Analytical Techniques:

Chromatography

Although spectrophotometry is exceedingly useful in the chemical identification of many organic materials, its optimum utilization requires that a material be in a relatively high state of chemical purity. Because the impurity levels of any given sample is often beyond the control of the analytical chemist, this criterion is often not met. For this reason, the analytical technique of chromatography is widely applied for the analysis of physical evidence. Chromatography is a means of separating and tentatively identifying the components of a chemical mixture. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture and allows it to be chemically isolated.

For example, illicit drugs and other controlled substances which are sold on the street are not manufactured to meet government labeling standards. Instead the may be “diluted” with practically any material that is readily available to the dealer in order to increase the quantity and associated street value of the product. Hence, the task of identifying an illicit drug preparation would be an arduous one without the aids of chromatographic methods to first separate the mixture into its components.  

The theory of chromatography has as its basis the observation that chemical substances have a tendency to partially escape into the surrounding environment when dissolved in a liquid or when absorbed on a sold surface. In short, the distribution or “partitioning” of a gas between the liquid and gas phases is determined by the solubility of the gas in the liquid. The higher its solubility, the greater the tendency of the gas molecules to remain in the liquid phase.

More importantly, if two different gases (analytes A & B) are dissolved in the same liquid, each will reach a state of chemical equilibrium independently of one another. Their solubilities will be determined solely by their respective solubilities in the liquid. 

The essence of chromatography is that some (but not all) substances will actually have a tendency to move in a specific direction over a stationary (or fixed) liquid phase in response to an applied flow. This motion is enhanced by forcing the motion of the air over the liquid.  

Interestingly, those components containing the highest number of molecules in the mobile gas phase (or present at higher number densities) will move more rapidly in response to the air flow. Eventually, when the entire gas phase (air + analytes) has advanced a reasonable distance, the component with the higher mobility will become entirely separated from the others.

The three different types of chromatography commonly used in the laboratory include: Gas Chromatography (GC), High-Performance Liquid Chromatography (HPLC), and Thin Layer Chromatography (TLC). We briefly outline those here.

Gas Chromatography (GC)

Gas chromatography (or gas-liquid chromatography) separates mixtures on the basis of their distribution between a stationary liquid phase and a mobile gas phase. A chemically inert carrier gas (e.g. nitrogen or helium) flows through a column made of stainless steel or glass. The liquid sample under investigation is injected into a heated injection port by syringe, where it is immediately vaporized and swept into the column by the carrier gas. As the carrier gas flows through the column, it carries along with it the components of a mixture that have been injected into the column. Components will be separated according to their relative mobilities (and corresponding solubilities) in the mobile gas phase.

As each component emerges from the column, it enters a detector. One type of detector uses a flame to ionize the emerging chemical substance, thus generating an electrical signal. The signal is recorded onto a strip-chart recorder as a function of time. This record is referred to as a chromatogram. 

A typical gas chromatogram will show a series of peaks, each one corresponding to a specific component of the chemical mixture. The time required for a component to emerge form the column from the time of its injection is known as the retention time. This experimentally determined parameter serves as a useful identifying characteristic of a material (tho it may vary for different instrument configurations).

Gas chromatography has an added advantage in that it is extremely sensitive and can yield quantitative results. The amount of substance passing thru the GC detector is directly proportional to the area underneath the recorded peak. The amount of sample can often be determined to within the nearest billionth of a gram.

One important extension of this application in forensic science is the technique of Pyrolysis GC. Many solid materials commonly encountered as physical evidence (paint chips, fibers, plastics, etc.) cannot be readily dissolved in a solvent for liquid injection. However, materials such as these can be heated or pyrolyzed to high temperatures (500 – 1000 degrees C) so that they will decompose into numerous gaseous products. Pyrolyzers have been designed in order to permit these gaseous products to enter the carrier gas stream, flowing into and thru the GC column.    

Liquid Chromatography (HPLC)

In the case of high–performance liquid phase chromatography (HPLC), the stationary phase is a finely ground particulate solid. The mobile (carrier) phase is a liquid that is pumped thru a column filled with fine solid particles whose surfaces have been chemically treated. The major advantage of HPLC is that the entire process takes place at room temperature. Examples of materials where HPLC may be the preferred method of choice may include organic explosives, which are generally heat sensitive. Also included are heat sensitive psycho-active drugs, such as LSD.

GC / MS

Many of the uncertainties associated with vapor phase chromatography (GC) have been overcome by linking the gas chromatograph to a mass spectrometer to yield an extremely powerful combination known as gas chromatography / mass spectrometry (GC / MS).

The separation of a mixture’s components is first accomplished on the gas chromatograph. A direct connection between the GC column and the mass spectrometer then allows each component to flow into the spectrometer as it emerges form the gas chromatograph.

In the mass spectrometer, the material enters a high-vacuum chamber where a beam of high-energy electrons is aimed at the sample molecules. The electrons collide with the molecules, causing them to lose electrons and to acquire a positive charge. These positively charged (+) cations are highly unstable and rapidly decompose into smaller fragments.

These fragments then pass through an electric or magnetic filed, where they are separated according to their masses. The advantage of this technique is that no two substances produce the same fragmentation pattern.   

Hence, this fragmentation pattern serves as a “fingerprint” of a specific chemical substance. It is also sensitive to minute concentrations. With the data obtained from a GC/ MS determination, a forensic analyst can, with one instrument, separate the components of a complex drug mixture and then unequivocally identify each substance present in the mixture. 

Thin Layer Chromatography (TLC)

The technique of thin-layer chromatography (TLC) incorporates a solid stationary phase and a mobile liquid phase to effect the separation of the constituents of a mixture. A thin-layer plate is prepared by coating a glass plate with a thin film of granular material, which is bonded to the plate with plaster of Paris.

Solid samples must first be dissolved and a few microliters of the solution spotted with a capillary tube or syringe onto the granular surface near the lower edge of the plate. A liquid sample may be applied directly to the plate in a similar manner. The plate is then placed upright into a closed chamber that contains a selected liquid, with care that the liquid does not touch the sample spot.

The liquid will slowly begin to rise up along the surface of the plate by capillary action. The rising liquid serves as the mobile carrier phase. As it moves past the sample spot, the components of the sample become distributed between the stationary solid phase and the mobile liquid phase. Those components with a greater solubility in the mobile liquid phase will travel up the surface of the plate at a faster speed. When the liquid front has moved a sufficient distance (usually 10 cm) the development is complete, and the plate is removed from the chamber and dried.

Because most components are colorless (exceptions: organic pigments such as chlorophyll and beta-carotene) no separation will be noticeable after development unless the materials are visualized. To accomplish this, the plates are placed under UV light, revealing those materials that fluoresce as bright spots on a dark background. For identification purposes, a high confidence level necessitates the use of experimental standards which can be compared to the results for confirmation of the suspect material’s chemical composition.  

Migration distances must therefore be matched with similar experiments on similar compounds using similar experimental configurations and apparatuses. If the distances are identical, then a tentative identification can be made. However, it must be cautioned that such identification cannot be considered definitive for the possibility always exists that numerous other substances can migrate the same distance up the plate when chromatographed under similar conditions. Thus, thin-layer chromatography cannot by itself provide an absolute and undisputed identification. Thus, TLC must be used in conjunction with other testing procedures in order to provide absolute proof of chemical identity.