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.

High temperature or High pressure?

High temperature in liquid chromatography

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’.

Ultra high pressure liquid chromatography

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.

Discussion

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?

In liquid chromatography liquid mobile phase flows through the column with stationary phase. The main principle of separation remains the same.

Compounds have different affinity to the stationary phase and are separated while flowing through the column. The compounds separated with liquid chromatography are disolved in the mobile phase. They have lower difussion coefficients than gaseous compound separeted with gas chromatography.

Except thin layer chromatography, majority of liquid chromatography is performed in high-pressure arrangement. The liquid is pushed through the column using high pressure pumps. In this case we are speaking about high pressure liquid chromatography (HPLC).

According the polarity of the mobile and/or stationary phase, the liquid chromatography separations can be divided in numerous methods for different kind of samples.

Chromatography is analytical chemistry method which is used (and useful) for the separation of complex mixtures of chemical compounds. The main mechanism of the separation is repeatable distribution of the tested compound in between two different phases.

Usually, one phase is solid, fixed in the separation device and the other is moving and flows through the unit. If gas is a second phase, we are referring to the gas chromatography, in case of liquid as a second phase the name is liquid chromatography.

The device where separation takes place is called chromatographic column. This cylindrical shape column is filled with the different kinds of materials – stationary phases. These materials are usually spherical silica particles with different, but well defined, surface chemistry.

The mobile phase flows through the column together with sample (mixture of compounds). Each compound has various affinity to the surface of stationary phase and therefore is separated form each other. In case of ideal state all compounds are eluted from the column in separated bands.

Various techniques are used to recognize these bands and transform them into the signal. In most of the cases the signal draws chromatographic peak – the “hill like” curve describing Gauss distribution.