Home wanted for cute, little peaklets: Combining many peaklets into single peaks in 2D GC

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  • Published: Oct 3, 2011
  • Author: Jon Evans
  • Channels: Laboratory Informatics
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Double separations

An effective and popular way to analyze complex samples is to separate them twice, using a slightly different separation mechanism each time. In such 2D separations, analytes that aren't separated during the first separation can often be separated during the second separation.

Now, this process may be fairly simple with electrophoresis, but it's quite a bit trickier with chromatography. In 2D gel electrophoresis, analytes are first separated on a strip according to their isoelectric point and then deposited on a gel where they are separated according to their mass. In 2D chromatography, analytes eluting out the end of a chromatography column with one kind of stationary phase are then sent down a chromatography column with a different stationary phase.

The process of transferring analytes between chromatography columns is a lot more complex than transferring them from a strip to a gel, with the analytes needing to enter the second column in the same order they elute from the first. In practice, this means repeatedly collecting the eluent from the first column, concentrating it and then injecting it into the second column.


Pesky peaklets

As a result, the same analyte may be split between sequential eluent packages, entering the second column several times and so producing multiple peaks, or peaklets, in the resultant chromatogram. Furthermore, because it usually makes sense just to place a detector at the end of the second column, only a single chromatogram is generated by the analysis.

Thus, even though the analytes are separated in two dimensions, the resultant chromatogram only displays them in a single dimension of time. In 2D gel electrophoresis, by contrast, the analytes are physically separated in two dimensions, with the analytes spread all over a square gel.

To get around these complexities, analytical chemists utilise software packages to combine the peaklets into single peaks representing individual analytes. What is more, these packages also transfrom the single, 1D chromatogram into a 2D chromatogram reflecting the separation by the two columns, based on knowing exactly when each of the eluent packages was inserted into the second column. The end result is a chromatogram version of an electrophoresis gel, with peaks representing single analytes spread across a square chromatogram.

Although effective, a limitation of these software packages is that they still need a fair amount of human direction to accurately combine the various peaklets into individual peaks. Now, however, Leo van Stee and Udo Brinkman at the VU University in Amsterdam, the Netherlands, have developed a software program, called 2DAid, that can do all this without any human involvement at all.


Complementary combination

The program uses two complementary methods to combine peaklets into a single peak. Based on analyte retention times, it first predicts peak shapes that should be present in the 2D chromatogram, and then combines all the peaklets in the region of the 2D chromatogram covered by the predicted peak shape. Next, it refines its findings by trying to find the identity of an analyte from its largest peaklet, based on matching with spectral libraries, and then seeing whether other peaklets in the region also match that analyte.

Van Stee and Brinkman tested 2DAid on two samples separated by 2D gas chromatography with mass spectrometry: a mixture of 34 anilines and phenols, and a mixture of 362 analytes belonging to a wide variety of compound classes. In both cases, the initial analyses produced 1D chromatograms consisting of thousands of different peaklets.

Using 2DAid, the two scientists were able to combine these thousands of peaklets into a few hundred peaks. Seeing as there were still many more peaks than known analytes, most of these peaks merely represented impurities and noise. To remove this noise, the scientists simply filtered out the smallest peaks. This eventually left behind 40 peaks for the first sample, including all the known analytes, and 450 peaks for the second sample, accounting for 350 of the analytes in the sample.

By removing any human involvement, 2DAid also worked very quickly, only taking around 20 seconds to combine all the peaklets into 2D peaks.


 


The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

Home wanted for cute, little peaklets: combining many peaklets into single peaks in 2D GC

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