19
Figure 2 [7] a b
demonstrates how high temperature
Figure 2: Mass Chromatogram (TIC) obtained from the LC-MS analysis of rat urine in positive ESI. A: elevated temperature analysis with a combined thermal and flow rate gradient, The thermal gradient started at isothermal conditions for 2 minutes at 50 °C, followed by a steep gradient (over 6 min) to the maximum applied temperature of 180 °C, where it was held for 1 minute before the re-equilibration step to the initial temperature (10 min). The “required” pressure was fixed at 9000 psi, and a model was used to derive flow rates, thus at the beginning of the run the flow rate was 0.25 ml/min, but at the end it was 0.54 ml/min B: conventional acetonitrile gradient in isothermal conditions (58 o
C). Initially and till 0.5 min acetonitrile content 0%, then
linear increase to 20% acetonitrile at 4 min, then linearly to 95% acetonitrile at 9 min where it was held isocratically for 1 min; finally an equilibration step for 3 min (0% Acetonitrile). The flow rate was 0.25 ml/min.
suggesting that the mode of interaction between the analyte and the stationary phase is consistent across the temperature range under investigation. It can also be seen from Figure 1 that some experimental data points are missing. It was not possible to detect the peaks eluting at these elevated temperatures and it was assumed that for hydroxyantipyrine, antipyrine, and phenacetin, there was some form of thermal degradation occurring in the column.
One interesting phenomenon that is highlighted in Figure 1 is that the elution order for caffeine and aminoantipyrine alters. Thus at temperatures below 103°C, the elution order for the pair is aminoantipyrine followed by caffeine, whereas above this critical temperature the elution order is reversed.
Figure 3: PCA scores plots (pc1 vs pc2) generated in SIMCA P, fromthe two data sets. A: thermal gradient in HPLC with (+ve) ESI detection; B: conventional RP-LC gradient with (+ve) ESI detection; Blue boxes represent lean rats; red triangles represent fat rats.
the surfacemorphologymight change for somematerials and that this in itself would result in different types of interactions between the analyte and the stationary phase.
It can be seen from Figure 1 that the relationship between 1/T and Ln k’ is linear,
One of the limitations of using an isothermal separation is that the run time will be dependant on the elution time of the first set of peaks and
also the last. If the temperature is too high then there will not be enough separation of the early eluters, whereas too low a temperature will result in the late eluting components of the testmixture eluting at a prohibitively long retention time.
liquid chromatography (HTLC) can be applied to the analysis of very complexmixtures. In this case, ametabolomic sample (rat urine) has been analysed using a conventional binary mobile phase gradient system, and also using an isocratic systemwith a thermal gradient to optimise the separation. The data fromthis experiment proved to be very useful, as there are substantial selectivity differences between methanol and water, even at elevated temperatures where water behavesmore like a lipophillicmobile phase. The differences in selectivity are incredibly useful inmetabolomic studies which are inherently looking for information hidden in a raft of data. The use of two potentially orthogonal separation techniques allows for the elucidation of this data in amuch easier format. This is demonstrated in the principal component analysis (PCA) plots shown in Figure 3 [7]
.
These were obtained from both the binary solvent chromatographic system and also from the isocratic, thermal gradient chromatographic system. In both approaches a clear discrimination between the two sample sets (lean and fat rats) is obtained using the PCA analysis. Although both approaches are able to separate between the two data sets, the data that is used to separate the two data sets is not the same allowing the separation scientist to unravel further information about the metabolome.
The final application is based around the use of HypercarbTM
(Porous graphitic carbon,
PGC). This is a unique chromatographic material made purely from carbon, which results in a highly retentive stationary phase. The manufacture of PGC involves a range of steps with varying degrees of extreme conditions from concentrated sodium hydroxide to temperatures in excess of 2000 K. This results in a stationary phase that it very inert and very stable to a wide range of chemical environments. In particular the use of elevated temperatures is ideally suited to this material as it can readily withstand very extreme temperatures beyond the temperatures used even in high temperature GC. One of the limitations of this material is that because of the amount of carbon present it is very hydrophobic, resulting in a highly retentive material. At low temperatures this can present a challenge as elution of hydrophobic analytes can be challenging, however as the temperature is increased so the elutropic strength of the mobile phase will increase resulting in a reduced retention. Unlike silica stationary phases, carbon does not have any active moieties that are
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