1. Chapter 32 Gas Chromatography 1

2. In gas chromatography, the components of a vaporized sample are separated by being distributed between a mobile gaseous phase and a liquid or a solid stationary phase held in a column. Two types of gas chromatography are encountered:  In gas-liquid chromatography (GLC), the mobile phase is a gas, and the stationary phase is a liquid that is retained on the surface of an inert solid by adsorption or chemical bonding.  In gas-solid chromatography (GSC), the mobile phase is a gas, and the stationary phase is a solid that retains the analytes by physical adsorption. 2

3. Gas-solid chromatography has limited application because of semipermanent retention of active or polar molecules and sever tailing of elution peaks. The tailing is due to the nonlinear character of adsorption process. Gas-solid chromatography permits the separation and determination of low-molecular-mass gases, such as air components, hydrogen sulfide, carbon monoxide, and nitrogen oxides. Gas-liquid chromatography has wider application than GSC. 3

4. Instruments for Gas-Liquid Chromatography The basic components of a typical instrument for performing gas chromatography are shown here. 4

5. The mobile phase gas in gas chromatography is called the carrier gas and must be chemically inert. Helium (He) is the most common mobile phase, although argon (Ar), nitrogen (N 2 ), and hydrogen (H 2 ) are also used. Pressure regulators, gauges, and flow meters are required to control the flow rate of the gas. Flow rates in gas chromatographs were regulated by controlling the gas inlet pressure. Inlet pressures usually range from 10 to 50 psi (lb/in 2 ), yielding flow rates of 25 to 150 mL/min with packed columns and 1 to 25 mL/min for open tubular capillary columns. Newer chromatographs use electronic pressure controllers both for packed and for capillary columns. 5 Carrier Gas System

6. In a classical soap-bubble meter, a soap film is formed in the path of the gas when a rubber bulb containing an aqueous solution of soap or detergent is squeezed. The time required for this film to move between two graduations on the buret is measured and converted to volumetric flow rate. Flow meter with digital readouts become common and is located at the end of the column. 6 Carrier Gas System

7. For high column efficiency, a suitably sized sample should be introduced as a “plug” of vapor. Slow injection or oversized samples cause band spreading and poor resolution. Calibrated microsyringes are used to inject liquid samples through a rubber or silicone diaphragm, or septum, into a heated sample port located at the head of the column. 7 Sample Injection System

8. 8 The sample port is usually kept at about 50 o C greater than the boiling point of the least volatile component of the sample. For packed analytical columns, sample sizes range from 0.1 to 20  L. For capillary column, sample size are 100 times smaller. A sample splitter is often needed to deliver a small known fraction of the injected sample, with the remainder going to waste. Splitless injection is also equipped for packed columns are used.

9. 9 Sample Injection System With autoinjectors, syringes are filled and the sample injected into the chromatograph automatically. In autosampler, samples are contained in vails on a sample turntable. Up to 150 samples can be placed on turntable. Standard deviation as low as 0.3% are common with autoinjection systems.

10. 10 Sample Injection System For introducing gases, a sample valve is often used instead of a syringe. With such devices, sample sizes can be reproduced to better than 0.5% relative. Liquid samples can also be introduced through a sampling valve.

11. The columns in gas chromatography are of two general types: packed columns or capillary columns. In the past, the vast majority of GC analyses used packed columns. But packed columns have been replaced by more efficient and faster capillary columns. 11 Column Configurations and Column Ovens Chromatographic columns vary in length from less than 2 m to 60 m or more. They are constructed of stainless steel, glass, fused silica, or Teflon. In order to fit into an oven for thermostating, columns are usually formed as coils having diameters of 10 to 30 cm.

12. Column temperature is an important variable that must be controlled to a few tenths of a degree for precise work. The column is normally housed in a thermostated oven. 12 Column Configurations and Column Ovens The optimum column temperature depends on the boiling point of the sample and the degree of separation required. A temperature equal to or slightly above the average boiling point of a sample results in a reasonable elution time. For samples with a broad boiling range, temperature programming whereby the column temperature is increased either continuously or in steps as the separation proceeds.

13. 13 Column Configurations and Column Ovens Optimum resolution is associated with minimal temperature. The cost of lowered temperature is an increase in elution time and the time required to complete an analysis. Derivatization is used to enhance detection or improve chromatographic performance. Isothermal at 45 o C Isothermal at 145 o C Temperature Gradient

14. The ideal detector for gas chromatography has the following characteristics: 1.Adequate sensitivity, (10 -8 ~ 10 -15 g solute/s). 2.Good stability and reproducibility. 3.A linear response to solutes that extends over several orders of magnitude. 4.A temperature range from room temperature to at least 400  C. 5.A short response time that is independent of flow rate. 6.High reliability and ease of use. 7.Similarity in response toward all solutes or, alternatively, a highly predictable and selective response toward one or more classes of solutes. 8.Nondestructive of sample. 14 Chromatographic Detectors

15. 15 Chromatographic Detectors No current detector exhibits all of the ideal characteristics. Here are the more common detectors used in GC:

16. 16 Flame Ionization Detector (FID) FID is the most widely used and generally applicable detector in GC. Effluent from the column is directed into a small air/H 2 flame and most organic compounds produce ions and electrons when pyrolyzed at the temperature of an air/H 2 flame. Compounds are detected by monitoring the current produced by collecting the ions and electrons. A few hundred volts applied between the burner tip and a collector electrode located above the flame serves to collect the ions and electrons. The current (~10 -12 A) is then measure with a sensitive picoammeter.

17. The flame ionization detector responds to the number of carbon atoms entering the detector per unit of time. It is a mass-sensitive rather than a concentration-sensitive device. FID has the advantage that changes in flow rate of the mobile phase have little effect on detector response. Functional groups such as carbonyl, alcohol, halogen, and amine, yield of fewer ions or none at all in a flame. FID is insensitive toward noncombustible gases (H 2 O, CO 2, SO 2, NO x ).  It is useful for the analysis of most organic samples including those that are contaminated with water and the oxides of nitrogen and sulfur. 17 Flame Ionization Detector (FID)

18. 18 Flame Ionization Detector (FID) Advantage:  a high sensitivity (~ 10 -13 g/s),  large linear response range (~10 7 )  low noise  generally rugged and easy to use Disadvantage:  destroys the sample during the combustion step  requires additional gases and controllers

19. 19 Thermal Conductivity Detector (TCD) TCD was one of the earliest detectors for GC and still finds wide application. TCD consists of an electrically heated source whose temperature at constant electric power depends on the thermal conductivity of the surrounding gas.

20. 20 Thermal Conductivity Detector (TCD) The heated element may be a fine Pt, Au, W wire, or a small thermistor. The electrical resistance of this element depends on the thermal conductivity of the gas. 4 thermally sensitive resistive elements are often used (Wheatstone Bridge). The effects of variations in temperature, pressure and electric power are minimized due to the two arms of a simple bridge circuit. The thermal conductivities of He and H 2 are roughly 6 ~ 10 times greater than those of most organic compounds.  Small amounts of organic species cause relatively large decreases in the thermal conductivity of the column effluent.

21. 21 Thermal Conductivity Detector (TCD) Advantage:  Simplicity  Large linear dynamic range (~10 5 )  General response to both organic and inorganic species  Nondestructive, solutes can be collected after detection Disadvantage:  Relatively low sensitivity (~10 8 g/s solute/mL carrier gas)  Low sensitivity precludes its use with capillary columns where sample amounts are very small.

22. 22 The electron capture detector (ECD) has become one of the most widely used detectors for environmental samples because this detector selectively responds to halogen-containing organic compounds, such as pesticides and polychlorinated biphenyls. Electron Capture Detector (ECD) In ECD, the sample eluate from a column is passed over a radioactive  emitter, usually Ni 63. An electron from the emitter causes ionization of the carrier gas (often N 2 ) 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 in the presence of organic molecules containing electronegative functional groups the tend to capture electrons.

23. 23 Electron Capture Detector (ECD) Compounds, such as halogens, peroxides, quinones, and nitro groups are detected with high sensitivity. ECD is insensitive to functional groups such as amines, alcohols, and hydrocarbons. Advantages:  Electron capture detectors are highly sensitive with highly selectivity for certain compounds and have the advantage of not altering the sample significantly. Disadvantages:  The linear response of the detector is limited to about two orders of magnitude.

24. One of the most powerful detectors for GC is the mass spectrometer. The combination of gas chromatography and mass spectrometry is known as GC/MS. A mass spectrometer measures the mass-to-charge ratio (m/z) of ions that have been produced from the sample. The flow rate from capillary columns is usually low enough that the column output can be fed directly into the ionization chamber of the mass spectrometer. 24 Mass Spectrometry Detector (MS) A typical GC/MS system is:

25. 25 Mass Spectrometry Detector (MS) Prior to the advent of capillary columns, when packed columns were used, it was necessary to minimize the large volume of carrier gas eluting from the GC. Various jet, membrane, and effusion separator were used for this purpose. No separator is needed for capillary column in GC/MS. The most common ion sources for GC/MS are electron impact (EI) and chemical ionization (CI). The most common mass analyzers are quadrupole and ion-trap analyzers.

26. 26 In GC/MS, the mass spectrometer scans the masses repetitively during a chromatographic separation. A computer data system is needed to process the large amount of data obtained by GC/MS mass spectrometers. The data can be analyzed in several ways. 1. The ion abundance in each spectrum can be summed and plotted as a function of time to give a total-ion chromatogram (TIC). 2. One can also display the mass spectrum at a particular time during the chromatogram to identify the species eluting at that time. 3. A single mass-to-charge (m/z) value can be selected and monitored throughout the chromatographic experiment, a technique known as selected-ion monitoring (SIM). Mass spectra of selected ions during a chromatographic experiment are known as mass chromatograms. Mass Spectrometry Detector (MS)

27. 27 Mass Spectrometry Detector (MS) An example of one application of GC/MS is shown: TIC SIM MS

28. 28 Mass Spectrometry Detector (MS) Mass spectrometry can also be used to acquire information about incompletely separated components. When multiple components are eluting at the same time, the mass spectrum of the front edge of a GC peak may be different from that of the trailing edge. With MS, we can not only determine that a peak is due to more than one component, but we can also identify the various unresolved species. GC has also been coupled with tandem mass spectrometers to give GC/MS/MS or GC/MS n, which are very powerful tools for identifying components in mixtures.

29. Thermionic Detector (or Thermionic Specific Detector, TSD) or (Nitrogen-Phosphorus Detector, NPD) is similar in construction to the FID. 29 Other GC Detectors Nitrogen- and phosphorous- containing compounds produce increased currents in a flame in which an alkali metal salt is vaporized. N and P can be selectively detected with a sensitivity that is 10 4 times greater than C. It is widely used for organo- phosphorous pesticides and pharmaceutical compounds.

30. 30 Other GC Detectors The electrolytic conductivity detector (ELCD) is for compounds containing halogens, sulfur, or nitrogen. Compounds are mixed with a reaction gas in a small reactor tube. The products are then dissolved in a liquid than produces a conductive solution. The change in conductivity as a result of the presence of the active compound is then measured.

31. 31 Other GC Detectors In the photoionization detector (PID), molecules are photoionized by UV radiation. The ions and electrons produced are then collected with a pair of biased electrodes, and the resulting current is measured. PID is often used for aromatic and other molecules that are easily photoionized.

32. 32 Hyphenated Methods GC is often coupled with the selective techniques of spectroscopy and electrochemistry. GC can be combined with MS, IR, NMR to provide component identification of complex mixtures. The combined techniques are called hyphenated methods. In early hyphenated methods, the eluates from chromatographic column were collected as separate fractions in a cold trap with a non-destructive non-selective detector. Each fraction was then investigated by NMR, IR, MS, or electroanalytical methods. Modern hyphenated methods can monitor the effluent from chromatographic column continuously.

33. 33 Gas Chromatographic Columns and Stationary phases In the early 1950s, GLC mainly used packed columns in which the stationary phase was a thin film of liquid retained by adsorption on the surface of a finely divided, inert solid support. The unpacked column has inside diameters of a few tenths of a millimeter which provides separations that were superior to packed column in both speed and column efficiency. In such capillary columns, the stationary phase was a film of liquid a few tenths of a micrometer thick that uniformly coated the interior of a capillary tubing. In the late 1950s, such open tubular columns were constructed, and the predicted performance were experimentally confirmed.

34. 34 Capillary columns did not gain widespread use in the first two decades due to some drawbacks: 1)Small sample capacities 2)Fragility of columns 3)Mechanical problems associated with sample introduction and connection of the column to the detector 4)Difficulties in coating the column reproducibly 5)Short lifetimes of poorly prepared columns 6)Tendencies of columns to clog 7)Patents (limited commercial development to a single manufacturer, the original patent expired in 1977) The most significant development is at 1979 when fused-silica capillaries were introduced. Gas Chromatographic Columns and Stationary phases

35. Capillary columns are also called open tubular columns because of the open flow path through them. They are of the following types: 1. Wall-coated open tubular (WCOT) are capillary tubes coated with a thin layer of the liquid stationary phase. 2. Support-coated open tubular columns (SCOT) have an inner surface lined with a thin film (<30  m) of a solid support material (ex: diatomaceous earth and liquid stationary phase is adsorbed).  SCOT has less efficiency but greater capacity than WCOT!! 3. Fused-silica open tubular (FSOT) columns are currently the most widely used GC columns. 4. Capillary columns with 530  m inside diameters, sometimes called megabore columns, are also used. 35 Capillary Columns

36. 36 Fused-Silica Capillary Columns Fused-silica capillaries are drawn from specially purified silica which contain minimal amounts of metal oxides. Fused-silica capillaries are given added strength by an outside protective polyimide coating. Advantages of fused-silica columns over glass columns: 1)Physical strength 2)Much lower reactivity toward sample components 3)Flexibility

37. 37 Fused-Silica Capillary Columns The popular inside diameters of fused-silica capillary is 250 and 320  m. The smaller inside diameter (150 or 200  m) gives higher resolution but need to reduce the size of sample injected and to have more sensitive detector. Capillary columns with 530  m inside diameters called megabore columns. The performance of megabore capillary columns are not as good as those of smaller diameter columns but significantly better than hose of packed columns.

38. 38 Properties of Typical GC Columns

39. 39 Properties of Typical GC Columns

40. Modern packed columns are fabricated from glass or metal tubing. They are typically 2 to 3 m long and have inside diameters of 2 to 4 mm. These tubes are densely packed with a uniform, finely divided packing materials or solid support, that is coated with a thin layer (0.05 to 1  m) of stationary liquid phase. Tubes are usually formed as coils with diameters of 15 cm so that they can be placed in a temperature-controlled oven. 40 Packed Columns

41. 41 Solid Support Materials The packing, or solid support, in a packed column serves to hold the liquid stationary phase in place so that as large a surface area as possible is exposed to the mobile phase. The ideal support: 1) small, 2) uniform, 3) spherical particles with good mechanical strength, 4) surface are of at least 1 m 2 /g, 5) inert at elevated temperature, 6) uniformly wetted by the liquid phase. Packed Columns Packings for gas chromatography were prepared from naturally occurring diatomaceous earth, which consists of the skeletons of thousands of species of single-celled plants. These support materials are often treated chemically with dimethylchlorosilane to reduce the tendency of adsorbing polar molecules as well as to give a surface layer of methyl group.

42. 42 Packed Columns Particle Size of Supports The efficiency of a GC column increases rapidly with decreasing particle diameter of the packing. The pressure difference required to maintain an acceptable flow rate of carrier gas varies inversely as the square of the particle diameter. The usual support particles are 250 to 170  m or 170 to 150  m.

43. Desirable properties for the immobilized liquid phase in a gas-liquid chromatographic column include: (1) low volatility (ideally, the boiling point of the liquid should be at least 100  C higher than the maximum operating temperature for the column) (2) thermal stability (3) chemical inertness (4) solvent characteristics (such that k and  values for the solutes to be resolved fall within a suitable range) Many liquids have been proposed as stationary phases but fewer than a dozen are commonly used now. 43 Liquid Stationary Phases

44. 44 Liquid Stationary Phases The retention time for an analyte on a column depends on its distribution constant. To separate various sample components, their distribution constants must be sufficiently different to accomplish a clean separation. To have a reasonable residence time on the column, an analyte must show some degree of compatibility (solubility) with the stationary phase especially with respect to the polarities of the analyte and the immobilized liquid. The polarity of the stationary phase should match that of the sample components. When the match is good, the order of elution is determined by the boiling point of the eluents.

45. 45 Liquid Stationary Phases Polar stationary phases contain functional groups such as –CN, –CO, and –OH. Hydrocarbon-type and dialkyl siloxanes are non-polar. Polyester phases are highly polar. Polar analytes: alcohols, acids, amines. Analytes with medium polarity: ethers, ketones, aldehydes.

46. 46 Liquid Stationary Phases Most of the liquid stationary phases have the general structure of polydimethyl siloxanes Polydimethyl siloxane with –R as –CH 3 gives relatively nonpolar property. A fraction of the methyl groups are replaced by functional groups such as phenyl (– C 6 H 5 ), cyanopropyl (– C 3 H 6 CN), and trifluoropropyl (– C 3 H 6 CF 3 ). And these substitutions increase the polarity of the liquids to various degrees. Polyethylene glycol has the structure: – HO – CH 2 – CH 2 – (O – CH 2 – CH 2 ) n – OH

47. 47 Liquid Stationary Phases Bonded and Cross-linked stationary phases The purpose of bonding and cross-linking is to provide a longer lasting stationary phase that can be rinsed with a solvent when the film becomes contaminated. The untreated columns slowly lose their stationary phase due to “bleeding”. Chemical bonding and cross-linking inhibit bleeding. Cross-linking is to incorporate a peroxide into the original liquid.

48. 48 Liquid Stationary Phases Film Thickness Commercial columns are available having stationary phases that vary in thickness from 0.1 to 5  m. Film thickness primarily affects the retentive character and the capacity of a column.  Thick films are used with highly volatile analytes because such films retain solutes for a longer time, thus providing a greater time for separation to take place.  Thin films are useful for separating species of low volatility in a reasonable length of time. For most id 250 – 320  m column, film thickness is typically 0.25  m. For megabore columns, 1 to 1.5  m of film thickness are often used.

49. GLC is applicable to species that are appreciably volatile and thermally stable at temperatures up to a few hundred degrees Celsius. 49 Applications of Gas-Liquid Chromatography A large number of important compounds have these qualities. OV-1 OV-3 OV-17 OV-210 OV-275 Carbowax 20M

50. Gas chromatography is widely used to establish the purity of organic compounds. Contaminants, if present, are revealed by the appearance of additional peaks in the chromatogram. The areas under these extraneous peaks provide rough estimates of the extent of contamination. The technique is also useful for evaluating the effectiveness of purification procedures. In theory, GC retention times should be useful for identifying components in mixtures. 50 Qualitative Analysis

51. 51 Because a chromatogram provides but a single piece of information about each species in a mixture (the retention time), the application of the technique to the qualitative analysis of complex samples of unknown composition is overcome by linking chromatographic columns directly with UV, IR, MS. Although a chromatogram may not lead to positive identification of the species in a sample, it often provides sure evidence of the absence of species. Qualitative Analysis

52. Quantitative GC is based on comparison of either the height or the area of an analyte peak with that of one or more standards. If conditions are properly controlled, both of these parameters vary linearly with concentration. Peak area is independent of the broadening effects; therefore, area is a more satisfactory analytical variable than peak height. If no equipment is available for peak area measurement, a simple method that works for symmetric peaks of reasonable widths is to multiple peak height by the width at one-half peak height. 52 Quantitative Analysis

53. Calibration with Standards A series of standard solutions that approximate the composition of the unknown is prepared. Chromatograms for the standards are then obtained, and peak heights or areas are plotted as a function of concentration to obtain a working curve. A plot of the data should yield a straight line passing through the origin; quantitative analyses are based on this plot. 53 Quantitative Analysis

54. The Internal Standard Method The highest precision for quantitative GC is obtained using internal standards because the uncertainties introduced by sample injection, flow rate, and variations in column conditions are minimized. A carefully measured quantity of an internal standard is introduced into each standard and sample and the ratio of analyte peak area (or height) to internal standard peak area (or height) is used as the analytical parameter.  The internal standard peak has to be well separated from the peaks of all other components in the sample.  The internal standard should be absent in the sample to be analyzed. 54 Quantitative Analysis

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57. 57 Although GC is a very mature technique, there have been many developments in recent years in theory, instrumentation, columns and practical applications. High-Speed Gas Chromatography We often focused on achieving ever-higher resolution in order to separate more and more complex mixtures. Critical Pair: the difficult-to-separate pair of components. The basic idea is that, for many separations of interest, higher speed can be achieved at the expense of some selectivity and resolution. The principles of high-speed separations can be demonstrated by: where k n is the retention factor for the last component of interest in the chromatogram. Advances in GC

58. High-Speed Gas Chromatography If we rearrange the equation and solve for the retention time for the last component of interest, we obtain: The advantage is faster separations by using short columns, higher- than-usual carrier gas velocities, and small retention factors. The disadvantage is reduced resolving power caused by increased band broadening and reduced peak capacity. 58 Advances in GC

59. 59 High-Speed Gas Chromatography A tunable column is a series combination of a polar and a nonpolar column. Figure shows the separation of 12 compounds prior to initiating a programmed temperature ramp. Advances in GC The separation of 19 compounds are after temperature program begun. Total analysis time is 140s.

60. 60 Miniaturized GC Systems Miniature GC systems are useful in space exploration, in portable instruments for field use, and in environmental monitoring. Microfabricated columns were designed using substrates of silicon, metal and polymers. Relatively deep, narrow channels are etched into the substrate. And these channels have low dead volume to reduce band broadening and high surface area to increase stationary phase volume. The miniature GC could be deployed in the field and was capable of sub ppb detection of the sample, such as the volatile organic compounds (trichloroethylene vapor) from contaminated soil or groundwater. Advances in GC

61. Multidimensional Gas Chromatography In multidimensional GC, two or more capillary columns of differing selectivities are connected in series. One might contain a nonpolar stationary phase and the 2 nd might have a polar stationary phase. Multidimensional GC can take several forms. In one implementation, called heart cutting, a portion of the eluent from the first column containing the species of interest is switched to a second column for further separation. In another methodology, comprehensive two-dimensional GC or GC  GC, the effluent from the first column is continuously switched to a second short column. Although the resolving power of the second column is necessarily limited, the fact that a column precedes it produces high-resolution separations. 61 Advances in GC

62. Gas-solid chromatography is based on adsorption of gaseous substances on solid surfaces. Distribution coefficients are generally much larger than those for gas-liquid chromatography, hence it is useful for separating species that are not retained by gas-liquid columns. 62 Gas-Solid Chromatography Ex: components of air, hydrogen sulfide, carbon disulfide, nitrogen oxides, carbon monoxide, carbon dioxide, and the rare gases. Gas-solid chromatography is performed with both packed and open tubular columns. For the open tubular column, a thin layer of the adsorbent is affixed to the inner walls of the capillary and they are sometimes called porous layer open tubular columns, or PLOT columns.