June 30, 2014

GC Analysis - Part I. Separation Principles

Alexis St-Gelais - Popularization

I have explained in a previous post the main theoretical principles of chromatography. Today, I will detail one of its practical applications, widely used by PhytoChemia: gas chromatography (GC). It lends itself to the analysis of volatile molecules - that is to say molecules capable of passing to gas phase at reasonable temperatures (typically less than 325 °C) without being degraded. Essential oils are therefore particularly suited to GC analysis, as they are by definition composed almost exclusively of volatile substances.

As all chromatographic techniques, GC requires a mobile phase and a stationary phase. The stationary phase is found usually under the form of a capillary column (although packed columns also exist, I will not discuss it here). The capillary column is a very thin hollow metal tube, typically 30 to 60 m long, with a thin film of stationary liquid coating the inner surface. This liquid constitutes the stationary phase. The mobile phase, in GC, is a stream of gas (helium or hydrogen), called carrier gas, flowing through the capillary column. At the exit of the column, a detector which can take different forms is found. This important aspect will be covered in further entries of this blog.

Figure 1. Inside of a GC apparatus, with the hollow capillary column within the high-precision oven.
The principle of separation GC is largely based on temperature. Most of the volume of the apparatus is in fact a high-precision oven, wherein the capillary column is placed (Figure 1). When a sample is injected, it is vaporized by a heater and carried away by the gas to the beginning of the capillary, which is typically held at a relatively low initial temperature (e.g. 40 °C). Molecules with a high boiling point will then interact strongly with the stationary phase and progress through the capillary tube at low speed, while those that are more volatile will remain in the gas phase and be transported by the carrier gas more rapidly towards the outlet of the column. In the case of essential oils, we gradually increase the temperature of the oven, and less volatile molecules progressively lose affinity with the stationary phase to finally elute out of the column. Thus, the typical elution order obtained is as follows: monoterpenes, oxygenated monoterpenes, sesquiterpenes, oxygenated sesquiterpenes, diterpenes. This matches quite well the trend in boiling points for these classes of molecules.

Another parameter affects the separation: the type of stationary phase. Various different liquids can be used as a stationary phase, with increased or decreased affinity for certain molecules. In the case of PhytoChemia, two capillary columns are used simultaneously, one apolar (DB-5) and the other polar (Solgel-Wax). While overall, the fact that compounds with lower boiling points elute earlier remains true, the resolution of the separation and exact elution order between the two columns vary due to the different interactions between the molecules of the mixture and the stationary phase. Oxygenated molecules are for instance more strongly retained on a polar column. The use of two columns is interesting for a few reasons, and I will introduce this aspect in another entry.

Overall, GC is an effective chromatographic analytical method, which provides excellent separations of somewhat complex mixtures in a reasonable amount of time. Its main drawback is that it is not suitable for all mixtures, since the analyst is always dependent on the volatility and thermal stability of the molecules being studied. That being said, GC is definitely the tool of choice for the study and quality control of essential oils and related products as offered by PhytoChemia.

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