Shining light tells left from right

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  • Published: Aug 17, 2009
  • Author: Jon Evans
  • Channels: Detectors
thumbnail image: Shining light tells left from right

Even fairly young children are able to tell left from right, but until recently analytical scientists have tended to struggle, at least at the molecular level. Now, however, there are a number of methods for separating and purifying molecules that can exist in two mirror-image structural configurations. Such molecules are called chiral and their left-handed and right-handed versions are known as enantiomers.

Many biochemicals, such as amino acids and sugars, are chiral, with nature tending to prefer one enantiomer over the other (for example, amino acids are left-handed but sugars are right-handed). As are about half the drug compounds in use today, which can cause major problems.

Because the body is inherently chiral, it tends to respond differently to different enantiomers. So a drug's beneficial effects are often due to just one enantiomer, or one enantiomer may be safe and another toxic; hence the need to separate different enantiomers.

High performance liquid chromatography and capillary electrophoresis (CE) form the centrepiece of current separation methods. A molecule that binds with the two enantiomers at different rates, such as the oligosaccharide cyclodextrin, is incorporated into the stationary phase or added to the separation buffer, causing the enantiomers to migrate through the column or capillary at different rates.

Normally, this is all that is needed to distinguish between two enantiomers, but occasionally they can't be cleanly separated or the separation is highly variable between runs. To help in these instances, it would be useful to have a detector that also responds differently to different enantiomers. This is what a team of analytical chemists from Vrije University in Amsterdam, the Netherlands, led by Freek Ariese, has now come up with.

Their work builds on the recent discovery of an unexpected effect that occurs when a naturally-phosphorescent chiral molecule binds with cyclodextrin. Like fluorescence, phosphorescence is the emission of light by an excited molecule, but in phosphorescence the light is emitted for a longer period of time (more than 10 nanoseconds). What has been discovered is that when the bound molecules are excited by light of the right wavelength, one enantiomer emits more light than the other, in terms of both the intensity and length of the phosphorescence.

This effect is obviously down to the slightly different way that the cyclodextrin molecule binds with each enantiomer. But rather than enhancing the phosphorescence, the cyclodextrin molecules appear to protect the phosphorescent enantiomers from the surrounding solution, preventing it from quenching the phosphorescence. And they are better at protecting one of the enantiomers than the other.

Ariese and his colleagues have now used this effect as the basis for a novel chiral detector, which uses a light emitting diode to stimulate phosphorescence and a photomultiplier tube to detect the emitted light. Attaching this detector to the end of a capillary tube filled with a cyclodextrin-containing buffer, they tested its ability to detect the two enantiomers of the naturally-phosphorescent chiral molecule camphorquinone after separation by CE.

As expected, the right-handed enantiomer migrated faster than the left-handed enantiomer. It was also more phosphorescent, emitting light for 400µs compared with just 149µs for the left-handed enantiomer. The two enantiomers therefore produced distinct, different-size peaks in the subsequent electropherogram, allowing them to be detected at levels of around 1µM.

Ariese and his colleagues then tested their detector on samples containing just one enantiomer of camphorquinone, which showed that these supposedly pure samples were actually contaminated with the other enantiomer at concentrations of 0.1-0.2%. They are now busy trying the detector out with other chiral compounds.

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

Left hand, right hand

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