Modern spectrophotometer manufacturers produce these instruments in three general optical configurations. Single beam spectrophotometers use only one beam of light for measurements. With these spectrophotometers, a blank or reference solution is first measured. Then, the sample solution is measured. The difference in absorbance (or transmission) values between the sample and reference cuvettes at the measuring wavelength yields the sample^s absorbance.
In double-beam spectrophotometers, the light beam is split in two after passing through a monochromator so that two light beams of identical wavelength and intensity pass through a reference and sample cuvette. These instruments generally use a mirrored rotating chopper wheel to alternately direct light through the sample and reference cuvettes. The measured absorbance is the difference in light intensity between the two transmitted beams, and can be measured with a single photodetector. Many double-beam spectrophotometers also contain scanning monochromators for absorption spectra generation. Some double-beam spectrophotometers have dual-wavelength capabilities, in which two separate monochromators generate two different wavelengths of light.5 These two monochromatic light beams pass through a single sample so that a difference in absorbance between the two wavelengths is measured. Dual-wavelength spectrophotometry is well suited for measuring induced absorbance changes in macromolecules or dyes whose absorbance is altered when oxidants or reductants are added. The third optical configuration involves spectrophotometers with PDA detectors that can produce simultaneous measurement of a sample at multiple wavelengths. In array-detector spectrophotometers, incident light first passes through the sample, then the diffraction grating, and finally activates the array detector. The separated wavelengths are detected by different pixels of the PDA or CCD array. These spectrophotometers are ideal for the rapid measurement of photosensitive samples, or where high spectral resolution is not required (i.e., HPLC detectors). Absorption Spectroscopy Applications Scientists have developed a nearly endless variety of applications for absorption spectroscopy. The most common use is to quantify the relative concentrations of proteins, nucleic acids, sugars, or lipids. The heterocyclic bases of DNA and RNA contain conjugated double bonds that absorb UV light near 260 nm. Similarly, the aromatic amino acids tyrosine and tryptophan absorb near 280 nm. Researchers can also measure protein concentration using colorimetric methods such as the Bradford, Biuret, Lowry, and bicinchoninic acid assays.4 Other colorimetric assays exist to measure DNA, carbohydrates, hexose amines, and sterols such as cholesterol.3 Another application is molecular identification, using the characteristic spectral shapes and absorption maxima of compounds such as cytochromes, coenzymes such as NADH, visual pigments, iron-sulfur proteins, and chlorophylls.
Scientists can measure enzymatic activities with absorption spectrophotometry as well. For example, the reporter enzyme b-galactosidase cleaves the chromogenic substrate o-nitrophenyl-b-D-galactopyranoside to form the yellow product o-nitrobenzene, and hydrolyzes chlorophenol red-b-D-galactopyranoside to produce a dark red product.6 Similarly, researchers can use absorption spectroscopy to assay enzymatic reaction kinetics. If the substrate or product of a reaction does not absorb appreciably in the UV or visible range, that reaction can be linked to a second reaction whose substrate or product can easily be monitored. Finally, scientists can apply absorption spectroscopy to examine the function of redox proteins in mitochondria and chloroplasts. Difference spectra can detect small absorbance changes, or spectral shifts of absorbance peaks, against a high background absorbance. This approach is widely used to examine proteins that undergo absorbance changes in the visible region, as well as photosensitive pigments such as rhodopsin. Several important factors should be considered when purchasing a spectrophotometer. These include the instrument^s detection range, optical configuration, and sensitivity, as well as the required spectral bandwidth and the need for wavelength scanning capabilities. Some optional accessories include add-ons to enable kinetic measurements, temperature control, or small volume measurements. For example, some spectrophotometers can measure absorbance in sample volumes as low as one microliter. Although some instruments are basic machines that display only the absorbance value on an LCD display, others are equipped with computers and software that perform common analyses, such as calculating the concentration, melting point, and molecular weight of an oligonucleotide of known sequence. An additional concern is whether the device is intended for high-throughput studies. Generally scientists can use spectrophotometers to analyze sample absorbance either in a cuvette or in a microtiter plate, but not both. Thus, it is important to determine how the instrument will be used prior to making a purchase. Finally, technically savvy users have one additional option: A number of companies sell the various components necessary to assemble a build-your-own spectrophotometer. Thus, researchers can design instruments containing every desired feature without having to pay for those that will not be used. .
References
1.A. Thackray, M. Myers Jr., Arnold O. Beckman: One Hundred Years of Excellence, Philadelphia: Chemical Heritage Foundation, 2000.
2.J. Lederberg, "An appreciation of Arnold Beckman," The Scientist, 14[5]:6, March 6, 2000.
3.D.B. Gordon, "Spectroscopic techniques: I. Atomic and molecular electronic spectroscopy," In: K. Wilson, J. Walker, eds. Principles and Techniques of Practical Biochemistry, 5th ed. Cambridge, UK: Cambridge University Press, 2000, pp. 453-97.
4.F.M. Ausubel et al., Current Protocols In Molecular Biology, New York: John Wiley and Sons Inc., 1988, pp. 10.1.1-10.1.3.
5.B. Chance, " principles of differential spectrophotometry with special reference to the dual wavelength method," Methods in Enzymology, 24:322-35, 1972.
6.H.E. Sussman, "Choosing the best reporter assay," The Scientist, 15[15]:25, July 23, 2001.