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temperatures is that a higher flow rate can be utilised, however for this to be of benefit the detector has to be capable of coping with the specified flow rate.
Figure 4 : Column: Hypercarb™ 5 µm, 100 x 4.6mm, mobile phase: H2O , flow rate: 2.0
ml/min Temp. gradient: 150 to 200°C@15 °C/min, hold at 200°C, detection: UV@254 nm Analytes in order of elution; Cytosine, Uracil, Thymine, Hypoxanthine,Guanine, Xanthine
susceptible to thermal degradation at extremes of temperature and as a result it is more inert.
Figure 4 shows a chromatogram obtained using this alternative experimental arrangement. A thermal gradient is used to elute the series of purines and pyrimidines from the column, and it can be seen that all of the series are eluted from the column with good resolution within twelve minutes. The use of the thermal gradient reduces the overall retention time of the last eluting peak and also means that a purely aqueous mobile phase can be employed. The other advantage of running at the elevated
Conclusions It has been demonstrated that the use of elevated temperatures within the field of liquid chromatography has substantial benefits. The importance of characterising the physical parameters of the experimental setup was discussed. It can be seen that the relationship that exists between the pressure, temperature and the flow within the column can be readily utilised to ensure that a separation can be achieved on a column using elevated temperatures which would not be feasible at a lower
temperature due to the physical constraints of the pumping systems. It should be noted however that there are some disadvantages of using this approach when employing sub 2 micron material, namely that there can be a loss of efficiency dependant on the flow that is employed. Examples were then given of temperature gradients and the advantages that using these can give the separation scientist. The use of isobaric thermal gradients is an interesting concept and one that manufacturers of pumps could develop to allow simple optimisation of a separation.
Hypercarb is clearly seeing renewed interest as the general interest in this type of
technology increases. This is a unique stationary phase that is ideally suited to the use within HTLC. Its ability to naturally withstand the elevated temperatures, and be chemically inert make it a very robust phase. At low temperatures it can suffer because of the degree of hydrophobicity, however at elevated temperatures even non polar compounds can be eluted from it.
So where will this technology go? The advent of thermally stable stationary phases has demonstrated that there are applications that HTLC is suited to. However, for further acceptance of this technology there has to be a step change in the mind set of the separation scientist, to accept that not all compounds are thermally labile and that clever utilisation of thermal gradients will result in a thermally stable HPLC system. It is certain that whatever developments happen, it will always be a hot topic of conversation.
References
1. A.M. Edge, I.D.Wilson, S. Shillingford, Chromatographia 66 (2007) 831
2. A.M. Edge, S. Shillingford, C. Smith, R. Payne, I.D.Wilson
J.Chromatogr. A 1132 (2006) 206
3 K. Hartonen,M-L. Riekkola TrAC 27 (2008) 1
4 T. Greibrokk, T. Andersen J. Chromatogr. A 1000 (2003) 743
5 T. Teutenberg, Anal. Chim. Acta 643 (2009) 1
6 P.T. Jackson, P.W. Carr J. Chromatogr. A 958, (2002) 121
7. H. Gika, G. Theodoridis, J. Extance, A.M. Edge, I.D.Wilson J. Chromatogr. B 871 (2008) 279
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