Life scientists have traditionally used UV-Vis spectrophotometers for evaluating the quantity and quality of proteins and nucleic acids, as well as a few other more specialized applications. “
Specs” have been in life science labs at least since the 1940s when Arnold Beckman introduced the
DU Spectrophotometer, yet the concepts underlying the enabling instrumentation have changed surprisingly little.
Spectrophotometry is a fairly mature field, and the “spec” as we know it will be around for a long time, predicts Jerry Messman, director of Fort Collins, Colorado-based Stranaska, a metrology consulting and education firm. “It can be used for a lot of things.”
That’s not to say that there have been no innovations in the basic hardware itself over the years, nor in its interfaces and accessories – quite the contrary. Ingenious adaptations have allowed for a variety of assays beyond quantitation and quality control. Footprints have become measurably smaller. Detection systems have become more sensitive, and new ones have been introduced. Computerization has made chart recorders and manual data analysis things of the past. Some accessories have allowed for time- and temperature-controlled measurements, while others have extended the spectrophotometer’s reach beyond the sample cuvette. And a global trend toward smaller volumes has given rise to instruments capable of assaying less than a single microliter of sample.
How it Works
When light is shone onto a sample in a spectrophotometer it will typically allow most of that light to pass through, preferentially absorbing a portion of only certain wavelengths. Because transmittance and absorption are inversely correlated, measuring the light that comes through the sample yields a quantitative measure of how much light the sample has absorbed.
Absorbance at specific wavelengths, in turn, is directly proportional to the quantity of biological material in the sample. And because each macromolecule has a characteristic absorbance spectrum, not only can the quantity of the macromolecule be assessed, but its purity can be assayed as well – typically by looking at absorbance at several different points on the UV-Vis spectrum. Similarly, specs are routinely used to count bacterial cells by measuring turbidity of the liquid cell cultures.
Traditional spectrophotometers employ a diffraction grating that allows only a narrow bandwidth of the source light to reach a liquid sample. All the light transmitted through the sample is collected and measured by a detector. In “scanning” spectrophotometers, the grating can generally be progressively adjusted to measure transmission at other wavelengths.
A variation on the theme uses an array detector. In this case, the full spectrum of light is directed to the sample. The light that passes through is then dispersed by a diffraction grating. Distinct bandwidths are collected by separate regions of the array, allowing for the entire spectrum to be assayed together.
Control
These days, most instruments are controlled by sophisticated software, with pre-programmed modules for common functions like DNA, RNA, and protein quantification, and even assessing the efficiency of microarray probe labeling; modules can be downloaded, purchased, or custom-written to accommodate more specialized applications. The data acquired – including that from a reference (or “blank”) sample – can be stored for future reference, and statistical measures performed. This all allows for what Jasco’s Richard Larsen considers “black box” operation of data acquisition and interpretation: the user loads a sample, pushes a button, and gets an answer.
The software may either be built in to the spec or reside on a PC, with some instruments offering a choice. Even on stand-alone units, options are often available to export data to a portable storage device such as a flash drive, or even directly to a PC through a USB or wireless (Bluetooth) connection.
Accessorize
Microprocessors can be used to manage more than just data acquisition and analysis. For example, many instruments offer programmable accessories such as thermal control – both electronic and water-controlled – modules to monitor the absorbance versus temperature. “DNA denatures when it’s heated, and the strands separate,” explains Luis Moreno, senior product manager for spectroscopy at Hitachi High Technologies America. When that happens, its absorbance properties change.
Automated sampling accessories such as sippers are becoming popular for more high-throughput applications. “You’re able to basically walk up with a vial of solution, push a button, and it sucks up that solution into a cell,” notes Larsen. “You get a reading and it pushes [the sample] out and gets the next one.”
A wide variety of cuvettes (and cuvette holders) are available for many instruments. For example, among Hitachi’s offerings are cylindrical and rectangular long-path cuvettes, tandem cell holders, and micro-sample cell holders and cells. The latter enable users to read samples as small as one-microliter.
Fiber optics extend the reach of the spectrophotometer to outside the box. Dip probes, for example, let the user determine the absorbance of solutions (or take reflectance readings of powders) in situ, without the need to transfer them to a cuvette, explains Jeff Prevatt, vice president of S. I. Photonics. And by attaching a fiber optic cable to the C-mount of a microscope, spectra can be obtained directly from the stage, he adds.
Using fiber optics comes with a price, though, warns Larsen. “There’s no fiber optic system out there that’s 100% efficient,” he says. “When you’re talking about an analysis where there is a very small difference in the amount of light that’s getting to the detector, any losses along the way will be significant.”
Maligned?
As a rule, fiber optics accessories are used mainly by array-based spectrophotometers. Although now an accepted part of the research world, these instruments didn’t always hold the place they do today in the biomedical research community.
Shortly after their introduction in the 1980s, a lot of vendors jumped on the bandwagon, producing inexpensive products with considerable drift and instability, that weren’t suitable for a QC department, says Prevatt. “They kind of tainted the market, … and unfortunately left a bad taste in people’s mouth.”
That stigma is dying away as people become more familiar with the array technology that is responsible for the digital camera revolution, Prevatt continues. His company and most of the field’s major players – including Agilent, Beckman Coulter, Hitachi, and PerkinElmer – now offer research-grade array spectrophotometers on a par with the best single-beam scanning instruments.
Some array-based specs even boast capabilities that go beyond the more traditional instruments.
NanoDrop
“NanoDrop is exciting,” exclaims Messman. “It’s a whole new approach.”
Like other array-based instruments, the NanoDrop 1000 measures the entire spectrum – in this case, from 220 to 750nm – virtually instantaneously. Yet its unique sample retention system allows for a single, undiluted, one-microliter sample to be assayed in a matter of seconds. The drop is placed on an optical fiber (embedded in stainless steel); when the arm – a second optical fiber – is lowered, it sandwiches the drop and holds it in place by surface tension. The exact volume of the sample – just as in cuvette-based instruments – is not a factor in assessing absorbance. Because the system reads a sample “at-strength” directly on the probe, explains NanoDrop’s technical marketing manager Philippe Desjardins, “We remove the human error” associated with preparing buffers, making dilutions, and caring for cuvettes. The company – purchased by ThermoFisher Scientific in October 2007 – also offers a higher–throughput version capable of assaying eight samples at a time.
An analogous technology – in this case, sandwiching a one microliter drop between an optical fiber and a mirror – is the basis of the Implen’s competing system, the NanoPhotometer.
The trend toward ever smaller sample volumes in continuous, yet Larsen doesn’t see it dropping into the nanoliters volume just yet. “When we get to that point we’re going to have to re-design the instrumentation, he speculates. “Just because there’s only so much light that you can squeeze down into a small volume.”
