Normal phase chromatography is not very popular with most analytical HPLC users. Poor reproducebility of the results is often cited as an argument against normal phase chromatography. Most problems, however, can be avoided when some precautions are taken, enabling the user to take advantage of the features of this technique such as the ability to separate positional and stereo isomers. Before starting, you should check, probably with the pump manufacturer, whether the pumps of the system have to be modified, by exchanging the seals, etc because of compatibility problems with the non polar solvents that are used.
One of the major reasons for the problems of reproducibility is well known but often neglected. When bare silica is used as the stationary phase, water is always adsorbed onto the silica surface and occupies sites of interaction, which are then not accessible to the analytes. Variable water content in the eluent (and also in the sample) may cause dramatic changes in the retention and separation. In order to avoid these drawbacks, it is recommended to blend the solvents from a water-saturated and a dried (by molecular sieve, sodium, etc.) portion. This gives the opportunity to keep the water content constant or to regulate it to a defined value. The latter approach may be the key to successful method development, as the separation sometimes reacts very sensitively to the concentration of water present in the mobile phase.

The user should keep also in mind that normal phase supports, especially bare silica, requires distinctive longer equilibration times, sometimes up to 10-fold compared to reversed phase materials. This must be taken in account especially when method development is done. Because the separations are often very sensitive to temperature fluctuations the use of a column oven is recommen-dable.

By careful consideration of these characteristics of normal phase chromatography, one can achieve excellent results, as shown by the baseline separation of β- and γ-tocopherol with high resolution; this is very difficult to achieve with reversed phase chromatography:

For further information, please feel free to contact Dr. Wolfgang Schräder, YMC Europe GmbH, Telefon: +49 (0)2064 427-290
Telefax: +49 (0)2064 427-120 or visit www.ymc.de