Wilkes University - CHM D Gas Chromatography (or 3D with MS Detector) A powerful separations tool for complex volatile mixtures of heat-stable samples [information shamelessly taken from Wikipedia and other sources – see last slide]

History Comprehensive 2-D gas chromatography (GC)  (abbreviated GC X GC)  a mature technique with commercial products on the market  technique has tremendous separation power  uses simple robust hardware  similar analysis times to temperature-programmed high- resolution capillary chromatography Comprehensive 2-D LC (LC X LC)  still in its infancy  more complex to perform  driven by user needs, especially in proteomics

The Concept additional physico-chemical criterion employed for the separation of the mixture of analytes (sample)  resolution and quality of chromatographic separation can be increased  higher specifity of the separation capability is obtained  separation of compounds indistinguishable by 1-D chromatography Gas-Phase Chromatography, 2-D [an illustrative example]  coupling a second, short column to the first long column  shock-freeze eluents in order of elution from the 1 st column  reheat them in order of elution for release into the 2 nd column  transit time through the 2 nd column needs to be shorter than the time until the next sample is reheated & released Gas-Phase Chromatography, 2-D [an illustrative example]  one column is used to separate analytes  followed by Time-of-Flight Mass Spectrometer (TOFMS) detection as the second dimension  TOF-Mass Spectrometers used in gas chromatography can be very short [ due to the limited range of m/z required]

The Instrument - Background

The Instrument Requirements two pieces of hardware are added to a conventional GC  a second column  an interface between the second column and the first (the modulator) the time for analysis on the second column is very fast (short column) the interface repetitively samples the effluent from the first column, and injects it onto the second

The Instrument Requirements R t on the two columns may be thought of as lying perpendicular to one another 2 nd column stationary phase different from the 1 st  retention mechanism different from that of the 1 st  retention mechanism are “uncorrelated”, “independent”, or “orthogonal”  molecules are separated on the basis of independent chemical properties in the 1 st and 2 nd columns Ex. 1 st column separates based on the basis of “molecular size” (volatility/b.p.) 2 nd column separates on the basis of polarity  the molecular property “polarity” is largely independent of the molecular property “size,” or volatility  Polar funct. groups can be attached to compounds of any size

The Instrument Requirements Modulator performs 3 tasks (in repetitive cycle):  accumulates sample eluting from 1 st column period of time equal to 1/3 to 1/5, of the duration of an individual peak from the first column Ex. a first column peak is 9 seconds wide at the base: modulator will accumulate material every 2 (or 3) seconds, thereby “chopping” the peak eluting from the first column into “cuts”  focuses the material collected from each cut into a narrow “band”, “plug”, or “chemical pulse”. by flash-freezing (with, for ex., a cold jet of CO 2 )  “launches” or injects the sharp chemical pulses sequentially onto the second column a series of high speed gas chromatographic separations occur one separation for each chemical pulse launched onto the second column

Data interpretation each vertical column of the image may be integrated and plotted as a function of 1 st column elution time  the conventional gas chromatogram, or “first-dimension chromatogram,” appears scanning downward from each “peak” in the first dimension chromatogram, one can count the number of coeluents,  visible as discernable second-dimension peaks, of which the conventional one-dimensional peak actually consists

Data interpretation Most of the colored spots -- the chromatographic peaks -- in images such as Figure 4 (above) are believed to represent one, or a very few, of the chemical species present in the sample. In the case of the diesel oil appearing in Figure 4, some 5,000 peaks are discernable. Even with this very high peak count, co-elutions still occur at the higher carbon number region on the right hand side of the image. Nonetheless, valuable information is still available from rich and chemically significant peak patterns. In the example of Figure 4, chemical classes are clearly visible. Column bleed products eluting from the first column are also clearly distinguishable from the sample matrix. Diagonal sub-bands appear throughout the chromatogram, corresponding to groups of isomers – the so-called “roof-tile” effect.

Data interpretation – the 3 rd D

Liver – Drug induced damage

Data interpretation – the 3 rd D

For more info... Visit Zoex Corporation for info taken from their technical note LECO Corporation – product flyer PegasusHT LECO Corporation – technical notes cation_notes.htm Visit this site for info on comprehenisive LCxLC