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[23] What do Chromatograms tell us? Peak elution has changed while I was using a Similar column and Similar conditions..

27 Oct 2013

Chromatograms are like fingerprints.  If you can “read” chromatograms well, you often can find a plausible cause. In this series, we will show a series of GC-chromatograms that are obtained from users and discuss some potential causes for the phenomena. Then we can move into some solutions for improvement.

The two dioxin chromatograms of the tetra isomers shown in fig 1 shows a different peak elution order. Customer was thinking he was buying a similar column, with similar film thickness. The 2,3,7,8 isomer on column 1 elutes BEFORE the 1,2,3,9 and on the new column 2 AFTER the 1,2,3,9 isomer. The mass spectrometer does not detect the difference as all tetra isomers will have the same mass spectrum.   It was discovered by running some standards.

Fig. 1 Two columns, both "5" type, produce different dioxin elutions..

This is a problem that many times can happen as the two phases used were not the same.  It is caused by a difference in stationary phase structure.  In figure 2 the phase structures are added and the Restek phase names are added also.

The sil-phenylene type phase, where the phenyl group is not pendant, but integrated in the main chain, will form a more robust polymer backbone.  The result is that such phases have increased thermal stability and have reduced bleed. Both phases can be used for similar application fields, however the chromatograms show completely different. As seen with dioxins, some peaks even may swap. Both phases are considered “5” type, but are very different.

Fig 2. same as fig 1 but now with phase composition structures

Fig. 3 shows a comparison of pesticides using similar column dimensions and same temperature program. Also here complete different chromatograms (ad RI values) are obtained

Several blogs have been published by my colleague, Jack Cochran, who has run many times into this challenge, see here some more examples

Pesticides:  Selectivity differences in arylene GC phases...pesticides and zebras.; Maybe a rose is a rose is a rose, but a “5” is not a “5”, when it comes to pesticide analysis...

Drugs : Selectivity differences for drugs of abuse when using Rxi-5ms and Rxi-5Sil MS GC columns: pendant diphenyl versus silarylene stationary phases.

Fig.3 When more peaks are present, differences between phases are much easier to observe..

This is the reason why Restek makes 2 separate phases available: The Rxi-5ms, which is a 5% phenyl 95% methyl polysiloxane, and the Silphenylene stabilized phase, called Rxi-5Sil MS. 

If the same peak elution is preferred, it is important is to choose the right stationary phase, see fig 4. If a phase is not listed, make sure you find the structure, so you can choose the correct phase and have the same peak elution order. Fig. 5 shows an example for a highly complex sample: choosing the correct Rxi-equivalent will provide the same elution order besides having a lower bleed and improved peak shape.Blog23-fig4
Fig.4 Phase equivalents to secure similar elution order

If there is freedom of choice, the Rxi-5Sil MS is always the preferred. It is more robust because of the Sil-phenylene structure, and has lower bleed. 

Peak elution for the same column can also change in other situations, which will be discussed in other blogs.

Biggest impact is seen when elution temperature changes.  The elution temperature can have a big effect on peak elution order.  This can happen when there is a change of:

  • Oven temperature program;
  • Column linear velocity or flow;
  • Carrier gas;
  • Film thickness or column length;
    Fig 5 when same phase composition is chosen, even very complex samples will show the SAME elution order..

Note that also an existing column can “change” in time. If the phase is stripping off, the film reduces and elution temperatures will change. I have also seen that accumulation of matrix causes the column to separate differently.