Addition of Water in Carbon Dioxide Mobile Phase for Supercritical Fluid Chromatography

Supercritical fluid chromatography (SFC) may be defined as a form of chromatography in which the mobile phase is subjected to pressures and temperatures near or above the critical point for the purpose of enhancing the mobile phase solvating power. During its relatively short existence, supercritical fluid chromatography (SFC) has become an attractive alternative to GC and LC in certain industrially important applications. SFC gives the advantage of high efficiency and allows the analysis of non-volatile or thermally labile mixtures. Carbon dioxide is widely used as a primary mobile phase in SFC because of its low critical temperature, low toxicity, chemical inertness and nonflammability. However, because of its nonpolar nature, neat CO2 is limited in its ability to elute polar compounds. Therefore, a major objective of research into SFC has been directed towards increasing the range of solute polarity that can be handled by the technique. To bring the SFC technique into routine use, mobile phases that are more polar than the commonly used CO2 are necessary. Polar mobile phases such as NH3 exhibit useful properties, but a more practical way to extend the range of compounds separable by SFC is the use of mixed mobile phases. The addition of modifiers (generally organic solvents) to supercritical CO2 changes the polarity of the mobile phase and also leads to a deactivation of the column packing material. Janssen and co-workers explained the effects of adding polar modifiers to supercritical fluid CO2 mobile phase in three different ways: (A) increasing mobile phase polarity, (B) increasing mobile phase density, (C) deactivation of active sites on the surface of the stationary phase. In capillary SFC, most separations are carried out with pure CO2 because of its compatibility with a flame-ionization detector (FID); indeed, except for formic acid and water the addition of any common modifier precludes the use of an FID. Although it is desirable to use FID in SFC, only water and formic acid produce acceptably low background noise and enable the use of this universal detector. Water and formic acid have been suggested by some investigators as very useful modifiers in packed column SFC because they significantly improve the separation of some polar compounds. One of the simple and effective ways for the addition of modifiers to supercritical fluid mobile phase reported in the literature is to use a saturator column which is usually a silica column saturated with polar modifiers. In our laboratory, μ-Porasil column and highly porous stainless-steel filters which are saturated with polar modifiers have been used successfully as a modifier mixing device. One problem with these kinds of saturation type mixing devices is that the amount of polar modifiers dissolved in supercritical fluid CO2 can not be changed easily because the principle of mixing is based on the saturation. In this paper, a newly developed mixing device was used to demonstrate the effects of addition of water in carbon dioxide mobile phase. The new mixing device in which small mixing chamber with magnetic bar was used to generate water-modified carbon dioxide mobile phase was applied for the separations of free fatty acids. For the analysis of fatty acids, GC often is not the method of choice because of the low volatility of fatty acids. HPLC is not helpful either, because free fatty acids cannot be detected easily by a UV detector or one of the other HPLC detectors. In this case, SFC is an alternative to GC or HPLC for the analysis of fatty acids. The ability to analyze fairly polar compounds with supercritical CO2 is demonstrated in this paper; however, a modifier must be used. The influence on the retention behaviour of adding a modifier depends on the nature of the substrate, the stationary phase, and on the modifier itself. Yonker et al., report that at CO2/methanol mixtures at 50 C UV absorbance maxima shifts for 2-nitroamisole. When dealing with the use of modifiers, it should be mentioned that some problems arise. First, a binary mixture of eluents can contaminate the instrument. The modifier remaining in a injector, tubing, expecially pump can be eluted slowly during the