4 August / September 2016

How to Design a Column Screening Kit for Preparative SFC Separations?

by Matt Przybyciel, PhD*

ES Industries Inc, 701 Route 73 South, West Berlin, NJ 08091. * Corresponding author:

Preparative supercritical fluid chromatography (SFC) is a powerful tool for the purification/isolation of both chiral (1-3) and achiral (4,5) compounds. Many chemicals can potentially be used as a supercritical mobile phase (6) in SFC, however virtual all current practitioners of SFC use CO2

which offers several advantages, particularly when compared to preparative liquid chromatography. CO2 has the potential to act as both a weak Lewis

acid and Lewis base, and it can participate in conventional or nonconventional hydrogen bonding interactions. In addition, it is miscible with a wide range of organic solvents, non-flammable, and has little UV absorbance at lower wavelengths. CO2

-based SFC is particularly well suited to the area

of preparative chromatography where it can be easily removed after fractionation, enabling the rapid recovery of isolated, pure compounds. In addition, any residual amounts of CO2

in isolated products are considered to be non-toxic.

Other advantages of SFC include the fact that diffusion coefficients of solutes in supercritical mobile phases have been shown to be 3-10 times higher than in normal liquids potentially allowing for very rapid separations through reduction of resistance to mass transfer, although it does not suffer from the solubility issues that are present with GC. The viscosity of mobile phases used in SFC are significantly lower than those used in LC producing much lower pressure drops across the column which allows the use of much smaller particles for both analytical and preparative separations. The use of the smaller particles enables either an increase in chromatographic efficiency in the same chromatographic run time or a decrease in chromatographic run time while maintaining the same efficiency experienced with larger particles.

One of the drawbacks of using CO2 as a mobile phase in SFC is that it is

relatively non-polar even though it has been described as a quadrupolar solvent because of its significant quadrupole moment (7). In order to modify the elution strength of CO2

to allow wider use with molecules of increased polarity, organic

solvent modifiers are mixed into the CO2 stream using a second high pressure HPLC pump. Commonly used modifier solvents are methanol or ethanol, but other organic solvents and solvent mixtures are also used.

Because of the non-polar behaviour of CO2 the stationary phase plays a very key role in SFC separations. In addition, many SFC

separations require the use of additives such triethylamine in order to diminish peak tailing and maintain acceptable retention factors, particularly when separating amines. Additives are difficult to remove and potentially alter the chemical properties for compounds being purified and isolated using SFC. As a result of these concerns, the use of additives is discouraged when the purifying and isolating compounds using SFC. In recent years SFC optimised stationary phases have been developed (8) to avoid the use of mobile phase additives while delivered desired chromatographic performance of ionisable compounds.

Chromatographers require a degree of flexibility when dealing with difficult to separate mixtures and thus they require a large variety of SFC stationary phases to cover the wide range of molecules encountered in the laboratory. Unfortunately, the large variety of stationary phases greatly complicates the column selection process for any particular separation. The large number of stationary phases available for SFC is the result of several contributing factors, firstly and chiefly, a ‘universal’ stationary phase has yet to be developed for SFC; in reversed phase HPLC, C18 is generally accepted as the ‘universal’ stationary phase. Secondly, SFC can separate mixtures containing a wide variety of chemical polarities from non-polar to extremely polar and in many cases exceeds the separation boundaries of reversed phase HPLC but

requires differing phases to accomplish this task. Finally, some of the stationary phases used in early practice of SFC are historical and predate the development of SFC optimised stationary phases. These phases were derived from normal phase liquid chromatography and include phases such as cyano, diol, silica and amino. The selection of the best SFC column for a given separation can be a daunting task and it is the focus of this article to design a set of screening columns that will aid the selection of the best preparative SFC column for a given separation.

The Column Screening Kit

Generally, most column screening systems use a set of analytical size columns (column diameters from 2 - 4.6 mm ID and lengths from 100 – 250 mm) to identify the best stationary phase for a particular preparative separation. In addition, most of these screening systems use an automatic switching valve to screen up to six columns. It would be, therefore desirable to limit a column screening kit to six columns. Ideally, one of the six columns in the screening kit would deliver the desired chromatographic performance and could be geometrically scaled up to perform the preparative separation. Preferably, the screening kit would utilise columns that would separate a wide variety of chemical polarities whilst maintaining acceptable peak shape without mobile phase additives being required.

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