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Please check out our Spectrophotometers section for more information or to find manufacturers that sell these products.
Turn on your kitchen sink. Is the water fit to drink? One way to make that determination is with your eyes. Is it turbid? Colored? The optical properties of the water can help you decide.
Researchers in fields from biology and chemistry to materials science and telecommunications use effectively the same trick—albeit far more precisely—to determine concentrations, measure reaction kinetics, and quantify the reflectance properties of the materials they work with and manufacture. Their method of choice: spectrophotometry.
"[Spectrophotometers] are pretty much the workhorse of a lab," says John Monti, spectroscopy product manager at Shimadzu Scientific Instruments. "If you are doing anything involving chemistry at all, whether it's biological chemistry all the way up to material properties for construction like window panes or automobile glass, you have to have some sort of spectrophotometer."
A spectrophotometer is an instrument that, depending on its configuration, measures how much light passes through, is absorbed by, or reflects off of a sample, whether in liquid, solid, or (rarely) gaseous form. Though they come in many shapes and sizes, all spectrophotometers contain a few basic components: a light source, wavelength and spectral resolution selectors, sample holder, and detector.
Spectrophotometer Light Sources
Spectrophotometers can probe a sample's optical properties over a large slice of the electromagnetic spectrum, from the ultraviolet (UV: 190 – 390 nm), through visible (VIS: 400 – 750 nm), to the near infrared (NIR: 750 – 2,500 nm). To generate these wavelength ranges, they use one or more lamps, or light sources: a deuterium lamp for UV, a tungsten/halogen lamp for visible and NIR, or a xenon flash lamp to cover both the UV and visible wavelengths.
According to David Haines, molecular spectroscopy product specialist at Varian , the different sources are not equivalent; they provide light intensities and noise at different parts of the spectrum. Most Varian and all Shimadzu spectrophotometers use both deuterium and tungsten/halogen lamps, though Varian's entry-level Cary 50 uses a xenon flash lamp.
In contrast, most UV-Vis spectrophotometers in Thermo Fisher Scientific's product line use xenon flash lamps. "We feel there is a strong advantage in many cases to the xenon lamp because it provides higher energy flux than combined sources across most of the spectrum, but especially in the UV range," says Gordon Bain, UV/Vis product specialist at Thermo; only the company's Evolution 600 and HELIOS instruments use deuterium and tungsten/halogen sources.
To deliver light to the sample, spectrophotometers adopt one of three basic configurations. The simplest is the single-beam architecture, typically reserved for low-end and teaching-lab equipment. Single-beam spectrophotometers have a single optical path, from light source, through sample, to detector, with no secondary "reference position" to account for fluctuations in source intensity. Shimadzu's UV 1240 and BioMini instruments are single-beam spectrophotometers.
Most research-grade spectrophotometers are double-beam instruments. A double-beam spectrophotometer splits the beam after the wavelength selector (usually a monochromator) using a rotating mirror called a "chopper," so that it can alternately pass through the sample or the detector. The result is that the instrument can keep track of source light intensity, thereby enabling it to correct for variations in source intensity.
"The problem with single-beam measurement is that, if there are any changes in the intensity of light from the lamp with time, the single beam instrument cannot account for that, whereas the double-beam instrument does," explains Haines.
In the third configuration, called "dual-beam," the source beam is physically bifurcated with a beam-splitter, such that the sample and reference positions each receive half of the original beam's energy, as well their own detectors. Shimadzu's UV 1800, Varian's Cary 50, and Thermo's HELIOS and GENESYS products are all dual-beam spectrophotometers.
Spectrophotometer Detectors
UV-Vis spectrophotometers can feature either silicon photodiode detectors or photomultiplier tube (PMT) detectors. The former, says Haines, "are more uniform than a PMT, but also less sensitive." On the other hand, he says, UV-Vis PMTs "are not sensitive at much above 900 nm." Also, adds Bain, PMTs are incompatible with xenon lamps, and so these lamps are coupled instead to silicon photodiode detectors.
Performance Characteristics
For some high-performance applications, it is important to consider how well the instrument can handle strongly absorbing (that is, low transmission) samples. This parameter is called photometric range and is expressed in terms of absorbance units (A). A spectrophotometer that can detect transmission of 10% has a photometric range of 1A, 1% is 2A, 0.1% is 3A, and so on. Varian's Cary 50 has a photometric range of 3.5A to 4A, meaning it can handle samples that absorb as much 99.99% of incident light. Shimadzu's UV 2450, says Monti, has a range of "5A against a background of 4A—an approximately 9-fold range."
Other critical performance characteristics of spectrophotometers include resolution (the ability to distinguish two closely spaced peaks), spectral bandwidth (an indication of the range of wavelengths exiting the monochromator), and the stray light level ("energy measured by the detector that can't be absorbed by the sample," according to Varian's Jim Steensrud).
Spectrophotometer Accessories
According to Bain, "the big differentiators for the customer" when it comes to choosing a spectrophotometer are not the lamps, the configuration, or the detectors—though obviously, these are important. Instead, it comes down to accessories.
While biologists may never need anything else, for many applications the standard quartz cuvette simply won't cut it. Some, for instance, need temperature control—both water-cooled and Peltier-based forms are available. Also available are sample holders for solid sample transmission analyses, as well as multicell holders and autosippers for higher throughput liquid work. Varian offers an 18-cell multicell holder for its Cary 50, says Haines, though six- or eight-cuvette formats are more typical.
For those conducting reflectance analyses on solid samples, both "diffuse reflectance accessories" and "specular reflectance accessories" are available. In a diffuse reflectance accessory, which usually incorporates an integrating sphere, the sample—a piece of cloth, say—is mounted on the outside of the sphere, opposite the light beam inlet hole. When light hits the sample, it deflects over a wide range of angles (that is, its reflection is "diffuse"), bouncing around inside the sphere until it hits the detector, which is located at either the top or bottom of the sphere. A specular reflectance accessory, in contrast, is simpler; it looks for specular reflection off the sample at an angle equal to the angle of incidence.
Companies offer several unique accessories, too. Shimadzu, for instance, sells an optional automatic stage for its UV 3700 (UV-Vis-NIR) spectrophotometer. The 3700 features a very large sample area, nearly three feet wide. "Within this huge sampling area you can have a stage which can move in both the X and Y directions, and also rotate," says Monti. "So, you can put a sample as large as the 12"-diameter wafers that are used in semiconductor manufacturing on the stage, set up a measurement method, and do complete characterization of the sample surface in both transmittance and reflectance modes."
Whatever vendor you opt to go with, when looking to make a purchase consider the following variables: application; wavelength range (UV, Vis); sample type (solid vs. liquid), volume, and throughput; measurement method (e.g. transmittance vs. reflectance); photometric range; space requirements (some models sit on the benchtop, whereas others are floor-standing); and accessories.
But, says Haines, don't forget to plan ahead. "They need to think about not just what they are doing now, but what they might be doing in a year's time. Because research can change quite rapidly, especially if the instrument is going to be used by a number of people in the lab."
Please check out our Spectrophotometers section for more information or to find manufacturers that sell these products.