Double Wavelength Spectrophotometry with a Rapid Scanning Instrument Robert Szentrlmay and Theodore Kuwana” Department of Chemistry, Ohio State University, Columbus, Ohio 432 10
A laboratory rapld scannlng spectophotometer employing an oscillating mirror for wavelength scanning was adapted to a rapid wavelength selection technique. Rapid wavelength selectlon (20 to 100 H r ) in the wavelength range 200 to 700 nm enables the application of thls system for the analysls of turbid solutions using double-wavelength spectrophotometry. This system Is applied to acquiring spectra of Intact mltochondria.
The basic principles, instrumentation, and fundamental applications of double-wavelength spectroscopy, including derivative spectroscopy, have recently been reviewed by T. J. Porro (1). In essence, the technique offers considerable enhancement in selectivity and sensitivity for the analysis of turbid samples in the UV-vis region. These may include for example solutions of high molecular-weight polymers, colloidal suspensions, powders, smoke, and biological samples, which scatter a large portion of the sampling light beam. Britton Chance, in his ingenious work on the study of the mechanism of cell respiration by mitochondria over the past three decades has developed a number of instruments and techniques utilizing the double-wavelength approach for spectral and kinetic studies (2-5) in highly turbid suspensions. In addition, a number of special techniques and quality instruments have been developed as a result of Chance’s original work (1, 6). The potential value of an oscillating-mirror rapid-scanning spectrometer as a highly versatile instrument for analytical applications has been recently emphasized in a number of reports. These applications include spectroelectrochemical studies (7), stopped-flow measurements (8), rapid spectral signal averaging techniques (9),and as a multiwavelength detector system for liquid chromotography (10)and microwave plasma emission spectrometry (11). The simplicity, compactness of design, large dynamic range (PMT detection), and flexibility of design make the system readily adaptable to numerous experimental applications. The oscillating mirror (galvanometer suspension) system can be readily adapted to computer control and data acquisition. Our current interest in the analysis of turbid solutions of mitochondrial respiratory particles has encouraged us to investigate the feasibility of utilizing this galvanometer based spectrometer as a tool for wavelength modulation techniques. Strojek et al. (12, 13) previously had shown the feasibility of using a more sophisticated version of the rapid scanning instrument for SO2 analysis (18 ppb for 10-m cell) by a combination of correlation and derivative spectroscopy. In this present application, we investigated some of the advantages and limitations of the galvanometer based spectrometer as a double-wavelength instrument for turbid solutions. We also investigated the possible uses in cases where rapid and versatile wavelength selection would be desirable.
EXPERIMENTAL Optics and Operations. The optical configuration for the laboratory built spectrometer was analogous to that described in a previous report (13)(RSS available from Harrick Scientific 1348
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Corp., Ossining N.Y.) and modification to those published previously (12). The light sources used were a 75-W Xenon Arc Lamp, Illumination Industries, Sunnyvale, Calif., and a Philips 13.2-V-100-W Rallye Tungsten Iodide Lamp (sports car headlamp, 5-mm filament). The source focusing mirror (32-cm radius) (see Scheme 1of ref. 13) focused the source image approximately 5 / 6 the distance between the source and the galvanometer mirror. A cylindrical mirror galvanometer (8.9-cm radius) focused the slit image approximately ‘/ the distance between the galvanometer and spherical mirror, Mz (radius 30.5 cm). M2 focused the galvanometer image on the grating and also on the exit slit. Four Bausch and Lomb “Certified Precision” gratings (300 l/mm blazed at 500 nm, 600 l/mm blazed at 300 nm, 600 l/mm blazed at 750 nm, 1200 l/mm blazed at 300 nm) were mounted on a rotating stand so they could be used interchangeably. The configuration resulted in a 3:l magnification of the entrance slit image at the exit slit. The galvanometer used was a self-contained 7-380 Galvanometer (Bell and Howell, Pasadena, Calif.) with an aluminized surface (0.15 inch wide X 0.20 inch high). The fluid damped galvanometer was specified to operate at 3.5 mA/degree beam rotation, terminal resistance of 75 ohms rt l o % , and at an undamped natural frequency of 575 i 10% Hz. Two RCA type 71512 head-on 3.8-cm diameter multi-alkali PMTs were used. The resolution of the instrument (1.2-nm width at half-height) had been previously determined by the use of a HeNe laser (633 nm) by replacing the light source with the laser. For this study the resolution was approximated by replacing the Xenon Arc Source with a Hg-Arc source (Illumination Industries, Inc.) and determining the width half-height of the 546-nm line. The Hg-Arc spectrum was taken utilizing the current output of only one of the two PMTs and measuring the voltage drop across a 100-kQ resistor t o ground. Spectrometer slits were set for a resolution of 1.6 nm for all spectra reported in this paper unless specified otherwise. Mitochondrial solutions were contained in a Lucite cell specially constructed for use in indirect coulometric titrations (1.25-cmpath length and 10-mL volume). Scattered light from the sample cell was focused onto the PMT with a double convex lens. The necessity of a conventional size stirrer prevented moving the cell closer to the PMT. A reference cell also containing a mitochondrial suspension which was not stirred during the course of the experiment, was placed near to the reference PMT (- 2 cm) and positioned to balance the baseline. The flatness of the baseline was thus limited by the mismatch between the sample and reference cells and their placement. With two matched 1-cm cuvettes a flat baseline within kO.01 absorbance unit could be achieved between 500-650 nm for the absorbance difference between fully reduced suspensions of mitochondria. Electronics. The circuit used for driving the galvanometer mirror is presented in Figure 1. OAl and OA2 are voltage followers (Fairchild pA 741) which track the input linear ramp (OAl) for wavelength. The relay (Teledyne 712-12) switches between these two voltages at a frequency imposed by an external square wave source (+12 V). OA3 (pA 741) serves as a unity gain amplifier in the dual wavelength mode and is used as a summing amplifier in the derivative or modulation mode. In the derivative mode the relay is closed to OAl (not switching) and a sine wave of the desired modulation amplitude is added to the linear ramp. Trim capacitor, C, is adjusted to give the optimal RC constant for galvanometer operation (damping). OA4 (Teledyne Philbrick 1009) and booster amp OA5 (Teledyne Philbrick 2001) serve as a current pump for the galvanometer coil (G). In this configuration the load current through the coil is proportional to the input voltage at OA4 and R. The computer operations were similar to
MODULATION INPUT
C
* -15V
Flgure 1. Circuit diagram of the electronics utilized for driving the rapid scanning galvanometer mirror
W A V E LENGTH (nm)
Figure 2. Absorbance spectra of a holmium oxide filter obtained using a 300 I/mm grating blazed at 300 nm: ( A ) Spectrum obtained in the normal simple sweep mode; ( 6 )spectrum obtained in the doublewavelength mode at 40 Hz modulation; scan time = 200 s and reference X at 510 nm those described previously (14). The output of a 12-bit Datal DAC-VR-12B Digital-to-Analog converter was applied directly to the input of OAl when the spectrometer was computer driven. The outputs of the two PMTs were balanced by adjusting the high voltage power applied to each. The PMT outputs were converted to log ratio output (absorbance) through a bipolar current log amp (Philbrick-Teledyne Model 4361) and processed directly by the Lock-in-Amplifier (PAR Model 126 with a Model 116 preamplifier. A lock-in time constant of 300 ms was used unless stated otherwise at the output stage. For transmitting data not processed by the lock-in to the computer, the signals were band pass filtered (RC = 0.1 ms) and amplified (gain of 5). The information from the lock-in was then directly plotted on a x-y recorder (Houston Model 2000) and sent in parallel to the computer when desired. A Tektronix Model 7613 storage oscilloscope with Polaroid Camera attachment was utilized to monitor modulation frequencies and responses.
RESULTS AND DISCUSSION Figure 2 A illustrates the absorbance spectrum of a holmium oxide optical filter obtained by the spectrometer described in the simple sweep mode. Figure 2B shows the same spectrum obtained in the dual-wavelength mode where a 40-Hz modulation was used. The signal was demodulated and rectified by the lock-in. A reference wavelength of 510 nm was used for the spectrum. This spectrum provided a serious test for the performance of the modulation technique because of the sharp absorbance peaks and the large wavelength excursions required by the galvanometer during the spectral scan. A careful analysis of the spectrum obtained in the dual-wavelength mode shows a slight loss in resolution (10-2070) probably due to harmonic vibrations of the galvanometer mirror and to the method of signal rectification
Figure 3. Driving and response waveforms for duaCwaveiength operation between 578 nm and the 536 nm peak of a holmium oxide filter: ( A ) Driving waveform for the galvanometer (output of OA3 in Figure 1) at 40 Hz with no galvanometer damping; ( 8 ) Driving waveform with damping (RC = 0.5 ms); (C) and ( D ) are the absorbance response signals for ( A ) and (B),respectively. Full scale time axis for all traces is 50 ms. For Figures A and 6full scale y-axis = 1.60 V. For Figures Cand Dfuil scale y-axis = 0.80 V (or 0.80 absorbance unit). The insets on the right of the figures are retraces for clarity of the transients for traces Cand D by the lock-in. Slight spectral distortions are also evident at large excursions of the mirror (i.e., see spectrum a t 350 nm). The resolution of this spectrum may be maximized and the spectral clarity optimized by adjusting the RC time constant (C) for the galvanometer drive waveform. This is most easily accomplished by monitoring one of the holmium oxide peaks in the dual-wavelength mode and adjusting the RC constant until a maximum peak height is observed. The reasons for these variations and approaches to possible improvements to the system will become evident during the discussion. Figures 3 A and 3 B show the oscilloscope traces of the galvanometer driving waveforms monitored as the input voltage to OA4. A similar type of voltage response can be monitored in parallel with the galvo coil. The corresponding spectral response for dual-wavelength operation as the mirror oscillates between 578 nm and the 536 nm peak of holmium oxide is shown in Figures 3C and 3 0 . If the driving waveform is undamped ( 3 A and 3C) three to four vibrations of the mirror are observed about the peak in the spectral response. Damping the driving waveform (RC ~ 0 . ms) 5 reduces the vibrations to a single oscillation and considerably improves the spectral characteristics and resolution (10-2070)obtained by the rectification procedure. In either mode of operation, the mirror usually requires approximately 2 ms to reach a constant position as determined from the PMT response. This response time should be a function of the galvanometers “natural frequency” (500 Hz) and might be improved by ANALYTICAL CHEMISTRY, VOL. 49, NO. 9, AUGUST 1977
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WAVELENGTH (nml Figure 5. Absorbance spectrum taken at 20-Hz modulation of an expanded portion of the holmium oxide filter shown in Figure 2. Arrows show which points (wavelengths) in the spectrum are monitored for a duration equivalent to the scan time (180 s). A 600 I/mm grating blazed at 750 nm was used
Figure 4. Transmission spectrum obtained of a Hg-Arc source utillzing a 1200 I/mm grating blazed at 300 nm; ( A ) Spectrum obtained in simple spectrum sweep mode; (B)spectrum taken with respect to 560 nm; (C) taken with respect to 598 nm; 40-Hz modulation; lock-in time constant = 100 ms; 200-s scan time
selecting higher frequency galvanometers and by utilizing more sophisticated damping procedures. Since the lock-in is sampling the entire waveform during rectification, this error contributes to an apparent loss in resolution and represents the most serious limitation to the spectroscopic procedure at present. Perhaps a clearer representation of the capabilities and limitations of the procedure employed is shown in Figure 4. The normal spectrum and double-wavelength spectrum are shown for the 579-577 nm doublet of a Hg-Arc used as a source for the spectrometer. Figure 4A shows the spectrum taken in the simple sweep-mode. Figures 4B and C depict the spectra obtained in the double wavelength mode taken a t a 4 0 - H ~modulation frequency. B and C were taken with respect to two different reference wavelengths for comparison. An approximate 20% loss in resolution between the two bands is observed. This can be decreased to =lo% by optimizing the RC time constant in the driving circuit (Figure 1). Further improvement of the system might be made using a type of demodulation enploying a sample and hold or boxcar type integration procedure to discriminate between the desired signal and the galvanometer response time. This type of cross-channel interference (Crosstalk) is a common problem for signal modulation and demodulation techniques (3-5). Two advantages for using the double-wavelength mode are in the enhanced sensitivity and selectivity afforded by the signal rectification and also in data representation. These have been summarized by T. J. Porro ( I ) . In turbid solutions or in solutions where background solution fluctuations (density, refractivity) occur, the double-wavelength approach introduces an added dimension of baseline stability. By the inherent 1350
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nature of the technique, the background fluctuations that are common to both wavelengths are eliminated in the demodulation procedure. However, care must be taken to account for the changes in the background (i.e., scattering) which are wavelength dependent. In addition, extreme caution and care must be taken in the interpretation of data obtained in the dual-wavelength approach. When the RSS spectrometer is operated in its usual split beam mode utilizing two PMTs, the low frequency noise and drift are caused primarily by the flicker and arc wander of the xenon source and by the fluctuations of the PMTs. The drift has been found to vary from 1 x to 1 x lo-' absorbance unit per hour. When using the double-wavelength approach, baseline drift was found to be within the limits of measurement imposed by the signal to noise (
