Gas chromatography-mass spectrometry (GC/MS)
Gas chromatography-mass spectrometry
Gas chromatography-mass spectrometry (GC/MS) combines the fine separating power of GC with the uniquely powerful detection capabilities of MS.
GC/MS with capillary column separation and electron ionisation (EI) is available within the Mass Spectrometry Facility.
This powerful technique is particularly suitable for the analysis of mixtures of volatile and low relative molecular mass compounds (< 800) such as hydrocarbons, fragrances, essential oils and relatively non-polar drugs. Chemical derivatisation, e.g. trimethylsilylation, can often be employed to increase the volatility of compounds containing polar functional groups (-OH, -COOH, -NH2, etc) thereby extending the range of suitable analytes to such compounds as steroids, polar drugs, prostaglandins, bile acids, organic acids, amino acids and small peptides.
A GC/MS analysis will usually consist of the following steps:
Samples such as water, soil, urine, blood plasma, etc., have to be subjected to a "clean-up" procedure prior to analysis in order to extract and concentrate the more volatile, low molecular mass components. Extraction can be performed by organic solvents or by solid phase extraction (SPE).
Where considered necessary, the extract can be derivatised with a choice of special reagents. For example, thermally labile and polar carboxylic acids groups can be methylated with BF3
/methanol (or TSM-diazomethane) or trimethylsilated with a variety of commercially available reagents.
Typically, a solution of the analyte mixture (1 uL, containing perhaps several hundred nanograms of material) is injected on to the GC column via a heated injection port. As the port is normally held at 250-300 C to facilitate vaporisation, the GC/MS technique might be less suitable for the analysis of thermally labile components that can decompose prior to separation. Derivatisation can offer some protection, but the analyst should be well aware of such dangers.
Following volatilisation in the heated injector, the mixture is pushed by a pressurized carrier gas (usually helium) through the GC column, which is heated in an oven. The column has an inner coating of a special liquid (commonly a silicone) - hence the proper name GAS-LIQUID chromatography. The separation relies on the fact that different compounds dissolve to different extents in this liquid and move through the column at different rates depending on their partition between the stationary liquid and the mobile carrier gas.
The degree of separation of the components depends on many factors including the nature of the sample, carrier gas type and flow rate, column type, dimensions and stationary phase, and the temperature of the column.
The column temperature can be kept constant (isothermal) or may be programmed ("ramped") to increase at a predetermined rate, usually linear.
The detection method currently available in the facility is electron ionisation (EI). The MS detector can operate in 2 different modes: scanning and selected ion monitoring (SIM).
The scanning mode provides a fairly reproducible mass spectral fragmentation pattern ("fingerprint"). Mass spectra are recorded (scanned) at regular intervals (typically 0.5 - 1 per second) during the GC separation and stored in the instrument data system for subsequent qualitative or quantitative evaluation. From such patterns, it is often possible to deduce structural features (mass spectral interpretation) but this requires experience and can be very time-consuming, particularly as a complex mixture might contain hundreds of components.
Such "fingerprints" can also be compared with those stored in a standard database (mass spectral library) and several important databases are currently available in the Facility to assist with problem solving. Although considerable care must be exercised in interpreting the results of such comparisons, this is often a useful technique in helping to identify unknowns, or to quickly eliminate known compounds from further consideration.
Although library searching is a very useful and timesaving technique, it is important to remember that such searches do not identify compounds - analysts do!
The additional measurement of the GC retention index, related to the time it takes a component to travel through the column, provides an additional parameter and is becoming increasingly important in identification work. Software is available in the Facility to assist with these measurements.
Quantitative work can be performed by integration of selected ion chromatographic peaks. Software is available for this purpose.
Selected ion monitoring (SIM)
SIM is much more sensitive technique for trace quantitative analysis. Here, instead of scanning a whole spectrum, only a few ions are detected during the GC separation. This can result in as much as a 500-fold increase in sensitivity, at the expense of specificity. Stable isotope-labelled internal standards can be employed. Again, software is available for such analysis. Depending on the analyte, low picogram to nanogram amounts can be measured using this powerful technique.