It's all in the Chromophores
Ultraviolet-visible spectroscopy (UV-Vis), an absorbance-based analytical technique, measures and identifies chemicals that absorb in the contiguous 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.
Compounds analyzed by UV-Vis possess “chromophores,” or color-absorbing carbon-carbon, carbon-oxygen, or carbon-nitrogen 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 (i.e., toward the visible region).
Grant Drenkow, product manager for UV-Vis at Agilent (Loveland, CO), describes UV-Vis as “the screwdriver of the laboratory,” for its ubiquity in nearly all science- or engineering based industries.
Because compounds have unique UVVis spectra, including a maximum absorption wavelength (λmax or “lambda max”) and molar extinction coefficient (ε, absorbance per mole), UV-Vis can be used to identify the presence of chemicals and their concentrations. For this reason, UV-Vis is a popular technique in chemistry, foods, pigments, pharmaceuticals, polymers, and the life sciences, for basic or applied research as well as quality control.
For determining a chemical concentration, users typically scan the entire UV-Vis spectrum, although dedicated applications may only examine λmax and the absorbance. The latter approach is useful in measuring kinetics— the appearance or disappearance of a chemical species over time. However, the distinction between singlewavelength and full-spectrum analysis has become almost moot since modern instruments “scan” the entire UV-Vis spectrum in about one second.
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 single-beam, 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. Dual-beam spectrometers use a second solvent reference cell and perform the correction automatically. Single- and dual-beam benchtop instruments use a broadspectrum lamp as the light source, and most use a photomultiplier tube as the detector. Some instruments (e.g., from Agilent) employ photodiode array detectors.
Handheld instruments are usually dedicated to one wavelength and analyte, for example, for water testing in the field. Handhelds employ singlewavelength light-emitting diode light sources and photodiode detectors.
Array-based instruments use a very broad-spectrum tungsten lamp emitting between 200 and 1000 nm, and use a photodiode array or charge-coupled device as the detector. Array instruments do not scan the UV-Vis spectrum in the traditional sense, but rather send and collect the entire spectrum at once. Array instruments are useful when spectra must be acquired quickly, for example, from an HPLC trace or fast kinetics experiments.
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,” Drenkow says. Other factors entering into purchase decisions are cost, throughput, and the reliability of data.
Sample size (or lack thereof) has for some time been an ongoing issue in analytical science. “People want more from less,” notes Philippe Desjardins, scientific marketing manager at Nano- Drop (Wilmington, DE), a subsidiary of Thermo Scientific. From this need has emerged microvolume UV-Vis, which analyzes liquid samples in the 0.5 to 50-microliter range.
Several vendors offer microvolumeonly 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 occurs frequently in conventional UV-Vis, to remove the cuvette and dilute or concentrate the sample.
Different models possess varying capabilities to adjust the path length, spectrum acquisition time, light source, and detector. For example, instruments from NanoDrop employ a xenon flash lamp that delivers source light across the entire UV-Vis spectrum at once, so spectrum acquisition takes just a few seconds.