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Cuvette-free Spectophotometer Handles Low Volumes

Wed, 12/07/2011 - 9:37am
Sandra Lloyd-Lewis, BSc Hons, Product Manager Spectrophotometry; GE Healthcare Life Sciences, Cardiff, U.K.
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Ultraviolet-visible (UV/Vis) spectrophotometers are used for the detection and quantification of a range of molecules, including nucleic acids and proteins. Conventional spectrophotometry measures the amount of light absorbed or reflected by a sample loaded into quartz or disposable plastic cuvettes and exposed to specific wavelengths of visible or ultraviolet light. Minimal working volumes for standard cuvette-based UV/Vis spectrophotometers typically range from 200 µL to 10 mL. After each sample is analyzed the cuvette must be emptied, then cleaned or disposed of.

Genomic and proteomic studies often require the analysis of hundreds of samples and have been a driving force behind the industry-wide trend toward miniaturization of analytical processes and instrumentation. Low-volume methods allow researchers to conserve valuable sample stores. They also decrease the time and cost of analysis, with rapid sample transfer, fast read-times, and reduced reagent needs.

Low-volume spectrophotometry is a relatively recent advance, evolving from technological breakthroughs in miniaturization, detection, and quantification methods. Technology has eliminated the need for cuvettes and is making it possible to analyze sample volumes of about 1 µL. GE Healthcare Life Sciences offers a series of spectrophotometers including Ultrospec and the low-volume spectrophotometer, NanoVue Plus, which has a sample plate at the center, eliminating the need for cuvettes. Researchers can pipette a 0.5 µL to 5 µL sample directly onto the sample plate.

The sample plate’s hydrophobic coating facilitates sample recovery with a pipette after each analysis. Alternatively, users can wipe the surface clean in preparation for loading the next sample. Internal studies have demonstrated that there is no sample carryover when the plate is wiped clean, minimizing the risk of potential for contamination of the sample mechanism and improving data precision.

The pathlength—the distance light travels through a sample in a cuvette—is a critical factor in spectrophotometric analysis. For conventional quartz cuvettes with working volumes of 200 µL to 10 mL, the pathlength typically ranges from 10 mm to 50 mm. In contrast, the pathlength on the NanoVue Plus is either 0.5 mm or 0.2 mm (dependent on sample data entered by the user). This increases the dynamic range of the instrument, thereby reducing the need for sample dilution, saving sample preparation time and eliminating a potential source of error.

In genomic and proteomic research, speed, accuracy, and cost are key factors. Read times on the NanoVue Plus are typically less than 5 seconds for a full wavelength scan from 200 nm to 900 nm, with peak identification and on-peak confirmation. Cuvette costs and wash steps are eliminated.

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