Gas chromatography (GC) and mass spectrometry (MS) have been combined to become a popular way of analyzing mixtures in food science, forensics, and other research.
Image Credit: Pratchaya.Lee / Shutterstock.com
GC and MS provide distinct but complementary results; while GC separates components of a mixture, MS can analyze and identify these components. These methods were first used in tandem in the 1950s, and are still widely applied in clinics and laboratories worldwide.
GC-MS Chromatogram or Spectrum Development
As expected, the GC step comes before MS. For GC, the mixture of interest must be in gaseous form, so the procedure must begin with the conversion of the sample to gas, if necessary. Once in gaseous form, it is passed through a column, also called the “stationary phase”.
The column is lined with material to differentially attract various components of the gas mixture, thereby separating them. They then eluate (emerge from the column) at different times, and are ionized for the MS analysis. These combine into what is shown as peaks on a GC-MS chromatogram.
The chromatograms are shown as graphs, with the X-axis showing “retention time”, and the Y-axis showing “intensity counts”. The retention time is the time it takes for the component to reach the detector at the end of the column. This makes the results vulnerable to the settings used in the column, such as flow rate and injection temperature.
The same parameters must therefore be used, and comparison between different labs or analyses is difficult. MS also analyses the mass to charge ratio, which is also added on as an X-axis component. Therefore, the output of GC-MS can be depicted as a chromatogram, with retention time on the X-axis, or as a spectrum, with mass to charge ratio on the X-axis.
The Y-axis, showing intensity counts, is a measure of how much quantity of the component is present. The area of the peak will reflect how many counts the detector did when the sample reached the MS detection area. This can also give misleading results as different analytes can have different affinities for the detector.
Higher affinity will lead to a larger peak area, which can give a false indication of the amount present. However, this can be easily solved by running standard curves with known concentrations of analytes.
The GC and MS components of the analysis provide different bits of information. Retention time data provided by the GC forms a way of identifying the chemical properties of the component. These can be polarity, volatility, and even whether certain functional groups are present or not.
MS provides an indication of the amount of the component or analyte present, as well as its molecular weight through the electron ionization mass spectra. The molecular weight is indicative of atomic composition, which can be of use when deciding how many atoms of a component are present in the molecule.
In certain cases, the composition of the molecule can be pieced together from the GC-MS results. Many organic compounds will fragment at the MS ion source. These fragments are then read individually for mass. Measuring these fragments can indicate how a molecule’s components are connected.
When analyzing data from a mass spectrometry spectrum, two key concepts arise parent ion peak and base peak. The parent ion peak, which is also called the molecular ion peak, is the peak that stems from the loss of an electron from the molecule. This represents the mass of the original molecule, since the mass of the electron is so small it is essentially the same as molecular mass.
The base peak is the largest peak in the spectrum, which indicates the most abundant component. The base peak is almost always the most stable ion in the mixture because the abundance is also an indication of ionic stability in the source. The base peak and the parent peak can occasionally be the same.
There have been occasional reports of misidentification of the components using GC-MS. GC-MS is commonly employed in confirming drug test results from immunoassays, where urine is screened for the presence of drugs such as opiates. Pholcodine is an over-the-counter cough drug commonly available worldwide.
The structural difference between pholcodine and morphine is small, consisting of only a side chain. Because of this, it has been known to interfere with immunoassays used in drug testing, and can potentially give false positives for opiates. GC-MS has also been shown to generate false positives for opiates when presented with samples containing pholcodine.
When tested, the concentrations also exceed the UK workplace guidelines threshold, and the EU suggested threshold, meaning it can have profound effects on people’s lives. The moral is that caution is needed when interpreting results, especially in clinical cases.