alters, and hence the temperature and resistance of the wire change with a concomitant out of i balance signal which is amplified and recorded. * Rugged ;Wide dynamic range (10');Nondestructive; Insensitive (10! g/s) - non-uniform —}— Flow outElectron Capture Detector (ECD) The ECD ionizes the carrier gas by means of a radioactive source, The potential across two electrodes is adjusted to collect all the ions and a steady saturation current, is therefore, recorded, Electrons from b-source ionize carrier molecules capture electrons and decrease current ; Simple and reliable ; Sensitive (10"g/s) to electronegative groups (halogens, peroxides) ‘Largely non-destructive ; Insensitive to amines, alcohols and hydrocarbons ; Limited dynamic range (10°)Flame ionization (FID) Mass flow Electron capture (ECD) Concentration Nitrogen-phosphorus Mass ftow Photo-ionization (PID) Concentration Hydrogen and air Reference Makeup Hydrogen and: Hydrogen and air possibly oxrgen Make-up Most organic epds. Universal Hilides, nitrates, nitriles, perowides, anhyurides, ‘organometaltics Nitrogen, phosphorus Sulphur, phospharus, tin, boron, arsenic, getmanium, selenium, chromium Aliphaties, aromaties, ketones, esters, aldchyules, amines, heterueyclics, ‘organosulphurs, some ‘onganometaies Ing 50 fe 10 pe 100 pe 2pe. 10 lo 10Summary of common GC detectors * The effluent from the column is mixed with hydrogen and air, and ignited. * Organic compounds burning in the flame produce ions and electrons which can conduct electricity through the flame. * A large electrical potential is applied at the burner tip, and a collector electrode is located above the flame. The current resulting from the pyrolysis of any organic compounds is measured. FIDs are mass sensitive rather than concentration sensitive; this gives the advantage that changes in mobile phase flow rate do not affect the detectors response. * The FID is a useful general detector for the analysis of organic compounds; it has high sensitivity, a large linear response range, and low noise. It is also robust and easy to use, but unfortunately, it destroys the sampleumn temperature raised, vapor pressure analyte increases, eluted faster * Raise column temperature during separation — temperature programming - separates species with wide range of polarities or vapor pressuresEvaluation * HETP- It is the distance on the column in which equilibrium is attained between the solute in the gas phase and the solute in liquid phase. Larger the number of theretical plates/ smaller the HETP, the more efficient the column is for separation. HETP=Length of column/n ; Where n=number of theretical plates= 16 * x2/y2 * Retention Time: Time in minute from the point of injection to the peak maximum. * Retention Volume: (1) V,=tR *F (retained) (2) VM = tM *F (non- retained) * average volumetric flow rate (mL/min) F can be estimated by measuring flow rate exiting the column using soap bubble meter (some gases dissolving in soap solution) * But measured VR and VM depend on - pressure inside columnApplications of Gas Chromatography * Qualitative Analysis — by comparing the retention time or volume of the sample to the standard / by collecting the individual components as they emerge from the chromatograph and subsequently identifying these compounds by other method. * Quantitative Analysis- area under a single component elution peak is. proportional to the quantity of the detected component/response factor of the detectors. Separation of fatty acids derived from fixed oilsApplications of Gas Chromatography SSS SSS === * Miscellaneous-analysis of foods like carbohydrates, proteins, lipids, vitamins, steroids, drug and pesticides residues, trace elements * Pollutants like formaldehyde, carbon monoxide, benzen, DDT etc * Dairy product analysis- rancidity * Separation and identification of volatile materials, plastics, natural and synthetic polymers, paints, and microbiological samples * Inorganic compound analysis