Instrumentation

The basic spectrophotometer generally consists of a light source from which a given wavelength or range of wavelengths is selected by a wavelength selection device. The radiation selected is directed through the analytical sample and the transmitted light monitored by a detector. The light intensity measured by the detector is subsequently compared to that transmitted by a reference substance, the ratio being displayed usually as an absorbance but less commonly as a percent transmittance on a readout device.

A. Light Sources

There are two commonly used sources of light in UV-visible absorption spectro-photometry, hydrogen or deuterium discharge lamps and incandescent filament

Copyright 2002 by Marcel Dekker. All Rights Reserved.

lamps. The discharge lamps emit most of their light output in the range 200360 nm. They consist of a quartz envelope filled with hydrogen or deuterium at low pressure (approx 5 torr), into which project the closely spaced ends of an anode and a cathode. The passage of an electric current across the tips of the anode and cathode leads to excitation of the intervening gas molecules, which subsequently dissociate into photoexcited atoms that emit energy in the form of ultraviolet radiation. The light output with deuterium lamps is about 3-5 times brighter than that in hydrogen discharge lamps. In some instruments the light emitted is collected and focused into the wavelength selection device by use of a concave mirror.

Incandescent lamps commonly find use as sources of visible light. They consist basically of a metal wire filament, usually of tungsten, which is sealed inside an evacuated glass envelope. The filament is heated by the passage of an electric current and then emits a broad band of energy with a maximum dependent on the temperature of the filament. For example, a tungsten filament heated to 2860 K has a radiation maximum in the near-infrared at about 1000 nm. Only about 15% of the light output from the filament occurs in the visible region, with most occurring in the infrared. Generally, higher temperatures will shift the maximum to shorter wavelengths, but this does compromise the lifetime of the filament. Most of the current instruments have both kinds of lamps, with a switching device permitting scanning over the whole UV- visible range (200-800 nm) in one sweep. Improvements in lamp technology have made it possible to employ deuterium lamps over the whole range.

In some of the more sophisticated instruments, lasers have been employed as light sources. These have the advantage of generating a high-intensity monochromatic light output which is useful for high-absorbance samples; a consequence of this is improved sensitivity and resolution. Laser beams are also more easily focused than beams from conventional light sources. This is useful when the sample cell is small in size, as in flowing streams. Tunable diode lasers are most commonly used for spectrophotometry analyses. Unfortunately, due to their high costs, lasers are not commonly used as light sources for routine analysis by UV-visible spectrophotometry. Their availability has led, however, to the development of newer spectrophotometric techniques such as photoacoustic and thermal lens spectrometry.

High-intensity line sources still occasionally provide a simple source of high-intensity monochromatic light and have found use in portable instruments, designed for specific analyses.

B. Wavelength Selection Devices

The wavelength selection device in most instruments is a monochromator. The monochromator consists of an entrance slit, a dispersive device, a collimator, and an exit slit. The slits are narrow planar apertures that are used to isolate a narrow

Copyright 2002 by Marcel Dekker. All Rights Reserved.

band of light. The entrance slit is placed between the source and the collimator and the exit slit is placed between the focusing device and the sample. The widths of the slits are frequently variable and significantly influence the quality and accuracy of the analysis. Collimators are either lenses or concave mirrors which serve to align the incident light beam into parallel light rays which then impinge on the dispersive element.

There are two types of dispersive elements, prisms and gratings. The prisms are constructed of transparent material of known refractive index. In prisms the incident light is refracted to varying extents, depending on its wavelength, generating a range of wavelengths that impinge upon the exit slit. The different materials give rise to prisms of different angular dispersion. Prisms are generally good for the separation of light of shorter wavelengths, but they are less efficient for the separation of light of longer wavelengths. Furthermore, the resolving power (R) of prisms as defined by

where b is the base width of the prism and dn/dX is the dispersive power of the prism, which is characteristic of the prism material. From Eq. (8) it can be seen that it would require large prisms to efficiently separate long wavelengths of light. Such large prisms would not only be difficult to construct, but would result in unduly expensive instruments. As a result, grating monochromators are more commonly used nowadays. These may be either reflection or transmission gratings. A transmission grating consists of a series of finely spaced parallel lines on a transparent material, whereas a reflection grating consists of a series of equally spaced parallel grooves cut into a reflecting surface. Reflection gratings are more commonly used in UV-visible spectrophotometers, because the entire optical system can then be contained within a smaller volume.

In a grating the incident beam is diffracted at each of the surfaces generating an interference pattern. The different wavelengths of light undergo constructive interference at different angles, permitting wavelength selection by pivoting the grating through different angles, thereby focusing different wavelengths of light onto the exit slit. The higher-order interference patterns can be removed by the use of appropriate filters. Older instruments employed filters as a means of wavelength selection. These were either interference or absorption filters. Interference filters consist of a thin layer of transparent dielectric medium between two thin reflective metal films, whose thicknesses are carefully controlled. A portion of the light in the incident beam is transmitted through the first metal film and undergoes a series of reflections through the dielectric medium between the films, generating an interferometric pattern. Those constructive interferences of second or higher order emerge from the second metal surface. The filters typically allow through a small band of wavelengths of spectral band width 10-15 nm with a maximum percent transmission of about 40%. The higher orders of constructive interferometric light that emerge from the filter are less intense with increasing order and can be selected by the use of additional filters. The interference filters permit through more light than monochromators with the same bandpass, but lack the versatility of monochromators. Interference filters are usually available for the ultraviolet, visible, and infrared regions of the electromagnetic spectrum. Absorption filters, on the other hand, select a desired range of wavelengths by absorbing undesired wavelengths. They typically consist of a colored plate of glass. The filters may be bandpass or cutoff filters. Bandpass filters transmit a band of light of about 30-250 nm in width and generally transmit 5-20% of the incident light. Cutoff filters transmit essentially 100% of the incident light beyond a certain wavelength, cutting off light of shorter wavelengths in short-wavelength cutoff filters and longer wavelengths in long-wavelength cutoff filters. The cutoff wavelength is generally defined as the wavelength where the absorbance of the filter is unity (10% transmittance). Absorption filters lack the selectivity possible with monochromators and are available only for the visible region. They are used, however, for eliminating unwanted orders of light emerging from interference filters.

C. Sample Cells

Sample cells are commonly made of quartz or fused silica and readily transmit light of 200- to 800-nm wavelength. However, less expensive cells, constructed of high-quality glass and, more recently, plastic are available but are useful largely in the visible region of the spectrum, though some plastics do permit measurements at wavelengths as short as ~300 nm. The cells are available in various shapes and sizes, many of which are constructed for specialized applications. The two most common shapes are cylindrical and rectangular. The cylindrical cells are often used in low-cost filter photometers, whereas the rectangular cells are used in high-precision instruments. Cell path length is largely dependent on the application and ranges from 5 |m to 5 m. Smaller sample volumes with extremely long path lengths have also been employed. In these cases the increased path length at constant cell volume provides additional analytical sensitivity. Flow-through cells have found use in flow injection analysis and liquid chroma-tography as well as in process analysis. In flowing streams the cell volumes employed can vary considerably, ranging from 5 to 50 |L depending on the analytical sensitivity required. However, the path length employed is commonly 1 cm. The choice of cell shape or size is largely dependent on the instrument employed and the application.

Autosampling devices have also been developed for automated instruments. These are often microprocessor-controlled and have found use in the analysis of large samples, eliminating the operator error often associated with long analysis periods.

D. Detectors

1. Photoemissive and Photomultiplier Tubes

The photoemissive tube consists of an anode and a cathode enclosed in a transparent envelope. The cathode consists of a semicircular metal sheet plated with a thin layer of photoemissive alkali metal (cesium or rubidium). A negative potential of about 90 V is applied across the gap between the cathode and anode, which facilitates electron migration to the anode following electron release by the impact of light photons on the cathode. The magnitude of the current is proportional to the intensity of the incident light. Photomultiplier tubes have the additional feature of possessing multiple anodes, referred to as dynodes, which are coated with an electron-rich material of low ionization potential such as BeO, GaP, or CsSb. Across each subsequent dynode an increasing potential is applied, which serves to both accelerate the electrons and enhance electron yield. As a result, high electronic gains can be achieved. A stable and regulated power supply is important for consistency of yield for a given light intensity. Both photoemissive and photomultiplier tubes generate amplifiable currents. However, they have limited ranges of optimum spectral response and are both limited by thermal and shot noise.

2. Photovoltaic Cells

The photovoltaic cell consists of a metal plate enclosed in a plastic case. Sandwiched between the plate and a glass window is a thin layer of semiconducting material such as selenium, plated with a thin layer of gold or silver. When light impinges upon the silver layer, it emits electrons by the photoelectric effect. These electrons pass through the selenium layer and onto the metal plate. The current generated by this effect is proportional to the intensity of incident light and can be measured. Despite the advantage of not requiring an external power source, the signal is not easily amplified due to the low internal impedance. As a result, photovoltaic cells find limited use in portable instruments. Other disadvantages include limited sensitivity, limitation of their usefulness to dilute samples, and the fact that they have slow response times and exhibit fatigue with time.

3. Photodiode Array Detectors

Photodiode array detectors are an offshoot of semiconductor technology. In semiconductors, impurities have been added to pure silicon to create two classes of materials. The addition of arsenic, bismuth, phosphorous, or antimony creates a pentavalent material («-type) that is able to function as a donor of electrons. The addition of trivalent elements such as aluminium, boron, gallium, indium, etc., to silicon gives rise to the p-type material, in which the trivalent material is able to accept electrons to make up for its electron deficiency. The trivalent metals have only three electrons in their outer shells. Application of a potential difference to n-type material causes electron movement from negative to positive potential (cathodic). With p-type material the applied potential difference is manifested as apparent movement of positive charge from positive to negative potential (anodic).

Photodiode array detectors consist of an array of p-n type semiconductor diodes mounted onto a semicircular plate facing the light source. In these instruments the transmitted light beam from the sample is split into its component wavelengths by a dispersive element, and the various wavelengths of light fall onto different photodiodes on the semicircular plate. Interaction of a photon with the semiconducting layer of the diode generates a flow of current, the magnitude of which is proportional to the intensity of transmitted light. The advantage here is that temporal scanning is no longer necessary and the whole spectrum can be obtained almost instantaneously. The quality of spectra obtainable with diode array instruments is, however, limited by the spectral wavelength range as well as the number of diodes covering the entire spectral range. Diode array instruments, despite having a slower response time, have found application as detectors in chromatographic separations, where the spectrum of the emerging solutes may be used for semiquantitative characterization of the solutes. The diode array detectors are significantly useful in multiple signal detection, peak identification, and peak purity determinations.

E. Readout Devices

Readout devices convert the electrical signal emerging from the detectors to an analog or digital signal that is more understandable to the operator. The electrical signal is either converted to a plot of voltage versus time or digitized as numerical values of voltage versus time. For scanning instruments the time can be read as wavelength for a consistent known scan rate. In the diode array instruments all measurements are recorded simultaneously, and can be issued as a single spectrum or incorporated into a three-dimensional profile with time. The use of microprocessors in data acquisition and handling has facilitated the development of simultaneous multicomponent determinations over a range of wavelengths as opposed to single-wavelength measurements. They have also facilitated extensive manipulation of the data obtained from the instruments, such as smoothing, overlaying, derivatization, etc.

F. Instrument Configuration

The simplest instrument configuration is the single-beam configuration, in which a single light beam is transmitted from the source through the described modules

Fig. 6 Schematic diagram for the layout of a single-beam UV-visible spectrophotom-eter.

to the detector (Fig. 6). In double-beam instruments the light beam emerging from the source is split into two separate beams for the sample and the reference paths, respectively. This modification is associated with increased instrumental cost and lower light energy throughput as a result of the splitting of the source beam (Fig. 7). The double-beam instruments have largely superceded the single-beam instruments because in the double-beam instruments it is possible to eliminate the instability and drift arising from temporal differences in scanning the reference and sample cells.

Fig. 7 Schematic diagram for layout of a double-beam UV-visible spectrophotometer.

wavelength selection dev ;s

Fig. 7 Schematic diagram for layout of a double-beam UV-visible spectrophotometer.

Forward and reverse optical designs have also been tried. In the reverse optical arrangement the wavelength selection device is placed between the sample cell and the detector, as opposed to being between the source and the sample cell in the forward optical arrangement. The reverse optical design is reported to improve detection limits as a result of greater light throughput, but does not eliminate stray light as well as does the forward optical arrangement.

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