Couple of days ago, I mentioned on Chromatographer’s facebook page article by Peter Carr about speed in HPLC published in Analytical Chemistry. There are two parts in the article: (i) critical comparison of different approaches how to reach speed in HPLC and (ii) theoretical background of speed and efficiency optimization in high performance liquid chromatography.
The last CASSS Discussion group focused on the possible advantages and disadvantages of high temperature and/or high pressure in a liquid chromatography. The Discussion group was hold as a debate – two experts against each other. The high temperature approach was defended by Nebojsa M. Djordevic (SANO CRO) and Michael W. Dong (Genetech) advocated the use of ultra high pressure in HPLC.
The first speaker was Nebojsa Djordevic. First of all, he started with short introduction of the influence of the temperature on the separation in HPLC. The most important equation in the liquid chromatography – the resolution equation – is temperature dependent. Change in the temperature causes change in all three parts of the equation: efficiency, selectivity and retention.
The higher temperature also decreases the mobile phase viscosity. With lower viscosity of the mobile phase, the pressure of the system decreases and then we can use higher flow rates (= faster analysis). At elevated analysis temperature the solubility of the samples increases and it is not necessary to use high concentration of the organic modifier in the mobile phase. Thus, high temperature liquid chromatography is another step to green chemistry.
Using a high temperature liquid chromatography one has to consider also some limitations. The secondary equilibrium (pH) changes, the kinetics varies (chiral separations) and the conformational changes of the sample can occur.
Using a high temperature is not only “heating” a column. The instrumental demands have to be also considered. The heater itself can form radial and axial temperature gradients, the solvent needs to be preheated; unheated detection cell can causes the precipitation of the sample, etc. Last but not least, the column and sample stability can change significantly using a high temperature.
The main advantage of high temperature HPLC is possible control of the elution selectivity. The high temperature can switch the elution order of (critical) peak pair and help to separate compounds which are not separated at ambient temperature. As Nebojsa Djordevic rightly mentioned ‘you don’t need a hundred thousands of plates if you have good selectivity’.
The next speaker, Michael W. Dong focused on the ultra high pressure liquid chromatography. His presentation was devoted mainly on the instrumental aspect of high pressure in HPLC. According M. Dong, high pressure instruments together with a low dispersion are new platform of HPLC. Currently, all main chromatography manufacturers offer the UPLC systems with pressure limit around 80 – 130 MPa (12 – 19 000 psi).
The UPLC allows fast and selective separation with high resolution for complex mixtures, enhanced peak capacity and fast method development. On the other hand, one has to take special care about injection precision, detector sensitivity (column bleeding) and method portability. Another issues rise from the high pressure safety, viscous heating of the mobile phase and system costs.
The main application of the UPLC system is connected with the high throughput, repeatability and speed, e.g. pharmaceutical industry. On the end of his presentation, M. Dong mentioned, that HPLC systems will be fully replaced by the UPLC instrumentation.
In the following discussion the pros and cons of both high temperature and high pressure systems were compared. While the high pressure allows only increase in the efficiency (and the increase in pressure is still more the penalty we have pay with using small particles), the elevated temperature changes also selectivity and retention of the separation. And if you are able to control the selectivity (the peak resolution) you don’t need (super) high efficiency.
The significant argument for the temperature is financial expenses. The implementation of the high temperature in HPLC instrumentation can be done easily and cheaply then in the case of the high pressure application.
To conclude, the elevated temperature in liquid chromatography was slightly forgotten during last couple of years. With proper implementation, however, the high temperature can bring significant improvement of current and future separations. Cheaply.
What do you think?