Chromatography

Chromatographic Theory
IUPAC defines chromatography as “a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction”. The possible mobile and stationary phases can differ greatly, depending on the type of chromatography being performed and the nature of the samples being analysed. For instance, gas chromatography utilises a gaseous mobile phase and a solid or liquid stationary phase, whereas liquid chromatography uses a liquid mobile phase. These phases are contained within some kind of column. The mobile phase passes through the column, carrying the substance to be analysed.

Regardless of the finer details, the basic theory behind chromatography is that different components will interact with the mobile and stationary phase to a different extent, thus having a different retention time (relating to the time spent in the chromatography column) and so allowing the components to be distinguished from one another. Following the ‘like dissolves like’ principal, a substance that is similar to the stationary phase (for instance, both are of the same polarity) will dissolve into the stationary phase with greater ease and so will be retained for longer. Conversely, those substances that have a greater affinity for the mobile phase than the stationary phase will not be retained (or will be poorly retained) and will elute (leave the column) more quickly. Depending on the detection system used, chromatography can be both a qualitative and quantitative technique.

There are a number of measures of how effective a chromatography system is, factors that can be determined in order to establish how well components have been separated and detected. Resolution relates to the degree of separation between two components. For instance, if two components elute from the analytical column very closely together, they will be poorly resolved from one another. Selectivity is a factor that measures a chromatography column’s ability to discriminate between two components. Efficiency relates to the width of a chromatographic peak, with a narrower peak being much preferred. This is often measured in terms of Height Equivalent to Theoretical Plate (HETP). Certain mathematical equations have been developed to assess column efficiency, such as the Van Deemter equation and the Golay equation.

Planar Chromatography
Chromatography at its most basic level includes planar techniques such as thin layer chromatography (TLC). This involves the use of a thin layer of stationary phase (typically silica, alumina, cellulose or a gel permeation material) on a flat carrier sheet, onto which the sample is spotted. Analyte components are drawn through the matrix by capillary action at different rates, thus allowing their distance travelled to be measured in order to calculate their retention factor (Rf). UV light may be required to visualise the spots. In forensic science, planar chromatography may be utilised as a quick and inexpensive method of distinguishing between inks, dyes and drugs, though realistically more complex chromatographic techniques are better suited.

High Performance Liquid Chromatography
High performance liquid chromatography (HPLC) utilises a solid stationary phase housed inside an analytical column, through which a liquid mobile phase carries the analyte of interest. Typically columns contain a silica-based stationary phase, with different bonded substances depending on the type of analytes to be separated. This is equally the case with the type of mobile phase used (the liquid used to carry the sample through the column). For instance, normal phase HPLC will use a polar stationary phase with a non-polar mobile phase, whereas reverse-phase chromatography uses the opposite. HPLC instruments will be coupled with some kind of detector, commonly UV/Vis, fluorescence or mass spectrometry detectors. The purpose of this is to detect components of the sample as they leave the HPLC column, allowing the analyst to identify and quantify the components of the sample.

Gas Chromatography
Gas chromatography (GC) is a method of separating compounds in order to aid in the identification and quantification of a substance. There are numerous possible routes of injection of a sample, each of which may be considered depending on the type and amount of sample available for analysis. As the sample is introduced into the GC, it is vaporised and swept onto the analytical column by the carrier gas. The carrier gas is the mobile phase in gas chromatography, and is always an inert substance such as hydrogen, helium or nitrogen. The gas supply lines are often coupled with various devices to trap moisture, oxygen and hydrocarbon impurities that can interfere with samples and damage instruments, a feature particularly vital in forensic analyses. Within the column is a stationary phase – most commonly in gas chromatography a capillary column is used in which the stationary phase coats the inner walls of the column. As the analytes pass through the column, components will interact with the gaseous mobile phase and the stationary phase to a different extent, resulting in components remaining in the column for varying lengths of time depending on certain properties, thus having different elution times (the time taken for a component to leave the column and be detected).

The process of gas chromatography alone is not particularly beneficial, thus it is often coupled with some form of detector, which will allow for the eluting components to be represented by a peak in a chromatogram. The retention time of these peaks can be useful in identifying the compound, and the size (typically peak area rather than height) can be used in quantification. Common GC detectors include the flame ionisation detector (FID) and the mass spectrometer (MS).

Forensic Applications
As a very basic analytical test, planar chromatography can be applied to the analysis of inks and dyes. An analyst may need to establish whether two pieces of writing were written using the same ink, and chromatography is one way of, at the very least, acting as a presumptive test for this. The ink samples will be spotted onto the separation medium and allowed to travel through the matrix. Two different ink samples may travel at a different rate through the stationary phase, thus indicating that they may not be from the same source. The same principal can be applied to dyes. For example, banknotes may be marked with dye which, if this gets onto the hands of a criminal (eg a thief), chromatography may be used to indicate if the dye on the hands of a suspect is the dye used to mark the stolen money under scrutiny. Of course further analysis would be necessary to confirm this.

High-performance liquid chromatography is often utilised in the analysis of materials used to make explosives. If dealing with a substance that is suspected to have been used in the production of an explosive device, HPLC can be used to provide qualitative analysis to aid in the identification of the material. Different substances will travel down the HPLC column at a different rate, having unique retention times. These retention times can be used to identify individual components in a sample mixture. HPLC can also be used in drug analysis, as it was in the investigation of professional skier Alain Baxter, who was accused of drug use.

Perhaps the most confirmative chromatographic technique used in forensic science is gas chromatography or liquid chromatography coupled with mass spectrometry, which can allow for substances to be identified. Libraries of mass spectra may be used to aid the analyst in identifying a sample, along with their own expertise. The technique will often be used alongside other tests. For instance, in the analysis of suspected illicit substances, presumptive tests may be used to give the analyst a clue as to the kind of drug they are dealing with. But these tests do not confirm identity, thus LC-MS and GC-MS may then be used to provide a more certain identification.

 

Carlin, M G. Dead, J R., 2013. Forensic Applications of Gas Chromatography. Florida: CRC Press.

Wood, M et al (2006). Recent applications of liquid chromatography-mass spectrometry in forensic science. J Chromatogr A, 1130(1), pp. 3-15.