Alexis St-Gelais – Popularization
The first part of this series dealt with the principles of separation of volatile molecules upon analysis by GC. After their separation, the analyst has yet to be able to detect molecules as they leave the capillary column, either to identify or quantify them. At PhytoChemia, we mainly use two types of detectors to do so: the flame ionization detector (FID) and the mass spectrometry detector (MS). This post focuses on the first of these detectors.
The FID is one of the simplest detectors used in organic chemistry. It relies on a flame generated by the combustion of a flow of hydrogen mixed with ultrapure air. The carrier gas coming out of the capillary column passes through the flame. When an organic molecule carried by the gas reaches the flame, it is oxidized and generates electrically charged ions. Electrodes near the flame measure the electrical current generated in real time, which is plotted on a graph called a chromatogram (Figure 1). This animation from Chromedia allows to visualize the process.
As a detector, the FID is relatively universal, which is a very useful property. Indeed, almost all organic molecules that are suitable for GC analysis will oxidize and produce an electric current. Only very small molecules (e.g. formaldehyde) or those that are not prone to oxidation by ionization will not give good results in FID. In short, in the case of essential oils, for example, all the compounds in the mixture produce an electrical signal.
The FID is also very sensitive. The quantity of essential oil that we inject for analysis at PhytoChemia is thus very small. Taking into account all the parameters of injection, just over 0.2 nanoliter of essential oil is introduced in each capillary column. And we are able to detect compounds representing less than 0.05% of that sample!
The FID has another useful advantage for the analysis of essential oils. The more abundant a molecule is, the more fragments it generates. The recorded electrical signal is thus relatively proportional to the concentration of the compound in the starting sample. It is therefore considered, by convention, that the total recorded electric current (all peaks in the chromatogram) represents the total number of molecules of the essential oil. The proportion of the total signal that each peak represents is the estimated concentration of the corresponding molecule in the sample (1,2). That is why our reports always refer to percentages. This convention avoids having to establish a calibration curve for every analysis and for each compound.
We must be aware that this is an approximation, not an absolute quantification. The response factor* of each analyzed molecule varies anyhow (although to a lesser extent for structurally related compounds, such as non-oxygenated monoterpenes (3)). I will come back to this aspect later on. That being said, for most essential oils norms (1) and for analysis certificates in the volatile products sector (2), as well as in the majority of scientific publications in related fields, the approximation “concentration ≈ % of total integration” is the way to go.
In short, the FID is an effective detection method, which is both sensitive and polyvalent. It is particularly indicated for the analysis of volatile natural compounds.
*The response factor is the constant by which the signal recorded by a detector must be divided to quantitate a molecule in mass units. For example, if 2.0 µg of a molecule passing through the detector producdes a signal of 50000 units (absorbance of light, electric current, etc.), the response factor of 25 000 units/µg.
(1) ISO 11024-2:1998(F). « Directives générales concernant les profils chromatographiques – Partie 2: Utilisation des profils chromatographiques des échantillons d’huiles essentielles », via AFNOR.
“Using the information provided by the data analysis system used for chromatogram B, according to the area normalization quantification method (internal normalization method, as of ISO 7609), ensure that the concentrations (considered as being equivalent to the percentage of total signal corresponding to the peaks of interest) or the concentration ratios fall between the minimal and maximal values indicated under the “Chromatographic profile” heading of the norm corresponding to the studied essential oil.” [Translated by author]
(2) IOFI Working Group on Methods of Analysis, 2011. Guidelines for the quantitative gas chromatography of volatile flavouring substances, from the Working Group on Methods of Analysis of the International Organization of the Flavor Industry (IOFI). Flavour Fragr. J. 26, 297-299.
(3) Raffa, K. F., Steffeck, R. J., 1988. Computation of response factors for quantitative analysis of monoterpenes by gas-liquid chromatography. J. Chem. Ecol. 14 (5), 1385-1390.