Gas chromatography (GC).
Theoretical principles
Gas chromatography (GC) is a highly efficient, sensitive method, which is used to analyse complex mixtures of substances. The procedure is described in European Pharmacopoeia Ph. Eur. 2.2.28. It is a chromatographic technique that uses gas as the mobile phase. The gas is routed through a column of a defined diameter containing the stationary phase, a highly viscous liquid. Nowadays most capillary columns are made of fused silica, have an internal diameter of between 0.1 and 0.5 mm and are between 5 and 60 m long. The inner column wall is coated with the stationary phase, whereby the film is 0.1 to 5 µm thick.
Hydrogen, helium or nitrogen is usually used as the carrier gas, whereby hydrogen offers significant advantages in terms of separating efficiency and analytical speed.
One basic prerequisite for gas chromatography is that the substance can be vaporised without decomposing - assuming that the substance is not available in gaseous form. Substances with inadequate volatility can be derivatised selectively to produce more volatile substances, such as trimethylsilyl derivatives. The sample can be introduced into the capillary column by directly injecting a liquid sample or by extracting from the gas space of a sample vial. The latter method, referred to as headspace gas chromatography, is a special technique which offers a means of detecting low concentrations of highly volatile substances in liquid or solid samples.
In its simplest form, the chromatographic separation of a mixture of substances takes place on non-polar separating phases as a result of the different boiling points of the individual substances in the mixture. The polarity of the separating phases can be varied or the phases modified to achieve an interaction between the analysed substance and the stationary phase in order to separate the substances. This means, for example, that enantiomers which have the same boiling point can be separated by virtue of the different degrees to which they interact with special cyclodextrin derivatives.
Having separated the mixtures of substances using a capillary column adapted to the respective separating problem, the separated components can be detected and identified using various detectors. The gas chromatogram enables identification and quantitation of the analysed components by comparing their retention times with those of the reference or calibration substances.
Gas chromatography can also be combined with mass spectrometry to produce what is referred to as the GC-MS method, which is capable of detecting extremely small quantities of a substance and obtaining information concerning its structure.
Our service
We use gas chromatography to examine volatile plant constituents, such as essential oil components, fatty acids, steroles, triterpene saponins, pyrrolizidine alkaloids and volatile components as defined by the German regulation on flavourings [Aromenverordnung] (thujone, asarone, estragole, methyl eugenol, safrole, coumarin) and the Cosmetics Directive 76/768/EEC (allergenic substances of natural origin). On the other hand, gas chromatography also constitutes an indispensable technique in the field of residue analysis. In this respect our analytical spectrum includes the quantitative analysis of solvent residues, pesticides, PCBs and wood preservatives (halogenated phenols), fumigants (ethylene oxide and phosphin). Another measuring technique that has become an essential element in the work performed at our laboratory is GC-MS, which enables us to conduct specific measurements, to confirm positive findings from contaminant analysis and to identify unknown substances.
Instruments and equipment
Our 20 gas chromatographs are equipped with the conventional injection systems, such as split/splitless, on-column, PTV, headspace sampler and purge and trap. We use the universal FID measuring technique for such specific detectors as FPD, NPD, ECD or MSD according to the respective application. Apart from 4 GC-MS units, our facilities have also included a GC-MS/MS unit for some time now. Analytical selectivity is improved several times by this enhanced tandem mass spectrometry technique (MS/MS). The selected mass fragments are fragmented again in a second stage, giving rise to highly specific signals for an isolated substance, and offering a means of masking out matrix disturbances.
Pesticide analysis
We use gas chromatography in conjunction with various selective detection techniques to analyse pesticides and we work in accordance with the extended and revised version of the DFG-S19 method, which is described as modular multiple method L-00.00.34 in the collection of methods as per § 64 of the German Food and Feed Code (LFGB. GC-ECD offers a highly sensitive method of measuring organochlorine pesticides and pyrethroids, and we use selective P-FPD to analyse the organophosphorus pesticides. GC-MS is used for the analysis of other classes of substances, as well as GC-MS/MS in special cases.
Release and stability testing of plant-based drugs
Many of the herbal raw substances used in finished herbal remedies contain volatile components, which can be determined by means of gas chromatography. We develop and validate gas chromatographic techniques for the batch release and stability testing of these products. They may include marker substances from the essential oil component category of substances, for example, or from the fatty acids, the phytosterols or from other triterpene compounds. When determining the fatty acids and phytosterins, derivatisation must be carried out prior to the gas chromatographic analysis in order to obtain sufficient volatile compounds.
Ph. Eur. methods
Of the gas chromatographic methods described in the European Pharmacopoeias (Ph. Eur), we perform the following analyses as part of our routine work:
- Identification and purity testing for essential oils and oil-based herbal drugs in accordance with the most diverse monographs
- Quantitative determination of fatty acids in accordance with method 2.4.22 or various monographs, such as saw palmetto fruit
- Quantitative determination of methanol and isopropanol in accordance with method 2.9.11
