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Please see our UV VIS Spectrophotometer section to find manufacturers that sell these products
A UV-VIS spectrophotometer, as the name implies, measures the intensity of light absorbed (or reflected) by, or transmitted through, a substance at specific wavelengths between about 190 and 900 nm—the ultraviolet and visible spectra. In its simplest form, it consists of a light source, a way to separate out the light into its spectrum (like a prism), and a way of detecting the light after it passes through (or bounces off) the test sample.
A typical spec (as it’s commonly called) is a box that sits on a bench. For most laboratories a UV-VIS spec “is a commodity, like a pH meter or balance: you use one to measure things in your lab,” says Gordon Bain, who manages the product line for Thermo Fisher Scientific (www.thermofisher.com). They should be easy to select and buy—most scientific equipment catalogs and distributors carry them—and easy to use.
Applications of UV-VIS spectrophotometers
UV-VIS specs are used in a wide range of industries for an assortment of applications, with quality assurance and quality control (QA/QC) accounting for the largest market segment. “Primarily you’d be doing quantitation, biological applications, color analysis, film thickness analysis, coating analysis…,” says Mark Talbott, Spectroscopy Product Coordinator at Shimadzu Scientific Instruments (www.ssi.shimadzu.com). For example, “it finds application in the photovoltaic industry for solar panels, as well as in the semiconductor industry for the analysis of wafers, and any type of lens manufacture.”
About 70% of what people do with specs is look at liquids, Talbott says. Most of this is measuring absorbance (also known as optical density) and—since the quantity of light blocked by a material is inversely proportional to the amount it allows to pass through—transmittance as well.
“Then you move into a completely different application, not as well-known unless you’re in the industry, which is the ability to analyze reflective surfaces,” allowing for an exact assessment of color, coating thickness, and other parameters, he notes. Specular reflections like those bouncing off a mirror are relatively easy to directly collect and measure using a simple accessory. Diffuse (scattered) reflections like those coming off cloth, on the other hand, need to be gathered up from all directions with more specialized equipment like integrating spheres.
Analysis of DNA, RNA, and protein concentration and quality has largely moved to small, customized specs that rapidly measure high concentrations of very small volumes, without the need for a cuvette (in the case of the Picodrop [Picodrop, www.picodrop.com], the sample can be measured while in a micropipet tip, allowing for full recovery of the sample), and without the need to generate a calibration curve. Such measurements can also be made using a standard UV-VIS spec with a short pathlength cell, but “it’s just not quite as easy or as rapid,” Talbott notes.
Range of instrumentation
Bain divides UV-VIS specs into standard (forward) monochromator and spectrograph (reverse) optical systems. “The optical layout in the two will be different, but they accomplish the same general thing,” he says. In the former, the source light travels through a monochromator, allowing it to be separated into its spectrum. A narrow portion of that spectrum—selected by turning a grating in the monochromator—is then transmitted to the sample. Light that is not absorbed is transmitted to a detector, which counts the photons hitting it.
In a reverse optical system, the entire “white” source light hits the sample. Light that is not absorbed is then transmitted through a slit to a dispersion grating, from where that rainbow is directed to a one-dimensional array of pixels. “Because nothing is moving you can calibrate it,” Bain explains, “and calculate which pixel number corresponds to what wavelength.”
UV-VIS specs will typically use either a tungsten lamp to produce visible light combined with a deuterium (D2) lamp for UV, or a xenon flash lamp to cover both spectra. Tungsten and D2 bulbs need time to warm up, and have relatively short lives, requiring replacement once or twice per year (which can be done by the user). Xenon lamps, on the other hand, are instant-on and last for years, but the instrument needs to be sent back to the factory for recalibration when they are replaced.
A split-beam or double-beam instrument utilizes a second (reference) pathway for light to travel—bypassing the sample—allowing the instrument to keep track of and correct for a change in lamp intensity over time. In a true double-beam setup, a cuvette with sample matrix can be placed in the path. The change of absorbance of that matrix can also be tracked and corrected for, a boon for kinetic experiments, for example.
Because xenon lamp intensity varies slightly from flash to flash, it is imperative to use it with a reference beam “to get really good stray light control and stability,” says Bain.
Most research-grade instruments utilize either a photomultiplier tube (PMT) or a photodiode array (PDA) detector. The former tend to be more expensive but are considerably more sensitive to very low light levels, as would be found when querying very highly absorbent samples. PDAs, on the other hand, will capture the entire spectrum at once, and can extend the range of the instrument to about 1100 nm (in the near-IR [NIR] range). Other detectors, such as indium gallium arsenide (InGaAs) and lead sulfide (PbS), are also sometimes used by UV-VIS specs to detect even further into the NIR.
Not to be outdone, detectors can also capture the entire spectrum (or selected parts of it) by allowing the monochromator to scan stepwise, recording the intensity of output at each wavelength.
Purchasing considerations
A high-end research instrument will likely be a double-beam, with two monochromators (the second helps to further reduce stray light); have one or more PMTs; be more than a meter wide (the long distance between optical components also helps to control stray light); and cost more than $30,000. It may have the ability to measure up to 8 or 10 AU (absorbance units—meaning 1/108 to 1/1010 of the light gets through). But “not many people or applications require that high level of performance,” says Bain.
On the other end, an entry-level, nonscanning, single-beam instrument can have a small footprint and cost under $3000, notes Bain. But he warns that instruments at the very low end may not have the photometric range to accurately measure over about 1.5 AU, which is not high enough for a typical analytical lab. Photometric range may be listed in the instrument’s specifications as “photometric linearity,” “absorbance range,” or the like.
Spectral bandwidth
Spectral bandwidth is a measure of how narrowly the chosen wavelength is being measured. For example, setting a monochromator with a 2-nm bandwidth slit to 450 nm will allow light between 449 and 451 nm through. A narrower bandwidth enables close or overlapping peaks to be better resolved. The tradeoff is that less energy gets through the smaller slit, thus reducing the signal-to-noise ratio and increasing the effective amount of noise, points out Derek Hodgeman, Production Manager at Buck Scientific (www.bucksci.com). Higher-end instruments may compensate by using higher-energy light sources and more sensitive detectors. Some manufacturers offer interchangeable slits, or a wheel with multiple slit sizes.
Cuvette holder
A typical UV-VIS spec will come with a single cuvette holder. An automated multicuvette changer, standard on the Thermo Fisher GENESYS™ 10S, is often available as an option. Similarly, throughput can be boosted with a sipper accessory that allows the user to spend less time filling and rinsing cuvettes.
User interface
The user interface is another consideration. Most specs come with a touchpad, with the software to run the instrument built in. More sophisticated operations and applications can generally be run from a PC. “Some vendors include software with the instrument, and some sell it as an option,” warns Bain. “The level of sophistication of what you can do on a local [built-in] control also varies.”
Applications and accessories
Of course, there are varied applications for which a UV-VIS spec will be used, and a host of accessories are available to cater to them, depending on the instrument. These include shakers, heaters, and coolers, as well as specific measurement tools like different relative specular reflectance accessories for different degrees of incident light.
So think about what you will need from a UV-VIS spectrophotometer, both now and in the foreseeable future:
- What will you be measuring, and what will you need to measure it?
- How accurate and precise do you need to be, and over what range of wavelengths and absorbance?
- Will you be processing just a few, or many, samples at a time?
- How much bench space are you willing to dedicate?
- What is your budget?
Then you can determine which spectrophotometer can best meet those needs.
Josh P. Roberts has been a full-time biomedical science writer for more than a decade. After earning an M.A. in the history and philosophy of science, he went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology; e-mail: [email protected]
Please see our UV VIS Spectrophotometer section to find manufacturers that sell these products