Gas Chromatography

1. Gas Chromatography

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. A sample containing the materials to be separated is injected into the gas chromatograph. A mobile phase (carrier gas) moves through a column that contains a wall coated or granular solid coated stationary phase. As the carrier gas flows through the column, the components of the sample come in contact with the stationary phase. The different components of the sample have different affinities for the stationary phase, which results in differential migration of solutes, thus leading to separation

3. Martin and James introduced this separation technique in 1952, which is the latest of the major chromatograhpic techniques. However, by 1965 over 18000 publications in gas chromatography (GC) were available in the literature. This is because optimized instrumentation was feasible. Gas chromatography is good only for volatile compounds or those, which can be made volatile by suitable derivatization methods or pyrolysis. Thus, about 20% of chemicals available can be analyzed directly by GC.

4. Gas chromatography can be used for both qualitative and quantitative analysis. Comparison of retention times can be used to identify materials in the sample by comparing retention times of peaks in a sample to retention times for standards. The same limitations for qualitative analysis discussed in Chapter 26 also apply for separations in GC. Quantitative analysis is accomplished by measurement of either peak height or peak area

5. 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. The mobile phase is, of course, a suitable carrier gas. This gas chromatographic technique is most useful for the separation and analysis of gases like CH4, CO2, CO, ... etc. The use of GSC in practice is considered marginal when compared to gas liquid chromatography.

6. 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. Instrumentation It may be wise to introduce instrumental components before proceeding further in theoretical background. This will help clarify many points, which may, otherwise, seem vague. It should also be noted that a detector will require special gas cylinders depending on the detector type utilized. The column temperature controller is simply an oven, the temperature of which can be varied or programmed

8. To Waste or Flow Meter Two-Stage Regulator Syringe Detector Injector Flow Controller Carrier Gas Cylinder Column

9. Three temperature zones can be identified: • Injector temperature, TI, where TI should allow flash vaporization of all sample components. • Column temperature, Tc, which is adjusted as the average boiling points of sample components. • Detector Temperature, TD, which should exclude any possible condensation inside the detector. Generally, an intuitive equation can be used to adjust all three zones depending on the average boiling point of the sample components. This equation is formulated as: TI = TD = Tc + 50 oC

10. The Carrier Gas Unlike liquid chromatography where wide varieties of mobile phase compositions are possible, mobile phases in gas chromatography are very limited. Only slight changes between carrier gases can be identified which places real limitations to chromatographic enhancement by change or modification of carrier gases

11. A carrier gas should have the following properties: • Highly pure (> 99.9%) • Inert so that no reaction with stationary phase or instrumental components can take place, especially at high temperatures. • A higher density (larger viscosity) carrier gas is preferred. • Compatible with the detector since some detectors require the use of a specific carrier gas. • A cheap and available carrier gas is an advantage.

12. Longitudinal Diffusion Term This is an important factor contributing to band broadening which is a function of the diffusivity of the solute in the gaseous mobile phase as well as the molecular diffusion of the carrier gas itself. HL = K DM /V Where; DM is the diffusion coefficient of solute in the carrier gas. This term can be minimized when mobile phases of low diffusion, i.e. high density, are used in conjunction with higher flow rates.

13. The same van Deemter equation as in LC can be written for GC where: H = A + B/V + CV The optimum carrier gas velocity is given by the derivative of van Deemter equation Vopt = { B/C }1/2 However, the obtained velocity is much greater than that obtained in LC.

14. The carrier gas pressure ranges from 10-50 psi. Higher pressures potentially increase compression possibility while very low pressures result in large band broadening due to diffusion. Depending on the column dimensions, flow rates from 1-150 mL/min are reported. Conventional analytical columns (1/8”) usually use flow rates in the range from 20-50 mL/min while capillary columns use flow rates from 1-5 mL/min depending on the dimensions and nature of column. In most cases, a selection between helium and nitrogen is made as these two gases are the most versatile and common carrier gases in GC.

15. 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.

16. Syringe Septum Carrier Gas Vaporization Chamber To Column

17. Column Configurations and Ovens The column in chromatography is undoubtedly the heart of the technique. A column can either be a packed or open tubular. Traditionally, packed columns were most common but fast developments in open tubular techniques and reported advantages in terms of efficiency and speed may make open tubular columns the best choice in the near future. Packed columns are relatively short (~2meters) while open tubular columns may be as long as 30-100 meters

18. Packed columns are made of stainless steel or glass while open tubular columns are usually made of fused silica. The temperature of the column is adjusted so that it is close to the average boiling point of the sample mixture. However, temperature programming is used very often to achieve better separations. The temperature of the column is assumed to be the same as the oven which houses the column. The oven temperature should be stable and easily changed in order to obtain reproducible results.

19. Several detectors are available for use in GC. Each detector has its own characteristics and features as well as drawbacks. Properties of an ideal detector include: High sensitivity Minimum drift Wide dynamic range Operational temperatures up to 400 oC. Fast response time Same response factor for all solutes Good reliability (no fooling) Nondestructive Responds to all solutes (universal) Detection Systems

20. a. Thermal Conductivity Detector (TCD) This is a nondestructive detector which is used for the separation and collection of solutes to further perform some other experiments on each purely separated component. The heart of the detector is a heated filament which is cooled by helium carrier gas. Any solute passes across the filament will not cool it as much as helium does because helium has the highest thermal conductivity. This results in an increase in the temperature of the filament which is related to concentration. The detector is simple, nondestructive, and universal but is not very sensitive and is flow rate sensitive.

23. Note that gases should always be flowing through the detector including just before, and few minutes after, the operation of the detector. Otherwise, the filament will melt. Also, keep away any oxygen since oxygen will oxidize the filament and results in its destruction. Remember that TCD characteristics include: Rugged Wide dynamic range (105) Nondestructive Insensitive (10-8 g/s) Flow rate sensitive

24. b. Flame Ionization Detector (FID) This is one of the most sensitive and reliable destructive detectors. Separate two gas cylinders, one for fuel and the other for O2 or air are used in the ignition of the flame of the FID. The fuel is usually hydrogen gas. The flow rate of air and hydrogen should be carefully adjusted in order to successfully ignite the flame.

27. The FID detector is a mass sensitive detector where solutes are ionized in the flame and electrons emitted are attracted by a positive electrode, where a current is obtained. The FID detector is not responsive to air, water, carbon disulfide. This is an extremely important advantage where volatile solutes present in water matrix can be easily analyzed without any pretreatment.

28. Remember that FID characteristics include: • Rugged • Sensitive (10-13 g/s) • Wide dynamic range (107) • Signal depends on number of carbon atoms in organic analytes which is referred to as mass sensitive rather than concentration sensitive • Weakly sensitive to carbonyl, amine, alcohol, amine groups • Not sensitive to non-combustibles – H2O, CO2, SO2, NOx • Destructive

29. Electron Capture Detector (ECD) This detector exhibits high intensity for halogen containing compounds and thus has found wide applications in the detection of pesticides and polychlorinated biphenyls. The mechanism of sensing relies on the fact that electronegative atoms, like halogens, will capture electrons from a b emitter (usually 63Ni). In absence of halogenated compounds, a high current signal will be recorded due to high ionization of the carrier gas, which is N2, while in presence of halogenated compounds the signal will decrease due to lower ionization.

31. Remember the following facts about ECD: 1. Electrons from a b-source ionize the carrier gas (nitrogen) 2. Organic molecules containing electronegative atoms capture electrons and decrease current 3. Simple and reliable 4. Sensitive (10-15 g/s) to electronegative groups (halogens) 5. Largely non-destructive 6. Insensitive to amines, alcohols and hydrocarbons 7. Limited dynamic range (102) 8. Mass sensitive detector

32. 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 mm diameter), which is coated with stationary phase. However, glass tubes are also used for large-scale separations.

33. Several types of tubing were used ranging from copper, stainless steel, aluminum and glass. 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.

34. Capillary/Open Tubular 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.

35. 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.

40. It should be noted that since capillary columns are not packed with any solid support, but rather a very thin film of stationary phase which adheres to the internal surface of the tubing, the A term in the van Deemter equation which stands for multiple path effects is zero and the equation for capillary columns becomes H = B/V + CV

41. Capillary columns advantages compared to packed columns • higher resolution • shorter analysis times • greater sensitivity Capillary columns disadvantage compared to packed columns • smaller sample capacity

42. Solid Support Materials The solid support should ideally have the following properties: • Large surface area (at least 1 m2/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

43. 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

44. Weight of liquid stationary phase * 100% Weight of stationary phase plus solid support % Loading = Increasing percent loading would allow for increased sample capacity and cover any active sites on the solid support. These two advantages are very important, however increasing the thickness of stationary phase will affect the C term in the van Deemter equation by increasing HS, and therefore Ht.

45. Generally, thefilm thickness primarily affects the retention character and the sample capacity of a column. Thick films are used with highly volatile analytes, because such films retain solutes for a longer time and thus provide a greater time for separation to take place. Thin films are useful for separating species of low volatility in a reasonable time. On the other hand, a thicker film can tolerate a larger sample size. Film thicknesses in the range from 0.1 – 5 mm are common.

46. Liquid Stationary Phases In general, the polarity of the stationary phase should match that of the sample constituents ("like" dissolves "like"). Most stationary phases are based on polydimethylsiloxane or polyethylene glycol (PEG) backbones:

47. The polarity of the stationary phase can be changed by derivatization with different functional groups such as a phenyl group. Bleeding of the column is cured by bonding the stationary phase to the column; or crosslinking the stationary phase. Liquid Stationary Phases should have the following characteristics: • Low volatility • High decomposition temperature (thermally stable) • Chemically inert (reversible interactions with solvent) • Chemically attached to support (to prevent bleeding) • Appropriate k' and a for good resolution

48. Bonded and Crosslinked Stationary Phases The purpose of bonding and cross-linking is to prevent bleeding and provide a stable stationary phase. With use at high temperatures, stationary phases that are not chemically bonded or crosslinked slowly lose their stationary phase due to bleeding in which a small amount of the physically immobilized liquid is carried out of the column during the elution process. Crosslinking is carried out in situ after a column is coated with one of the polymers

50. In summary, stationary phases are usually bonded and/or crosslinked and the following remarks are usually helpful: 1. Bonding occurs through covalent linking of stationary phase to support 2. Crosslinking occurs through polymerization reactions to join individual stationary phase molecules 3. Nonpolar stationary phases are best for nonpolar analytes where nonpolar analytes are retained preferentially 4. Polar stationary phases are best for polar analytes where polar analytes are retained preferentially