Gas Chromatography

Packed columns contain a stationary phase (see below), either loaded directly into the column if it is a solid at its operating temperature, or coated onto the surface of a solid support if it is liquid at its operating temperature. Thus, the operating principle of GC can be distinguished as either gas–solid chromatography (mainly an absorptive process by the stationary phase) or gas–liquid chromatography (GLC; mainly partition of the analytes between the mobile and stationary phases), based on the physical characteristics of the stationary phase. Capillary columns, introduced in the early 1980s, have now replaced packed columns for most applications. The original glass columns were fragile and have been superseded by fused silica capillary columns. Fused silica is high–purity synthetic quartz, with a protective coating of polyimide applied to the outer surface. Since these columns retain their flexibility only as long as the coating remains undamaged, their operating temperature must be maintained below 360° for standard columns (400° for high–temperature polyimide coatings). The first capillary columns were 0.2, 0.25 or 0.32 mm internal diameter (i.d.) and between 10 and 50 m long. Subsequently ‘megabore’ (0.53 mm i.d.), ‘minibore’ (0.18 mm i.d.) and ‘microbore’ (0.1 or 0.05 mm i.d.) columns have evolved. When coated with a heat–resistant polymer, these have the advantages of flexibility and strength, and can be threaded with ease through intricate pipe work. A single column can be fitted into almost any manufacturer’s GC. Capillary columns provide improved resolution, sensitivity, durability, less bleed with increasing temperatures, ease of maintenance and repair and yield reliable and highly reproducible separations, typically over many hundreds or thousands of injections. This has reduced the number of columns required to achieve a satisfactory separation of quite complex mixtures.

The internal surface of the silica is deactivated by a variety of processes that can react silanol groups (Si–OH) on the silica surface with a silane reagent (usually a methyl or phenylmethyl surface is created). For gas–solid capillary chromatography, a fine layer (usually less than 10 μm) of stationary phase particles is adhered to the tubing (porous layer open tubular, or PLOT, columns).For gas–liquid capillary chromatography, the stationary phase may be coated or bonded directly on to the walls of the column (wall–coated open tubular, or WCOT), or on to a support (e.g. microcrystals of sodium or barium chloride) bonded to the column wall (support–coated open tubular, or SCOT).Stainless–steel capillaries are usually reserved for applications that require extremes of temperature, or where the possibility of column breakage cannot be tolerated. Nowadays, the internal surface is specially deactivated chemically or by lining with fused silica, which allows for more flexibility and greater durability.

Column performance, whether capillary or packed, is made on the basis of its efficiency (the narrowness of a peak), the peak shape (whether it tails or fronts) and its ability to resolve compounds. This section deals with separation theory, and the reader may find it useful to refer at intervals to Fig.3.

If gas chromatographic retention data are to be exchanged between laboratories, they must be independent of the instrument used. The concept of RI has been shown to be more reliable than that of relative retention time (i.e. the retention time of the solute relative to that of a reference compound). The RI system uses a homologous series of compounds (i.e. a series of compounds that increase in size by an additional methylene unit) to provide the reference points on the scale. The most commonly used is the system described by Kovats (1961) using straight–chain saturated hydrocarbons (n–paraffins or n–alkanes). For any column temperature and stationary phase, the elution times of members of a series of n–alkane homologues are assumed to increase by an index of 100 for each additional methylene unit. On this scale, H₂ has an index of zero, methane has an index of 100, ethane 200, and so on up the scale of alkanes. The RIs of unknown substances are measured against this scale, obviating the need to correct data between laboratories because of variations in retention time. The method is illustrated in Fig.4, in which phenobarbital has a retention time of 4.5 min and a RI of 1957.

where t_R is the retention time, P_z is the carbon number of the smaller n–alkane, P_z+n is the carbon number of the larger n–alkane and x is the unknown solute.

The inlet system provides the means of introducing the specimen into the GC. Obtaining a narrow sample band at the start of the chromatographic process is critical to achieve good resolution, since broad sample bands usually produce broad peaks, especially for analytes that elute early. The choice of injector depends on the characteristics of the specimen or residue, the quantity and characteristics of the analytes to be separated, the temperature and nature of the stationary phase and the column. Solids may be dissolved in a suitable solvent and injected with a micro–syringe. It is best to keep the solution as concentrated as possible to reduce the size of the solvent peak. Liquids can be injected using a micro–syringe, but with sensitive detection systems the sample should be dissolved in a suitable solvent to reduce the sample size and avoid overloading the detector. Gases and vapours may be introduced by injection through the inlet port septum using a gas–tight syringe.

The four main types of GC injectors are Megabore direct (or packed column), split, splitless and cold on–column. In reality, splitless is an extreme example of split injection and both are carried out using the same hardware. Conventional glass syringes of 1 to 10 μL volume with stainless steel needles can be used on the packed column, or vaporisation capillary injectors, and the injection is made by piercing a silicone rubber septum. Care must be taken to select septa that have low bleed characteristics at the operating temperature, and those with Teflon backs are most reliable in this respect. Unstable materials can be decomposed by the high temperature of the injection system, particularly if the system is constructed of metal. For labile substances cold on–column injection is preferred, but clean extracts must be used to minimise column contamination.

In splitless injection, all the carrier gas passes to the column. This is useful for very volatile compounds, for low sample concentrations or for trace analysis. The flow rate in the injector is the same as that in the column (1 to 4 mL/min), and the only path for the injection to take is into the column, since the split vent is closed. At a fixed time after injection (usually 15 to 60 s), the injector is purged by opening the split vent to introduce a much larger flow of carrier gas through the injector (typically 20 to 60 mL/min) and any remaining sample in the injector is discarded through the split vent. Since the rate of sample transfer onto the column is so slow (because of the low gas flow), peaks are usually somewhat broader than for split injections. Temperature conditions can be adjusted to narrow or focus the sample band at the top of the column. Splitless injections should therefore be made with the initial column temperature at least 10° below the boiling point of the solvent (see Table 28.6), and the initial temperature should be held at least until after the purge activation time. Solvent condenses on the front of the column and traps the solute molecules, which focusses the sample into a narrow band (known as the solvent effect).Individual solutes with a boiling point 150° above the initial column temperature condense and focus at the top of the column in a process known as cold trapping. Either the solvent effect or cold trapping must occur before efficient chromatography can be obtained. Some newer chromatographs have the option of a pulsed splitless injection. In this mode, the column head pressure is increased immediately upon injection (typically to 174 kPa) and held there for 30 to 60 s, before returning to the normal operating pressure. This facilitates band sharpening and, while the process is not guaranteed to increase the fraction of the injection delivered onto the column, sensitivity is often improved because of improved chromatography.

Glass liners for split and splitless injectors come in a variety of shapes and volumes and it is prudent to start with a straight liner, and to investigate some of those that cause turbulence (e.g. the inverted cup style) later if this is unsatisfactory. A plug of deactivated glass wool in the liner helps prevent the deposition of non–volatile or particulate material on the column, but may cause some peak discrimination, and for the best results needs to be placed at a consistent position in the liner. Packing splitless injection liners with deactivated glass wool may decrease the chromatographic performance, but this must be weighed against the potential for damage to the stationary phase from the repeated injection of non–volatile or particulate material.

Some detectors respond to almost all solutes, while others (selective detectors) respond only to solutes with specific functional groups, atoms or structural configurations. Additional functional groups can often be added to solutes, generally after extraction (see below under derivatisation), to achieve a response from a selective detector and gain additional sensitivity and selectivity. The use of detectors such as the ECD to identify amenable compounds, and the NPD to detect compounds that contain phosphorus and nitrogen, removes many of the extraneous peaks frequently observed when using non–selective detectors, such as the FID. However, these selective detectors have also led to the detection of substances such as plasticisers from blood–collection tubes or transfusion lines, which interfere in many toxicological analyses. Detectors that detect the presence of a solute and also give information about its structure are increasingly popular and MS, Fourier transform infra-red spectroscopy and atomic emission spectrometry have been invoked to achieve this goal. Detector sensitivity is measured as signal–to–noise ratio, in which the signal corresponds to the height of the peak, and the noise to the height of the baseline variability. A signal–to–noise ratio of 8 to 10 is considered sufficient to confirm the presence of a peak. Each type of detector has a linear operating range in which the response obtained is directly proportional to the amount of solute that passes through, although this can be modified slightly by the nature of the solute and the chromatographic conditions (mobile phase type and flow, detector temperature). The linear operating range is considered to be exceeded when the incremental response obtained from the detector varies by more than 5% from that expected.

Most detectors (except MS) rely on gas other than the mobile phase (combustion, reagent or purge gas) for their operation. Usually, a total flow of at least 30 mL/min is necessary to sweep the solute molecules physically through the body of the detector at sufficient speed to prevent refluxing and produce narrow peaks. Thus, the addition of a ‘makeup’ gas is invariably required with capillary columns. Recommended gases and their flows for each detector are included in the manufacturer’s instruction manuals, and it is important to follow these guidelines (and those on maintenance) to achieve the stated performance.

Here, only the most widely used detectors are considered in detail. Several other types of detectors are available; for a more detailed discussion, the reader is referred to the text by Scott (1996).

The ECD is a selective detector with a very high sensitivity to compounds that have a high affinity for electrons; for many compounds, the sensitivity of the ECD often exceeds that of MS, and sometimes even that of the NPD. Compounds that contain a halogen, nitro group or carbonyl group are detected at concentrations of 0.1 to 10 pg, 1 to 100 pg and 0.1 to 1 ng, respectively. This makes it very useful for compounds such as the benzodiazepines or halogenated pesticides and herbicides. Alternatively, the great sensitivity of the detector may be utilised by preparing derivatives with halogenated reagents, such as trifluoroacetic, heptafluorobutyric or pentafluoropropionic (PFP) anhydrides. Linearity (at best only two or three orders of magnitude) is a limiting factor for quantitative analysis. In older models, the addition of a small amount of quench gas, such as methane, improves stability and linearity, and is essential if argon or helium carrier gas is used. Newer models can be operated successfully with helium as both carrier and detector gas. The ECD, because of its high sensitivity, can be contaminated easily: an impure cylinder of gas can damage a detector beyond repair in a matter of only a few hours. Cleaning is difficult, although some material can be removed by heating the detector to its maximum operating temperature overnight, and the injection of water in 100 μL aliquots through an empty glass column can also help. However, if contamination is avoided, it is virtually maintenance free. The radioactive source requires special handling procedures that may be subject to federal regulations. More recently, it has been shown that this detector can work with greater sensitivity and operate over an increased linear range using a helium plasma in place of the radioactive source.

In the Fourier–transform infra-red detector (FTIRD), the column effluent is conducted through a light pipe and swept by a scavenging gas into the path of an infrared light beam that has been processed by an interferometer. The interferometer directs the entire source light to a beam splitter, which sends the light in two directions at right angles. One beam takes a fixed pathlength to a stationary mirror, while the other takes a variable pathlength to a computerised moving mirror. The two beams are recombined, and the difference in path lengths creates constructive and deconstructive interference, or an interferogram. The recombined beam is then passed through the sample. Analyte molecules absorb light energy of specific wavelengths from the interferogram, and the sensor reports variation in energy versus time for all wavelengths simultaneously. For molecules to be infra-red active they must be able to undergo a change in dipole moment with the transition to their excited state. As a result, many compounds that are symmetrical do not respond.

Fourier transform refers to the mathematical computation that converts the data from an intensity versus time plot into an intensity (% transmission) versus frequency spectrum. Each dip in the spectrum corresponds to light absorbed, and can be interpreted as characteristic of specific functional groups in the molecule. Computer libraries allow for easy and rigorous comparison of spectra. FTIR can be fully quantitative, but it is relatively insensitive (10 ng range). Its advantages are that it is non–destructive, and it can distinguish between isomers (MS cannot). Because of the logistical difficulties of combining FTIR with GC, this combination of techniques has started to emerge only recently.

Atomic emission detector

With the atomic emission detector (AED), carrier gas that elutes from the column delivers solutes into a high–temperature helium plasma, where heat energy is absorbed by the constituent elements. In returning to their ground state, energy is emitted as light, the wavelength of which is characteristic for each element. Emitted light is focussed by a quartz lens and spherical mirror onto a diffraction grating, and the dispersed light focussed onto a diode array that is continuously scanned (wavelength usually 170 to 800 nm). Typically, some 15 elements can be monitored simultaneously, and each is plotted against time. The composite chromatogram allows the percentage elemental composition of each peak to be determined. Sensitivity is very good, but the detector is complex and expensive to operate and is not widely used.

A GC is an almost ideal inlet device for quadruple MS. The detector is maintained under vacuum, and in the most common technique of electron impact (EI) the column effluent is bombarded with electrons. Compounds absorb energy, which causes them to ionise and fragment in a characteristic and reproducible fashion. The resultant ions are focused and accelerated into a mass filter that allows fragments of sequentially increasing mass to enter the detector stepwise. The mass filter scans through the designated range of masses (usually up to about 700 amu) several times per second. The abundance of each mass at a given scan time produces the mass spectrum, which can be summed and plotted versus time to obtain a total ion chromatogram. The MS detector can be operated either in full scan mode (collecting all the ions within a given mass range) or selected ion monitoring (SIM) mode, which collects only pre–selected masses characteristic for the compound under study. Sensitivity for the two modes of operation is quite different, 1 to 10 ng for full scan, increasing to 1 to 10 pg in SIM because of the dramatic decrease in background noise. The linear range is excellent and often spans five or six orders of magnitude. Recent advances in computer technology, coupled with improved detector design, have revolutionised the use of the MS detector from a research tool to one of routine application.

The simultaneous use of a combination of a universal detector (FID) with a specific detector to monitor the effluent of a column can provide useful information about the properties of functional groups and substituents in a molecule. The FID response is roughly dependent on the number of carbon atoms in a molecule and is quite predictable. However, the ECD response varies widely for different compounds, is dependent on the electron–deficient part of the compound, and is difficult to predict. The NPD response of a compound depends to some extent on the number of phosphorus or nitrogen atoms in a molecule, but it also depends on their environment. Thus, by using the FID as a reference, and measuring the ECD or NPD response relative to it, another characteristic for identification is obtained in addition to retention behaviour.

Dual detector systems can be used in several ways. The column can be split at the detector end and the effluent passed into two different detectors that operate in parallel. This approach allows the most flexibility, since the choice of detectors is wide, and the effluent can be split in proportion to the sensitivity required from each detector. For capillary columns this is accomplished easily with zero dead volume press–fit tee connectors, but it is a more complicated operation for packed columns. Additional makeup gas may be required to ensure a good flow through the detectors, and care should be taken to use tubing of a smaller or equal total area to the analytical column to avoid loss of peak shape through refluxing at the detector. Alternatively, the GC oven houses two completely separate, but identically matched columns, each connected to a single detector. This is not an ideal approach, as matching columns is difficult and has to be checked at frequent intervals. Another approach is to stack the detectors in series, and some manufacturers deliberately provide detectors in identical modules for this purpose. There are limitations to the choice of possible detector combinations, as the first detector must always be a non–destructive detector, such as the ECD, AED or FTIRD.

Prior to chromatography, it is usually necessary to isolate the compound(s) of interest from either a biological matrix (plasma, urine, stomach contents, hair and tissue) or some other matrix, such as soil, air or water. Removal of extraneous material and concentration of the compounds of interest usually take place simultaneously. The high water solubility of some drug metabolites (e.g. glucuronide conjugates) requires chemical conversion to a less polar entity to permit isolation from water–based samples, and a hydrolysis procedure is often used for this purpose.

Derivatisation enables the analysis of compounds that otherwise could not be monitored readily by GC. To some extent the availability of stable polar stationary phases in capillary columns and the use of temperature programming has negated the requirement for derivatisation, although it is still widely used. Choice of reagent is based on the functional group that requires derivatisation, the presence of other functional groups in the molecule and the reason for performing the reaction. Although the retention characteristics are changed, the order of elution of a series of derivatives will be the same as that for the parent compounds. The preparation of derivatives modifies the functionality of the solute molecule to increase (or sometimes decrease) volatility, and thereby shortens or lengthens the retention time of a substance, or to speed up the analysis.

Another common reason for derivatisation is to improve resolution and reduce tailing of polar compounds (hydroxyl, carboxylic acids, hydrazines, primary amines and sulfydryl groups). For instance, hydroxylated compounds often have long retention times and column adsorption causes tailing, which results in low sensitivity. However, they readily form silyl ethers and these derivatives show excellent chromatography, and sensitivity can often be improved by a factor of 10 or more. Derivatisation can also help to remove the substance peak away from interfering material. For example, the reaction of amfetamine with acetone enables successful differentiation from methylethylketone on most stationary phases. Derivatives may also be used to make the molecule amenable to detection by selective detectors, or can be used to improve the fragmentation pattern of the compound in the mass spectrometer.

The reaction may be carried out during extraction (e.g. extractive alkylation), on the dry residue after solvent extraction (e.g. silylation) or during injection (e.g. methylation). In choosing a suitable reagent, certain criteria must be used. A good reagent produces stable derivatives without harmful by–products that interact with the analytical column, in a reaction that is almost 100% complete. Poor reagents cause rearrangements or structural alterations during formation, and contribute to loss of sample during reaction. Most manufacturers of derivatising reagents provide information on the potential uses of each product, along with standard operating instructions. Entire texts, such as that by Blau and Halket (1993), are devoted to this topic.

When attempting a new analysis, it is advisable first to review published literature for a method that can be copied or for a method that involves a similar type of compound and can be adapted. Column manufacturers’ catalogues are a useful source of information and invariably show examples of separations performed with their columns. Data on boiling points and RI (see monographs in Part Two) are also useful indicators. If the review is not helpful, a start can be made with a standard column, selecting either a 100% methyl-PSX capillary column (25 m with a 0.5 μm film) or a OV1–packed column (1.7 m with a 3% loading on 100 to 120 mesh support) and using standard flow conditions (1 to 2 mL/min helium for a capillary or 30 to 60 mL/min for packed). The oven temperature should be taken from 80 to 300° at 10°/min (or started at 200 or 250° if only an isothermal oven is available). A solution of the compounds of interest in ethanol or methanol should be injected with the injector temperature set at 250°. If a peak tails, derivatisation or using a more polar stationary phase should be considered. Fine–tuning is carried out once some peaks have been obtained. Having established the chromatography, the extraction and concentration steps can be determined. Manufacturers’ catalogues are again a useful source for both derivatisation and solid–phase extraction procedures.

Good preventative maintenance is essential. The injector (or liner) should be cleaned periodically, and any glass wool changed regularly (approx. every 100 to 1000 injections, depending on the quality of the extracts). For capillary columns, the performance is improved by periodically removing the first 5 to 10 cm of capillary tubing (a retention gap could be considered for dirty samples), and for packed columns by replacing the glass wool and first few centimetres of packing. It is advisable to monitor performance by selecting certain performance criteria (e.g. a certain response size or amount of acceptable separation between two closely eluting components) to indicate when maintenance is required. The manufacturers’ instructions for cleaning detectors should be followed.

The presence of traces of contaminants in the carrier gas supply shortens the column life drastically, and also causes detector deterioration. In–line filters (to remove oxygen, hydrocarbons, etc.) and molecular sieves (to remove water vapour) are strongly recommended, and the use of stainless–steel gas tubing minimises further contamination. Carrier–gas flow should be optimised for a particular column and a particular carrier gas. This is most important for capillary columns. Fig. 6 shows the relationship between efficiency expressed as the HETP versus carrier-gas velocity (van Deemter plot) for a 28 m by 0.25 mm i.d.WCOT OV-101 column. Modifying the mobile phase in GC has very little effect compared with that observed with HPLC or thin–layer chromatography (TLC) and, in general, affects efficiency rather than selectivity. Nitrogen gives higher efficiency, but at the expense of longer analysis time, while the less dense, but more hazardous, hydrogen gives lower efficiency, but faster analysis. In practice, nitrogen is usually used for packed columns and helium for capillary columns. Certain detectors impose restrictions on the choice of carrier gas, but an additional supply of gas can be added to the column effluent to purge the detector. Experimenting with higher flow and a lower operating temperature (or vice–versa) can give rewarding results for the separation of compounds that elute closely. This effect is particularly noticeable for two compounds that have different polarities, as the retention of the more polar compound is influenced to a greater extent the longer it resides in the column (non–polar compounds elute in boiling point sequence). Conditions of constant flow improve the efficiency of late–eluting peaks and produce faster chromatography than do constant pressure conditions.

For a particular separation, the lowest temperature compatible with a reasonable analysis time should be used. In general, retention times double with each 20° decrease in temperature. If the time is excessive, it is generally better to reduce the stationary phase loading or use a shorter column than to increase the column operating temperature. There is a maximum temperature at which a column can be operated and there is also a minimum temperature below which efficiency drops sharply. Manufacturers give the temperature operating ranges for each of their stationary phases (see Table .3). The stationary phase must be a liquid at the temperature of operation, and if a column is run at too low a temperature to obtain longer retention times the stationary phase may still be in the solid or semi–solid form. When using temperature programming, experimentation with a faster initial ramp followed by a slower subsequent ramp or an isothermal period can help resolve problematic separations.

Efficiency can also be improved by decreasing the column diameter or increasing the column length. The resultant increase in analysis time (particularly if the flow must be reduced to accommodate the increased pressure demand imposed by a narrower column), can usually be offset by using a slightly higher operating temperature (temperature increases affect retention time much more than do increases in gas flow). As shown in Table .8, reducing the diameter of a capillary column markedly increases efficiency, but the retention time remains constant only as long as the same phase ratio is maintained. Therefore, unless there is a simultaneous reduction in film thickness, retention increases in direct proportion to the phase ratio.

The solvent used for the sample can sometimes produce unexpected derivatives that give different retention times (traces of acetic anhydride that remain in butyl acetate avidly derivatise primary amines at room temperature). An inert non–polar solvent should be used if possible to minimise the co–extraction of unwanted contaminants. Acetone, other ketones, ethyl acetate and carbon disulfide readily form derivatives with primary amines and should be avoided.

The choice of injector type and injection solvent also play an important part in the chromatography. A solvent volume should be chosen that does not expand to exceed the capacity of the injector (see Table .6), otherwise backflush and irreproducible results are obtained. Split injection significantly reduces the amount of solvent and associated contaminants that enter the column and, although the analyte response is reduced, the improvement in the signal–to–noise ratio often results in enhanced sensitivity.

The use of a selective detector, such as an ECD (with the preparation of a strongly responsive derivative if appropriate), can improve sensitivity typically up to 100–fold. Similarly, switching from full scan to selected ion monitoring in MS improves the sensitivity, usually by a factor of ten. However, selective detectors should not be used as a substitute for cleaning up of sample extracts, as loading contaminants onto the column affects the chromatography adversely, even if the selective detector does not respond to the compounds. Increasing the detector temperature may also improve sensitivity.

Fronting or splitting of peaks indicates column overload. If the detector sensitivity permits, the best option here is to inject a smaller sample volume (or a more dilute sample), rather than to increase the column loading or diameter, otherwise efficiency is also affected.

If trace impurities are sought in the presence of a preponderant component, a number of stationary phases of differing polarities should be tried. Trace impurities are seen easily if they emerge before the main component of a mixture, while they may be lost completely in the tail if they elute just after the large peak. Early peaks are also sharper and thus, for the same peak area, higher – an effect that can contribute enormously to the successful detection of trace substances.

The most commonly used general screening system is a 100% dimethyl-PSX (methyl-PSX or X-1) capillary column (for packed columns, SE-30, OV-1 or OV-101 are equivalent). This should always be used for screening purposes, since it has the best chance of eluting any compound of interest. Analysts have collaborated to compile comprehensive lists of retention indices using this system (de Zeeuw 2002), some of which are included in Part 3 (see Index of Gas Chromatographic Data).

Most of the data are for the drugs themselves, but thermal decomposition may occur and the peak observed may be for the decomposition product (referred to as ‘artifact’) rather than the original drug. Where the drug is known to chromatograph badly, or to decompose, data are given for suitable derivatives [e.g. methyl or ethyl esters for the sulfonamides, and trimethylsilyl (TMS) derivatives for hydroxides]. Wherever possible, the RI of the drug is given, since this is a more reproducible parameter than retention time or relative retention (see discussion above). However, if a laboratory prefers routinely to use the latter parameters, the RI data can be converted easily after chromatography of a few representative drugs and using a regression analysis of RI against either retention time or relative retention. RIs for some additional non–drug substances that might interfere with toxicological analyses, but are not included in the monographs, are located in Part 3. A nitrogen phosphorus (alkali flame ionisation) detector is the best detector for nitrogenous drugs and phosphorus–containing pesticides, but a FID should also be used, since some drugs do not contain nitrogen (e.g. some anti–inflammatory agents).ECDs are excellent for benzodiazepines and halogen–containing compounds, such as some phenothiazines and herbicides. Extra selectivity can always be obtained by using element–specific detectors (e.g. those for phosphorus and sulfur for compounds that contain these elements). Additional specificity or confirmation of identity can be obtained by using a mass–selective detector, such as MS or ion–trap detector. In any analysis for an unknown compound, the data obtained from complimentary techniques, such as TLC, HPLC or colour tests, should always be assessed for compatibility with the GC result. (In the tables of retention indices given here, a dash indicates that no value is available for the compound, not that it does not elute.)