The New Look of a Mature Platform

Ultraviolet-visible (UV-Vis) spectrophotometry is arguably the most common as well as one of the oldest forms of absorption-based analysis. UV and visible regions of the electromagnetic spectrum are contiguous: UV wavelengths range from 10 to 4000 angstroms; they are visible from 4000 to 7000 angstroms.

According to data from Strategic Directions International (Los Angeles, CA), the market for scanning UV-Vis spectrophotometers is approximately $400 million per year, with an annual growth rate of six percent.

“Overall miniaturization, speed, ease of use, reliability, and mobility will be the ongoing drivers for growth in UV-Vis spectroscopy over the next decade,” says Thomas Sahiri, president of Implen (Westlake Village, CA). He predicts that ongoing innovation will make these instruments relatively inexpensive, easy to use, and fast for diagnostics, day-to-day routine lab applications, and on-site testing. “New optical components are enabling important new features such as easier cleaning, consumable-free operation, and automatic virtual dilution, which significantly increases the instrument’s dynamic range.”

Additional factors fueling innovation in UV-Vis instrumentation are shrinking lab space, increasing demand for small-volume UV (driven mainly by life science markets, specifically nucleic acid and protein analysis), and the pressure to shorten analysis times.

Sahiri notes that photodiode array spectrophotometers are becoming less expensive, smaller, and more feature-rich. “The introduction of fiber optics in spectrophotometers has increased the system flexibility significantly, while manufacturers are constantly improving the performance of the small, fast systems without moving parts. USB stick-type mobile spectrophotometers will provide a new step to miniaturization that will open new application fields for diagnostics, off-site research, forensic analysis, and food testing.”

Replacing More Costly, Time-Consuming Assays

“HPLC detectors are the most common application of UVVis technology,” notes Merlin K.L. Bicking, Ph.D., president of ACCTA (St. Paul, MN), an analytical chemistry consulting and training company. “But conventional spectrophotometers still play an important role in many laboratories across a variety of industries.”

UV-Vis has been an integral part of testing for content uniformity and dissolution in the pharmaceutical industry. Dissolution tests are conducted to determine if a pill or tablet will dissolve under physiologic conditions. The process involves taking samples from the dissolution test at specific time points and analyzing them for levels of active ingredients. The standard analysis by liquid chromatography involves preparing calibration standards, collecting and filtering samples, and then transferring all the solutions to autosampler vials for analysis. The typical HPLC cycle time is 5 to 10 minutes per sample, plus time for sample prep, mobile phase preparation, and solvent disposal.

“If the formulation does not contain any unusual components and the active ingredient has a relatively unique absorbance spectrum, a UV-Vis is an attractive alternative,” according to Dr. Bicking. Fully automated systems allow direct transfer of the dissolution media to the UV-Vis instrument. “Using a diode array spectrophotometer, results can be obtained within one or two minutes of collection. Furthermore, software is available to automatically calculate the desired dissolution parameters. Even without automation, these systems require considerably less time and effort than HPLC.”

Other “replacement” applications exist throughout industry. The requirement is that the target analyte shows reproducible UV-Vis absorption without interference from other species.

Although UV-Vis is a mature method, its speed and power continue to support cutting-edge research. At her lab at the University of Kansas, Melinda Toumi, Ph.D., uses UV-Vis spectroscopy to ensure quality and accuracy in several of her workflows. “We perform UV absorbance measurements at 280 nm for protein concentration as well as Bradford assays that use the visible region of the spectrum.” Toumi also relies on UV-Vis to assure low endotoxin levels in purified DNA. “These early experimental steps are vital to obtaining meaningful results since many subsequent reagent quantities are calculated in relation to the protein concentration.”

Streamlining Plate-Based Assays

When Ronald Earp, Ph.D., president of BMG LABTECH (Cary, NC), was in graduate school, he used non-scanning UV to capture surface plasmon resonance data. In 2004, Earp spearheaded the use of non-scanning UV-Vis detection in microplate readers. “Full-spectrum capture existed for cuvettetype readers but not for microplate readers,” he tells Lab Manager Magazine. The benefit of non-scanning spectrum acquisition is speed—the entire UV-Vis spectrum is captured in microseconds, including all assay-relevant wavelengths.

This is the basic idea behind Thermo Fisher Scientific’s NanoDrop UV-Vis instrument, in which a sample droplet obviates the need for a cuvette.

Instead of using a monochromator or filters, the fullspectrum approach excites the sample with a fullspectrum xenon lamp emitting from around 200 nm to 1000 nm. An array of charge-coupled device detectors (similar to sensors in digital cameras) on the collection side are tuned to receive transmitted light 1 nm apart along the entire spectrum. No mirrors, gratings, scanners, or moving parts are involved.

Conventional microtiter plate assays involving three wavelengths require three separate measurements and operator involvement. The non-scanning approach provides absorption or transmission at all three wavelengths, with optical density ratios or full spectra as needed. The dynamic range for BMG’s UV-Vis readers are approximately three optical density units, which according to Dr. Earp is sufficient for most biological assays. BMG’s readers also have a cuvette port for situations that demand the more traditional format.

For example, nucleic quantitation or purity assays employ the ratio of absorbance at 260 nm to the absorbance at 280 nm, with an additional measurement at 340 nm for cellular debris. DNA is considered pure when the A260/280 is 1.8 or higher. For pure RNA, the ratio must be two or higher. Similarly, the nonscanning approach can quantify the conversion of NADH to NAD+ at 340 nm while simultaneously quantifying protein at 280 nm.

“Traditionally, doing these types of microplate assays with UV required reading the plate many times,” Dr. Earp comments. Alternatively, fluorescence assays are more sensitive than UV but require separate reporter reagents for each analyte or event, plus associated preparation.

Angelo DePalma is a freelance writer living in Newton, NJ. You can reach him at angelo@adepalma.com.

For additional resources on UV-Vis spectrophotometry, including useful articles and a list of manufacturers, visit www.labmanager.com/spectrophotometers