Ultraviolet-visible spectroscopy (UV-Vis) measures and identifies chemicals that absorb in the ultraviolet and visible regions of the electromagnetic spectrum. UV wavelengths range from 10 to 400 nanometers, while the visible spectrum—colors visible to the human eye—lie from approximately 400 to 800 nanometers. UV-Vis can be used to identify the presence of chemicals and their concentrations. It’s a popular technique in chemistry, foods, pigments, pharmaceuticals, polymers, and the life sciences.
Compounds analyzed by UVVis possess “chromophores,” or color-absorbing carbon-carbon, carbon-oxygen, or carbonnitrogen double bonds, as well as carbon-halogen single bonds. Depending on the extent and type of unsaturation, particularly whether the bonds are conjugated (alternate with single bonds), compounds may absorb in the UV or visible region of the spectrum. Chemicals such as carotene and certain dyes, which have extensive conjugation, are brightly colored and absorb in the visible region. Conjugation causes a shift in absorbance to higher wavelengths.
At one time, vendors routinely offered separate instruments for UV and Vis, but today the two tend to be combined. Some manufacturers add a third capability, near-infrared (NIR), which is immediately above the visible region.
UV-Vis spectrometers come in four basic models: low-cost singlebeam, dual-beam, array-based, and handheld. Single-beam techniques must apply a correction for the loss of light intensity as the beam passes through the solvent. Dualbeam spectrometers use a second solvent reference cell and perform the correction automatically. Single- and dual-beam benchtop instruments use a broad-spectrum lamp as the light source, and most use a photomultiplier tube as the detector. Some instruments employ photodiode array detectors. Handhelds employ single-wavelength light-emitting diode light sources and photodiode detectors.
UV-Vis instruments have become much faster, more compact, and feature-laden. Array instruments, in particular, provide a level of detail and throughput that was previously unavailable. “Although they may have originally been interested in just one or two wavelengths, users love capturing the entire spectrum in seconds,” says Grant Drenkow, UV-Vis product manager at Agilent (Loveland, CO). Other factors entering into purchase decisions are cost, throughput, and the reliability of data.
Sample size is an ongoing issue in analytical science. “People want more from less,” notes Philippe Desjardins, scientific marketing manager at NanoDrop (Wilmington, DE). From this need has emerged microvolume UV-Vis, which analyzes liquid samples in the 0.5 to 50 μl range.
Several vendors offer microvolume- only instruments or microvolume cells for conventional UV-Vis spectrophotometers. In its most elegant configuration, a microdrop of sample is deposited atop a small pedestal and a second pedestal immediately approaches to complete the optical path by contacting the sample’s meniscus. Embedded in each pedestal is an optical fiber—one serving as a conduit for the light source, the other for the detector. The sample stays in place due to surface tension, but the path length may be optimized by adjusting the distance between 0.05 mm and 1 mm, depending on the sample concentration. This eliminates the need, as in conventional UV-Vis, to remove the cuvette and dilute or concentrate the sample.
• Photodiode array (PDA) light path scans a complete spectrum from 190 to 1,100 nm in less than one second
• Fewer moving components reduce the need for recalibration
•Uses a deuterium discharge lamp for the full UV and visible range
• Ideal for quantitation of nucleic acids or fluorescent-labeled nucleic acids and protein analysis
• Sample mounting, measurement and cleaning are all performed by the device
• Analysis can be performed with 1 µl or 2 µl samples
• Analysis results can be converted to PDF or CSV files
GENESYS™ 10S and BioMate™ 3S
• GENESYS instruments are capable of producing scan data at speeds up to 4,200 nm per minute, and feature a unique optical design that reduces stray light
• No warm-up time is required with the BioMate 3S, and samples are not constantly exposed to UV light
•BioMate 3S includes pre-configured test methods for nucleic acid, protein and cell growth measurements
Thermo Fisher Scientific
• Feature a color touch screen, USB connection, optional Bluetooth for data transfer and an optional built-in printer
• Includes application-based software for all stand-alone and PC-controlled instruments
• Equation Editor allows users to program specific methods that include calculations based on measured data