The Ice Men Cometh

A cool way to separate biological compounds is being developed by Japanese researchers.

A stationary phase made of ice and water has been used to separate oestrogen, amino acid derivatives and poly(oxyethylene) oligomers. Yuiko Tasaki and Tetsuo Okada from the Department of Chemistry at the Tokyo Institute used this phase to achieve a much larger theoretical plate number [(N) = 1,500] than previous ice experiments [(N) = 250] which allowed these applications.¹ This novel stationary phase could be used to provide better separation selectivity for polar groups than silica gel, Okada told LCGC Europe.

The surface and bulk properties of the ice stationary phase can also be modified by adding functional groups that allow separation by partition and adsorption mechanisms. Adsorption and partition mechanisms are usually clearly divided in liquid chromatography (LC) but in ice chromatography the two modes are interchangeable. According to Okada: "Switching between two modes alters the separation selectivity and enhances the retention of solutes in some cases. This means we have broader choices of separation modes to achieve the desired separation." The partition mechanism operates when a quasi-liquid layer (QLL) is formed between the stationary phase surface and the organic solvent is used as the mobile phase.² This layer was calculated to have a thickness of around 10 nm which increases directly with temperature. The adsorption mechanism is predominant at lower temperatures when the ice–water surface is solid. Salt-spiked ice stationary phases also allow adsorption–partition switching in thermodynamically predictable ways, says Okada.

The research project originated from the idea that ice crystals act as an adsorbent for some biological molecules, such as anti-freezing proteins. The researchers then began to explore the potential of an ice–water phase as an "environmentally friendly" stationary phase to separate biological molecules. Another benefit of this research is that it offered insight into the nature of ice–water interactions in stratospheric reactions, including depletion of the ozone layer and meteorological phenomena, such as melting polar ice-caps.

"Many researchers are interested in ice crystals," says Okada. "More than 13 types of ice crystals are currently known, but new ones are always being investigated. Some of them are formed under high pressures or low temperatures, but this is not always the case. By studying the ice–water interface we have discovered the nature of hydrogen bonds formed between solutes and the ice surface, evaluated the chemistry of pre-melting ice surfaces and also estimated the size of aqueous phase droplets developed in salt-doped ice particles."

As well as investigations into the surface chemistry of the ice, the separation efficiency of ice chromatography has also been improved significantly over previous attempts. This is because the researchers devised a method to prepare fine ice particles with a diameter of less than 10 µm.

Despite the fact that experimental conditions need to be kept below freezing point, that mobile phases that dissolve ice–water cannot be used and that the columns are not suitable for long-term use, Okada says there is potential to develop this technique further. "Even the ice stationary phases used at the moment are useful for routine separation and could replace preparative silica gel columns that are widely used in organic chemistry labs. Also, if we add more functions to ice stationary phases, they can be possibly be used in other areas of analytical chromatography."

Okada admits the low durability of the ice stationary phase is a serious drawback to the technique and that for practical applications, a longer life-time is essential. A more durable ice stationary phase would also allow the use of an aqueous mobile phase, and the researchers are currently working on ways to impregnate ice into porous supports as well as investigating the use of monolithic ice columns to create a more durable phase. "I do not want to suggest specific applications for ice chromatography at present but do believe that a number of applications will be exploited after further investigation," he says.

1. Y. Tasaki and T. Okada, Anal. Chem., 81, 890–897 (2009).

2. Y. Tasaki and T. Okada, Anal. Sci., 25, 177–181 (2009).