Gas chromatography (GC) is a technique used to separate, identify and quantify analytes in a mixture. A gas chromatograph consists of an injector, column (packed or capillary), oven, and detector. The main manufacturers of gas chromatographs supply the following types of detectors: micro-electron capture detector (micro-ECD), flame ionization detector (FID), flame photometric detector) (FPD), mass selective detector (MSD), nitrogen chemiluminescence detector (NCD), nitrogen-phosphorus detector (NPD), sulfur chemiluminescence detector (SCD). When the gas chromatograph is coupled to a mass selective detector, the system is known as GC-MS. GC-MS is widely used in modern laboratories for research and analytical purposes. Manufactures provide sophisticated MS detectors equipped with triple quadrupole systems that can detect compounds at femtogram levels. However, this type of GC-MS apparatus is very expensive. A femtogram level sensitivity is not essential for all types of analyses, and researchers and analysts can thus use GC-MS systems that are equipped with single quadrupoles or ion traps that are cheaper and give nano to picogram sensitivity.
Sample preparation
A sample is very rarely directly injected into a GC-MS system. An aliquot of the sample is extracted with organic solvents such as methanol, hexane, dichloromethane and ethyl acetate. Solid samples are usually ground to ensure that analytes are quantitatively extracted. Thorough mixing is essential for heterogeneous samples. The extract is dried over anhydrous sodium or magnesium sulfate, and the solvent evaporates on a rotary evaporator. In case there are interfering compounds, the extract is redissolved in a small amount of solvent for subsequent clean-up (silica gel, florisil or other cartridges are normally used). The residue is dissolved in an appropriate solvent and transferred into a vial for GC-MS analysis. For quantitative analysis, loss of extract should be avoided at all stages of the sample preparation and the volume of the reconstituted sample should be properly noted. The reconstituted sample must be properly sealed in vials, and exposure to bright light and temperatures must be avoided. Reconstituted samples are normally stored in a refrigerator when GC-MS analysis cannot be done immediately.
Mode of operation of a GC-MS instrument (Kemp, 1991; University of Colorado, 2011)
The sample is manually or automatically loaded on the injector that is kept at a fairly high temperature. The analytes vaporize at a high temperature and enter a heated capillary or packed column. In the column, there are non-covalent interactions between the analytes and column materials. A carrier gas such as helium carries the analytes through the column. In principle, separation of compounds takes. The volatility and polarity of compounds, column temperature, column packing polarity, a flow rate of gas, length of the column all affect the separation of compounds and their retention times, (RT, the time it takes for an analyte to travel from the injection port to the detector. Volatile components travel faster through columns, that is, they have shorter retention times. Polar column retain polar compounds, while non-polar compounds retain bon-polar ones. Compounds that have similar physical and chemical properties do not separate very well, hence a proper temperature programming is required to separate them. Sometimes longer columns are used to separate compounds in complex mixtures. Compounds that co-elute do not give clean spectra. Once separated, the compounds enter the mass spectrometer in the gaseous phase. When the compounds enter the ionisation chamber, they are bombarded by electrons (electron ionization, it is more frequently used because it gives more structural details) or charged ions (chemical ionisation). At this stage, molecular ions, also known as parent ions, are produced. However, due to the high energy content of the molecular ions, a fragmentation occurs, and smaller ions (daughter ions) are produced. The parent and daughter ions consecutively pass through electrostatic and magnetic analysers. The electrostatic analyser separates the positively and negatively charged ions, whereas the magnetic analyser separate the ions based on their m/z values. The resolution of the ions is greatly dependent on the technical features of these analysers. At the final stage, the ions reach the detector-recorder that measures the intensities of the ions. For every compound that passes through a mass spectrometer, a chart of relative abundance versus m/z is plotted. This chart is known as a mass spectrum and is unique to a particular compound. Ions that have elements such as chlorine give two peaks for every fragment due to the presence of two stable isotopes. Ions that contain a large number of carbon atoms clearly show signals corresponding to carbon-12 and carbon-13 isotopes. For small fragments, the signals corresponding to carbon-13 are not clearly visible. Stereoisomers and diastereomers give similar MS spectra, and caution must exercised in terms of assignment of configuration. An analyst must use other techniques to confirm the configuration of stereoisomers and diastereomers.
Analysis of difenoconazole and lambda-cyhalothrin by GC-MS
Various pesticides are used to control weeds, insects and fungi in plants, fruits and vegetables. However, when pesticides are used excessively and incorrectly, there is a danger of getting an accumulation of these chemicals in plant materials and higher up in the food chain, which in turn constitutes of a health hazard. A proper monitoring is hence required to control the quality of food products. The quality monitoring process has to be systematic and unbiased, and involves quantitative extraction of the pesticides, cleaning and analysis by GC, GC-MS, high performance liquid chromatography (HPLC), HPLC-MS and (ultraviolet) UV spectrophotometry (The Royal Society of Chemistry, 1991; Alder, Greulich, Kemper, G and Viett, 2006).
Many research and analytical laboratories use the GC-MS technique to screen pesticide residues in food products. Difenoconazole (C19H17Cl2N3O3, MW of 406.30) and lambda-cyhalothrin (C23H19ClF3NO3, MW of 449.8) are two such pesticides that can be analysed by GC-MS. In two published reports (Siecke, 2011; Whitney and Lyle, 2011), these two compounds were extracted from fruits and vegetables by organic solvents and analysed by GC-MS. They were both detected at concentrations that were below ppm levels. The extraction and analytical procedures maintained the integrity of the products, and the GC-MS technique was found to be appropriate. The GC-MS technique can be also used to analyse natural organic compounds that have been extracted from plants, fruits, vegetables, and marine and other biological samples.
Advantages and disadvantages of a GC-MS technique
Difenoconazole and lambda-cyhalothrin can be also analysed by other techniques such high performance liquid chromatography, HPLC-MS and UV spectroscopy. However, high performance liquid chromatography and UV spectroscopy cannot confirm the identity of a compound. Moreover, solvents are not required to run a GC-MS apparatus, that is, a minimum amount of waste is generated, and the negative impact on the environment and analyst is minimized.
The disadvantage of GC-MS instrument over high performance liquid chromatography and UV systems is that it is more expensive.
References
Alder, L., Greulich, K., Kemper, G and Viett, B., 2006. Residue analysis of 500 high priority pesticides: better by GC-MS or LC-MS/MS? Mass Spectrometry Reviews, 25, pp. 838-865.
Kemp, W., 1991. Organic Spectroscopy. 3rd ed. Hong Kong: The Macmillan Press Ltd.Siecke,C., 2011. Lambda-cyhalothrin (146).
The Royal Society of Chemistry, 1991. The Agrochemicals Handbook. 3rd ed. Cambridge. The Royal Society of Chemistry.
University of Colorado, 2011. Gas Chromatography. Web.
Whitney, W. and Lyle C. R., 2011. Rapid Multiresidue Pesticides Analysis Using Fast-GC/MS and Chromatogram Deconvolution Software. Web.