Introduction
The term ‘Forensic toxicology’ covers any application of the science and study of poisons to the elucidation of questions that occur in judicial proceedings. All the techniques of analytical chemistry used by the forensic toxicologist, from color tests to mass spectrometry, are based on the molecular structure of the compounds involved, whereas toxicity is related to the dose. Toxicity of a poison is a biological concept, as anything can become a poison if it exceeds the threshold limit of the organism’s ability to deal with it. If the poison is not specified by name, the request to ‘test for poisons’ is a major problem for the forensic toxicologist because there is no single chemical method of analysis capable of detecting all the various poisons. The forensic toxicologist needs a repertoire of standard methods that can be modified according to the nature of the investigation, the type and amount of material for analysis, and the time and resources available. Gas chromatography is one specific form of the more general separation process of chromatography.
Fundamentals
Gas chromatography (GC), like other forms of chromatography, is a method of separating mixtures of substances of analytical interest, either from each other or from an extraction residue. The separation is performed on a column containing the stationary phase, either solid or liquid, which is maintained at a defined temperature in an oven and at a defined flow of carrier gas (mobile phase). When a mixture of substances is injected at the inlet, each component is swept towards the detector and is partitioned between the stationary phase and the gas phase. Molecules with greatest affinity for the stationary phase spend more time in that phase and consequently take longer to reach the detector. The detector produces a signal dependent on the structure of substance passing through it. Each substance passing through the column will have a characteristic retention time, which is defined as the time from the injection to peak maximum.
Columns
The GC columns most widely used fall into two distinct categories: packed and wall-coated open tubular (WCOT) columns, commonly known as capillary columns because their inner diameters are small. Packed columns were developed first, but since about 1980 the commercial availability of highly efficient and rugged fused silica WCOT columns has resulted in their use dominating the gas chromatography coupled to mass spectrometry (GC-MS) field, especially for the analysis of trace amounts of specific organic compounds in complex mixtures.
Packed columns
Packed columns do not possess the high efficiencies and separating capabilities of WCOT columns, but they do have capacities which simplify sample introduction techniques and provide a larger quantity of sample component for introduction to the detector. Typical packed columns for analytical work are 2-5 m in length and have an internal diameter of 2 mm. They use carrier gas flows of 20-50 ml min-1. The most common support particles are formed from diato-mites, which are skeletons of single-celled algae.
These are prepared by molding diatomaceous earth into bricks and drying them in an oven. The bricks are then crushed and screened to particles in the 80-120 mesh size (80-120 openings per inch (2.54cm) in a screen). These particles are very strong,with a high specific surface area (1-20m2g_1) and good pore structure (1-2 um).
WCOT columns
WCOT columns were originated in 1956 when Golay first showed them to be theoretically ideal. They underwent major advances when pioneering work on high performance glass WCOT columns occurred in Europe between 1975 and 1980. These columns are now made of pure silica and are extremely rugged when an external coating of polyimide polymer is applied. The technology of producing high quality columns of controlled internal diameter and stationary film thickness has advanced considerably. Commercially available fused silica WCOT columns with polar and nonpolar stationary phases have a consistently high efficiency and give excellent analytical results. They are produced in lengths of 10-100 m with internal diameter of 0.20-0.35 mm and use carrier gas flow rates of 2-5mlmin_1. Fused silica WCOT columns have the additional advantage over glass or metal columns of being physically flexible,so much so that they may actually be tied into a knot without breaking. To separate volatile compounds correctly,it is better to use a large stationary film thickness (alkanes C1 to C15 with 5 u.m). In contrast, a lower stationary film thickness (0.12 u.m) will optimize separation of C10 to C40.
Capillary columns offer other advantages over packed columns. Their superior resolution can be used to separate complex mixtures or to increase the certainty that a single compound is correctly identified. The high efficiency results in tall narrow peaks, which considerably enhance the signal-to-noise ratio and consequently the detection limits. Short columns (2-10 m) can be used to give similar resolution to packed columns,but in a shorter time.
Choice of the Stationary Phases
Over 700 substances have been used as stationary phases. GC may be divided into gas-solid chromato-graphy (mainly adsorptive processes) and gas-liquid chromatography (mainly partition),depending on whether the stationary phase is a solid or a liquid at its operating temperature. If the stationary phase is a liquid,it must be coated on a support for packed column chromatography. For capillary column chro-matography,the stationary phase may be coated directly on to the walls of the column,or on to a support which is bonded to the glass walls. The stationary liquid phase may be chosen from over 100 phases available,but in practice fewer than 10 are in common use (Table 1). Some examples of phases in gas-solid chromatography are given in Table 2.
In general,nonpolar compounds chromatograph best on nonpolar phases,and polar compounds on polar phases,but this is not necessarily the decisive factor. For example,alcohols,and particularly etha-nol,are well separated on polar columns,such as polyethylene glycol. Nonpolar phases,like polydiphe-nyldimethyl siloxane,also give good results: separation is obtained using the volatilization temperature of the different constituents of the mixture after concentration of the analytes in the head column. In this case, a partition process is followed by an adsorptive one. Ethanol and 1-propanol are well separated (due to their different boiling points),in contrast with results using a polar stationary phase.
Optimization of Oven Temperature
For a particular separation,the lowest temperature compatible with a reasonable analysis time should be used. If the time is excessive,it is generally better to reduce the stationary phase loading than to increase the column temperature.
Table 1 Some examples of phases in gas-liquid chromatography
| Support materials | Separated compounds |
| Apiezon L (hydrocarbon grease) | Barbiturates, amphetamines |
| SE-30, OV-01, OV-101 (dimethyl silicone polymers) | Separation on the basis of molecular weight |
| Apolane-87 (high temperatue non-chiral hydrocarbon phase) | Many drugs |
| Carbowax 20 M (polyethylene glycol) | Alkaloids, basic drugs, amphetamines |
| OV-17 (phenyl methyl silicone, moderately polar silicone phase) | Many drugs |
| XE-60 (cyanoethyl silicone); OV-225 (cyanopropyl phenylmethyl silicone) | Steroids |
| Polyester | Fatty acid esters, barbiturates |
| Polyamides (Poly A 103) | Barbiturates, ternary amine tricyclic antidepressants |
| Chirasil-Val | Optical enantiomers (amino acids, polar drugs) |
| Mixed phases | Anticonvulsant drugs |
Table 2 Some examples of phases in gas-solid chromatography
| Stationary phase | Separated compounds |
| Molecular sieve | Inorganic gases, 02,N2, CO, in blood |
| (4A, 5A, 13X) | |
| Silica gel | Inorganic gases, C02, CO, H2,N2 |
| Chromosorb/Porapak | Fatty acids, amines, alcohols |
| Tenax | Trapping of volatile substances |
| Carbopak B and C | Hydrocarbons C1to C10, ethanol in |
| blood, substances abused by | |
| ‘glue sniffers’, ethylene glycol in | |
| blood |
In a screening procedure for complex mixtures with components of widely varying retention char-acteristics,it may be very difficult or impractical to select a column temperature that will allow all the components to be resolved. It is therefore necessary to vary the column temperature throughout the analysis, starting with a low temperature and finishing with a higher value. For mixtures with components of the same family,the oven temperature can start at a higher level and separation can also be obtained under isothermal conditions.
There is a maximum temperature at which a column can be operated,and there is also a minimum temperature below which the efficiency will drop sharply. 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 semisolid form.
Gas Pressure and Flow Control
In order to perform accurate and reproducible GC,it is necessary to maintain a constant carrier gas flow. Under isothermal conditions,simple pressure control is adequate for packed or capillary columns. Flow control is highly desirable,if not essential,during temperature programming with packed columns and can be used to advantage with on-column injectors on capillary columns. Carrier gas flow should be optimized for a particular column and a particular carrier gas.
Introduction of Samples
Sample introduction in a GC analysis is critical. Poor introduction technique can reduce column resolution and the quality of quantitative results. The sample must be injected as a narrow band on to the head of the column and contain a composition truly representative of the original mixture.
Introduction of the sample to the column is the injector’s function. Gases can be injected using a rotary valve containing a sample loop of known volume. Solid samples can be dissolved in a suitable solvent and converted to a vapor by the temperature at the inlet upon injection. The most common technique is liquid injection through a self-sealing septum into a heated injection port.
Alcohol can be introduced to packed columns by injecting a sample of blood directly diluted with water. The glass fiber insert will trap all nonvolatile compounds and the total flow of carrier gas will pass through the column. The injector temperature selected must be as low as possible to avoid column pollution or contamination. One limitation is the frequent change of insert necessary.
Split-splitless injection
Split and splitless injectors are more conventional today. For split injection,a flow of carrier gas will purge the septum and another will pass through the vaporization chamber. This later will be separated between column flow and purge. The ratio of these two flows (the split ratio) is the proportion of injected sample that reaches the column. The function of the splitter is not to reduce sample volume but to ensure that the sample enters the column as a plug and is not exponentially diluted,and to prevent overloading of the column with sample.
Although the split method of injection does prevent column overloading,the fraction which reaches the column may not be representative of the original sample because it is a flash vaporization technique: higher molecular weight (low volatility) components of the sample (like cannabinoids) in contact with the metal surface of the syringe plunger are not expelled from the syringe with the same efficiency as compounds (volatile compounds like amphetamines or solvents) whose boiling points are at or below the injection temperature. Since low injection volumes are generally used for WCOT columns (as compared with packed columns),a significant fraction of the sample is in contact with the metal surface and split discrimination may result. To prevent this,inlet liners are available to provide efficient heat transfer and thorough mixing of the sample to minimize discrimination.
Split injection is used for volatile compounds or for diluting the sample. It is also used to analyze compounds eluted rapidly after the front of solvent,and for solvent analyses after headspace preparation in order to reduce injection time. In forensic toxicology,split injection is largely used for the analysis of medications,drugs of abuse and powders. For tox-icological analyses of biological samples,splitless injection is preferred,while the split technique is most beneficial for samples containing compounds at high concentrations.
In forensic toxicology,more components are present at trace levels,and particularly in alternative matrices like hair,saliva or sweat,and target substances may be undetected because most of the sample is vented to the atmosphere and does not reach the detector. For analysis of trace compounds,the split-less injection technique is generally used.
Splitless injection may be either on-column,using a needle fine enough to enter the column bore,or off-column using a low-volume heated block. In either case,the top of the column is held at a low temperature to condense the sample contained in the solvent. Without reconcentration,the volume of the injection region will increase the band widths of eluting peaks and reduce the efficiency of the separation.
One method of reconcentration is known as the solvent effect. This occurs because the front of the solvent plug which enters the column mixes with the stationary phase and is more strongly retained than the rear of the solvent plug. Sample components therefore encounter a barrier which has the effect of condensing components at the head of the column. This applies only to compounds with a boiling point near those of the solvent. When a solvent starts to volati-lize,compounds with a similar boiling point are concentrated. It is preferable to choose solvents with low volatility (i.e. hexane,toluene,octane). For example, the use of toluene for amphetamine and iso-octane for cannabinoids is recommended. Because of the interaction of the solvent and the stationary phase,some columns can be damaged if polar or aromatic solvents are used. To accomplish this solvent effect,it is necessary that the column temperature at injection is low enough to prevent the solvent from migrating too rapidly from the head of the column. This requires a column temperature of 20-40°C below the boiling point of the solvent,and may require auxillary cooling of the oven.
A second means of solute reconcentration is cold-trapping. In this method,the initial column temperature must be about 150°C below the boiling points of the components to be trapped. Compounds with lower boiling points require a solvent effect for reconcentration.
If left in the splitless condition,the time to sweep all of the solvent vapor on to the column would be sufficient to cause a large solvent tail,which would interfere with early-eluting peaks. Therefore,after a specified time (split-valve off-time),the operation of a solenoid valve causes conditions to change so that the inlet flow is greater than the column flow,which remains constant. Any solvent or sample remaining in the injector is back-flushed with carrier gas and will be purged.
On-column injection
The injection modes previously described all require flash vaporization of the sample,sometimes leading to sample discrimination or decomposition of thermally labile compounds like lormetazepam or loprazolam, two benzodiazepines. These can be overcome by injecting the sample directly on to the WCOT column through a cool injection port (at the same temperature as the column). This method of injection for WCOT columns (internal diameter 0.25-0.35 mm) was not prominent among early injector designs,owing to the mecanical difficulties of aligning the syringe needle with the column. Like splitless injection,on-column operation requires cold-trapping or the solvent effect to concentrate the sample at the head of the column. On-column injection syringes have needles which are too fine to penetrate a septum and,in order to minimize carrier gas loss,valve assemblies are used which grip the needle or which only open when the needle is in the narrow entrance channel.
Solid injection
Solid injection is used when solvent interference is serious. The ‘moving needle’ injector has found application in steroid analysis and for the determination of anticonvulsant drugs. A solution of the material to be injected is placed on the tip of the glass needle with a syringe. A small flow of carrier gas sweeps the solvent out of the top of the device to waste. The dry residue is then introduced by moving the needle into the heated injection zone of the chromatograph with a magnet. This form of injection can only be used with drugs that will not volatilize with the solvent.
Programmed temperature volatilization injection
The programmed temperature volatilization (PTV) mode of injection exhibits the advantages of split-splitless and on-column injectors. The sample is introduced in a cold vaporization chamber to avoid sample degradation and loss of compounds. The injector is warmed slowly to evaporate the solvent and to concentrate the sample (there is the possibility of concentration without the conventional extraction step with an organic phase and concentration by evaporation). The injector is then heated rapidly and split or splitless injection is operated. Degradation of analytes is minor and comparable with the on-column injection technique.
Headspace introduction
Headspace analysis permits the detection of volatile substances in a liquid or solid sample and minimizes column contamination. A small volume of the sample is placed in a vial sealed with a septum disk and this vial is equilibrated at an appropriate elevated temperature. A sample of the vapor is removed with a syringe and is then injected on to the column. This technique is used,for example,in the assay of ethanol and other solvents in blood and for complex household preparations,such as polishes,which contain volatile substances.
In forensic toxicology,biological fluids are easily and directly analyzed (without sample preparation), but tissue such as lung (identification of abused volatile substances),intestines (to determine the method of administration) and muscles may also be analyzed. Muscles are used to confirm carbon dioxide intoxication when biological fluids,and particularly blood,are not available at autopsy (for instance,if the body has been dried by the high temperature of a fire).
Methyl and ethyl mercaptans are used to document alkane intoxication (methane,propane,butane). After fatal massive ingestion of ethanol,ketones from an overloaded liver metabolism may be identified (i.e. isopropanol,acetone).
For headspace analyses,it is necessary to choose a stationary phase polarity similar to volatile polarity, for better focalization,and a large film thickness for an optimal separation of the volatile compounds.
Simple injection-double detection
Simple injection followed by double detection is possible by placing a Y quartz connector between the retention gap and two different analytical columns,each coupled to a detector. This technique can be used to analyze the large family of psychotropes (benzodiazepines,tricyclic antidepressants (TCA), neuroleptics,etc.).
Detectors
In GC,the detection problem is that of sensing a small quantity of organic compound in an inert carrier gas. To detect these compounds under such a wide variety of conditions,a number of detectors have been developed. The ideal detector will have high sensitivity, wide linear dynamic range and a small cell volume so that the GC peak is not distorted. Those in most common use are the flame ionization and electron-capture detectors.
Flame ionization detector (FID)
This detector is probably the most widely used of all the detectors because it responds to nearly all classes of compounds. Carbon compounds detectable must be capable of undergoing oxidation (hydrogen,nitro-gen,water,hydrogen sulfide,sulfur dioxide,ammo-nia and carbon dioxide will be not detected). The principle is simple. The effluent from the column is mixed with hydrogen and the mixture burnt at a small jet in a flow of air (H2/O2 flame at 2000°C). Above the jet is the collector electrode. A polarizing potential (positive voltage) is applied between the jet and the electrode to measure the created ions (negative ions) when an eluted component is burnt in the flame. The FID response depends upon the numbers of ions produced by a compound.
Nitrogen-phosphorus detector (NPD) or alkali flame ionization detector (AFID)
The introduction of alkali metal vapors into the flame of a FID confers an enhanced response to compounds containing phosphorus and nitrogen. Modern versions of this detector have an electrically heated rubidium silicate source of metal ions. The detector is particularly useful for drug analysis because most drugs contain nitrogen,while the solvent and the bulk of the coextracted material from a biological sample do not. This detector is also especially useful for the detection of pesticides containing phosphorus.
Electron-capture detector (ECD)
From the time of its discovery in 1960,the ECD has enjoyed a steady growth in development and use. This is a selective detector which is highly sensitive to all electron reacting compounds containing halogen, nitro group or carbonyl group (i.e. benzodiazepines, pesticides,halogenated solvents,anesthesic gases). The detector consisted of a small chamber with two electrodes parallel to each other and a radioactive source,usually 63Ni,placed close to the cathode to ionize the carrier gas. A potential applied to the electrodes produces a steady background current. When an electron-capturing substance appears in the chamber,some of the electrons are removed and a fall in the detector current results. The response of the detector is therefore a loss of signal rather than an increase in signal,as given by most other detectors.
Mass selective detector (MSD)
Generally,the courts of justice only recognize chemical-toxicological analysis results in cases where they have been confirmed by a second independent method. The second analytical method employed is often GC-MSD. Today,GC-MSD is the method of choice as it is the more powerful tool for the identification of xenobiotics and their metabolites in specimens because of its separation ability and detection sensitivity. Mass spectrometry is based on the fact that when a molecule is ionized in a vacuum,a characteristic group of ions of different masses is formed. A mass spectrum is produced by separating these ions and recording a plot of ion abundance versus ionic mass. Mass spectrometers are classified according to the principle used to separate ionic masses. The most commonly used mass spectrometers fall into two broad classes: quadrupole and ion trap.
The most widely used method of ionization is that of electron impact (EI),in which the vaporized sample molecules are bombarded with a stream of high energy electrons. The energy absorbed causes fragmentation of the analyte,producing both negative and positive ions.
The simplest process which may occur initially is the removal of a single electron from the intact molecule to give the positively charged molecular ion (M+). M+ may rearrange and/or fragment into ions of lower mass-to-charge ratio (m/z),which may fragment further. The beam of ions is directed through a set of resolving slits to a detector,usually an electron multiplier. The most important piece of information that may be obtained from a mass spectrum is the molecular weight. However,certain classes of compounds do not show molecular ions.
‘Soft ionization’ techniques have been developed to generate a molecular ion or ‘quasimolecular ion’ and fragmentation is kept to a minimum. The most commonly used technique is chemical ionization (CI),in which the sample is mixed with a large excess of a reagent gas,such as methane,ammonia or isobutane. The mixture is then bombarded with high energy electrons,as in EI ionization. The reagent gas undergoes preferential ionization and the primary ions so produced react with further reagent gas molecules. These secondary ions subsequently react with the sample molecules to produce new ions. Usually,few fragmentation/rearrangement ions are observed and this quasimolecular ion is the most intense ion in the spectrum. Electronic impact and chemical ionization are complementary techniques,thus providing both molecular weight and fragmentation information. The majority of the work done has involved the study of positive ions,but interest in the negative chemical (NCI) technique has increased in recent years. For example,the CI mode of detection for negative ions is the technique of choice for detecting benzodiazepines because they possess halogen groups (electronegative functional groups) located on aromatic rings with high negative density that will give more stability to the anions formed in the ion source.
The advantage of MSD over other commonly used analytical techniques lies in the wide range of samples (blood,urine,tissues,bile,hair,saliva,gastric con-tent,sweat,etc.) that may be examined and the amount of information that may be obtained. The main disadvantages lie in the complexity and cost of this technology.
Tandem mass spectrometry
Although GC-MS is in an advanced stage of development,new techniques,applications and instrumentations are continually being introduced. Many of these are minor modifications of existing methods, or are for specialized uses such as extended mass range and the analysis of substances of low volatility.
Among the most significant recent development is the technique of tandem mass spectrometry (MS-MS). MS-MS retains the advantages of two separations of sample components. It can be applied to a wider range of samples than GC-MS,as analytes do not have to be volatile enough to pass through a GC column. Analysis of known analytes in simple mixtures can be performed in a matter of minutes without chromatographic separation or other chemical treatment to remove interferences. To obtain increased specificity,a gas chromatograph can be added to the MS-MS configuration.
GC-MS is still the method of choice for identification of unknown compounds in a mixture. Since comparison of unknown and reference spectra is required,the many variables and configurations of MS-MS are not necessarily advantages. Spectra must be obtained under the same conditions for comparison,and large MS-MS reference files do not yet exist. For the present,MS-MS is best regarded as a specialized form of MS that is suited for certain applications not amenable to GC-MS or difficult to solve by GC-MS alone.
Pyrolysis-Gas Chromatography
There are many complex substances of forensic interest that do not have sufficient vapor pressure at the normal operating temperatures of the gas chromato-graph (up to 300°C). These include hairs and fibers, paints,plastics,adhesives and other substances that are polymeric in nature. The technique of pyrolysis-GC can be conveniently used to characterize these substances.
A typical pyrolysis apparatus consists of a wand that contains a platinum coil or strip that can be heated under controlled conditions to temperatures exceeding 1000°C. The sample can be introduced directly on to the platinum strip or inserted into a quartz tube blocked at both ends with quartz wool. The tube is then inserted into the platinum coil. The wand is then introduced into the injector of the gas chromatograph,usually via a heated interface. A controller attached to the wand controls the heating rate,duration of heating and final temperature of the pyrolysis. Since the injector and interface are continuously bathed by an inert carrier gas,the analyte does not burn (no oxygen available). Instead it decomposes into simpler molecules. If the pyrolysis is performed under constant conditions and the sample size and topography are similar each time,then the resultant pyrogram will be quite reproducible with respect to the number and relative abundance of the fragmentation. Pyrolysis-GC is also sensitive to small changes in the composition of the polymers that make up the sample,so that similar substances such as nylon 6-11 and 6-12 can be distinguished.
Applications of Gas Chromatography to Forensic Science
There are a large number of applications of GC to forensic science. The use of pyrolysis with GC has served to extend these uses.
Drugs
Drug analysis was one of the earliest uses of GC in forensic science. All sorts of ‘street’ drugs can be separated and quantified by GC. A few,such as amphetamines,have to be derivatized if silicon columns are employed. Blood and other body fluids are also analyzed for drugs by GC after suitable extraction processes have been carried out.
Fire residues
GC is the only major method for the analysis of fire residues. After a suitable method of concentration is applied to free the accelerants from the fire residues, GC is used to separate the components of the accelerants. In the vast majority of cases,petroleum products are used as accelerants in fires and the peak patterns from the GC analysis can be used to identify the type of product (e.g. gasoline) that was employed in the fire.
Alcohol
With the advent of computers and autosamplers,the use of GC for the detection and quantitation of alcohol in drunk-driving cases,for example,has become widespread. This is a high volume operation at many toxicology laboratories and,in many cases, the samples are loaded in the evening and run all night. If blood is used,the headspace above the blood is often analyzed,thus avoiding an extraction process.
Trace evidence
Materials such as hairs,fibers,paints,plastics and other polymers are conveniently characterized by pyrolysis-GC. This technique is very sensitive to minute differences in the chemical content of such materials,and is quite reproducible,thus aiding in comparisons of known and unknown samples.
Perspectives
Looking to the future,it is reasonable to expect continued evolutionary development in:
• sample inlets,such as automatic solid phase injectors,which do not require preparation of the specimens (hydrolysis,extraction,concentration);
• column quality,with reduction of the internal diameter in order to increased efficiency and sensitivity;
• fast separation processes using higher head-column pressure;
• detector performances - continuous improvement in sensitivity and resolving power (faster acquisition).
