Spectrophotometers: An Absorbing Tale

In 1940, nearly 30 years after Danish physicist Neils Bohr explained how light energy affects the electrons orbiting atomic nuclei, Coleman Instruments produced an instrument to take advantage of this principle. The device was an attachment to the company's pH meter that could measure absorbance of light in the ultraviolet (UV) region.1 In the 61 years since, absorption spectroscopy has become one of the most widely used analytical techniques in scientific research. Investigators routinely emplo

Oct 15, 2001
Gregory Smutzer
In 1940, nearly 30 years after Danish physicist Neils Bohr explained how light energy affects the electrons orbiting atomic nuclei, Coleman Instruments produced an instrument to take advantage of this principle. The device was an attachment to the company's pH meter that could measure absorbance of light in the ultraviolet (UV) region.1 In the 61 years since, absorption spectroscopy has become one of the most widely used analytical techniques in scientific research. Investigators routinely employ this method to measure the concentration of nucleic acid and protein samples, and UV-visible spectrophotometers are considered standard equipment for molecular biology labs.

The modern spectrophotometer is not actually based on Coleman Instruments' design, but rather on one by Arnold Beckman.1,2 He developed a mechanism to accurately control wavelength selection from a quartz prism, and integrated the optics and electronics of his spectrophotometer into a single unit that greatly simplified its use. Beckman's Model DU spectrophotometer was introduced in 1942, and its production had an immediate impact not only on scientific research,2 but also on the war effort. The DU spectrophotometer was extensively used in the mass production of penicillin.1 Scientists also employed the device in the analysis of crude oil, which contains benzene--used to make synthetic rubber--and toluene, a starting material for trinitrotoluene (TNT).1

In 1913, Bohr described the fundamental physical principles on which spectrophotometers are built. He proposed that atoms exist in only a limited number of energy levels, which represent the energy states of electrons in an atom. When one of these electrons absorbs radiant energy, it undergoes a transition from a lower energy state to a higher energy state, causing the electron to jump from a lower orbital to a higher one. Only the exact amount of energy that is equivalent to the difference in energy levels of an excited and ground state electron (a quanta of energy) will be absorbed by an atom or molecule. After absorption, these excited electrons return to their ground state with loss of energy, either as heat, or in certain cases, as light (fluorescence or phosphorescence).3

Molecules contain many covalent bonds whose atoms vibrate about their bond axes. These motions cause the stretching and bending of the molecule's covalent bonds, producing a variety of vibrational and rotational energy sublevels. Each of these different energy sublevels can absorb light of a discrete wavelength. Thus, when a molecule is subjected to light at ambient temperature, this subdivision of energy sublevels results in an absorbance spectrum that consists of a smooth curve rather than a series of discrete lines that are emitted from individual atoms of many elements.

Biological molecules such as nucleic acids and proteins absorb light energy in the UV and visible region of the electromagnetic spectrum. This region of the electromagnetic spectrum is therefore ideally suited for biological absorption spectroscopy applications. Because of the absorption of UV light by atmospheric gases, the practical short-wavelength limit for an absorption spectrophotometer is approximately 190 nanometers.3 The long-wavelength limit of spectrophotometers is usually determined by the wavelength response of the detector, but some commercial UV-visible spectrophotometers extend the measurable range into the near infrared (780-3,000 nm).

Spectrophotometer Components

Courtesy of WPA Ltd.

WPA Ltd.'s Lightwave UV-Vis spectrophotometer

A spectrophotometer consists of a light source, a monochromator for wavelength dispersion, a transparent sample holder, a light detector, and a device for measuring output from the detector. Light sources generate either UV or visible light, and can be either a tungsten halogen lamp for emission of visible radiation (380-780 nm), a hydrogen or deuterium discharge lamp for UV radiation (190-350 nm), or a xenon lamp that generates a continuous output of light over the UV and visible spectrum. Xenon lamps are advantageous because they do not emit the strong emission spikes in the UV that are characteristic of UV lamps, and can also emit light well into the infrared.

To measure the sample's absorbance at a defined wavelength, polychromatic light must be separated into monochromatic light. This is the monochromator's function, to disperse the light into a spectrum of varying wavelengths and to enable specific wavelength selection. Monochromators have an entrance slit, collimating and focusing mirrors, a movable light dispersion device, and an exit slit, which together function to determine the spectral resolution of incident light.

The monochromator's dispersion device can be either a prism or a grating. The former splits light by refraction (bending), whereas the latter does it by diffraction (interference). However, neither device actually produces truly monochromatic light. Instead, they allow for selection of a narrow range of wavelengths, called a bandpass. Generally, a diffraction grating is superior to a prism, because a grating produces a more linear dispersion of light, and produces a narrower bandpass. Thus, gratings have replaced prisms in most fields of spectrophotometric analysis. A glass or crystal prism splits white light into its component parts, as different wavelengths of light travel different paths when passing through it. In contrast, diffraction occurs on gratings by reflectance of light off the grating's engraved surface.

When a user selects a specific wavelength, the spectrophotometer responds by rotating the dispersion device so that that wavelength passes through the narrow exit slit and into the sample. By measuring absorbance data as the dispersion device is rotated, the instrument collects information over a wide spectral range, producing an absorbance spectrum.

The light exiting the monochromator passes through the sample and into a photodetector that measures the portion of light that is not absorbed by the sample. Several types of detectors are used in absorption spectrophotometry, and include photomultiplier tubes, photodiodes, photodiode arrays (PDAs), and charge-coupled devices (CCDs). Photomultiplier tubes are photocell detectors with a cathode surface. Incoming photons cause the ejection of electrons from the cathode, generating a current that is proportional to the incident light's intensity. Photomultiplier tubes are highly sensitive, have fast response times, and are especially useful for measuring low levels of light. Furthermore, the composition of the cathode's photosensitive layer can be manipulated to increase sensitivity in either the UV or visible region.

Photodiodes are semiconductor sensors that generate a current when illuminated by UV, visible, or near infrared light. The outer silicon P-layer and inner silicon N-layer of the photodiode form a P-N junction that is depleted of current carriers. On illumination, electrons are pulled into the N-layer of the diode, producing a relative positive charge in the P-layer. This electron flow generates a current that is proportional to the intensity of incident light that activates the photodiode, and allows the photodiode to function as a photoelectric converter.

Some spectrophotometers use multichannel detectors, such as PDAs and CCDs, which activate photosensitive elements known as pixels. In a PDA, each individual diode measures a portion of the spectrum as light is dispersed onto the linear diode array. Activating these diodes produces a photocurrent, which in turn charges a storage capacitor. The instrument electronically scans these charged capacitors to generate an absorption spectrum. PDAs permit almost instantaneous spectral acquisition without mechanical scanning by a monochromator. They are also less expensive than CCDs, but generally exhibit greater noise. Silicon-based photodiode arrays are sensitive to light in the 200-to-1100 nm range, whereas indium-gallium-arsenide arrays are sensitive to light in the near infrared region.

Optical Configurations

Courtesy of MiraiBio Inc.

MiraiBio's Gene Spec III spectrophotometer

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.

Gregory Smutzer (smutzer@hotmail.com) is a freelance writer in Lansdowne, Pa.
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.

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