Researchers seeking a new ultraviolet spectrophotometer face no shortage of choices. Since making their first appearance on lab benches more than 60 years ago, UV specs have expanded and morphed into instruments that can handle a breadth of research applications, from the simplest single-wavelength measurements to high-performance, multispectrum analyses. Scientists must therefore keep their laboratories' specific needs in mind when making their choices. Features to consider include the intended applications, optical configuration and light sources, detection methods, sample format, and data management.
Traditionally, UV spectrophotometers come in either a single- or double-beam configuration. As their name suggests, single-beam instruments rely on one beam of light to make measurements. Light at a given wavelength passes first through a reference cell, then through the actual sample solution; the difference between the two measurements is the absorbance result.
Double-beam instruments split a light beam in two with a mirrored chopper wheel,...
LIGHT SOURCES AND DETECTION
The instrument's spectral range must also be considered. Labs that wish to quantify only nucleic acids and protein or to measure bacterial growth can often save money by buying a dedicated instrument, such as the GeneQuant II from Amersham Biosciences of Piscataway, NJ. The instrument measures absorbances at 230, 260, 280, 320, 595, and 600 nm. For greater flexibility, researchers should consider a higher-end instrument capable of broad spectral measurements. These instruments can be programmed to read and analyze ELISAs and other colorimetric assays.
UV instruments typically cover the range between 190 nm and 380 nm, often using deuterium arc lamps for illumination. Some specialized instruments, however, offer wavelength ranges that delve deeper into the ultraviolet spectrum for photonics and semiconductor applications. Examples include the Cary Deep UV from Varian of Palo Alto, Calif., and the U-7000 Automated Vacuum UV System from Hitachi High Technologies of Tokyo.
Some instruments contain multiple lamps to offer optimum illumination in the ultraviolet, visible, and even near-infrared (780 nm to 3,000 nm) regions of the spectrum. Tungsten and halogen lamps typically cover the visible portion of the spectrum (between about 380 nm and 800 nm), while xenon lamps can cover both the UV and visible regions.
Instrument bandwidth depends largely on a spectrophotometer's monochromator slit width, which casts a band of light from which the spectral bandwidth can be derived, and should be considered when deciding on the laboratory's accuracy needs. A tight bandwidth allows the instrument to make high-resolution absorption measurements in complex sample mixtures. Variable slit widths allow researchers more freedom to manipulate one spectrophotometer to meet multiple experimental needs.
To detect absorbance, instrument manufacturers typically use either photo-multiplier tubes (PMTs) or photodiodes. PMTs offer quick response times as well as good sensitivity and can be tuned to specific target ranges in the UV spectrum. But some instrument manufacturers rely on the increased dynamic range of photodiodes and even assemble them into photodiode arrays that take full-spectrum measurements in seconds while others take minutes.
Among the more traditional sample formats, spectrophotometers accept sample cells, cuvettes, sippers, and microtiter plates, as well as combinations of these. Microplates naturally lend themselves to the high-throughput requirements of large-scale laboratories. But even in smaller labs, a single cuvette or cell system can prove a bottleneck. Some manufacturers therefore offer multiple-container changers to increase throughput and cut experimental run times.
Cuvette sample volumes can range from 5 ml down to 1 μl, and several instruments can be equipped with a variety of sample holders to suit changing needs. Some instrument manufacturers also allow for additional flexibility, including temperature controls to preserve perishable solutions during longer measurements, for instance.
Most standalone spectrophotometers include their own onboard software that drives the instrument and manipulates data. Higher-performance instruments, though, are often designed for use with a PC, requiring additional software from manufacturers. Users can sometimes also pick and choose specific software modules and upgrades to match their analysis needs.
Another factor for consideration should be the data's ultimate end use. While independent research laboratories may be solely interested in experimental results, larger pharmaceutical agencies must consider regulatory guidelines such as the US Food and Drug Administration's rule 21 CFR 11 for paperless record keeping, and the instrumentation demands set for European Pharmacopoeia applications.
Tariq J. Malik