topic/ in chemical injtrument~tion
edited by
FRANKA. SETTLE. JR. virginiaMitarynstitute Lexington. VA 24450
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Evolution ofiiistiimentation for UV-VisibleSpectrophotometry Part l lnes R. Alternose Milton Roy Company, Analytical Products Division, 820 Linden Avenue, Rochester, NY 14625 Historically, the development of analytical methods has proceeded hand in hand with the introduction of new measurine instruments. The earliedl were gravrmetric and volumetric. By the end of the 19th century, with the inventiun of thespertruscopr. spectroscopic methods were added to the list of available procedures. At first used only qualitatively and semiquantitatively, the introduction of electrical methods allowed precise quantitation. Since the 1930's, rapid development of vacuum tube amplifiers, photoelectric tubes, transistors, integrated circuits, and semiconductor devices has enahled increasing levels of sophistication. Nearly any physical property that is characteristic for a substance can be the basis of a n analytical method. In the case of spectroscoov. a t soecific ... the ahsorotion of lieht " wnveleneths is the orooertv used for mea~~~-~ surement. Spectrophotometry refers t o measurements of relative amounts of light as a function of wavelength. Generally measurements are made by comparison with some scale, so that in nearly all cases, the instrument directly or indirectly acts as a comparator. The unknown is evaluated relative t o a standard. The measurement is relarive hecause the wading ohmined frum a sample material is ratiod to the reading ohtarned from a refrrence matrrinl. The reference material in spectrophotometric measurements is usually a colorless material such as distilled water (1). A more detailed discussion of the principles of spectrophotometry and associated instrumentation can be found in references 14. In order to understand how spectrophotometric measurements are made, a brief discussion of light and its properties is necessary. Several centuries ago, Newton discovered t h a t sunlight passing through a n aperture and then a prism produced all the
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bes RueIius Alternose holds a master's degree in pharmaco agy hom ma Univwscy of R m h e s I e ~and a oache or r degree from Cotby CO.ege n Watewiiie. Maine. Prevtous work experience includes positions at Pennwalt Laboratories and Bausch & Lamb.
Soliens Division, as a research scientist. She has also taught high school biology and chemistrv.
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colors of the rainbow. He correctly theorized that white light is a composite of all of the colors of the rainbow. After Newton's discovery, it was found that light traveled in waves, consisting of peaks and valleys, similar t o waves of water. The size of the wave, or wavelength, is the distance covered hy one peak and adjacent valley. The color perceived is a function of wavelength. For instance, red light has a wavelength of 760 nm, while violet light has about half that value. This variation in wavelength is what gives the appearance of color. The spectrum visible to the human eye, ie., whitelight, ranges from violet a t 380 nm t o red a t 760 nm. Electromagnetic radiation above and below this region is in the infrared and ultraviolet regions, respectively, and is not visible. Radiant energy actually covers a wide range, from 5 X 10W to 1X 1012nm, with thevisible spectrum eomprising only a small portion of the entire range. Although some molecules naturally ahsorb visible radiation, many others do not. In order t o exploit the capability of a spectrophotometer t o measure absorbance, these molecules must be made to combine with reactive compounds to form a complex that will ahsorh visihle lieht. These reactive chemicals arc referred t u nrlorimetrir re. agents and thc resultant light-nhmrhing complex is a chromophore
The Color Comparator One of the first instruments t o use absorbance of light to determine eoneentration was the color comparator. To perform this function, the comparator made use of the Beer-Lambert Law (also known as the BeerBouger Law). This law states that the amount of light absorbed hy a solution (A) is proportional to: a, its absorbance index (a characteristic of the absorbing material which varies with wavelength); b, the pathlength of the light through the solution; and e , the eoneentration of the solution. Mathematically, the law is stated: A = abc. The color comoaratar comnared the intensity of the light trauimit~edthrough a standard solutirm of knuwn cuncentratim with that of a solution of unknown concentration. The lightsource for hoth thesample and the standard was the same. The user visually compared the transmitted Light and adjusted the pathlength until t h e light transmitted from hoth solutions appeared the same in intensity. By measurement of
the pathlength, theeoncentration of the unknown could be computed using the BeerLambert Law. The obvious inherent difficulty in using this method is that it is onlv as mod as the analyst's ability co match the htmuity of lrght trnnsmitted through the twosulutiund The development of photodetectors (1-3) presented the advantage of replacing the human eye with a much more sensitive detector of light intensity. The filter phatometer represented a n instrument that eombined the principles of spectroscopy and photodetection.
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The Fllter Photometer The filter photometer employs filters to isolate a hand of wavelengths t o he passed through the sample. The light that is transmitted faUs upon a photodetector which converts the radiant energy to an electrical signal that is relayed t o a meter calibrated in percent transmittance units andlor absarbance. In this instrument the Beer-Lamhert Law formula (A = obe) is also used. The nathleneth. .. . however. remains constant. making b wnsutnt. If all solutions ore ana: ly,cd at the same wavelength, a hecomes a constant The al,rurhance, A, therefore vnries a t a constant rate with eoneentration ( e ) . This is the basis far the generation of a standard curve of absorbance versus eoneentration. If various concentrations of a substance in solution are prepared, analyzed for absorbance, and plotted, the relationship between absorbance and concentration (for solutions t h a t follow the Beer-Lambert Law) is a straight line. The rate a t which absorbance varies with concentration can be computed from the slope of the straight line, and unknown concentrations of the same substance that fall in the linear region can be determined. ~
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The Spectrophotometer The chief limitation of filter photometers is the breadth of the wavelength hand used far analysis, which can cause deviations from Beer's Law. In the 1930's, a new type of instrument was developed that was considerably improved in terms of accuracy, yet still relatively easy to use. This new instrument was called a colorimeter or spectroohotometer. Basicallv. ,. a soectroohotometer uses n grating or prism, rather than n filter. 10 irulate a speciirc wavelength fur nnnlgris (Continued on page A218)
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inrtrumentation (refer to section on themonochromator). An example of this type of instrument is the Spectronie 20 spectrophotometer, a popular basic instrument widely used because of its simplicity and ruggedness.
Evolution of Optical Systems The minimum requirements for an instrument to study absorption spectra (is., a spectrophotometer) are: a radiation source, a monochromator, a sample compartment, and a means of detecting and measuring the light intensity. This section will discuss the evolution of the optical components of a spectrophotometer-the light source, the monochromator, and the photosensor, as well as double- and split-beam optics.
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WAVELENGTH CAM Figure 1. Specbonic 20 optical diagram (Milton Roy Co./Analytical Products Division, formerly a division of sausch 8 Lomb).
The Light Source The optical path must start with a light source: in the visible range an incandescent tungsten lamp. Figure 1is an optieal system schematic diagram of the Spectronie 20, an example of a general-purpose single-beam spectrophotometer. The reference phototube serves to compensate for fluctuations in lamo outout. The lieht is then focused hv a lens and passes through the entmnre slit. A s t w n d lrnr fu~uvesthe light hefore it ir dispersed. The visible and UV-visible models of the Spectronic 21 spectrophotometer represent an advance in the evolution of optical systems. Thisoptical system has been designed to provide a wide wavelength range, low stray light, and a moderately narrow handpass (10 nm). In the visible model (Fig. 2) white light from the tungsten lamp is chopped by a chopper fan, providing alternating lightldark cycles of light to compensate for any dark current in the photodetector. The chopped light is focused on the entrance slit of the monochromator by a fused silica condenser lens. Four filters are positioned near the entrance slit to eliminate higher-order wavelengths and reduce stray radiant energy to a low level. The filters are automatically programmed to intercept the light heam at the proper wavelengths. After passinathrouah the entrance slit, the light is incident on a focusing mirror and theneonverees onto the diffraction eratine. The use olmirrom rnrher than 1enrt.r rrpresenrs nn irnpnwemenr in optical derign because they focus lryht m the same place at all navelengths, therehy preventing chromatic aherration. Until 1963, few instruments were usable below 210 nm. This limitation was imposed principally due to the absorption of ultraviolet radiation bv. ootics made of natural quartz. Specially manufactured vitreous silica optics now permit the use of wavelengths as low as 190 nm, enabling the observation of certain chemical douhle bonds and other chromophares in this region. The UV-visible models of the Spectronic 21 (Fig. 3) have two lamps: tungsten for the visible range and deuterium for the UV. Light from the proper lamp is selected by positioning the lamp interchange mirror to either pass the deuterium light or to reflect the tungsten light. (Continued on page A22I)
CHOPPER FILTERS
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Figure 2. Spectronic 21 Model DV optical diagram
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Figure 3. Specbonic 21 Model UVD optical diagram.
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The next level of sophistication in optical design is demonstrated by the Spectronic 5011601 and the 1001 (refer t o optical diagram in Fig. 4). The visible source lamp is a tungsten-halogen lamp which has the advantages of long life and more energy. In a lain tungsten lamp, the tungsten evaporated from the filament willcoat the glassenvelope, thereby decreasing the energy output. In tungsten-halogen lamps, the halogen combines with the evaporated tungsten to form a compound that is redeposited on the filament: hence, when the lamp fails i t does so all a t once, rather than slowly deteriorating. An additional advantage of the tungsten-halogen lamp is an increased life span. The UV source for the Spectronic 601 is a deuterium lamp. This has evolved as the best choice for all UV-VIS instruments over other gaseous sources, because i t provides a continuous, convenient, high-energy light source with a long Life expectancy. The lamp has a narrow band source, from 190 t o 350 nm, and is the best choice for operation a t 340 nm because the lack of longer wavelengths gives reduced stray radiant energy when compared to a tungsten lamp. The source selection mirror (Fig. 4) presents two major improvements in spectrophotometer optics: a toroidal design and silica eoatine. Unlike the soherical desien used prwic~trsly,this mirror hos ruo drfferent radii of rurvature. The sharper curve rr oriented w r t m l l y and cancels most uf the off-axi* astigmatism arising from the large angle between incoming and outgoing rays. This mirror focuses the sources a t the monochromator entrance slit with more energy in a smaller area. The second develoment, the SiO* overcoatine. ~. ... which is used on all of the mirrors, renders rhcm imperviour to deara duriun hyrhrmrrakanu pern~it~eleaning hy the user. ~~~
The Monochromator The heart of every spectrophotometer is the monochromator. Its function is to eliminate all hut one wavelength (or as close t o one wavelength as possible), so that only a single wavelength is passed through the sample for analysis. Generally, a monochromatar consists of a system of slits, lenses, and mirrors, together with a dispersing device. T h e important characteristic of a monochromatar is its ability t o produce parallel, highly monochromatic light. There are three common types of dispersing devices: filters, prisms, or gratings (1-3). Filters act to eliminate unwanted wavelengths by absorption or interference, Filters represent the simplest means of isolating a band of wavelengths. Far greater resolution, prisms or diffraction gratings are employed. Gratings are generally preferred over prisms because they offer several advantages as dispersing elements: I ) their dispersion is nearly constant with wavelength; 2) there is better dispersion from the same size dispersing element: and 3) reflection gratings can work in the far UV and near IR where absorption prevents the use of prisms.
Figure 4. Spectronic2000 optical diagram, a daubie-beam UV-VIS instrument.
Gratings
Bandwidth
A grating is a highly polished surface with a large number of parallel, equally spaced grooves. For several decades master gratings have been produced that are ruled on special ruling machines to remarkable aecuracies in groove spacing and geometry. The ruling process consists of burnishing the grooves, one a t a time, in a thin layer of aluminum deposited on an optically flat piece of glass. No cutting is involved. The specially shaped diamond tool glides over the surface under constant pressure and impresses its shape in the form of a triangular groove typically 0.83 pm wide. After each stroke, the blank is indexed by exactly one groove spacing, taking care to keep periodic indexing errors to less than 0.010 pm. Under certain conditions, especially in the 350-nm region of the wavelength range, light source and detector problems combine and the stray radiant energy of even the best mechanically ruled gratings can become performance limiting. However, with the development of high-powered lasers and modern photoresists, it became possible to produce gratings by an optical technique. These gratings, known as blazed halographic gratings, have the same high spectral efficiency as the ruled master gratings, hut hecause they are made by a purely optical technique, are free of the small residual mechanical errors that eive rise t o most of the " stray radiant rneruy, whim is nn impormnt ronsidrmrion in I'V-\'IS sprztmphotometry (7,8). To make master gratings is a time-cansuming, expensive process, therefore a method of making copies of the master gratings was developed. Today nearly all spectrophotometers use replica gratings as the spectral dispersing device. These gratings are copies of the master (ruled or holographic) made by a liquid resin casting process, which preserves the optical accuracy of the master gratings to virtual perfection. This applies to the overall figure (flatness), the shape of individual grooves, and even stray light characteristics. There is no optical test that can distinguish between master and replica, no matter whether the master was ruled or made in photoresist by holographic techniques. (7).
One of the most important specifications of the monochromator is the spectral bandwidth or bandpass. This value (e.g., 20 nrn for the Speetranie 20) describes the purity of the light passed through the sample. In practice, a hand of wavelengths passes through the sample, rather than a single waveleneth. The bandwidth is defined as the v a n of monorhnmator settings in nnnbtnerrri ncrdtd tu nwvc the itwage ofthe rnrrancr .lit urrvss thc exit r l i t There are. in grnrral, three tbctwr limitinl: the energy and conrrqucntially the bandpasr: the light inrrnsitvoutvut of thesuurcr,theeffi~iencv ~. of the system used t o isolate wavelength bands, and the sensitivity of the detector. The narrower the bandwidth, the greater the resolution and efficiency of the measurements. However, with narrower bandwidths, there is a decrease in intensity. This trade-off between resolution and intensity is a consideration in choosing an instrument. Generally, if quantitative analyses are being made a t fixed wavelengths, the need for a low handwidth is not as stringent (2,3,
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Exit Slit The grating disperses the light and a speetrum is formed in the plane of the exit slit. As the grating is rotated, different wavelengths become incident on the exit slit. The exit slit isolates the selected narrow hand of wavelengths. In order to focus the desired wavelength on the exit slit, the grating must he moved to the proper position. This is accomplished in the Spectronic 20 by a wavelength cam (Fig. 1). In a n instrument such as the Spectronic 21 (Figs. 2 and 3), a sine bar controls the movement of the " erating. For many years this was the only method available. Microprocessors are now used to control such functions. An example is the Spectronic 5011601 (Fig. 4). The monochromatar has its own microprocessor t o control
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lamn selection and to oosition the filter wheel, the grating, and the lamp interchange mirror. A sine function in the mieroprocessor eliminates the need for a sine bar in the monochromator to make linear wavelength changes and replaces the wavelength cam in simpler instruments.
Monochromator Design Monochromator design, as well as components, bas undergone evolution. The Spectronic 20 (Fig. 1) represents a relatively simple arrangement. The Spectronic 21UVD (Fig. 3) is a more sophisticated design providing the higher o p t i d quality characteristics required to cover the wider wavelength range. The optical arrangement employed is called a folded crossover CzernyT u r n e r configuration, utilizing t h r e e mirrors. A collimating mirror renders the light parallel (collimated or parallel light will not go out of focus); the focusing mirror focuses the filament image onto the exit slit; and the foldine mirror nrovides a eeometric configuratron identical to that I'm theviarble mudel. The use uf mirrors instead of lenaea presents three advantages: no change in focal length with color (chromatic aberration), work at any wavelength, and more compact design. The monoehmmator of the Speetronic 5011601 and 1001 (Fie. 4) is a svmmetric e r i s h x s e d Czernv-Turner eonkeuration , -~ that avoids problems due to doubly refracted light and also enables a more compact design. Additionally, it is easier to baffle and to separate mechanically the light source from the detector. The mirrors are larger so that the beam does not catch the edge of the optics and cause stray radiant enerev. Internal baffline has heen added to absorb rero-order light and reduce stray radiant energy. The munuchromator is larger ihan past designs, which allows for bigger slits and therefore more energy. ~
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T h e Photodetector The instruments used to illustrate the evolution of optical design have similarly undergone an evolution of photodetection devices. After passing through the sample, the light impinges upon the detector, and the resultant signal passes through an amplifier to the meter. Typically, early spectrophotometers such as the Spectronic 20 used a vacuum phototube which will analyze in the range of 340600 nm. However, the detection range is capable of extension to 950 nm hy s change to a phototube of different energy sensitivity.
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Figure 5. Spectronic 1001 opticai diagram, a splii-beam UV-ViS instrument. Top view showing entire optical path and side view showing beam spliner and subsequent optical path.
More recent designs employ specifically designed silicon chips which convert the light energy to an electrical signal for detection. The use of a silicon chip for photodetection represents improvements in range, accuracy, speed, and size. In modern UV-VIS instruments with a wide wavelength range, such as the Spectronic 5011601 and 1001 (Fig. 4),twosensors are preferable because they provide an excellent costfvalue trade-off. A single silicon photocell lacks sufficient sensitivity to the UV, and a single wide range photomultiplier tube (PMT) is expensive and lacks sensitivity in the far red. The compromise is to use a moderately priced PMT for wavelengths below 630 nm, where high sensitivity is needed; and a silicon photocell above 630 nm, where it is most sensitive and there is plenty of radiant energy.
Doubie-Beam Optics Modern single-beam instruments provide excellent quantitative measuring capability. For qualitative measurements, however, a douhle-beam instrument has been historically regarded as the best choice. A doublebeam enables simultaneous measurement of a sample and its reference and therefore is ideal for obtaining an absorbance profile for a substance over the entire workine waveIengrh range The douhle-heam compensates for all but the shortest-term electrreal fluctuatmns, as well as other time-depen-
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dent irregularities in the source, the detector, and the amplifier. The optics of a double-beam instrument are similar to that of a single-beam up to the point of the heamsplitter (Fig. 5). Double-beam light is achieved by splitting the light beam leaving the monochromator and alternately switching it from reference to sample. The beams are then recombined, though separated in time, to fall on asingledetector. This results in an alternating signal from the detector whose amplitude is proportional to the difference in intensities in the two channels (2, 3).
Split-Beam Optics In recent years the unique split-beam optics, as featured in the Spectronie 1001 (Fig. 4), provide a good cost-effective compromise in design between single- and doublebeam. Approximately 8% of the Light is reflected from the beam-splitter plates and is used as a reference beam. The instrument measures the ratio of the light coming through the two channels independently of the variations in light level leaving the monochromator. This design represents significant improvements in energy level, bandpass uniformity from instrument to instrument, and stray radiant energy over that of previous instrumentation. I t is ideal far use when the solvent is stable and does
not require a constant reference. In such instances the split-beam design has the advantage of higher energy, while maintaining excellent resolving capabilities with its 2nm bandpass.
Evolution of Instrument Control The first generation of spectrophotometers, exemplified hy the Spectronic 20, have manual controls for three functions: wavelength selection, zero ahsorbance1100% transmittance adjustment, and the "dark current" adjustment (occluder). All of these controls are made by means of knobs to adjust dial settings on the outside of the instrument. In a semi-automated instrument, such as the Spectronic 21, there are manual controls for zero absorbanee/lOO% transmittance, wavelength, selection of lamp, sensitivity (for electronic gain, high absorbance requires a low sensitivity setting, and vice versa) and s factor set button for data readout in concentration, Automated settings include zero transmittance adjustment by the chopper and selection of appropriate filters. Microprorcsror-controlledspectrophotom r t r r ~represent the use of sldtr-of-the-art clerrronirs ru perform mans of the ilperations and functions previously done manually. Basically, a micropr~lessoris a single integrated circuit chip that can read data from inputs, perform computations, and control outputs. The ahility to perform these various functions in a desired sequence allows microprocessors to handle ~~~
most laboratory data acquisition requirements and to control different instruments. In the past decade, the development of microprocessor-controlled analytical instruments has ex~eriencedohenomend erawth. The micropnwssor has been used in these inrtrumrntv t u tompletelyautl~rnateanal~z. ieal procedures from sampling to recording of results, or to provide step-by-step guidance through an experimental operation. Microprocessor control of spedrophotometers is through a keyboard and typically communication with the user is via an alphanumeric display or CRT. The following functions are under the control of the microprocessor: control of the optical system (lamps, filters, wavelength drive) d a t a mode selection (absorbance, transmittance, or concentration) automatic zero and wavelength seleetion control of an accessory test parameter storage monitoring the operation of the instrument (e.g., self-tests, error messages, diagnostics)
Literature CHed (11 Ewing, George W."Instrumentsl Methoda of Chemicd Analpia," 5th ed.: McGraw-Hill: New York, 1985: Chap 3. (21 Willard, Hobart H.; Memitt, Lynne L.: Dean, John A,:
Settle.Frank"lnstrumcntalMethadsofAoal~~ia,"Gfh
(41 Judd, Deane B.; Wyszeeki. Guenter. "Color in Business, Science, and Industry." 2nd ed.; Wiley: New York, 1963: pp 9E-106. (51 Hazen, James. "Introduction to Spcetroecapy"; Bauseh& Lomb: Rochester, NY. (61 '"FundamenUllr of SpeeVophotomeW Bausch & Lomh: Rochester, NY. (7) "Diffraction Grating H a n d h m k B a w h & Lomb: Roehwfer. NY. 1970. Bsrtle, L.SPIE 19811.240.27. (81 Loewon, E.G.;
General Reference laitinen, HerhertA.;Ewing. Galen. W. "A HimryofAns-
lyticalChemistry";Divisionofhal~calChemistryof the American Chemical Society: Chap 111.
The use of microprocesson to control spectrophotometers has lead to ageneration of instruments now termed as beine "user friendlv." This means that the instrument is easv ro (uss and rommunirnres wirh rhe user. Mirroproresrara in nperrrophormwrrrr represent a major evolutionary step not only in instrument control hut in data manipulation and sample handling capabilities as well. The evolution of these systems will be the subject of Part I1 of this article.
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