Gas Chromatography ( GC ) for Pharmacy students

Gas chromatography
Introduction, types, principles, sample preparation, derivatization techniques, instrumentation, applications.

GC consists of:
GSC (gas solid chromatography)
GLC (gas liquid chromatography)

Gas → Mobile phase
Solid / Liquid → Solid phase

1. DISCOVER . LEARN . EMPOWER Mr. Yunes Alsayadi Assistant Professor of Pharmaceutical Analysis, Chandigarh University, Mohali, Punjab UNIVERSITY INSTITUTE OF PHARMA SCIENCES Pharm D GC 1

2. GAS CHROMATOGRAPHY Separation of gaseous & volatile substances , s Simple & efficient in regard to separation . GC consists of : GSC (gas solid chromatography) GLC (gas liquid chromatography) Gas → Mobile phase Solid / Liquid → Solid phase GSC not used because of limited no. of Solid phase GSC principle is ADSORPTION GLC principle is PARTITION 2

3. 3 The organic compounds are separated due to differences in their partitioning behavior between the mobile gas phase and the stationary phase in the column. Princip l e 3

4. 4 Sample to be separated is converted into vapor And mixed with gaseous M.P Component more soluble in the S.P → travels slower Component less soluble in the S.P → travels faster Components are separated according to their Partition Co-efficient Criteria for compounds to be analyzed by G.C VOLATILITY: THERMOSTABILITY : 4

5. The father of modern gas chromatography is Nobel Prize winner John Porter Martin, who also developed the first liquid-gas chromatograph. (1950) 5 5

6. 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. The mobile phase is, of course, a suitable carrier gas. M ost useful for the separation and analysis of gases like CH 4 , CO 2 , CO, ... etc. The use of GSC in practice is considered marginal when compared to gas liquid chromatography. 10/03/2023 8 6

7. Gas - Liquid Chromatography (GLC) The stationary phase is a liquid with very low volatility while the mobile phase is a suitable carrier gas. GLC is the most widely used technique for separation of volatile species. The presence of a wide variety of stationary phases with contrasting selectivities and easy column preparation add to the assets of GLC or simply GC. 7

8. PRACTICAL REQUIREMENTS Carrier gas Flow regulators & Flow meters Injection devices Columns Temperature control devices Detectors Recorders & Integrators 8 8

9. Syringe Injector Det e ctor Carrier Gas C ylinder Colu m n T o W aste o r F low Meter Flow Controller T w o - Sta g e Regulator 9 9

10. Requirements of a carrier gas Inertness Suitable for the detector High purity Easily available Cheap Should not cause the risk of fire Should give best column performance 10 10

11. CARRIER GAS » Hydrogen ( H 2 ) better thermal conductivity D isadvantage: it reacts with unsaturated compounds & inflammable » Helium ( He) excellent thermal conductivity it is expensive » Nitrogen ( N 2 ) reduced sensitivity it is inexpensive 11 11

12. 12 11/2 / 2017 Flow regulators & Flow meters Deliver the gas with uniform pressure/flow rate flow meters:- Rota meter & Soap bubble flow meter Rota meter placed before column inlet it has a glass tube with a float held on to a spring. T he level of the float is determined by the flow rate of carrier gas

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14. Soap Bubble Meter Similar to Rota meter & instead of a float, soap bubble formed indicates the flow rate 14 11/2 / 2017

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16. Sample preparation The prerequisite in GC separation is that all solutes being separated must be: (a) fairly volatile, and (b) thermally stable. (c) Usually, the solute should be dissolved in a non-aqueous matrix (H 2 O changes column behavior). Lack of volatility prevents the direct use of GC for many solute. One way to overcome this difficulty is to derivatize the solutes into more volatile forms. Cl Cl O O H O 2,4-dichlorophenoxyacetic acid (A cancAer suspect agent). Silylation 16

17. Derivatization of a solute can be used for any of the following reasons To increase the volatility of the solute. To increase the thermal stability of solute To improve the response for the solute on certain detectors (e.g., incorporating halogen atoms into a solute so that it can be detected using an electron capture detector). To improve the separation of the solute from other sample components (i.e., changing the structure of a solute will also affect its retention on the column) 17

18. 25 4. Most derivatization reactions can be classified into one of three group: Silylation Alkylation Acylation Most of these reactions are performed using minimal amount of sample and reagents (i.e., 0.1~2.0 mL) are typical carried out at room temperature. Some, however, do require heating to moderate temperatures (60 ~ 100 O C). 18

19. Silylation This is the most common type of derivation techniques used in GC. It involves replacing an active hydrogen on the solute (i.e. R-OH, RCOOH, R-NH 2 , etc.) with an alkylsilyl group (usually –SiMe 3 ). The result of this reaction is that the solute is converted into a less polar, more volatile and more thermally stable form. The most common reagent used in silylation is trimethylchlorosilane (TMS). Examples of its use are shown below: Cl Cl O O + Cl Si Me 3 Cl Cl O SiMe 3 O Cl Si Me 3 OH R OH + R Si O Me 3 + HCl The resulting Product of this reaction is usually just referred to as a TMS- / d 2 / e 2 r 1 i v 7 a tive. 19

20. Injectors Septum type injectors are the most common. These are composed of a glass tube where vaporization of the sample takes place. The sample is introduced into the injector through a self- sealing silicone rubber septum. The carrier gas flows through the injector carrying vaporized solutes. The temperature of the injector should be adjusted so that flash vaporization of all solutes occurs. If the temperature of the injector is not high enough (at least 50 degrees above highest boiling component), band broadening will take place . 20

21. Carrier Gas Syringe V apori z ation Chamber To Column Septum 21

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29. 29 Gas Chromatographic Columns and Stationary Phases Packed Columns These columns are fabricated from glass, stainless steel, copper, or other suitable tubes. Stainless steel is the most common tubing used with internal diameters from 1-4 mm. The column is packed with finely divided particles (<100-300  m diameter), which is coated with stationary phase. However, glass tubes are also used for large-scale separations .

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31. 31 Stainless steel is the most widely used because it is most inert and easy to work with. The column diameters currently in use are ordinarily 1/16" to 1/4" 0.D. Columns exceeding 1/8" are usually used for preparative work while the 1/8" or narrower columns have excellent working properties and yield excellent results in the analytical range. These find excellent and wide use because of easy packing and good routine separation characteristics. Column length can be from few feet for packed columns to more than 100 ft for capillary columns.

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33. Capillary Columns 33 Open tubular or capillary columns are finding broad applications. These are mainly of two types: Wall-coated open tubular (WCOT) <1 mm thick liquid coating on inside of silica tube Support-coated open tubular (SCOT ) 30 mm thick coating of liquid coated support on inside of silica tube] These are used for fast and efficient separations but are good only for small samples. The most frequently used capillary column, nowadays, is the fused silica open tubular column (FSOT), which is a WCOT column.

34. 34 The external surface of the fused silica columns is coated with a polyimide film to increase their strength. The most frequently used internal diameters occur in the range from 260-320 micrometer. However, other larger diameters are known where a 530 micrometer fused silica open tubular column was recently made and is called a megapore column, to distinguish it from other capillary columns. Megapore columns tolerate a larger sample size.

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40. 40 Capillary columns advantages compared to packed columns higher resolution shorter analysis times greater sensitivity Capillary columns disadvantage compared to packed columns smaller sample capacity Need better experience

41. 41 Solid Support Materials The solid support should ideally have the following properties: Large surface area (at least 1 m 2 /g) Has a good mechanical stability Thermally stable Inert surface in order to simplify retention behavior and prevent solute adsorption Has a particle size in the range from 100-400 mm

42. Selection of Stationary Phases General properties of a good liquid stationary phase are easy to guess where inertness towards solutes is essential. Very low volatility liquids that have good absolute and differential solubilities for analytes are required for successful separations. An additional factor that influences the performance of a stationary phase is its thermal stability where a stationary phase should be thermally stable in order to obtain reproducible results. Nonvolatile liquids assure minimum bleeding of the stationary phase 42

43. 43 Detection Systems Characteristics of the Ideal Detector: The ideal detector for gas chromatography h a s the following characteristics: Adequate sensitivity Good stability and reproducibility. A linear response to solutes that extends over several orders of magnitude. A t em p e r a tu r e r a n g e f r o m r o o m t empe r a tu r e t o at least 400 o C.

44. 44 Characteristics of the Ideal Detector A short response time that is independent of flow rate. High reliability and ease of use. Similarity in response toward all solutes or a highly selective response toward one or more classes of solutes. Nondestructive of sample.

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46. 46 Thermal Conductivity Detectors(TCD) A very early detector for gas chromatography, and one that still finds wide application, is based upon changes in the thermal conductivity of the gas stream brought about by the presence of analyte molecules. The sensing element of TCD is an electrically heated element whose temperature at constant electrical power depends upon the thermal conductivity of the surrounding gas. The heated element may be a fine platinum, gold, or tungsten wire or a semiconducting thermistor.

47. Thermal Conductivity Detectors(TCD) The advantage of the thermal conductivity detector is its simplicity, its large linear dynamic range(~10 5 ), its general response to both organic and inorganic species, and its nondestructive character, which permits collection of solutes after detection. A limitation of the katharometer is its relatively low sensitivity (~10 -8 g solute/mL carrier gas). Other detectors exceed this sensitivity by factors 61 47

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49. 49 Flame Ionization Detectors (FID) The flame ionization detector is the most widely used and generally applicable detector for gas chromatography. The effluent from the column is mixed with hydrogen and air and then ignited electrically. Most organic compounds, when pyrolyzed at the temperature of a hydrogen/air flame, produce ions and electrons that can conduct electricity through the flame. 49

50. A potential of a few hundred volts is applied. The resulting current (~10 -12 A) is then measured. The flame ionization detector exhibits a high sensitivity (~10 -13 g/s), large linear response range (~10 7 ), and low noise. A disadvantage of the flame ionization detector is 11/ 1 t / 2 h 1 a 7 t i t i s dest r uct i ve of sa m pl e . 64 50

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52. 52 Electron-Capture Detectors(ECD) The electron-capture detector has become one of the most widely used detectors for environmental samples because this detector selectivity detects halogen containing compounds, such as pesticides and polychlorinated biphenyls (PCB) The effluent from the column is passed over a  emitter, usually Nickel-63. An electron from the emitter causes ionization of the carrier gas and the production of a burst of electrons. In the absence of organic species, a constant standing current between a pair of electrodes results from this ionization process. The current decreases markedly, however, in the presence of those organic molecules that tend to capture electrons. 52

53. The electron-capture detector is selective in its response being highly sensitive to molecules containing electronegative functional groups such as halogens, peroxides, quinones, and nitro groups. It is insensitive to functional groups such as amines, alcohols, and hydrocarbons. An important application of the electron-capture detector has been for the detection and d / 2 e 1 7 te r m i nation of chlo r ina t ed in s ect i cide s . 53

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55. 55 Temperature Zones in GC Three temperature zones should be adjusted before a GC separation can be done. 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, which depends on the operational mode of the separation. The detector temperature should be kept at same level so as to prevent any solute condensation in the vicinity of the detector body. 55

56. Temperature Programming Gas chromatographs are usually capable of performing what is known as temperature programming gas chromataography (TPGC). The temperature of the column is changed according to a preset temperature isotherm. TPGC is a very important procedure, which is used for the attainment of excellent looking chromatograms in the least time possible. 56 56

57. Interfacing GC with other Methods Chromatographic methods (including GC) use retention times as markers for qualitative analysis. This characteristic does not absolutely confirm the existence of a specific analyte as many analytes may have very similar stationary phases. GC, as other chromatographic techniques, can confirm the absence of a solute rather than its existence. When GC is coupled with structural detection methods, it serves as a powerful tool for identifying the components of complex mixtures. A popular combination is GC/MS . 57

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