Running hot and cold: Analyzing oligosaccharides with temperature gradient CE
- Published: Mar 20, 2017
- Author: Jon Evans
- Channels: Electrophoresis
There are many different ways to improve the resolution of capillary electrophoresis (CE). They include varying the separation current, altering the composition of the background electrolyte, incorporating additives into the electrolyte and coating the capillary walls.
One approach that isn’t generally taken is varying the operational temperature. This is because raising the temperature generally increases the migration speed and leads to band broadening, both of which cause analyte peaks to overlap and thus reduce resolution. Now, though, Andras Guttman and Marton Szigeti from the University of Debrecen in Hungary have shown that varying the temperature could be just the trick for improving the resolution when analyzing oligosaccharides by CE. This discovery could help in the search for oligosaccharide-based disease biomarkers and the ongoing development of oligosaccharides as novel drug treatments.
Branched and linear
The first evidence for the benefits of temperature on the separation of oligosaccharides by CE came in 2015. This is when Guttman and Szigeti discovered that the migration behaviour of branched and linear oligosaccharides varied in different ways at different temperatures. As a consequence, they were able to improve the separation of branched and linear oligosaccharides by simply varying the temperature. Investigating this effect, they found it was probably due to the change in temperature causing minor changes in the shape of the branched and linear oligosaccharides, sufficient to alter their migration speed in CE.
So if temperature could help to separate branched oligosaccharides from linear oligosaccharides, perhaps it could also help with the more challenging task of separating different branched oligosaccharides. To find out, Guttman and Szigeti first tried separating a commercially-available mixture of nine different branched oligosaccharides, including afucosylated, fucosylated and high mannose oligosaccharides, by CE at a range of temperatures, from 15°C to 45°C at 5°C intervals.
Although the resolution fell for many of the individual oligosaccharides at the higher temperatures, partly because the resulting increased migration speed caused the oligosaccharides to bunch together, it did improve the resolution for some of them. Once again, this appeared to be due to the oligosaccharides changing shape at higher temperatures. Interestingly, the resolution improved for some of the slower migrating oligosaccharides, which raised the possibility of optimizing the resolution for all the oligosaccharides by applying a temperature gradient during CE.
This is exactly what Guttman and Szigeti did next. They separated the same mixture of oligosaccharides by CE, but after six minutes they steadily increased the temperature from 15°C to 45°C over two minutes and then held it at this temperature for the next 10 minutes. In this way, they were able to separate all nine oligosaccharides, which wasn’t possible when conducting CE at a single temperature.
Given these successful results, Guttman and Szigeti conclude that “temperature gradient CE can open up a new chapter in high-resolution glycan analysis”. In order to see whether there is scope for further improving the separation resolution, they are now planning to conduct more detailed investigations into the effect of temperature on the kinetic and transport properties of oligosaccharides during CE. They are also intending to investigate whether temperature gradients could improve the separation of other analytes as well.
Analytical Chemistry, 2017, 89, 2201−2204: "High-resolution glycan analysis by temperature gradient capillary electrophoresis"
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.
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