about 1.4 micron-’, while in S X Q it is 1.3 micron-’ in the pure liquid and slightly larger in nonpolar solvents. (This is a sufficiently large gap to slow internal conversion and allow fluorescence to occur.) Also, both azulene and the 8MQ zwitterion are not phosphoi’nscent, indicating strong coupling between the lowest excited singlet state and the ground state via the lowest triplet. Therefore, v;e propose that the fluorescence in SMQ arises from fluorescence of the second excited singlet state of the zwitterionic species. We recognize that this proposal must be regarded as a tentative assignment awaiting confirmation from life-time and polarization studies.
LITERATURE CITED
(1) Albert, A., Barlin, G. B., J . Chem. SOC.1959, p. 2384. (2) Anderson, P. D., Ph.D. thesis, hlassachusetts Institute of Technology, 1966. Buttery, R. G.. G., J . (3) Badger, G. M.,Buttery. Chem. SOC. 1956, p. 3236.
(4) Banfield, J. E., J . Org. Chem. 25, 300 (1960). (5) Bankovskii, Yu. A., Chera, L. M., Ievinsh, A. F., J . Anal. Chem. U.S.S.R. 18, 577 (1963). (6) Ibid., 19, 380 (1964). (7) Barkovskii, U. F., Kharkover, M. Z., Proc. Akad. Nauk. SSSR 153, 979 (1963). (8) Bayliss, N. S., McRae, E. G., J . Phys. Chem. 58, 1002 (1954). (9) Beer, M., Longuet-Higgins, H. C., J . Chem. Phys. 23, 1390 (1955). (10) Bhatnagar, D. C., Forster, L. S., Spectrochim. Acta 21, 1803 (1965). (11) Corsini, A., Fernando, Q., Freiser, H., ANAL.CHEM.35, 1424 (1963). (12) Gordy, W., Stanford, S. C., J . Am. Chem. SOC.62, 497 (1940). (13) Lang, L., “Absorption Spectra in the Ultraviolet and Visible Region,” Vol. 11, Academic Press, New York, 1961 _I__.
(14) Lee, H. S., Freiser, H., J . Ora. Chem. 25, 1277 (1960). (15) Mason, S. F., in “Physical Methods in Heterocyclic Chem&try,” A. R. Katritzky, ed., Vol. 11, Chap. 7, Academic Press, New York, 1963. (16) Mason, S. F., J . Chem. Soc., 1959, p. 1253. (17) Mataga, N., Kaifu, Y., J . Chem. Phys. 36, 2804 (1962). (18) Mat,aga, N., Kaifu, Y., Koizumi,
M., Bull. Chem. SOC.Japan 29, 465 (1956). (19) Mataga, N., Tsuno, S., Ibid., 30, 368 (1957). (20) Ibid., p. 711. (21) Melhuish, W. H., J . Phys. Chem. 65, 229 (1961). (22) Orloff, M. C., Ph.D. thesis, University of Pennsylvania, 1964. (23) Pariser, R., J. Chem. Phys. 24, 250 (1956). (24) Pariser, R., Parr, R. G., Ibid., 21, 466 (1953). (25) Ibid., p. 767. (26) Parr, R. G., “Quantum Theory of Molecular Electronic Structure,” W. A. Benjamin, Inc., New York, 1964. (27) Perkampus, H.-H., Kortum, K., 2. Anal. Chem. 190, 111 (1962). (28) Popovych, O., Rogers, L. B., Spec584. trochim. Acta 1954 (29) Streitweiser, A.: “Molecular Orbital Theory for Organic Chemists,” Wiley, New York, 1962. (30) Viswanath, G., Kasha, M., J . Chem. Phys. 24, 574 (1956). (3:) Weissburger, A,, Proskauer, E. S., Organic Solvents,” Interscience, New York, 1955. RECEIVEDfor review May 18, 1966. Accepted August 11, 1966. Work supported in part by the U. s.AtomicEnergy Commission Contract AT(30-1)-905.
Sensitive Spectrophotometer for Measuring Kinetics of Reversible Systems P. A. LOACH
and R. J. LOYD
Biochemistry Division, Department of Chemistry, Northwestern University, Evansfon, 111.
b Details are given for construction of a sensitive spectrophotometer whose components include a double monochromator for achieving a high spectral purity in the detecting beam and a feedback circuit for stabilizing the light emitted from the detecting beam source. The instrument is capable of quantitatively measuring changes as absorbance unit, small as 5 X has a resolution of 1 A. over much of the spectrum (2600 to 12000 A.), and can measure precisely kinetic parameters which may vary from second to minutes. Typical lightinduced absorbance changes for the photosynthetic bacterium Rhodospirillum rubrum are presented, and the importance of using a low intensity detecting beam for such systems i s underscored.
M
easurement of fast photochemical reactions in solution was first achieved by use of a high-intensity flash lamp (14-16) for rapid (10-6 to second) excitation followed by a second low-intensity flash for measuring absorbance changes. Extension of this method (23-26) has been employed for
studies of photosynthetic systems. to While more rapid changes second) may be followed, the present limitation in sensitivity is of the order of 5X absorbance unit. A more sensitive method takes advantage of the availability of commercial digital memory averagers as signal improvement and storage devices (8). However, the fastest reactions that can be followed are of the order of second. One of the most rewarding applications ( 7 , 9, I O ) is the measurement of the small changes in absorbance displayed by photosynthetic material as a response to light excitation. Such light-induced absorbance changes were first discovered by Duysens (4). The photosynthetic systems are ideal for signal improvement because they are rapidly excited by radiation in the visible region of the spectrum, the quantum yield for excitation is near 1 ( d , 2 2 ) ,there is a wide range of absorbance changes that can be observed (from 200 to 1300 mp, some positive and some negative), the dark decay times may vary from a few milliseconds to many seconds depending on the conditions, and most changes are fully reversible (21). From performance characteristics of
previously reported kinetic spectrometers which utilize the signal averaging technique (7, I O ) , absorbance unit of reversible change can be detected with a peak to peak S I N = 10 after a few minutes averaging time. Each of these instruments uses a single monochromator for its detecting beam and operates with a typical resolution of approximately 50 A. The kinetic spectrometer reported herein has a 10-fold increased sensitivity, uses a double monochromator for its detecting beam having a resolution of less than 1 A. over much of the spectrum (260 to 1200 mp), and is capable of measuring precisely kinetic parameters which may vary from second to minutes. EXPERIMENTAL
Methods and Materials. A schematic drawing of the optical arrangement used is shown in Figure 1. T h e detecting beam light source is a tungsten-halogen lamp powered b y a programmable supply (Hewlett-Packard 6367A) having less than 0.5-mv. ripple and 5 mv. per hour drift. The double monochromator is that of a Cary 14R recording spectrophotometer with the scattered transmission attachment No. VOL. 38, NO. 12, NOVEMBER 1966
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1710
ANALYTICAL CHEMISTRY
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1462 (Applied Physics Corp., Monrovia, Calif.). The chopper mot.or for ordinary double beam use is switched off to allow a single d.c. beam pass through the sample cell and into a n appropriate multiplier phototube. About 15y0 of the detecting beam is deflected by a precision ground quartz plate (H. S. Mart,in and Sons, Evanston, Ill.) placed before the cuvette. This deflected beam is focused onto a matching multiplier phototube tjo provide a feedback signal to the detect'ing lamp supply. Kot shown in the figure are filters which are often placed in front of this second multiplier photot,ube. The source lamp feedback circuit is shown in Figure 2. To speed construction, commercially available units were used wherever possible. The GE 1958 quartz-iodine lamp used requires 28 volts a t 5.5 amperes to operate. The output of the reference multiplier phototube is only about 10 to 15% that, of the sample beam, so a voltage gain of about 200 was required to produce the desired corrections. The gain was raised until oscillation of the loop occurred and then reduced below this point. A control loop more sophisticated
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than a conventional proportional system did not seem justified. A Philbrick P2A operational amplifier had the necessary high-input impedance with low drift rates and was employed in a n inverting configuration. An Electronics Research Model SR 28P5 power supply was used to set the 28-volt operatsing point. A second Philbrick amplifier (P55) was used as meter amplifier so that the error signal could be observed, and this was reset as necessary. The meter scale indicates from -0.5 volt to +0.5 volt. The overvoltage limit in t.he Hewlett-Packard supply was reduced to 30 volts so that errors in setting the feedback zero does not significantly shorten the lifetime of the lamp. I n this way the lanip is stabilized against changes in intensity of longer than 3-msec. duration. Because without the use of this feedback circuit the most troublesome noise has been long-term drift in the detecting beam light source, this modification improved the sensitivity of the instrument nearly ten-fold. The intensity and half width of the detecting beam passing through the cuvet,te can be cont,rolled by appropriate
manual adjustment of the slit width control on the Cary. The useful intensity range is from lo3 ergs per sq. cm. per second to 10-' erg per sq. cm. per second as measured by an 8-junction, air-type Eppley thermopile (Yo. 5721) having a circular bismuth silver couple with a lamp black coating and a Keithley Model 149 millimicro voltmeter. The thermopile calibration was checked against a 50-watt NRS standard carbon filament lamp (Eppley Laboratory, Newport, R. I., No. E 6327). After passingthrough thesamplecuvette, the detecting beam passes through a narrow band pass filter (Baird Atomic B-9 or B-11) and onto an end window multiplier phototube whose supply voltage (Fluke 405 B) is varied from -700 to -2000 volts depending on the sensitivity required. The negative direct current output from the multiplier phototube is summed to zero through a resistive network attached to a positive voltage stabilized power supply (Electronic Research Associates SR 28P5). Any deviation from thiq zero is amplified with a Northern Scientific 301 d.c. amplifier and registered in a digital memory averager [either a VOL 38, NO. 12, NOVEMBER 1966
1711
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Photo-induced absorbance change at 4 3 3 R. rubrum cells
mp in 2-week-old
Absorbance at 880 rnp = 0.01 9
Northern Scientific digital memory oscilloscope (NS-513) or a Nuclear Data Enhancetron]. Presently available averagers are not able to measure changes faster than about lo-* second. A repetitive change in absorbance is caused by a second beam of light a t a right angle to the detecting beam (Figure 1). The source for this beam is either a 1000-watt tungsten projection lamp or a 150-watt Xe lamp (Hanovia 901 C). The light is collected with a pair of condensing lenses, caused to converge to a focal point at the chopper, and then appropriately projected onto the sample cuvette. A xenon flash lamp has also been used for excitation ( 1 1 ) . The chopper motor is a pen motor (LIFE Model R-4-154) with a 45' excursion. It is driven by a 28-watt direct coupled Hi-Fi amplifier (20) which has enough internal feedback to dampen the oscillation a t the end of its excursion. About 10 msec. are required for a n attached 2-gram blade to complete its total excursion. By adjusting the length of the chopping blade and the size of the focal image, the exciting light can be chopped as fast as 10 psec. (IS),but for most experiments the time is near 1 msec. The fastest repetition rate which has been used is 3 per second, although about 40 per second are possible. The timing unit used for triggering the z-axis sweep of the digital memory averager unit, coincident with either moving the chopping blade out of the light path or into it, consists of a Beckman four decade present counter (Model 542R) and a unijunction transistor oscillator (UJT) used as a time base as shown in Figure 3. The UJT oscillator time base is a typical transistor manual unit, powered by a 8.4-volt mercury cell, and produces two time intervals selected by a switch. The time intervals were chosen to have no multiples of 60 He or its near harmonics, so that such pickup is minimized by the averager. The time periods used were 14 msec. and 1.53 seconds,
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ANALYTICAL CHEMISTRY
reproducible to about 0.1%. The values are easily changed. A bistable multivibrator was incorporated into the counter, and the counter modified such that two decades preset the on time and the other two preset the off time. This is accomplished by feeding timing pulses to both counter sections through 6AS6 gate tubes. The plate voltages of each section of the multivibrator are applied to the gate tube grids, with each half controlling one gate tube. I n this way one inpiit is always cut off. The counter which is receiving pulses accumulates to its preset number, then its reset signal is used to toggle the multivibrator. The gate tubes reverse conduction and the second counter section begins to accumulate, with the first counter section being reset. The output of the niultivibrator is also sent to a n amplifier stage and cathode follower to furnish the power for the various outputs. The following trigger voltages are provided: 6-volt pulse with 0.2-psec. rise time for the Enhancetron using a n external Schmidt trigger; 5- to 10volt pulse for 20-msec. duration for the Northern Scientific digital memory oscilloscope; 1 2 volts for the driving unit of chopper motor; a thyratron switch for a xenon flash lamp (Kemlite S-65R, Chicago).. When a sufficient number of repetitive scans is recorded in the memory of the totalizer or averager unit the data are recorded in a Moseley 2D-2A XY recorder. Preparation of the photosynthetic materials examined has been described (12) as has the spectrum of lightinduced changes (1, 4 , 5, 10). RESULTS A N D DISCUSSION
The instrument described has been most useful in obtaining data of the type indicated in Figure 4,where the maximal change in absorbance was 0.001. For this experiment, two 7-69 Corning color filters were placed in the exciting beam
filter holder, a 4-96 Corning color filter over the end window of the reference multiplier phototube, and a Baird Atomic B-9 narrow band pass interference filter, maximally transmitting at 433 mp, over the end window of the measuring beam multiplier phototube. Both multiplier phototubes were RCA C31000. The sample was placed in a four-sides clear 1-cm. cuvette in air a t 25" C. The vertical arrow indicates when the exciting light was blocked from reaching the sample. The exciting light was allowed to fall on the sample a t zero time; 4 minutes' totalizing; total dark period, 30 seconds. The detecting beam intensity was approximately 5 ergs per sq. cm. per second with a bandwidth of about 3 A. Typical data obtained in the ultraviolet region of the spectrum are to be published (13). For this experiment, 2-week-old R. TUbrum cells were suspended in 0.05A1f phosphate buffer a t pH 7.5 and 25' C. The decay of the photoproduced absorbance change has a typical life time for whole cells. Quantitative data for this magnitude of change can be obtained within a few minutes. Smaller changes of the order of 10-4 absorbance unit may be of equal interest to the larger (0.001 to 0.05 absorbance unit) changes normally measured. One of the major reasans for improving the sensitivity and reliability of this instrument is so that small changes can be measured quantitatively. Figure 5 shows the results of 16 minutes' totalieing of a reversible change of 5 X 10-6 absorbance unit. The instrument's AA. sensitivity is at least 1 X Filters, multiplier phototubes and other conditions for Figure 5 were as described for Figure 4. Total dark time was 0.7 second. The onset of light and dark periods is indicated by the vertical arrows.
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Figure 6. Comparison of apparent decay kinetics at two wavelengths for a constant detecting beam intensity Two wavelengths indicated
One of the chief advantages of using a double monochromator is the high resolution and purity of light which is equal to that of the Cary 14R. Coupled with the high-gain and low-noise multiplier phototubes such as the RCA C31000 (potassium-cesium antimonide photocathode) and C70007 A (S-1 response), a low-intensity detecting beam may be employed-sufficiently low to have no measurable effect (10, 13, 18) on the system being studied. Figure 6 shows the effect of the detecting beam a t constant energy (4.5 X lo2 ergs per sq. cm. per second) but different wavelength when absorbance changes in chromatophores from R. rubrum are measured between 790 and 870 mp. Chromatophores were prepared from 4day-old R. rubrum cells. Absorbance a t 880 mp = 0.60. A 4-96 Corning color filter was placed in the exciting beam and two 7-69 Corning color filters were placed on the front of the measuring multiplier phototube. The traces were initiated on the light off signal. Totalizing time employed was 110 seconds. Total light period was 3 seconds and the dark period was 20 seconds. Sufficient exciting light intensity was used to saturate the changes as observed a t 810 mp. At 865 mp only about one third of the total photochange is actually observed because of excitation by the detecting beam. One multiplier phototube was used for this experiment, an RCA C70007A. Detecting beam intensity was 4.5 x lo2 ergs per sq. cm. per second and its band width approximately was 50 A. The sample was in a 1-cm. cuvette at 25’ C. and pH 7.5 (0.05M potassium phosphate buffer). Even a t this relatively low intensity the slow decay time in these systems allows partial excitation by the detecting beam, resulting in apparently faster decay times a t wavelengths where the light is more effectively absorbed and more efficiently used. At detecting beam intensities of several hundredfold lower magnitudes all decay times are identical at the various wavelengths studied (13).
Such excitation by the detecting beam or, alternatively, too short a dark period for the exciting beam may allow the steady state level of excitation to become sufficiently high that only the more rapid early portion of the decay can be observed. Part of the variation in kinetics observed in different laboratories (10,I S , 19) may be explained by such phenomena. The utilization of a feedback loop to stabilize the detecting beam light source brings the sensitivity nearly to the limit imposed by the characteristics of the various power supplies employed. During the course of development of this instrument, the detecting beam lamp stability proved to be a most important consideration. When a typical tungsten 6-volt, 18-ampere lamp (GE-PHI18 ATIDP) was used, the intensity fluctuation, even with a well stabilized power supply, was often several percent of the total intensity. Thus, even with the employment of a differential amplifier (r),relatively large fluctuations of the detecting beam light intensity limit the accuracy of the data recorded. We found an approximate 10-fold increase in stability upon using a tungstenhalogen lamp, and an additional 10-fold stabilization employing the feedback system. The use of a single multiplier phototube for both detection of absorbance changes and light stabilization was not practical because the chopping time for a split beam would have to be fast (
