Gas Chromatography by mirola

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Published on March 5, 2014

Author: MirolaAfroze

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GAS CHROMATOGRAPHY By Mirola Afroze, SO, DRiCM, BCSIR

Prepared by Mirola Afroze, SO, DRiCM, BCSIR INTRODUCTION Chromatography is probably the most powerful and versatile technique available to the modern analyst. In a single step process it can separate a mixture into its individual components and simultaneously provide an quantitative estimate of each constituent. Samples may be gaseous, liquid or solid in nature and can range in complexity from a simple blend of two entantiomers to a multi component mixture containing widely differing chemical species. Furthermore, the analysis can be carried out, at one extreme, on a very costly and complex instrument, and at the other, on a simple, inexpensive thin layer plate. Gas Chromatography Page | 2

Prepared by Mirola Afroze, SO, DRiCM, BCSIR DEFINITION OF CHROMATOGRAPHY The term chromatography is difficult to define rigorously because the name has been applied to such a variety of systems and teclmiques. All of these methods, however, have in common the use of a stationary phase and a mobile phase. Components of a mixture are carried through the stationary phase by the flow of a gaseous or liquid mobile phase, separations being based on differences in migration rates among the sample components. Gas Chromatography Page | 3

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Chromatography may be defined as a separation process that is achieved by distributing the components of a mixture between two phases, a stationary phase and a mobile phase. Those components held preferentially in the stationary phase are retained longer in the system than those that are distributed selectively in the mobile phase. As a consequence, solutes are eluted from the system as local concentrations in the mobile phase in the order of their increasing distribution coefficients with respect to the stationary phase; ipso facto a separation is achieved. Gas Chromatography Page | 4

Prepared by Mirola Afroze, SO, DRiCM, BCSIR HISTORY OF CHROMATOGRAPHY The first scientist to recognize chromatography as an efficient method of separation was the Russian botanist Tswett (1), who used a simple form of liquid-solid chromatography to separate a number of plant pigments. The colored bands he produced on the adsorbent bed evoked the term chromatography for this type of separation (color writing). Although color has little to do with modern chromatography, the name has persisted and, despite its irrelevance, is still used for all separation techniques that employs the essential requisites for a chromatographic separation, viz. a mobile phase and a stationary phase. Gas Chromatography Page | 5

Prepared by Mirola Afroze, SO, DRiCM, BCSIR The technique, as described by Tswett was largely ignored for a along time and it was not until the late 1930s and early 1940s that Martin and Synge(2) introduced liquid-liquid chromatography by supporting the stationary phase, in this case water, on silica in a packed bed and used it to separate some acetyl amino acids. In their paper, they recommended replacing the liquid mobile phase by a suitable gas, as the transfer of sample between the two phases would be faster, and thus provide more efficient separations. In this manner, the concept ofgas chromatography was created but again, little notice was taken of the suggestion and it was left to Martin himself and A. T. James to bring the concept to practical reality nearly a decade later Gas Chromatography Page | 6

Prepared by Mirola Afroze, SO, DRiCM, BCSIR BASIC CHROMATOGRAPHIC TERMINOLOGY Chromatograph: Instrument employed for a chromatography. Stationary phase: Phase that stays in place inside the column. Can be a particular solid or gel-based packing (LC) or a highly viscous liquid coated on the inside of the column (GC). Mobile phase: Solvent moving through the column, either a liquid in LC or gas in GC. Eluent: Fluid entering a column. Eluate: Fluid exiting the column. Gas Chromatography Page | 7

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Elution: The process of passing the mobile phase through the column. Chromatogram: Graph showing detector response as a function of a time. Flow rate: How much mobile phase passed / minute (ml/min). Linear velocity: Distance passed by mobile phase per 1min in the column (cm/min). tm: time at which an unretained analyte or mobile phase travels through the column. Adjusted retention time : tr = tr-tm Gas Chromatography Page | 8

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Relative retention (separation factor) α= t’r2/t’ 1 a ratio of relative retention times α > 1,indicates quality of the separation; ↑α = greater separation Capacity factor k = (tr-tm)/tm ↑k = greater retention used to monitor performance of the column α = k2/k1 Retention time tr: Retention time is the time elapsed between the point of injection of the sample and the point of appearance of the peak of the chromatogram. Retention volume: Under a given set of operating conditions a constant volume of gas is required to elute a component from the column. Thus, the volume measured from the point of injection to the projection of the peak of chromatogram is known as retention volume Gas Chromatography Page | 9

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Gas Chromatography Page | 10

Prepared by Mirola Afroze, SO, DRiCM, BCSIR GAS CHROMATOGRAPHY Gas chromatography (GC) is a powerful and widely used tool for the separation, identification and quantitation of components in a mixture. In early 1900s, Gas chromatography was discovered by Mikhail Semenovich Tsvett as a separation technique to separate compounds. Gas Chromatography Page | 11

Prepared by Mirola Afroze, SO, DRiCM, BCSIR In this technique, a sample is converted to the vapor state and a flowing stream of carrier gas (often helium or nitrogen) sweeps the sample into a thermally-controlled column. This technique uses a gas as the mobile phase, and the stationary phase can either be a solid or a non-volatile liquid in which case small inert particles such as diatomaceous earth are coated with the liquid so that a large surface area exists for the solute to equilibrate with. GC is divided into two major categories: • Gas Solid Chromatography (GSC) • Gas Liquid Chromatography (GLC) Gas Chromatography Page | 12

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Gas Solid Chromatography (GSC): GSC utilizes as the stationary phase a glass or metal column filled with powder adsorbent where the adsorbent is a solid of large surface area. In GSC, retention of solutes is dependent largely upon differences in adsorption properties of the solutes for the powder adsorbent as they pass through the stationary phase. Gas Liquid Chromatography (GLC): GLC where the adsorbent is a non-volatile liquid coated on an inert solid support or capillary column in which the inside wall will be coated with nonvolatile liquid. In GLC, the retention of solutes is dependent largely upon the partition co-coefficient of the solutes for the nonvolatile liquid of the stationary phase. In both cases, the mobile phase or the carrier gas is an inert gas which is made to flow at a constant rate through a packed column that is a small diameter tube containing the adsorbent. Gas Chromatography Page | 13

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Figure: Schematic of a Gas Chromatography Gas Chromatography Page | 14

Prepared by Mirola Afroze, SO, DRiCM, BCSIR WORKING PRINCIPLES OF GAS CHROMATOGRAPHY For separation or identification the sample must be either a gas or have an appreciable vapour pressure at the temperature of the column – it does not have to be room temperature. The sample is injected through a self sealing disc (a rubber septum) into a small heated chamber where it is vaporised if necessary. Although the sample must all go into the column as a gas, once it is there the temperature can be below the boiling point of the fractions as long as they have appreciable vapour pressures inside the column. This ensures that all the solutes pass through the column over a reasonable time span. The injector oven is usually 50–100 °C hotter than the start of the column. Gas Chromatography Page | 15

Prepared by Mirola Afroze, SO, DRiCM, BCSIR The sample is then taken through the column by an inert gas (known as the carrier gas) such as helium or nitrogen which must be dry to avoid interference from water molecules. It can be dried by passing it through anhydrous copper(II) sulphate or self indicating silica (silica impregnated with cobalt(II) chloride). Unwanted organic solvent vapours can be removed by passing the gas through activated charcoal. The column is coiled so that it will fit into the thermostatically controlled oven. The temperature of the oven is kept constant for a straightforward separation, but if there are a large number of solutes, or they have similar affinities for the stationary phase relative to the mobile phase, then it is common for the temperature of the column to be increased gradually over a required range. This is done by using computer control, and gives a better Gas Chromatography Page | 16

Prepared by Mirola Afroze, SO, DRiCM, BCSIR separation if solute boiling points are close and a faster separation if some components are relatively involatile. The solutes progress to the end of the column, to a detector. The sample components can be detected by a suitable detector at the exit. With a suitable differential detector at the exit of the column, the signal obtained is proportional to the instantaneous concentration of the dilute component in the binary gas mixture Once a mixture has been separated by GC its components need to be identified. The material to be identified by GC is run through the column so that its retention time (the time for the components to pass through the column) can be determined. Gas Chromatography Page | 17

Prepared by Mirola Afroze, SO, DRiCM, BCSIR INSTRUMENTATION OF GAS CHROMATOGRAPHY The instrumentation of gas chromatography includes i. Carrier gas ii. Sample port iii. Column iv. Detector v.Monitor & Recorder Gas Chromatography Page | 18

Prepared by Mirola Afroze, SO, DRiCM, BCSIR CARRIER GAS The carrier gas plays an important role, and varies in the GC used. Carrier gas must be dry, free of oxygen and chemically inert mobilephase employed in gas chromatography. Typical carrier gases include helium, nitrogen, argon, hydrogen and air. Which gas to use is usually determined by the detector being used, for example, a DID requires helium as the carrier gas. When analyzing gas samples, however, the carrier is sometimes selected based on the sample's matrix, for example, when analyzing a mixture in argon, an argon carrier is preferred, because the argon in the sample does not show up on the chromatogram. Gas Chromatography Page | 19

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Safety and availability can also influence carrier selection. The purity of the carrier gas is also frequently determined by the detector, though the level of sensitivity needed can also play a significant role. Typically, purities of 99.995% or higher are used. Carrier gases that are used in gas chromatography: Helium is generally used because of its Excellent thermal conductivity Inertness Low density Greater flow rate Limitations It is highly expensive Gas Chromatography Page | 20

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Hydrogen Better thermal conductivity Lower density Limitations It may react with unsaturated compounds It creates fire and explosive hazards Nitrogen It is inexpensive Limitations It gives reduced sensitivity Air Air is used as carrier gas only when the oxygen in the air is useful for the detector or separations Gas Chromatography Page | 21

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Gas Chromatography Page | 22

Prepared by Mirola Afroze, SO, DRiCM, BCSIR SAMPLE PORT /SAMPLE INJECTION SYSTEM The sample injection system is very important because very small amount of sample is used. The system must introduce the sample in a reproducible manner and must vaporize instantly so that the sample will enter the column easily. Liquid samples are generally introduced but hypodermic syringe and solid sample must be dissolved in volatile liquids (acetone) for direct introduction Syringe containing sample is introduced through a septum, injection port oven temperature heated to temperatures that ensures fast volatilization of sample, i.e. above the b.p. of all sample components, usually 275º C. Gas Chromatography Page | 23

Prepared by Mirola Afroze, SO, DRiCM, BCSIR 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 Gas Chromatography Page | 24

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Gas Chromatography Page | 25

Prepared by Mirola Afroze, SO, DRiCM, BCSIR SEPARATION COLUMN The columns can be constructed of metal or glass tube. It can be any length from a few centimers to over a hundred meters. It can be coiled, straight or bent. The choice of column depends on the sample and the active measured. The main chemical attribute regarded when choosing a column is the polarity of the mixture, but functional groups can play a large part in column selection. The polarity of the sample must closely match the polarity of the column stationary phase to increase resolution and separation while reducing run time. The separation and run time also depends on the film thickness (of the stationary phase), the column diameter and the column length. Gas Chromatography Page | 26

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Two types of columns are used in GC: Packed columns are usually made of stainless steel or glass and contain a packing of finely divided, inert, solid support material that is coated with a liquid or solid stationary phase. In GSC, columns are packed with size graded adsorbent or porus polymers while in GLC, the packing is prepared by coating the liquid phase over a size graded inert solid support. The nature of the coating material determines what type of materials will be most strongly adsorbed. Thus numerous columns are available that are designed to separate specific types of compounds. Gas Chromatography Page | 27

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Capillary columns have a very small internal diameter, on the order of a few tenths of millimeters, and lengths between 25-60 meters are common. The inner column walls are coated with the active materials (WCOT columns), some columns are quasi solid filled with many parallel micro-pores (PLOT columns) and solid supported coated with stationary liquid phase (SCOT). Most capillary columns are made of fused-silica with a polyimide outer coating. These columns are flexible, so a very long column can be wound into a small coil. Gas Chromatography Page | 28

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Figure: Capillary and Packed Columns Figure: Capillary columns Gas Chromatography Page | 29

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Gas Chromatography Page | 30

Prepared by Mirola Afroze, SO, DRiCM, BCSIR The Stationary Liquid Phase The column is the heart of the gas chromatograph, and the success or failure of a particular separation depends to a large extent on the choice of the stationary liquid phase. A number of points should be considered when selection of a liquid phase is made: i. The solute solvent interaction forces that contribute to the selectivity of the liquid phase. Solution properties such as the polarity, chemical interactions, and hydrogen bonding and other cohesive forces will affect the separation. ii. Prediction of retention behavior Types of solution, their concentration and use of multistage columns, mixed columns determines the prediction of retention times. Gas Chromatography Page | 31

Prepared by Mirola Afroze, SO, DRiCM, BCSIR iii. The temperature limitations of the liquid phases The liquid phase should have low volatility and no tendency to decompose at the operating temperature. iv. The possibility of irreversible reactions on the column Irreversible reactions between the solute and the liquid phase or its impurities or degradation products may limit the usefulness of certain solvents. The solute solvent interaction A. Cohesion forces: Dipole-dipole interactions- results from the interaction between two permanent dipoles. Induction forces- results from an interaction between a permanent dipole in either solute or solvent and induced dipole in the other. Gas Chromatography Page | 32

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Dispersion forces- arise from electric field produced by the very rapidly varying dipoles formed between nuclei and electrons. Dispersion forces are always present in any solute-solvent system. B. Hydrogen bonding: Hydrogen bonding is one type of orientation force that is very important in GC. Hydrogen bonds are intermediate in strength between the strong chemical bonds. It can be classified into five groups-Class (I) consists of compound capable of forming networks of multiple hydrogen bonds, Class (II) composed of compounds containing both a donor atom (ONF) and an active hydrogen atom, Class (III) consists of compounds having donor atoms but no active hydrogen, Class (IV) made up of molecules containing active hydrogen atoms but no donor atoms and Class (V) compounds of this group show no hydrogen bonding at all. Gas Chromatography Page | 33

Prepared by Mirola Afroze, SO, DRiCM, BCSIR C. Polarity In general, polar solutes will be retained to a greater extent as the polarity of the solvent is increased. Conversely, non-polar solutes are retained to a greater extent as the polarity of the solvent is decreased. Thumb rule: Polar solvents dissolve polar compounds and non-polar solvents dissolve non-polar compounds Gas Chromatography Page | 34

Prepared by Mirola Afroze, SO, DRiCM, BCSIR DETECTORS The detector is that device and associated equipment used to sense and measure the small amounts of the components present in the carrier gas stream leaving the chromatographic column. The impulse received from the elute of the column in the form of solute vapor is sensed by the detector. It in turn converts this impulse into an electrical signal proportional to the concentration of the solute in the carrier gas. This signal is then amplified and recorded as a peak in the chromatograph. In all types of detectors, when the carrier gas passing they give a zero signal but when a component is eluted, it is detected and a signal proportional to the concentration of that component is produced. Gas Chromatography Page | 35

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Classification of detectors: Depending upon selectivity detectors are classified as Non-selective detectors: Responds to all compounds except the carrier gas (e.g. TCD, FID, (PID) Selective detectors: Respond to a range of compounds with common physical and chemical properties (e.g. FID, ECD, PID, N and P detector, Sulfur chemiluminiscence) Specific detectors: Respond to a single chemical compound Detectors are also classified as I. Integral and differential II. Destructive (FID, NPD, FPD) nondestructive (e.g. TCD, ECD, PID) Gas Chromatography and Page | 36

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Characteristics of an ideal detector Functional • Sensitivity • Stability • Versatility • Proportionality • Reactivity • Response Time • Signal Recording Non Functional • Simplicity • Cost and availability • Robustness • Safety Gas Chromatography Page | 37

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Detectors that are used in GC • Thermal conductivity detector (TCD) • Flame ionization detector (FID) • Electron capture detector (ECD) • Discharge ionization detector (DID) • Flame photometric detector (FPD) • Hall electrolytic conductivity detector (HECD) • Helium ionization detector (HID) • Nitrogen phosphorous detector (NPD) • Photoionization Detector (PID) • Mass selective detector (MSD) Gas Chromatography Page | 38

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Flame Ionization Detection (FID) The most commonly used detector is the flame ionization detector (FID) it is a general carbon detector. It does not detect compounds that do not contain carbon such as nitrogen(N2), oxygen(O2), or water. The presence of N, O, or S in a carbon compound will tend to decrease the response of the FID. The Carbon atoms (C-C bonds) are burned in a hydrogen flame. The hydrogen can be supplied ether from a cylinder or from an electrolytic hydrogen generator. The hydrogen must be pure to avoid background noise. A charcoal filter is often placed in the hydrogen supply line to remove any organic contaminants. Gas Chromatography Page | 39

Prepared by Mirola Afroze, SO, DRiCM, BCSIR The response of the detector depends on the flow of the hydrogen, air and the makeup gas (if it is used). A certain amount of inert gas is needed for optimum response of the detector. Generally the flow from a capillary is too low so a makeup gas is used to provided the inert gas flow. The makeup gas has other beneficial effects such as stabilizing the detector, prolonging the lifetime of the jet, and purging any unswept areas of the detector. It is also very important to adjust the air and hydrogen gas flows for optimum response. Gas Chromatography Page | 40

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Mechanism: Compounds are burned in a hydrogen-air flame. Carbon containing compounds produce ions that are attracted to the collector. The number of ions hitting the collector is measured and a signal is generated. Selectivity: Compounds with C-H bonds. A poor response for some non-hydrogen containing organics (e.g., hexachlorobenzene) Sensitivity: 0.1-10 ng Linear range: 105-107 Gases: Combustion - hydrogen and air; Makeup - helium or nitrogen Temperature: 250-300°C,and 400-450°C for high temperature analyses Gas Chromatography Page | 41

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Figure Flame ionisation detector Gas Chromatography Page | 42

Prepared by Mirola Afroze, SO, DRiCM, BCSIR The Nitrogen–Phosphorus Detector (NPD) The NPD is fundamentally similar to the flame ionisation detector. They both work by forming ions and subsequently detecting them as a minute electrical current. However, a major difference arises in the way the ions are formed. The eluate (exit gases) of the GC is forced through a jet in the presence of air and hydrogen gas. The mixture passes over the surface of a heated rubidium salt in the form of a bead. The excited rubidium atoms (Rb*) selectively ionise nitrogen and phosphorus. Gas Chromatography Page | 43

Prepared by Mirola Afroze, SO, DRiCM, BCSIR The ions formed allow a small electric current to flow between two charged surfaces which, under different operating conditions, gives a response to either nitrogen or phosphorus containing compounds, or both. To differentiate between nitrogen and phosphorus containing compounds the retention time of each solute in the column is used. Mechanism: Compounds are burned in a plasma surrounding a rubidium bead supplied with hydrogen and air. Nitrogen and phosphorous containing compounds produce ions that are attracted to the collector. The number of ions hitting the collector is measured and a signal is generated. Gas Chromatography Page | 44

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Selectivity: Nitrogen and phosphorous containing compounds Sensitivity: 1-10 pg Linear range: 104-10-6 Gases: Combustion - hydrogen and air; Makeup - helium Temperature: 250-300°C Gas Chromatography Page | 45

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Figure The nitrogen–phosphorus detector Gas Chromatography Page | 46

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Thermal Conductivity Detector (TCD) Measures the changes of thermal conductivity due to the sample. A detector cell contains a heated filament with an applied current. As carrier gas containing solutes passes through the cell, a change in the filament current occurs. The current change is compared against current in a reference cell. The difference is measured and a signal is recorded. When a separated compound elutes from the column , the thermal conductivity of the mixture of carrier gas and compound gas is lowered. The filament in the sample column becomes hotter than the control column. The imbalance between control and sample filament temperature is measured and a signal is recorded. Gas Chromatography Page | 47

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Mechanism: A detector cell contains a heated filament with an applied current. As carrier gas containing solutes passes through the cell, a change in the filament current occurs. The current change is compared against the current in a reference cell. The difference is measured and a signal is generated. Selectivity: All compounds except for the carrier gas Sensitivity: 5-20 ng Linear range: 105-106 Gases: Makeup: same as the carrier gas Temperature: 150-250°C Gas Chromatography Page | 48

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Figure: Thermal conductivity detector (TCD) Gas Chromatography Page | 49

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Electron Capture Detector (ECD) Another useful detector is the electron capture detector (ECD) It is an excellent detector for molecules containing an electronegative group such as Cl or F etc. (or derivitized molecules) It is probably the second most common detector after the FID. It is most often used for the trace measurement of halogen compounds in environmental applications for detecting insecticide and herbicide residues. The ECD uses a radioactive source such as Ni63 which produces Beta particles which react with the carrier gas producing free electrons. These electrons flow to the anode producing an electrical signal . When electrophillic molecules are present, they capture the free electrons, lowering the signal. The amount of lowering is proportional to the amount of analyte present. It is sensitive down to 10-15but the dynamic range is only about 104. Gas Chromatography Page | 50

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Mechanism: Electrons are supplied from a 63Ni foil lining the detector cell. A current is generated in the cell. Electronegative compounds capture electrons resulting in a reduction in the current. The amount of current loss is indirectly measured and a signal is generated. Selectivity: Halogens, nitrates and conjugated carbonyls Sensitivity: 0.1-10 pg (halogenated compounds); 1-100 pg (nitrates); 0.1-1 ng (carbonyls) Linear range: 103-104 Gases: Nitrogen or argon/methane Temperature: 300-400°C Gas Chromatography Page | 51

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Figure: Electron Capture Detector (ECD) Gas Chromatography Page | 52

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Photoionization Detector The photoionization detector (PID) uses a UV lamp (xenon, krypton or argon lamp, depending on the ionization potential of the analytes) to ionize compounds. The ionization produces a current between the two electrodes in the detector. The detector is non-destructive and can be more sensitive than an FID for certain compounds (substituted aromatics and cyclic compounds for example). Mechanism: Compounds eluting into a cell are bombarded with high energy photons emitted from a lamp. Compounds with ionization potentials below the photon energy are ionized. The resulting ions are attracted to an electrode, measured, and a signal is generated. Gas Chromatography Page | 53

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Selectivity: Depends on lamp energy. Usually used for aromatics and olefins (10 eV lamp). Sensitivity: 25-50 pg (aromatics); 50-200 pg (olefins) Linear range: 105-106 Gases: Makeup - same as the carrier gas Temperature: 200°C Figure: Photoionization Detector Gas Chromatography Page | 54

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Atomic Emission Detector One of the newest gas chromatography detectors is the atomic emission detector (AED). The AED is quite expensive compared to other commercially available GC detectors, but can be a powerful alternative. The strength of the AED lies in the detector's ability to simultaneously determine many of the elements in analytes that elute from the column. It uses microwave energy to excite helium molecules (carrier gas) which emit radiation which breaks down molecules to atoms such as S, N, P, Hg, As, etc. These excited molecules emit distinctive wavelengths which can be separated by a grating and sent to the detector (typically a photodiode array) which produces the electrical signal. The atomic emission detector is very sensitive (10-15) and has a dynamic range of 104. Gas Chromatography Page | 55

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Figure: Atomic Emission Detector Gas Chromatography Page | 56

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Thermal Conductivity Detector The thermal conductivity detector (TCD) consists of an electrically-heated wire or thermistor. The temperature of the sensing element depends on the thermal conductivity of the gas flowing around it. Changes in thermal conductivity, such as when organic molecules displace some of the carrier gas, cause a temperature rise in the element which is sensed as a change in resistance. Low molecular weight gases have high conductivities so hydrogen and helium are often used as carrier gases. Nitrogen and argon have similar conductivities to many organic volatiles and are often not used. However if nitrogen is used as a carrier gas, the detector can be used to measure hydrogen or helium. TCD’s are often used to measure lightweight gases or water (compounds for which the FID does not respond). Gas Chromatography Page | 57

Prepared by Mirola Afroze, SO, DRiCM, BCSIR The TCD is not as sensitive as other detectors but it is a universal detector and is nondestructive. However, modern detectors called micro-TCD’s have very small cell volumes, and new electronics that produce much higher sensitivities and wider linear ranges. Due to its increased sensitivity, and the fact that it is a universal non-destructive detector, it is again becoming more popular for certain applications. Figure: Thermal Conductivity Detector Gas Chromatography Page | 58

Prepared by Mirola Afroze, SO, DRiCM, BCSIR MASS SPECTROMETER (MS) The Mass spectrometer is the only detector which does not require higher temperature than is the temperature of GC column. The outlet of capillary column of GC is placed directly to ionization source. The MS employed consists of EI or CI ionization and RF analyzer such as quadrupole or ion trap. Mechanism: The detector is maintained under vacuum. Compounds are bombarded with electrons (EI) or gas molecules (CI). Compounds fragment into characteristic charged ions or fragments. The resulting ions are focused and accelerated into a mass filter. The mass filter selectively allows all ions of a specific mass to pass through to the electron multiplier. All of the ions of the specific mass are detected. The mass filter then allows the next mass to pass through while excluding all Gas Chromatography Page | 59

Prepared by Mirola Afroze, SO, DRiCM, BCSIR others. The mass filter scans stepwise through the designated range of masses several times per second. The total number of ions are counted for each scan. The abundance or number of ions per scan is plotted versus time to obtain the chromatogram (called the TIC). A mass spectrum is obtained for each scan which plots the various ion masses versus their abundance or number. Selectivity: Any compound that produces fragments within the selected mass range. May be an inclusive range of masses (full scan) or only select ions (SIM). Sensitivity: 1-10 ng (full scan); 1-10 pg (SIM) Linear range: 105-106 Gases: None Temperature: 250-300°C (transfer line), 150250°C (source) Gas Chromatography Page | 60

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Gas Chromatography Page | 61

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Table: GC Detectors Gas Chromatography Page | 62

Prepared by Mirola Afroze, SO, DRiCM, BCSIR APPLICATION of GAS CHROMATOGRAPHY Qualitative analysis Generally chromatographic data is presented as a graph of detector response (y-axis) against retention time (x-axis). This provides a spectrum of peaks for a sample representing the analytes present in a sample eluting from the column at different times. Retention time can be used to identify analytes if the method conditions are constant. Also, the pattern of peaks will be constant for a sample under constant conditions and can identify complex mixtures of analytes. Gas Chromatography Page | 63

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Qualitative analysis The area under a peak is proportional to the amount of analyte present. By calculating the area of the peak using the mathematical function of integration, the concentration of an analyte in the original sample can be determined. Concentration can be calculated using a calibration curve created by finding the response for a series of concentrations of analyte, or by determining the response factor of an analyte. Gas Chromatography Page | 64

Prepared by Mirola Afroze, SO, DRiCM, BCSIR High Efficiency of separation The technique has strong separation power and even complex mixture can be resolved into constituents Speed of separation The analysis is completed in a very short time The sensitivity in detection of compounds The sensitivity is quite high and it is micromethod and only a few mg of sample is sufficient for analysis. Accuracy It gives good precision and accuracy Gas Chromatography Page | 65

Prepared by Mirola Afroze, SO, DRiCM, BCSIR Application It has wide application for most groups of pharmaceutical agents Personnel The technique is suitable for routine analysis because the operation of a gas chromatograph and related calculations does not require highly skilled persons. Limitations of Gas Chromatography The primary limitation of gas chromatography is that the sample must be capable of volatilized/vaporiaed without undergoing decompositions. The samples must be thermally stable to prevent degradation when heated. In addition, sample cannot be used for further analysis once separated. Gas Chromatography Page | 66

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