18 Chapter 1631

18 Gas chromatography S Dawling, S Jickells and A Negrusz Introduction . 469 Quantitative determinations . 505 Gas chromatography Optimising operation conditions to columns . 470 customise applications . 506 Inlet systems . 483 Specific applications. 508 Detector systems . 493 References . 510 Specimen preparation . 498 Further reading . 511 Introduction When a mixture of substances is injected at the inlet, each component partitions between the Gas chromatography (GC) is applicable to a wide stationary phase and the gas phase as it is swept range of compounds of interest to toxicologists, towards the detector. Molecules that have greater pharmaceutical and industrial chemists, envir- affinity for the stationary phase spend more time onmentalists and clinicians. Adsorption GC was in that phase and consequently take longer to developed by a German scientist, Fritz Prior, in reach the detector. The detector produces a the late 1940s. In the early 1950s Archer J. P. signal proportional to the amount of substance Martin and Richard L. M. Synge, two scientists that passes through it, and this signal is from the UK, invented partition chromatog- processed and fed to an integrator or some other raphy, for which they received the Nobel Prize in recording device. Each substance that elutes chemistry in 1952. This marks a true beginning from the column has a characteristic retention of GC as a broadly used analytical technique. If time, defined as the time interval from injection a compound has sufficient volatility for its mo- to peak detector response. Figure 18.1 shows a lecules to be in the gas or vapour phase at or schematic of a GC system. below 400ЊC, and does not decompose at these Identification of components was traditionally temperatures, then the compound can probably based primarily on peak retention time, but it is be analysed by GC. becoming increasingly more reliant on the The separation is performed in a column nature of the response obtained from the (containing either a solid or liquid stationary detector. The analyst has two main goals: firstly, phase) that has a continuous flow of mobile to make each different compound appear in a phase passing through it – usually an inert discrete band or peak with no overlap (or co- carrier gas, but more recently supercritical fluids elution) with other components in the mixture, (SCFs) have been used for some applications – and, secondly, to make these bands uniform in maintained in a temperature-regulated oven. shape and as narrow as possible. This is achieved 470 Clarke’s Analytical Forensic Toxicology Gas supply Injector CarrierMake up Detector Regulators and dryers Sample tray Detector Oven Instrument controller Data Data Column processor monitor Storage Figure 18.1 A modern GC system. partly by judicious choice of the column provided much higher resolution efficiencies stationary phase and its loading, and partly by and smaller peak volumes, which provoked optimising the operating conditions of the further modifications in detector design. The column. In addition, the method of introducing latest column developments mean that peaks of the sample into the chromatograph, the choice only a few milliseconds are a real possibility in of detector, and chemical modification to both GC and liquid chromatography (LC). Since improve the volatility of the compounds also this matches the response time of the current contribute to converting a mediocre analysis sensor electronics, the ingenuity of the detector into a first-class one. design engineers is again being tested. There has been a continuous synergism between enhanced detector performance and column performance, each advance being recip- Gas chromatography columns rocally dependent on the other. Initially, high- sensitivity detectors permitted the development of a precise column theory that, in turn, enabled Packed columns made of glass or steel contain a the design of columns that had much higher stationary phase (see below), either loaded efficiencies. The improved efficiency of columns directly into the column if it is a solid at its oper- resulted in substances eluting from the separa- ating temperature, or coated onto the surface of tion columns as very low volumes compared to a solid support if it is liquid at its operating the volume of the detectors and dispersion of the temperature. Thus, the operating principle of GC substance into that relatively large volume can be distinguished as either gas–solid chro- resulted in reduced efficiency. Thus the effi- matography (mainly an adsorptive process by ciency of separations became limited by the the stationary phase) or gas–liquid chromatog- geometry of the detector, and not by its intrinsic raphy (GLC; mainly partition of the analytes sensitivity. Detector design was modified to between the mobile and stationary phases), incorporate smaller tube dimensions, and the based on the physical characteristics of the volume of the sensing cell was thereby reduced stationary phase. Capillary columns, introduced greatly. The introduction of capillary columns in the early 1980s, have now replaced packed suitable for routine use in GC in the mid-1980s columns for most applications. The original glass Gas chromatography 471 columns were fragile and have been superseded allows for more flexibility and greater durability by fused-silica capillary columns. Fused silica is and reduces the possibility of degradation of high-purity synthetic quartz, with a protective analytes which can be catalysed by the presence coating of polyimide applied to the outer of metal surfaces. surface. Since these columns retain their flexi- Both bonding and cross-linking impart bility only as long as the coating remains enhanced thermal and solvent stability to the undamaged, their operating temperature must stationary phase (often designated ‘DB’), and be maintained below 360ЊC for standard should be used if they are available. For con- columns (400ЊC for high-temperature polyimide ventional packed column chromatography, coatings). The first capillary columns were 0.2, however, the stationary phase must first be 0.25 or 0.32 mm internal diameter (i.d.) and coated onto the surface of a solid support. between 10 and 50 m long. Subsequently ‘mega- bore’ (0.53 mm i.d.), ‘minibore’ (0.18 mm i.d.) and ‘microbore’ (0.1 or 0.05 mm i.d.) columns Stationary phases and support materials have been developed. When coated with a heat- resistant polymer, these have the advantages of Solid stationary phases flexibility and strength, and can be threaded with ease through intricate pipework. A single In gas–solid chromatography, the stationary column can be fitted into almost any manufac- phase is an active solid at its operating tempera- turer’s GC apparatus. Capillary columns provide ture. A conventional packed column is filled improved resolution, sensitivity and durability, completely by stationary phase particles, but in a less bleed with increasing temperatures, and ease capillary column a fine layer (usually less than of maintenance and repair; they yield reliable 10 lm) of particles is adhered by proprietary and highly reproducible separations, typically processes to the inner surface of the tubing, to over many hundreds or thousands of injections. create a PLOT column. These solid phases may This has reduced the number of columns be inorganic materials (e.g. aluminium oxides, required to achieve a satisfactory separation of molecular sieves, silica gel, or graphitised quite complex mixtures. carbon), or they may be organic polymers The internal surface of the silica is deactivated such as styrene. Both packed and capillary by a variety of processes that can react silanol columns use similar solid-phase materials. groups (Si1OH) on the silica surface with a Sample compounds undergo a dynamic gas– silane reagent (usually a methyl or phenyl- solid adsorption–desorption process with the methyl surface is created). For gas–solid capillary stationary phase, and since the particles are chromatography, a fine layer (usually less than porous, size exclusion and shape selectivity 10 lm) of stationary phase particles is adhered to processes also occur. The carrier gas (mobile the tubing (porous layer open tubular columns, phase) merely serves to sweep towards the or PLOT columns). For gas–liquid capillary chro- detector those solute molecules that are not matography, the stationary phase may be coated currently adsorbed. The resultant columns are or covalently bonded directly onto the walls of highly retentive, and separations impossible the column (wall-coated open tubular columns, with liquid phases can be accomplished easily on or WCOT columns) or onto a support (e.g. PLOT columns above ambient temperature. microcrystals of sodium or barium chloride) These columns are generally reserved for the bonded to the column wall (support-coated separation of low-molecular-weight materials, open tubular columns, or SCOT columns). such as hydrocarbon and sulfur gases, noble and Stainless-steel capillaries are usually reserved for permanent gases, and low boiling solvents. Since applications that require extremes of tempera- PLOT columns occasionally shed particles, their ture, or where the possibility of column breakage use is not advised with detectors that are affected cannot be tolerated. Nowadays, the internal adversely by the intrusion of particulate matter surface of metal columns is specially deactivated (the mass spectrometer is particularly vulnerable, chemically or by lining with fused silica, which as the column interface operates under vacuum). 472 Clarke’s Analytical Forensic Toxicology Graphitised carbon black methanol to pentanol. Tenax-TA is a porous Carbopaks are graphitised carbon black, having polymer of 2,6-diphenyl-p-phenylene oxide, adsorptive surfaces of up to 100 m2/g. They are used both as a chromatographic phase and as a usually modified with a light coating of a polar trap for volatile substances prior to analysis. It is stationary phase. Difficult separations of the C1 also used for high-boiling alcohols, polyethylene to C10 hydrocarbons can be achieved rapidly. glycols (PEGs), phenols, aldehydes, ketones, Carbopak C with 0.2% Carbowax 20M has been ethanolamines and chlorinated aromatics.

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