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Photodiode Array Detectors in UV-VIS Spectroscopy: Part II In Part I, which appeared in last month's issue, the theoretical aspects of diode array UV-VIS spectroscopy were discussed. The second article in this two-part series describes the applications of diode arrays in analytical chemistry. Applications of the linear photodiode array (LPDA) spectrometer in the UV-VIS spectral region can be considered under four headings: (1) molecular spectroscopy, (2) rapidscanning detection for time-dependent phenomena, (3) rapid-scanning detection in HPLC, and (4) other ap-
plications, which include low-lightlevel applications and atomic spectroscopy. Spectre-electrochemistry The combination of electrochemistry and spectroscopy was first reported in 1964 (i). Since that time spectroelectrochemistry has been used to study the redox chemistry of inorganic, organic, and complex biological molecules. The redox reactions in these systems occur on the second to millisecond time scale. Consequently, the ability of the LPDA spectrometer to measure spectra on a millisecond
time scale is important. Although Raman, fluorescence, and internal- and specular-reflectance spectroscopy have been used in spectroelectrochemistry, the majority of applications involve light absorption or transmission in the UV to IR spectral region. Transmission and absorption spectroelectrochemistry require that the light first pass through an optically transparent electrode and its diffusional neighborhood, then through the solution, after which the transmitted intensity can be detected (Figure 1). Conventional electrochemical cells with transparent electrodes have path
Figure 1 . T y p i c a l s p e c t r o e i e c t r o c h e m i c a l c e l l s Adapted from Reference 2 0003-2700/85/A357-1207$01.50/0 © 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 11, SEPTEMBER 1985 · 1207 A
lengths on the order of 1 cm. Thin-layer electrodes (TLE) with smaller path lengths (< 0.02 mm) allow rapid electrolysis (20-120 s) of electroactive species (2). Optically transparent electrodes (OTE) are subdivided into three classes (3). The first electrode class consists of a very thin film of conductive material, such as platinum or gold, deposited (100-5000 À) on a transparent substrate such as quartz (UV-VIS) or glass (visible). The second is the minigrid class, which consists of a minigrid or micromesh of metal (Pt, Au, Ag, Cu, or Ni). The third class is a mercury electrode in which a thin film of mercury is deposited onto a Au or Ni minigrid or onto P t or carbon film electrodes. OTEs have advanced to the stage that routine spectrochemical measurements are possible, and the field is expanding to include OTE cells for HPLC (4). The small cell volume typically found in the TLE and resulting small absorbance values for dilute samples have resulted in a number of studies to maximize cell geometry, to use fiber-optic coupling, and to assemble averaged spectra from multiple experiments to improve S/N ratios. In a basic cyclic voltammetry experiment, the spectra are recorded for a series of applied potentials. Potentials are maintained until the solution reaches equilibrium and electrolysis ceases. The Nernstian equation and Beer's law can then be used to calculate the formal potential, the number of electrons involved in the reaction, and the ratio of the oxidized to the reduced form of electroactive species (5). This information is used to hypothesize the redox reaction pathway. The optically transparent thin-layer electrode can also be used in the study of reaction mechanisms. The oxidized state of the compound is converted in the redox reaction by the application of a potential for a specified time. In a manner analogous to stopped-flow and kinetic techniques, the spectral properties of the reduced state are monitored and treated mathematically with conventional kinetic equations. Unlike spectroelectrochemical measurements using scanning wavelength spectrophotometers with photomultiplier detection, which suffer from slow or inadequate detector and scanningsystem response time, the LPDA spectrophotometer can provide complete spectral characterization of redox intermediates. Actual spectral data can replace hypothesized reaction mechanisms and mathematical modeling. The LPDA spectrophotometer has been used in the spectroelectrochemical analysis of the biologically important class of compounds, metalloporphyrins (6, 7). With the complete
spectral information provided by an LPDA spectrophotometer system for mitochondrial b-cytochromes, the field of spectroelectrochemistry has been combined with mathematical data manipulations, such as singularvalue decomposition, to identify different b-cytochromes in beef heart mitochondria (8). The ability of current LPDA systems to skip diodes (thereby drastically reducing detector response time to less than 5 ms for the monitoring of predefined spectral regions of interest) is an area yet to be explored. HPLC During the past 15 years, HPLC (high-pressure liquid chromatography) has become one of the major analytical techniques for the analysis of complex mixtures of chemical compounds, such as pharmaceutical formulations, plant extracts, and drugs in human body fluids. With the advent of integrated circuits and inexpensive, powerful microprocessors, HPLC detection systems have become so refined that they are now commonly the most sophisticated equipment in an HPLC system. Yet even with the major advances in detector technology, the technique of HPLC has suffered from the lack of a universal detector that provides optimum sensitivity and flexibility. The U V detector is one of the most commonly used detection systems in HPLC. The UV-VIS detector can be used not only to detect that a component is eluting from the chromatographic column (qualitative analysis) but also to determine the concentration of a compound provided that a calibration curve from known concentrations of the compound of interest exists (quantitative analysis). The first HPLC photometric detectors were fixed-wavelength UV detectors using the strong 254-nm emission line of low-pressure mercury lamps. The low cost and the relative universality of this detector have made it the most common type on the market today; however, these detectors are severely limited in that they analyze only one wavelength. Chemical determinations that have depended on single-channel detection have often been limited in specificity because of the broadband absorption character of the chromophores. When compounds show little or no absorbance at 254 nm or when interfering substances exist, accurately quantifying a single compound at a single wavelength is impossible. These limitations led to the development of selective filter photometers that monitor a limited number of wavelengths and to variable-wavelength detectors offering a wide selection of UV and visible wavelengths.
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With conventional variable-wavelength detectors the eluent flow must be stopped to trap a peak in the flow cell while a UV-VIS spectrum is obtained. This approach presents the analyst with many wavelengths but does not provide spectrophotometric analysis at more than one of these wavelengths at any given time. The advent of the LPDA detector has eliminated the necessity of the single-wavelength approach to HPLC detection. Although an LPDA detector is still limited to compounds that absorb UV and visible light, this detector allows simultaneous acquisition of spectral information, without stopping the eluent flow. The LPDA HPLC detector has several advantages beyond simple component identification. These advantages occur both in the temporal and spectral domains. In the temporal domain, analysis time can be shortened because the wavelength dimension allows the analyst to observe all UVVIS-absorbing chromophores during a single elution. In the spectral domain, an improvement in detection level is gained by virtue of the availability of the total UV-VIS spectrum of the chromophore. The analyst can sum the output intensity over several discrete diodes or wavelengths (broadband integration); this provides a significant advantage over single-wavelength detectability. In most cases this can account for increases of 5-500 X in detectability (9). The analyst can obtain multiwavelength chromatograms from a single analysis of one sample. In situations where the solutes are not resolved by the column but where spectral overlap is minimal, all the components comprising a single elution profile can be determined simultaneously by monitoring each component at a wavelength that is free of interference. Simultaneous acquisition at different wavelengths also ensures the detection of all components of a complex mixture (universal detection). Additionally, when using an individual narrow-wavelength band, wavelengths can be selected that optimize sensitivity while minimizing interferences. The chromatographer can choose the wavelength of maximum absorbance to gain sensitivity or the wavelength providing the optimum signal relative to possible interfering substances to gain selectivity. Additional specificity can be obtained from information about peak position in both the wavelength and time domains. Identification of unknowns can be carried out concurrently with quantitative analysis of known species in the same chromatogram. Increases in sensitivity are obtained by adaptability in both wavelength and time aver-
and refractive index changes found in HPLC are similar to those found in stopped-flow kinetic experiments. Although both types of experiments involve flowing systems, the spectral data requirements of the two techniques place different demands on the LPDA spectrometer and associated computer. In the stopped-flow experiment, speed of acquisition is critical for the characterization of reactive intermediates. In the HPLC experiment, memory capacity of the data acquisition system is usually more important than spectral acquisition on a millisecond time scale. S t o p p e d flow Figure 2 . R e a c t a n t s A , B, C, and D; m i x e r s 1, 2, 3, and 4 Adapted with permission of Update Instruments
aging. Theoretical analyses show that significant increases in sensitivity are obtained by adapting the spectral bandwidth to the chromophore's natural bandwidth. With an HPLC-LPDA system the analyst can use data in the computer memory from other wavelengths rather than repeating analyses at different wavelengths. Simultaneous detection of a sample absorption band and an unabsorbed wavelength provides a means of signal correction. Ratioing the intensity of the absorbing wavelength to that of a minimally absorbing wavelength effectively removes system fluctuations and temperaturedependent refractive index changes. Typically, the error introduced by using a wavelength of minimal absorption is less than other sources of experimental error. Optical compensation with a separate photodiode measuring source intensity allows a dynamic reference and true spectral ratio measurement. H P L C d a t a processing
The commercial availability of HPLC-LPDA systems and the advent of the 16- and 32-bit microcomputer in the analytical laboratory have made it possible to use on a routine basis a number of data-processing techniques previously unavailable to the chromatographer. Peak purity has long been a validation problem for the analyst. It often requires at least two analyses, if not more, under various chromatographic regimes. Multiple analyses of a peak at best yield only single wavelengths per analysis. Under those conditions the analyst must rely on wavelength ratios as an indication of purity. The absorbance ratio of two carefully chosen wavelengths, when plotted as a function of time, changes through the peak only if the eluent is not a pure compound (8). Alternatively, with an LPDA system
the spectra obtained at the up slope, apex, and down slope can be used to determine coelution. If the eluting compound is pure, spectra taken anywhere along the envelope of the peak should exhibit changes only in the amplitude of the response. Shifts in the maxima or minima are an immediate indication of the presence of an impurity in the chromatographic peak. Superposition of these three spectra after normalization should show a net difference of zero if the chromatographic peak is pure. Automatic acquisition of spectra during elution can be achieved manually or automatically by computer software that can detect when the signal leaves or returns to the baseline or passes through up- and down-slope inflection points or peak apexes. Periodic acquisition of spectra at constant time intervals throughout an analysis can also be of value. Accurate and precise representation of the spectral information in HPLC is critical. Spectral quality can be severely affected by the choice of the detector integration time. The integration time must be sufficiently short to allow sufficient data points to be taken from the spectra to define the chromatographic peak clearly. An integration time that is too long will distort the location of the peak maxima. The integration time must be short enough so that no noticeable concentration change will occur during spectral acquisition. Short integration times result in a decreased S/N ratio; however, significant improvement can be obtained by time averaging a number of spectra or scans. On the basis of the Ramsey criterion, five data points must be taken across a peak to define the peak envelope minimally (with a cubic-spline fit algorithm). Some commercial instruments require that a minimum of 25 data points be taken across a chromatographic peak so that simpler fitting algorithms can be used. The effects of temperature control
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Fast-flow kinetic studies of chemical reactions have traditionally been conducted with single-wavelength spectrometers or, if the reaction time permitted, with conventional scanning spectrophotometers. Recently, UVVIS spectrometers using LPDAs combined with a commercially available stopped-flow device have been successfully used to study the oxidation of cytochrome c oxidase in the 450-920-nm region (10) and to study the catalytic cycle of molybdenumcontaining enzymes (11). The speed of spectral acquisition found in LPDA spectrophotometers has made possible the spectral detection and characterization of chemically reactive intermediates directly through spectral analysis rather than through the classical reaction rate techniques. Wavelength-dependent anomalies, Rayleigh scattering, and refractive index changes, which occur in stopped-flow experiments, are readily detected and corrected with the information contained in the total spectrum acquired with an LPDA spectrophotometer. The overall information contained in a nonabsorbing spectral region or a spectral region of minimal absorption can be effectively used, just as in HPLC, to correct for system artifacts. Rayleigh scattering is attributed to the presence of bubbles or macrocontaminants in the sample and appears as temporal random noise at the detector. This noise limits the dynamic range of the system. Refractive index changes are attributed to insufficient temperature control in some cases and the subsequent thermal lensing effects (12). One commercially available stopped-flow system (with a digitally controlled, positive-displacement, motor-driven ram) incubates both the optical cell and reactants in the same isothermal bath to minimize these refractive index anomalies (13). The ram drives the syringes in a programmable fashion and also responds to a stop command from the receiver syringe (14).
Figure 3. Block diagram of a stopped-flow system using a diode array spectrophotometer Adapted with permission from Update Instruments company literature
In a typical stopped-flow experiment (Figure 2), two syringes are loaded with reactants. The syringes are actuated, causing the reactants to flow at a constant velocity. Mixing occurs in the cuvette, which is located in the thermostated bath. In one mode of operation, the LPDA spectrophotometer is triggered to take data during a period of continuous flow (Figure 3). After a certain interval of continuous flow required to purge the cuvette, the flow is stopped. Spectra continue to be acquired after the flow is stopped. As many as four reactant syringes and
three mixers can be used. Spectral information is acquired on a millisecond time scale (15). In the past, because of the lack of LPDA systems, the study of reaction rates has led to speculation of reaction intermediates based on kinetic data. In the past the chemical "dead time" (16) has been used to characterize the performance of a stopped-flow instrument, based on the assumption that the reactants were homogeneously mixed in a very small volume. The question of homogeneity can now be tested by the LPDA stopped-flow sys-
Figure 4. Rapid-flow, rapid-scan spectra of an acid-base reaction with a dye, Bromcresol purple (a) Spectrum of remainder of last experiment being cleared from the cuvette; elapsed time, 12.5 ms. (b) Acid and base forms of the dye give a steady spectrum; elapsed time, 62.5 ms; flow stopped. (c) Decay of base form of the dye; elapsed time, 75 ms. (d) Base form of the dye decaying; elapsed time, 100 ms. Adapted with permission of Update Instruments 1212 A · ANALYTICAL CHEMISTRY, VOL. 57, NO. 11, SEPTEMBER 1985
tern. If a reaction is very fast with respect to the physical dead time, then the homogeneity of the sample can be detected by the LPDA spectrophotometer during steady flow. If the reaction is strictly second order and the mixing is complete, in light of the speed of the reaction, there should be no spectral evidence of reactants in the flow chamber or cuvette, and the products should be fully developed (Figure 4). General applications
Previously the use of the LPDA spectrophotometer in atomic spectroscopy was limited because the requirements for high resolution and large spectral coverage could not be readily achieved. The availability of longer (2048- and 4096-element) arrays makes the application of the LPDA more feasible for general atomic spectroscopy. Currently, the LPDA spectrometer is used as a diagnostic tool in the semiconductor industry for plasma etching of semiconductor wafers. Atomic lines of various plasma gases are monitored and used as a diagnostic parameter to determine the extent of wafer etching. Some work has begun in the application of the LPDA spectrometer to optical rotatory dispersion or circular dichroism (17). The study of lowlight-level phenomena in the UV-VIS region, such as chemiluminescence and fluorescence, requires an intensified LPDA. The same detector arrays are used with the addition of a microchannel plate image intensifier to the face of the detector. The sensitivity of the intensified LPDA compares favorably with that of a photomultiplier. The speed of the LPDA and intensified LPDA compared with conventional scanning spectrophotometers, which use photomultiplier tubes, al-
lows t h e characterization of t r a n s i e n t luminescent intermediates and the s t u d y of reaction decay lifetimes. T h e L P D A s p e c t r o p h o t o m e t e r is becoming t h e d e t e c t o r of choice in m a n y areas of U V - V I S spectroscopy ( H P L C , k i n e t ics, molecular a n d a t o m i c spectros copy, luminescence studies, a n d spectroelectrochemistry) in which t h e s p e e d of acquisition a n d completeness of s p e c t r a l information are i m p o r t a n t .
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On t h e technological front t h e n e x t horizon in a r r a y s p e c t r o p h o t o m e t e r s will be t h e t w o - d i m e n s i o n a l array. F u t u r e t r e n d s in research a n d i n d u s t r y for L P D A s p e c t r o p h o t o m e t e r s can be expected t o include improved software a n d c o m p u t e r systems t o t a k e a d v a n tage of t h e s p e e d of t h e L P D A a n d t h e w e a l t h of information available from t h e array, longer arrays, a n d d e d i c a t e d application-oriented L P D A s y s t e m packages. T h e m o s t i m p o r t a n t t r e n d of all is t h e i m p a c t t h a t t h e L P D A s p e c t r o p h o t o m e t e r h a s h a d a n d will c o n t i n u e t o h a v e on all areas of c h e m i s t r y — t h e ability t o acquire complete s p e c t r a l information on short-lived re action i n t e r m e d i a t e s . T h e a d v a n c e m e n t s in L P D A technology now place t h e analyst in a position t o i n t e r p r e t s t r u c t u r e in t e r m s of t h e resulting spectroscopy.
References (1) Kuwana, T.; Darlington, R.; Leedy, D. Anal. Chem. 1964, 36, 2023-26. (2) Heineman, W. R. Anal. Chem. 1978, 50,390-400 A. (3) Heineman, W. R. J. Chem. Ed. 1983, 60, 305-8. (4) Pinkerton, T. C ; Hajizadeh, K.; Deutsch, E.; Heineman, W. R. Anal. Chem. 1980,52,1542-44. (5) Fritz, M. L.; Durst, R. A. Talanta 1983, 30, 933-39. (6) Rhodes, R. K.; Kadish, Κ. Μ. Anal. Chem. 1981, 53,1539-41. (7) Kadish, K. M.; Rhodes, R. K. Inorg. Chem. 1981,20, 2961-66. (8) Reddy, K.V.S.; Hendler, R. W. J. Biol. Chem. 1983,258, 8568-81. (9) Dessy, R. E.; Reynolds, W. D.; Nunn, W. G.; Titus, C. Α.; Moler, G. F. J. Chromatogr. 1976,126, 347-68. (10) Armstrong, F.; Shaw, R. W.; Beinert, H. Biochim. Biophys. Acta 1983, 722, 61-71. (11) Barber, M. J.; Salerno, J. C. In "Mo lybdenum and Molybdenum-Containing Enzymes"; Coughlar, M. P., Ed.; Pergamon Press: New York, N.Y., 1980; Chap ter 18. (12) Chattopadhyay, K.; Coetzee, J. F. Anal. Chem. 1972,44, 2117-18. (13) Miller, M. L.; Gordon, G. Anal. Chem. 1976,48, 778-79. (14) Hansen, R. E., Update Instruments, personal communication, 1985. (15) Hansen, R. E.; Beinert, H. Anal. Chem. 1966, 38, 484-87. (16) Sturtevant, J. M. In "Rapid Mixing and Sampling Technique in Biochemis try"; Chance, B., Ed.; Academic Press: New York, N.Y., 1964; p. 89. (17) Aiello, P. J.; Enke, C. G. In "Multi channel Image Detectors, Vol. 2"; Talmi, Y., Ed.; ACS: Washington, D.C., 1983; Symposium Series 236, pp. 57-73.
