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Digital Camera Reads Chromatography Plates

Photonics Spectra
Apr 2006
Hank Hogan

Thin-layer chromatography is a standard laboratory method in chemistry used to identify compounds, assess purity and track a reaction’s progress. It involves an adsorbent film on a glass plate or other carrier on which samples of interest are placed. As the components of the sample travel through the film, they separate, enabling analysis.

The problem is that scanning densitometry, the standard measurement technique used with thin-layer chromatography, has some drawbacks. In densitometry, a light typically diffusely reflects off a plate and into a detector, quantifying absorbance and thereby capturing the chromatography results. But it is slow, taking up to 20 minutes per slide, and it can be done only off-line, after the sample spots stop moving.

Accent-30-to-35_img_6.jpg

The cover of the experimental setup for image acquisition in thin-layer chromatography has been removed, revealing the light box on the bottom and a thin-layer chromatography plate atop it. Suspended above the light box and plate are a lens, filter and CCD camera.

Working with colleagues at the University of York in the UK, chemistry professor David M. Goodall is developing an alternative for reading chromatography plates that promises to be much faster. In a demonstration system, the scientists fitted an Astromed TE4/A CCD camera with a Nikon lens and an Edmund Optics filter, and they used an 11-W fluorescent lamp to shine through the plate. They operated the camera at its maximum dynamic range and readout rate, capturing one image every few seconds during a run.

In processing the plate images, they subtracted a blank image, taken with the shutter closed, and then performed 2 × 2 binning of the data. Each pixel represented an area of  0.163 × 0.163 mm on the thin-layer chromatography plate. To provide a good signal reference, they divided the plate into narrow lanes that were free from spots, interspersed with wider ones that contained the sample. In this way, they created an absorbance baseline.

Accent-30-to-35_img_7.jpg
An image of the raw, uncorrected plate with a 10-ng sample spot shows the two reference lanes surrounding the sample lane that are used in the calibration of the images.

They also averaged multiple images. This, Goodall explained, solves the problem of local fluctuations in the scattering or absorbance properties of the chromatographic material. Such variations are indistinguishable from a thin-layer chromatography analyte, except in that they do not move. So the local fixed pattern noise can be removed by the superposition of multiple snapshots, after the appropriate shift to account for the displacement between shots.

By averaging 55 images together in this manner, the researchers achieved a limit of detection of 1 ng. A comparison of their results for thin-layer chromatography with dry and wet plates to off-line measurements performed using densitometry showed that the methods produced similar measurements.

Goodall said that the group plans to refine the approach for real-time ultraviolet imaging, using a phosphor to convert UV signals into something that can be detected by either a CCD or a CMOS sensor. Most analytes of interest absorb in this region, he explained.

Contact: David M. Goodall, University of York, York, UK; +44 1904 432 574; e-mail: dmg1@york.ac.uk.


GLOSSARY
chromatography
The chemical method of separating compounds dissolved in one phase (usually mobile) through its equilibration with a second phase (usually stationary). The mechanism of separation may involve partition, adsorption, permeation or exclusion, or ion exchange.
densitometry
The detection and analysis of the transmission and reflection properties of objects and photographic images.
digital camera
A camera that converts a collected image into pixels that are black or white digital or shades of gray. The digital data may then be manipulated to enhance or otherwise modify the resulting viewed image.
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