Most scientists used a spectrophotometer as students, maybe in an introductory biology lab, and many researchers continue to apply this technology. A quick look on PubMed revealed the term spectrophotometry in at least a few thousand articles each year since 1972—more than 7,000 in 2008. Simply put, a spectrophotometer measures how much light of a specific wavelength goes through a sample or is reflected. Therein lies the tricky part, because different platforms analyze light of different ranges of wavelengths.
This technology got started in 1940 with the release of the Beckman DU Spectrophotometer. That device came from the company—first called National Technologies laboratories then Beckman Instruments and now Beckman Coulter—started by U.S. chemist Arnold Beckman. U.S. engineer Howard Cary served as project leader in developing this device.
The first spectrophotometer and today’s follow the same general principle: The device directs light of a specific wavelength at the sample, and reflected and transmitted light get measured with sensors. Exposing a sample to various wavelengths of incident light creates a fingerprint of the sample. The light not reflected or transmitted is absorbed.
One of the most important improvements in the technology is the method of creating the light. In the first platforms, developers used prisms to create light of the desired wavelength. Today, the light comes from a variety of sources, such as deuterium or halogen lamps. The source of light depends, in part, on the kind of wavelength that a spectrophotometer must create.
One of the most important improvements in the technology is the method of creating the light.
Spectrophotometers use incident light of wavelengths from about 10–25,000 nanometers (nm). Moving across the spectrum from higher to lower energy, these spectral ranges are ultraviolet (UV; 10–380 nm), visible (Vis; 390–700 nm), near infrared (NIR; 800–2,500 nm), and IR (IR; usually mid-IR or 2,500–25,000 nm). As a quick aside: consider these numbers as approximations only, because different sources divide the ranges a bit differently—but usually within 10 or so nm of each other.
In most cases, spectrophotometers come as IR, Vis, UV-Vis, or UV-Vis-NIR. Although spectrophotometers could be grouped in many ways—single wavelength or multi-wavelength, angle of the incident light and so on—this buyer’s guide takes readers from lower to higher energy light sources, moving from longer to shorter wavelengths. From a functional perspective, this arrangement provides an easier approach from a researcher’s standpoint.
To aid scientists even more, this buyer’s guide also provides brief examples of recent applications of each kind of spectrophotometry. In that way, this article shows scientists what this technology can do, which usually reveals more than just telling researchers the instrument specs. Whether showing or telling, however, nothing short of a long list—or perhaps a book—could provide an inclusive tour of this area of instrumentation. Instead, the following provides an overview intended to help researchers plan their next steps toward adding a valuable new piece of equipment to a lab.
IR all around
In the IR category, spectrophotometers tend to be Fourier Transform, or FT-IR, platforms. One of the best-known devices is the Nicolet FTIR spectrophotometer, which comes in several models from Thermo Fisher Scientific. For example, the Nicolet iS 50 FT-IR can be set up as a simple benchtop device or a fully automated platform. It can also cover light from far-IR to Vis.
These devices can be used in many ways. For example, Shimadzu’s IRSpirt comes with 23 application programs. Plus, it’s small and light enough to take it to the sample. This spectrophotometer also accommodates third-party accessories, which can expand the applications. Similarly, Bruker’s ALPHA II platform uses the company’s QuickSnap sampling modules to test solid, liquid or gaseous samples.
Beyond the hardware, scientists also require software capabilities. Especially in regulated applications, such as pharmaceutical R&D or academic drug research, scientists will appreciate a device—such as Qualitest’s QualiFT-IR 5000 Spectrophotometer—that comes set to accommodate the needs of the U.S. FDA’s CRF 21 Part 11 requirements. This platform, and others, can also use optional software packages designed for specific applications.
Although spectrophotometers came into labs long ago, this technology applies to modern research across a wide range. In Iran, for example, scientists used FT-IR spectrophotometry—plus, spectrophotometry in other spectral ranges, as well as other methods of analysis—to study an experimental form of nano-sensor.1 The scientists built the nano-sensor from ligands attached to quantum dots, and used it to quantify copper ions in water samples. FT-IR spectrophotometry helped the researchers characterize the quantum dots.
Many healthcare tasks can also use this technology. At the school of pharmaceutical sciences at Brazil’s Universidade Estadual Paulista, scientists showed that FT-IR spectrophotometry can be used to quantify the antimicrobial daptomycin in a powder.2 The authors noted: “IR spectrophotometry stands out because it does not use organic solvents and … also allows the quantification of substances.”
So, a single FTIR spectrophotometry platform—if purchased with the desired features and options—can often perform many procedures as it comes, and it can be easily outfitted to do even more.
What we see
The visible spectrum replicates the wavelengths that humans can see. For many Vis spectrophotometers, though, the wavelength range extends a bit above and below our visual capabilities.
For example, Thermo Fisher Scientific’s Model 1200 provides a detection range of 325–1,000 nm, with a wavelength accuracy of ±2 nm. Although these platforms can be fairly simple, this one includes modes for absorbance, transmittance, and concentration. It also performs one-button automatic zeroing and blanking. This device can be run with Windows-based software to collect data in Excel. As the product description notes: “A tool-free and alignment-free lamp change, large sample compartment, and a variety of optional accessories, make this model ideal for any standard application.”
The V-1200 from VWR provides similar features, plus an 8-cell changer can be added as an option. VWR calls this instrument “reliable, robust, and easy to use.” In a Vis spectrophotometer, those are precisely the features that should be expected, because these are far from the most complicated platforms among spectrophotometers. In fact, they are some of the simplest, using a single lamp for incident light and a simple photodiode detector.
Some of these devices look just like the ones that I used in undergraduate labs almost 40 years ago, and I wouldn’t be surprised to hear that some of those are still in use. Why? They were old-school, rugged pieces of equipment that did a job without any unnecessary features. As an example, one Cole-Parmer Visible spectrophotometer gives a scientist a wavelength range of 335–1,000 nm, and the output is analog, which remains effective for many measurements.
Despite being simple, these devices can be used in many ways. Even in describing Cole-Parmer’s basic model, the company states: “These spectrophotometers suit a variety of general-purpose and quality control applications—ideal for biochemistry, environmental chemistry, food testing, and water testing labs. They combine value and simplicity with the accuracy of much more expensive instruments.”
Simple or not, these instruments still get used in advanced research. As an example, an international team of scientists recently used visible spectrophotometry to study quartz in concrete.3 They reported: “The visible spectrophotometer test was performed to evaluate the dissolution potential of the different samples of deformed quartz, which confirmed that the reactivity of the quartz increases as the deformation of the crystalline structure increases.”
A spectral sweet spot
Spectrophotometers that work in UV and Vis might get used more than any other type. This is also a segment of the market that delivers many options to researchers. That all started in 1947, when scientists could purchase the first commercial device, the Cary 11 UV-Vis Spectrophotometer, developed by Applied Physics Corporation, which was co-founded by American engineer Henry Cary.
That legacy lives on in Agilent’s Cary 60 UV-Vis. It covers wavelengths of 190–1,100 nm, and its lamp usually lasts about a decade. Add on its ability to scan that entire wavelength in 3 seconds, and this platform helps scientists collect lots and lots of data—all long before even thinking about changing the lamp. Also, scientists can skip the cuvette with this device, because it includes a fiber-optic probe that goes right in a liquid sample.
Some of today’s devices also work with very small sample volumes. For example, Thermo Fisher Scientific NanoDrop ND-2000 UV-Vis only requires 0.5–2 microliters of sample. This platform can be used to analyze cell cultures, nucleic acids, and proteins. Scientists can also develop custom methods.
It is even possible to quickly scan many small samples. Revvity's DropletQuant UV-VIS microvolume spectrophotometer, for example, can scan 96 samples—using just 2 microliters per sample—in less than five minutes. This system can also be used as part of an automated workflow.
In India, a team of scientists developed a method to remove pesticides from samples using graphene oxide-magnetic nanoparticles (GO-MNPs).4 They reported: “The advantages of the present method are use of simple UV-vis spectrophotometry for monitoring and low-cost use of GO-MNPs nanomaterial for the removal of pesticides from sample solution.”
Broad benefits
In some cases, scientists want a spectrophotometer that covers more options, and that’s just what an UV-Vis-NIR device does. It can scan samples with light that ranges from about 10–2,500 nm. This allows more samples to be analyzed in more ways.
The specific platform might not cover every nanometer of this range. For instance, Shimadzu’s UV-2600 covers 185–900 nm, and it can be enhanced with the ISR-2600Plus two-detector integrating sphere to scan into NIR, going as long as 1,400 nm. In describing that upgrade, Shimadzu’s product literature points out: “As a result, the UV-2600 can accommodate measurements of solar cell anti-reflective films and polycrystalline silicon wafers.”
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The broader range of incident light and detection makes these spectrophotometers more complex than some of the others. For example, it takes more than one detector to cover such a wide range. In Jasco’s V-780 UV-Vis-NIR platform, a photomultiplier tube detects UV-Vis light, and an indium-gallium-arsenic detector picks up the NIR light. Moreover, a monochromator and automatically exchanged gratings create the incident light. Combining all of these elements, this device covers 190–1,600 nm wavelengths.
With the ability to cover such a wide spectral band, scientists want a system that works in many applications. For the V-780, Jasco notes that—with the right accessories—this device can be used “for virtually any biological, material science, or routine QA/QC measurement.” To accommodate more sample formats, the options “include a wide variety of liquid cell holders, micro and ultra-micro cell holders, flow cell units, and accessories for solid samples.” Even automation accessories can be added.
In many cases, researchers use UV-Vis-NIR spectrophotometers in material sciences. A team of researchers in Germany, for example, used one of these platforms to characterize an iron-based metal-organic framework.5 They reported: “The semiconductor properties of the oxidized material were studied by diffuse reflectance UV/vis/NIR spectroscopy….”
Life science applications also benefit from this technology. One international team of scientists studied sunbird feathers, noting that “wavelengths outside of the visible spectrum that are reflected by coloured tissues have rarely been considered, despite their potential significance to thermal effects.”6 With UV-Vis-NIR spectroscopy applied to feathers from different anatomical areas of 68 species of sunbirds, this team found that “breast plumage was the most reflective and cap plumage the least” and that “differently coloured feathers consistently vary in their thermal, as well as obvious visual, properties.”
Concluding checklist
This overview of spectrophotometers reveals some of the many ways that these instruments can be used and some of the questions that they can answer. In addition, the brief product descriptions given above show that scientists can select from very basic to far more sophisticated models. The question is not which particular product is the best overall, but rather which one fits functionally with a specific application.
To help scientists make the best selection, Cole-Parmer created a checklist that is worth considering:
1. Density, shape, or size of the product you wish to measure
2. Detection limits
3. Wavelength range
4. Analytical working range
5. Sample throughput (single sample vs. multi-sample)
6. Cost of instrument and associated consumables
7. Data quality
8. Measurement time
9. Custom and/or pre-configured method options
10. Footprint of instrument.
In reviewing this list, some of the earliest comments about spectrophotometers come to mind: “a potential user must consider the implications of each principle very carefully in studying the performance of various instruments.” American physicist Van Zandt Williams wrote that in 1951, when he worked at Revvity.7 More than 65 years later, those words ring truer than ever.
In fact, today’s selection of instruments swamps the ones available when Williams was writing. Consequently, the “performance of various” instruments must be compared more carefully and completely.
The amazing thing is that 75-year-old technology proves as useful as ever, probably more so. The high-end instruments provide more power and potential, but even the simplest instruments can address important questions in current science. Sometimes, scientists require super-sophisticated devices; sometimes, something simple but robust works even better.
From basic teaching labs—where many of us first encountered these instruments—to advanced research projects, spectrophotometers of all sorts still make an impact. As this overview reveals, many labs would benefit from more than one of these instruments, and this technology will probably impact scientists for another 75 years or more.
References
1. Elmizadeh, H, et al. Ligand-capped CdTe quantum dots as a fluorescent nanosensor for detection of copper ions in environmental water sample. J. Fluoresc. 2017 [epub ahead of print; PMID: 28936785].
2. Tótoli, EG, Salgado, HRN. Fourier-transform infrared (FTIR) spectrophotometry: an ecofriendly method for the analysis of injectable daptomycin. J. AOAC Int. 100:1569–1576. 2017. [PMID: 28917263].
3. Techer, F, et al. Relationship between degree of deformation in quartz and silica dissolution for the development of alkali-silica reaction in concrete. Materials 10:E1022. 2017. [PMID: 28869559].
4. Shrivas, K, et al. Removal of endrin and dieldrin isomeric pesticides through stereoselective adsorption behavior on the graphene oxide-magnetic nanoparticles. Environ. Sci. Pollut. Res. Int. 2017. [epub ahead of print; PMID: 28918582].
5. Spirkl, S, et al. Single-crystal to single-crystal transformation of a nonporous Fe(II) metal-organic framework into a porous metal-organic framework via a solid-state reaction. Inorg. Chem. 2017. [epub ahead of print; PMID: 28960968].
6. Shawkey, MD, et al. Beyond colour: consistent variation in near infrared and solar reflectivity in sunbirds (Nectariniidae). Naturewissenschaften 104:78. 2017. [PMID: 28871351].
7. Williams, VZ. Principles of infrared spectrophotometry. Science 2925:51–51. 1951 [PMID: 14798382].
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