By finding a simple way to combine a sample concentration technique known as isotachophoresis (ITP) with standard electrophoresis, US chemists have come up with a speedy new technique for analysing tiny concentrations of amino acids.

Testing this technique on six amino acids that have been highlighted as possible markers for life on Mars, the chemists found it could detect them at concentrations as low as 200fM, making it 1000 times more sensitive than previous techniques, in around 10 minutes.

With the rise of microchip-based separation systems, the need to concentrate the analytes present in tiny samples has become ever more essential. One commonly used sample concentration technique is ITP.

This involves inserting a sample of charged analytes into a capillary between two different electrolytes. The electrolyte in front of the sample, known as the leading electrolyte (LE), consists of ions that move faster under an electric field than the charged analytes. The electrolyte behind the sample, known as the trailing electrolyte, consists of ions that move slower than the charged analytes.

Because of this difference in migration speeds, the sample is essentially trapped between the TE and LE. On applying an electric field, the analytes move and separate as in normal electrophoresis, but being trapped between the TE and LE concentrates the different analytes together, forming thin adjacent zones of pure analyte.

To separate these zones enough to allow the different analytes to be detected, non-fluorescent or non-absorbing ionic compounds are usually added as spacers. An alternative and simpler method is to separate the zones by electrophoresis, but this requires reversing the polarity of the electric field as the analytes have usually migrated to the far end of the capillary during the ITP stage.

But now Jonathan Shackman and his colleagues at Temple University in Philadelphia have found a way to combine ITP with electrophoresis that doesn't require reversing the polarity. To do this, Shackman took advantage of a novel version of ITP known as gradient elution ITP (GEITP), which he developed with David Ross at the US National Institute of Standards and Technology in Maryland.

This, in turn, came out of a novel form of electrophoresis they developed in 2007 known as gradient elution moving boundary electrophoresis (see Paddle your own canoe). By applying a pressurised (hydrodynamic) flow opposite to the analytes' normal direction of travel and then gradually reducing this flow, Ross and Shackman found they could get analytes to travel up a capillary one at a time, according to their migration speed.

In GEITP, the sample is mixed with the TE in the sample reservoir at one end of a capillary. By applying a hydrodynamic flow, LE is pumped from the other end of the capillary until it enters the sample reservoir. Then, while maintaining the flow to keep the sample, TE and LE in the sample reservoir, an electric field is applied to the capillary, inducing ITP in the sample reservoir and thereby concentrating the analytes into distinct zones. Finally, the flow is reduced, drawing the concentrated analytes into the capillary.

At this point, Shackman injects LE into the sample reservoir until it fills the entire capillary, allowing him to conduct standard electrophoresis. But even at this stage an opposite flow can still prove useful, by ensuring that the analytes only migrate to the end of the capillary after they have been successfully separated.

'An ideal separation would utilize gradients with baseline separation of all analytes, but this is rarely found in practice for multi-component mixtures,' explains Shackman. 'However, easily adjustable gradients (such as hydrodynamic flow) allow us to come much closer to this ideal and more efficiently use the analyst's time.'

After their success testing this method with the extraterrestrial amino acids, Shackman and his team are now looking at ways to improve this combined technique still further. 'We are currently developing a two-channel microfluidic device to automate solution switching and decouple the electrolyte solutions, which will allow for independent optimization of each stage,' Shackman told separationsNOW.

The chemists' ultimate goal is one-minute enrichment and one-minute separations of amino acids at femtomolar levels.

Related links:

• Analytical Chemistry, 2009, 81, 5442 - 5459: "Capillary and microfluidic gradient elution isotachophoresis coupled to capillary zone electrophoresis for femtomolar amino acid detection limits"
• Analytical Chemistry, 2007, 79, 6641 - 6649: "Gradient elution isotachophoresis for enrichment and separation of biomolecules" 

Article by Jon Evans

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.