Complete Absorption: No Problem With World Precision Instruments

It's a very common problem: You need to determine the concentration of a molecule of interest spectrophotometrically but have only a few microliters of sample. Until now, there were not a lot of options. In the last year, World Precision Instruments has developed three instruments for just these sorts of microscale absorbence measurements. Dr. Su-Yi Liu, R&D scientist at World Precision Instruments, said that the company was originally formed "to develop instruments for electrophysiology but has moved into fiber optics in the last few years." Two of the company's products, the DipTip and the SpectroPette, enable researchers to take absorbence measurements with only a few microliters of sample in a microfuge tube. A third device-the MicroVette-is a capillary microcuvette that has a volume of only 4 µl but has a path length of 2.3 cm (over twice the standard pathlength of a 1 cm wide cuvette). The longer path length means increased sensitivity. These micro-scale measuring instruments are constructed using fiber optic technology. The simplest and most economical of the instruments is the DipTip. This sensor consists of two optical fibers: one carries the incident light to the sample and the other returns unabsorbed light back to the spectrophotometer tube. The bottom of the probe has a small mirror to reflect the incident light back to the return fiber. The light traverses a total path length of 1 cm through the solution. For an accurate reading, the 7 mm tip of the probe must be completely immersed, for which several microliters of solution in a 0.5 ml microfuge tube is sufficient. For times when less sample is available, World Precision Instruments offers the SpectroPette. This instrument similarly uses two optical fibers to carry light to the tip of the instrument and return light to the spectrophotometer. However, instead of simply a probe, there is a plunger mechanism, much like a pipettor, to take up solution into a measuring chamber at the tip. Since the fluid is held in the chamber, less sample is required than with the DipTip probe. The measuring chamber fills with 0.6 µl of fluid, so an absorbence reading can be taken with less than a microliter of sample. According to Dr. Liu, the sensitivities of the DipTip and the SpectroPette are approximately equal. For extremely sensitive microscale measurements of very dilute samples, there is the MicroVette. The MicroVette is a synthetic silica capillary microcuvette. With this system, a fiber optic cable inserted into one end of the capillary introduces the incident light, which then travels to the receptor fiber in the other end. Dr. Gerald Lee Vogel, the project leader of the American Dental Association Health Foundation who collaborated with World Precision Instruments to develop this device, mentioned that other researchers have previously constructed capillary cuvettes. However, it was always difficult to load sample into these systems. Dr. Vogel devised a method to fill and empty the capillary so that the optical fibers do not need to be moved and multiple samples can be easily loaded, measured, and removed. In addition to having this loading feature, the MicroVette is coated with a material that has a lower refractive index than the silica tube. This layering of highly refractive material on the silica-which is designed on the same principle as glass fiber optic cables-means that light going through the sample is not lost through the sides of the tube but is reflected back into the sample. This coating enables a small amount of sample to create a long but very thin path length, so sensitive measurements can be made with very small volumes. Dr. Wesley Corson, associate professor of pathology and laboratory medicine, and of ophthalmology at the Medical University of South Carolina, found the SpectroPette instrument useful in his studies. He studies how vitamin A affects the visual receptors and, for these studies, he routinely needs to measure the amount of rhodopsin isolated from salamander retina, a model organism for this area. His preparations from single salamander retina typically yield approximately one picomole of pigment in less than 10 µl He routinely measures a complete absorption spectra using only 1 percent of the sample. He noted that the device is very easy to use. He also indicated that instrument he has is somewhat specialized since it cannot be used for UV readings. However, he acknowledged that World Precision currently offers a SpectroPette that measures in both UV and visible spectra. The author, Paul Diehl, can be reached at paul_vfocus@msn.com. For additional information on this product, please circle reader service #71 or contact World Precision Instruments at 941-371-1003 or at www.wpiinc.com

Complete Absorption: No Problem With World Precision Instruments

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