Gas chromatography- mass spectrometry (GC/MS)
Which compounds are suitable for the GC/MS analysis?
The analysis of mixtures of volatile and low relative molecular mass compounds (m/z< 800) such as hydrocarbons, fragrances, essential oils and relatively non-polar drugs. Chemical derivatisation, eg 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.
What information does GC/MS provide?
The instruments in the MS Facility can operate in two different modes, full scan and selective 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 library searching is a very useful and timesaving technique, it is important to remember that such searches do not identify compounds - analysts do!
Quantitative work can be performed by integration of selected ion chromatographic peaks. 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.
What sensitivity may be achievable
Depending on the analyte, low picogram to nanogram amounts can be measured using this powerful technique.
How many samples can be run in a day?
The run time per sample is typically 15-60 min depending on the nature of the sample and the GC method used. The column temperature can be kept constant (isothermal) or may be programmed ("ramped") to increase at a predetermined rate, usually linear. In a programmed method, about 5 min has to be allowed for cooling between injections. The autosampler has capacity of 100 samples. When calculating sample numbers, calibration samples, quality control samples, blanks and wash samples have to be included in some cases.
What is sample preparation?
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 Trimethylsilated with a variety of commercially available compounds.
How much sample will I need?
GCMS – the autosampler is capable of injecting 0.1-10 μL, so at least 10 μL of sample will be required. A typical injection volume is 1 μL, minimum of 20-50 μL of sample is required. Generally, no more than a microgram of dissolved material should be injected, depending on the capacity of the particular column. Overloading will cause the separation to deteriorate and can lead to serious contamination of the instrument. Concentrated samples can be diluted prior to injection or subjected to a “split” injection where only a specified proportion of the sample enters the column.