August 25, 2014

GC Analysis - Part IV. Retention Indices

Alexis St-Gelais - Popularization

I mentioned in the first post of this series on GC analyzes that PhytoChemia resorted simultaneously to two capillary columns to analyze essential oils, one polar (Solgel-Wax) and one non-polar (DB-5). The rationale behind this technique will reveal some of the art of interpreting essential oils profiles.

We have seen that the detection by FID gives an interesting estimate of the amount of a molecule in a mixture, but provides no information on its structure. How then can we identify it? Instead of using information from the signal itself, we rather refer to the separation behavior of the molecule during GC analysis.

As long as we use the same analytical method, a given compound will always be eluted at the same speed, and therefore have a relatively constant retention time. However, columns degrade over time, and different laboratories may use different methods. The best way to compare data between laboratories is to use a relative reference scale called retention index. To find the index, we first inject a series of reference molecules (called alkanes) using our current analytical method and capillary columns. Alkanes are unbranched carbon chains of different lengths, comprising between 8 to 36 carbons in the case of PhytoChemia. They will be eluted sequentially from the shortest to the longest chain length, following the increase of their boiling points. To the retention time of the 8-carbons chain (octane), a retention index of 800 is assigned. For the chain comprising 10 carbons (decane), a 1000 retention index is assigned, and so on up to 3600. 

Once this is done, we calculate the retention index for each of the molecules of an essential oil by comparing their retention times with those of the alkanes. For example, a compound eluting halfway between alkanes with 10 and 11 carbons will have an index of 1050. Regardless of the method used, these indices are relatively robust: β-pinene will always have the same retention index, around 965, provided that the column used is similar. Therefore, with a proper database and a literature survey, one can identify the molecules using their retention index on DB-5 column, which is the most widely used in the field of essential oils. We can find a large number of reference data in the flagship book of Robert P. Adams (1) or through the NIST website (2), again for DB-5 columns. 

There are however two problems if only one column is used. First, the retention indices are not 100% robust. Differences between methods, laboratories and analyzed matrixes can induce a change of several retention indices units (we have so far observed the β-pinene between 962 and 966, for example). Consequently, two molecules with close retention indices can be confused using a single DB-5 column, and the FID does not provide any more information to clarify the situation. Moreover, it so happens that more than one compound is eluted at any given time. A typical case in DB-5 is that of τ-muurolol + τ-cadinol, which are almost always superimposed on each other with a retention index of about 1635. It is thus impossible to know what proportion of the peak aera should be related to each compound, or even to check if one of them is missing.

This is where a second column proves to be useful. Since its selectivity is different, the Solgel-Wax column separates the compounds of the mixture in a distinct way. Retention indices of the molecules on the second column are thus different. Instead of relying on a single retention index from DB-5, PhytoChemia rather uses pairs of retention indices. Thus, a peak at 1635 on DB-5 will be assigned to τ-cadinol if visible at 2122 on Solgel-Wax, and to τ-muurolol if rather found at 2138. The use of pairs of indices greatly increases confidence in our identifications, and also frequently allows to solve the problems of co-elution in order to individually quantify the two compounds (Figure 1). 

Figure 1. Example of the usefulness of analysing with two columns in case of coelution. On the DB-5 column (above), the two compounds are severely superposed, which prevents their individual quantification. On Solgel-Wax column (below), both compounds are distinct. Using pairs of retention indices, it is possible to find which compound is which and to quantify them individually.
Interpreting a spectra can be a relatively complex task that requires experience (embodied in part in the database we built), vigilance and time. That is why it is important to deal with professionals. 

Retention indices should always be shown on your analysis reports, even if the analyst used a MS detector to identify the molecules (3). Indeed, several molecules can produce a quite similar mass spectrum. The use of retention index and literature often prevents misidentifications. A convincing identification in gas chromatography should always be based on the correlation of two elements: either the retention indices on two columns, or a mass spectrum coupled with a consistent retention index. 

(1) Adams, RP, 2007 Identification of Essential Oil Components by Gas Chromatography / Mass Spectrometry, 4th ed., Allured Publishing Corporation, 804 p. 

(2) National Institute of Standards and Technology, 2011. NIST Chemistry WebBook [Online]. "Standard Reference Data Program". URL: 

(3) Marriott, PJ, Shellie, R., Cornwell, C. Gas chromatographic technologies for the analysis of essential oils. J. Chromatogr. A, 2001, 936, 1-22.

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