Variable Temperature Computerized Dual-Beam, Rapid Scanning Stopped-Flow Apparatus for the Study of Air-Sensitive Systems and Transients in Enzyme Reactions Nicholas Papadakis,’ Richard B. Coolen,2 and James L. Dye3 Department of Chemistry, Michigan State University, East Lansing, MI 48824
A double-beam, vacuum tight, thermostated stopped-flow apparatus has been constructed and computer interfaced. A specially designed all-quartz mixing and observation cell equipped with a double mixer and two optical path lengths has been used. The syringes were made out of heavywalled precision bore Pyrex tubing. Steel plungers with adjustable Teflon tips were machined to fit them. The entire apparatus was constructed such that the solutions contact only Pyrex, quartz, and Teflon, while the flow system is surrounded by a thermostat bath. Quartz fiber optics were utilized to transmit the dispersed light from a Perkin-Elmer Model 108 rapid-scan monochromator to the flow and reference cells and then to a pair of photomultipliers.The performance of the system has been tested by studying a number of well characterized chemical reactions.
Since the initial measurement of the rate of reaction of the solvated electron with water in ethylenediamine ( I ) ,we have continued to develop stopped-flow systems for the study of air-sensitive reactions (2-6). The incorporation of a scanning monochromator ( 3 ) ,quartz mixing and observation chambers ( 7 ) , an FM tape recorder for data acquisition and subsequent analysis by computer ( 4 - 7 ) , and backpumped pushing syringes (6) were significant improvements. However, the kinetics studies were still limited by the necessity to work a t or near room temperature and by analog data storage. This paper describes the construction and testing of a variable temperature, vacuum-tight stopped-flow system which is on-line with a remote PDP8/I computer. The data-averaging and display routines and the data-acquisition system are described in a companion paper (8).To make the system useful for the study of transients in enzyme reactions, a major effort was made to extend the wavelength range into the UV region. Two path lengths were incorporated to permit study over a wide concentration range.
FLOW SYSTEM DESIGN Greased syringes and stopcocks cause problems with many solvents and with enzymes. Metals such as stainless steel catalyze the decomposition of metal-amine solutions. Therefore, the flow system was designed to permit contact of the solutions only with quartz, glass, and Teflon. The need to operate a t variable temperatures and to evacuate the entire system required the development of adjustable Teflon seals. Since temperature artifacts are common with flow systems ( 9 ) ,the entire flow apparatus was enclosed in a Plexiglas thermostat bath (10).Up to four stock solutions which had been prepared and de-gassed on a vacuum line Present address, Department of Chemistry, Cornel1 University, Ithaca, NY 14850. Present address, Erie County Laboratories, 462 Grider Street, Buffalo, NY 14215. Author to whom correspondence should be adressed. 1644
ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
could be admitted to each side of the system and accurately diluted or mixed before being introduced into the pushing syringes. Figure 1 gives an overall schematic diagram of the flow system. Flow Cells. I t is common to construct mixing chambers by drilling appropriate holes in metal or plastic. These are then either attached to optical cells or fitted with quartz windows. Our flow and reference cells were constructed entirely of quartz in order to be completely leak-free. The key to this technique is an Airbrasive unit (Model C, S. S. White Industrial Division, New York, NY) which rapidly “drills” holes in quartz by driving powdered aluminum oxide in a stream of nitrogen through an appropriate nozzle. We had previously used this technique (6, 7 ) to construct four-jet tangential mixers from heavy-walled 1- and 2-mm i.d. quartz capillaries (Engelhard Industries, Inc., Amersil Quartz Division, Hillside, NJ) which had been polished flat on two opposite sides (Precision Glass Products Co., Oreland, PA). The optical path length of the cells was limited to the diameter of the capillary. To achieve more complete mixing and to have two path lengths available, cells of the type shown in Figure 2 were constructed ( I 1 ) . With this design, the mixed solutions were divided into four streams which were recombined in a second four-jet mixer. A quartz rod was sealed into the tube prior to the drilling operation to serve as a plug. All holes were drilled to enter the central bore of the capillary tangentially. To prevent drilling into the wall of the capillary opposite the hole being drilled, a piece of polyethylene medical tubing was inserted into the central bore. The capillary was mounted in an indexing head so that the holes could be precisely located. The capillary was rotated 90’ between successive holes. By using the appropriate nozzle tip and positioning it a t the proper distance from the tube, we were able to drill holes with only a slight taper such that the entrance diameters were just under half the bore diameter. These were enlarged to exactly half the bore diameter with a tungsten rod in a drill press by using wet abrasive powder. For the long path length optical cell, another capillary was utilized. Two parallel holes of the same diameter as the bore of the capillary and a t a distance apart equal to the desired path length were drilled to make an angle of about 45’ with the central bore. The pieces were assembled in our glassworking shop. The mixer was finished by attaching four inlet tubes and two vacuum joints (Fischer-Porter 2-mm quartz “SolvSeal” joints) as shown in Figure 2. The appropriate holes were plugged by sealing ground quartz rods into them (heavy lines in Figure 2) and the capillary for the long path length cell was sealed to the mixer and to an exit tube. Finally, the two ends of the long path length capillary were ground until the inlet and outlet holes were just uncovered. Two flat optical windows were then sealed to the ends. For one cell, these windows were cut from -0.2-mm thick quartz cover slips. For a second cell, thicker windows were
Figure 2. Schematic diagram of the mixing and observation cell (A) Fischer-Porter 2-mm quartz Soiv-Seal joints, (B)-0.5 tion of mixers and stream-splitter
mm, (C) cross-sec-
Figure 1. Schematic diagram of the thermostated stopped-flow apparatus (A) rinsing ports, (8) reactant ports (four on each side), ( C ) thermostated burettes (four on each side), (D) reactant reservoirs, (E) mixing and observation cell, (F) reference ceii, (G)pushing syringes, (H)pneumatic pistons, (I) stopping syringe, (J) quartz light fibers, (K) Plexiglas thermostat bath, (L) to vacuum, (M)to vacuum and "waste", (1, 2, 3, 4, 5) flow valves in bath. (Valves outside of the bath are represented by solid areas in the tubes)
first sealed on and then ground and polished t o give a final thickness of -0.2 mm. The reference cell was constructed in the same way as the sample cell but without the mixers. Individual holders were machined from aluminum for each of the cells. The cells were wrapped with Teflon tape and provided with round (long-path) and slit-shaped (short-path) masks so that light could not by-pass the solution. Provision was made for entrance and exit quartz light fibers for each path length. The entire cell holder was made liquid-tight by the liberal application of Dow Corning 3110 RTV flexible silicone polymer to prevent leaks of the bath fluid into the optical path. Pushing and Stopping System. After testing a number of commercial "gas-tight'' syringes, we concluded that none would be truly leak-free over a wide range of temperatures. Therefore, we constructed syringes and plungers which eliminated leakage problems. The syringe bodies were made from heavy-wall precision bore tubing (Trubore 8'700-765, i.d. = 0.553 inch, Ace Glass, Inc., Vineland, NJ). T h e top of the syringe body was formed by fusing a flat plate across its diameter to minimize distortions and dead spaces. The plungers were machined from steel to fit these syringes. The design is shown in Figure 3. The Teflon wiper which forms the primary seal is forced against the wall by turning the threaded rod. In this way, vacuum-tight operation down to -30 "C can be achieved. To completely exclude air from the system (which can permeate through Teflon), the syringes were constructed to permit back-pumping between two "0"-rings via a sidearm. The sidearm is also used as an exit port t o permit rinsing of the system when solutions are changed. Special care was taken to prevent distortion of the syringe body when the sidearm was sealed on. A small pinhole was first
Figure 3. Syringe design (A) Teflon tip, (B)rod (with reverse threads at bottom), ( C ) Viton O-rings, (D) fill position, (E) rinse position, (F) locking nut
"drilled" with the Airbrasive unit and the sidearm was sealed to the body by a procedure that glassblowers refer to as a "stickseal". Although construction of the syringes is a painstaking task, the elimination of one of the major problems of stopped-flow systems-leaky syringes-makes the effort worthwhile. Mechanical System. The flow system is mounted vertically on a framework of aluminum plates supported by 3/sinch threaded brass rods and securely fastened to an angle iron table which also serves to hold the lamp, monochromator, and detectors. A Plexiglas bath with two removable sides surrounds the entire flow system and serves to support the flow valves (Kontes Teflon valves, K-826610, 0-4 mm, Kontes Glass Co., Vineland, N J ) and the glass waste lines. Two air-driven magnetic stirrers are used in the bath to mix the contents of the reactant reservoirs. The pushing and stopping syringes are securely mounted between aluminum plates, and are separated from the aluminum by Teflon gaskets. The plungers for the two pushing syringes are securely fastened t o an aluminum block which is constrained with guide rods to move only in the vertical direction. This block is attached to a pneumatic piston (Schrader, 2-inch bore and stroke, Scovill Div., Wake Forest, NC) which permits filling, removal to waste through the sidearm, or rapid pushing as desired. The pneumatic piston can be actuated either manually or automatically. In the latter case, it is actuated by the beginning of scan (BS) signal from the scanning monochromator. A miniature pneumatic cylinder operated with a manual valve is used to expel the waste solution from the stopping ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
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syringe. The plunger of this syringe travels about 1 cm before striking the aluminum stopping plate, thus providing three pushes for each filling of the pushing syringes (-1.5 ml total per push) while allowing for critical flow velocity to be attained. The plunger is fitted with two metal flags which interrupt two light beams prior to flow stop and give an accurate measure of the flow velocity and the true zero of time [see the companion paper for details (8)]. Solution-Handling System. The pushing syringes are connected to the flow control valves via two-turn spirals of 2-mm i.d. glass tubing. Additional spirals connect the flow valves to Solv-Seal joints (Fischer-Porter Co., Lab-Crest Div., Warminster, PA) which attach to the flow cell. These spirals are required for strain-relief and have been found necessary for low-temperature work ( I O ) . The top of the cell is connected via another joint to the waste line and the stopping syringe by means of a U-shaped piece of 2-mm i.d. capillary tubing. During assembly of the flow system, the glass parts are observed with a high-magnification telescope as the pushing plungers are moved. The aluminum plates and syringe-holders are adjusted until no movement can be seen. Up to four solution bottles can be mounted on each side of the apparatus with 5-mm Solv-Seal joints. Each bottle is attached to a 10-ml thermostated buret which has a Rotaflo adjustable Teflon valve (Lab Glass, Inc., Vineland, NJ) a t the bottom. The four buret tips are sealed through the top of a funnel-shaped tube which drains into the mixing and storage reservoir. Because of dead-spaces a t the valves and in the tubes, a small amount of solution of the desired concentration is prepared and run through the system before preparation of the solution to be studied. By evacuating the entire system prior to a run, bubble formation can be virtually eliminated. Since concentrated enzyme solutions tend to foam excessively under vacuum, it is necessary to handle such solutions at atmospheric pressure or above. In such cases, the evacuated system was filled with water prior to the run to exclude bubbles. I t is our opinion that trapped air bubbles are a major source of annoyance and improper stopping in stopped-flow systems. An evacuable and vertically mounted flow system minimizes such problems. Optical System. Quartz fiber optics (Schott Optical Co., Duryea, PA) transfer the dispersed light from the monochromator through the bath fluid to the cells and then to the photomultipliers. This eliminates alignment problems as well as errors resulting from movement of the mixing and observation cell. Double beam operation and analog conversion of intensities to absorbance are employed to permit a wide dynamic range of light intensities, to cancel out lamp intensity and P M tube voltage fluctuations, and to allow for the study of small absorbance changes over large background contributions. Recently a two-branch fiber beam-splitter has been installed which has a slitshaped end to match the monochromator exit slit. Although the individual fiber positions are not randomized, the cancellation of lamp fluctuations is much more effective than it was when separate fiber bundles were used for the sample and reference beams. Three light sources have been used with this system, a 50-watt tungsten (quartz-iodine) lamp, a 150-watt xenon lamp, and, most recently, a 1000-watt xenon lamp. The latter source, with appropriate optics matched to the monochromator, improved the signal-to-noise ratio (S/N) dramatically and has been used exclusively in our recent work. This lamp is housed in a Model C-60-50 Universal Lamp Housing and powered with a Model C-72-50 power supply (Oriel Optics Corp., Stamford, CT). The Perkin-Elmer Model 108 rapid scanning monochro1646
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ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
mator with a quartz prism (four-pass Litrow system) allows us to scan any desired spectral region between 250 and 1050 nm (depending upon the detector used) with 3 to 150 scans per second. This instrument, which was purchased in 1964 and reconditioned in 1974, is no longer commercially available. The wavelength range scanned is determined by the tilt angle of a rotating mirror and is independent of the scan speed. The original read-out device used to relate angle of rotation to wavelength has been replaced by a system which produces two kinds of triggering signals from phototransistors (GE L14B). One of them (BS signal) marks the beginning of each revolution of the mirror and is produced by reflecting a light beam from a polished notch in one of the gear teeth on the gear which is attached to the mirror shaft. The second (GT signal) is produced when the same light beam passes between the gear teeth to a phototransistor which is mounted behind the gear. In this way, each ,rotation of the mirror produces one BS pulse and 136 G T pulses. The GT signal is used to trigger the data-collection system after its frequency has been appropriately multiplied (8).Each rotation of the mirror produces both a forward and a backward scan and the scan rate (wavenumbers per second) is approximately a sinusoidal function of time. The use of either quartz-envelope P M tubes (RCA 6903, S-13 response) or EM1 P M tubes (EM1 9684 B, S-1 response) allows us to detect light in any spectral region between 220 and 1050 nm. T o reduce the rather substantial dark current of the red-sensitive EM1 tubes, we used the recommended magnetic lens assembly and mounted the PM tubes in a specially made Dry-Ice cooled housing. Since two fiber bundles are used for each P M T (short and long path length), a sliding bar was mounted in such a way that one beam a t a time can be isolated. Provision was also made for calibration filters to be inserted into the sample beam, and an adjustable V-shaped vane into the reference beam. The detectors are powered with a Furst Electronics Model 710-P regulated power supply. The outputs of the two P M tubes are input to an analog logarithmic ratio amplifier circuit which utilizes Philbrick/ Nexus operational amplifiers (types P-25 A, P L l P , and P65AU) and switch-controlled high frequency filters. The output of the absorbance circuit is amplified and biased to permit full utilization of the -5 to +5 volt range of the analog to digital converter (ADC). PERFORMANCE TESTS The performance of the computer and interface system is described in the companion paper (8). Therefore, we limit this section to a description of the calibration procedures and tests of the flow system performance. Calibration Procedures. Since all data are collected and analyzed by using computer techniques, calibration of both wavelength and absorbance can be easily done for each run. Scale expansion of the wavelength axis is simply a matter of changing the angle of nutation of the mirror, while the center of scan is determined by a calibration drum which sets the angle of a roof mirror in the instrument. Although both settings can be accurately made, we find it more convenient to insert a didymium and/or holmium oxide glass filter into the sample beam prior to each run. From the known peak positions and an appropriate sinusoidal interpolation formula, the relationship between wavenumber and point number (core location) can be accurately determined. Since peak positions were always reproducible to within f l core memory location, this calibration procedure was completely reliable. The analog circuitry was designed to provide a signal proportional to absorbance. The proportionality constant and any nonlinearities in response, even if wavelength de-
pendent, were determined by scanning the desired wavelength range with calibrated neutral density filters in the sample beam and with identical solutions in the sample and reference cells. Typically, neutral density filters (Optical Industries, Inc., Santa Anna, CA) with absorbances of 0.1, 0.3,0.7, 1.0, 1.3, and 2.0 were used. An appropriate program was written for the CDC 6500 computer which then automatically determined the true absorbance a t any wavelength and nominal absorbance reading by a multiple regression procedure. Because separate P M tubes are used for the reference and sample beams, the base line is not flat over the entire region of scan. I t is, however, a simple matter to correct for response differences by base-line subtraction, and this is automatically done by the PDP-8/1 computer upon request. In addition, absorbance a t infinite time or a t the zero of time may be subtracted as desired. Optical System. The Beer-Lambert law was used to determine the effective path lengths of the cell. For the short path length, freshly prepared aqueous solutions of potassium permanganate, calibrated with a Cary Model 15 spectrophotometer were used. Up to an absorbance of 2.0 a t 524 nm, the absorbance varied linearly with concentration, yielding a path length of 1.99 f 0.02 mm. (Note: all estimates of error listed in this paper refer to least-squares estimates of the standard deviation.) Similar tests for the long path length with aqueous solutions of 2,4-dinitrophenolate (12) a t 360 nm gave an effective path length of 1.850 f 0.005 cm. These tests showed that stray light which bypasses the solution was negligible. Scattered light, which caused some problems initially, has been virtually eliminated by resurfacing the monochromator mirrors. When scanning a t wavelengths below 290 nm, some scattered light has been observed. For example, about 5% of the light a t 275 nm is scattered light when no filter is used in the beam from the 1000-watt xenon lamp. However, the insertion of a mixture of 0.3M nickel sulfate and 0.2M cobalt sulfate in water (13) into the beam eliminates this problem without significant UV absorption. This solution also serves a second purpose by decreasing the dynamic range of light intensities when scanning from the UV to the red region of the spectrum. This prevents saturation of the P M tubes in the visible when the slits are opened wide enough to give acceptable S/Nin the UV region. Without such a filter, the signal intensity varies by more than two orders of magnitude over the range of 260 to 550 nm. The S/N depends, of course, upon the wavelength region examined and the slit-widths used, as well as upon the filtering or averaging bandwidth of the detection system. Therefore, it is not very meaningful to quote S/Nvalues. Two major noise sources are limiting a t the present time. One arises from vibrations of the scanning mirror and can be removed only by scanning more slowly or by collecting data a t fixed wavelength. The second source derives from the fact that the sample and reference fibers do not “see” identical regions of the xenon arc. Although the beamsplitter has greatly reduced this problem, it has not entirely eliminated it. An indication of the “worst-case” S/N is given in Figure 4, which shows a single 13.3-msec scan from 245 to 340 nm of a holmium oxide glass filter. The combination of lower source intensity, mirror reflectivity, fiber transmittance, and P M tube sensitivity in the UV compared with the visible region gives a signal intensity a t 260 nm which is only 0.2% that a t 470 nm. The S/N does not vary over such a wide range, however, since positional fluctuations of the xenon arc and mirror vibrations give essentially wavelength independent contributions. A completely randomized beam splitter would reduce these contributions substantially.
f 01 A
1 .... . ... ....,.,,,.’ ...,..
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WAVELENGTH ( n m l
Figure 4.
Spectrum of holmium oxide glass filter
Top: Single 13.3-msec scan over the wavelength range 250-340 nm. Bottom: Ten-fold expansion of the top scan minus the average of 100 scans
The Flow System-General Performance. The flags on the stopping syringe permit measurement of the average flow velocity and the time of stopping for each push. Flow times were reproducible; for example, 25 successive pushes gave an average flow velocity of 3.22 f 0.15 m sec-l. This is well above the critical velocity (-1 m sec-l) for turbulent flow for water a t 20 OC in a 2-mm tube (14). The mixing efficiency is good enough that we have not yet been able to measure any systematic deviations from 100% complete mixing a t the time of observation in the short path length cell. This was tested by mixing equal amounts of 2 X 10-4M p-nitrophenolate and 1 X 10-4M hydrochloric acid with observation a t a fixed wavelength of 400 nm. A flat straight line was observed with a residual absorbance corresponding to the unreacted p-nitrophenolate. Since this reaction occurs a t a diffusion-controlled rate ( 1 5 ) ,it is over before the solution reaches the observation tube and any deviations from a constant absorbance would be caused by poor mixing. The stopping time is reproducible and less than one millisecond. This was determined by following the absorbance a t fixed wavelength during flow and after flow had stopped for several reactions. No quantitative measure of the stopping time has been developed but the estimate of less than one millisecond is based upon the “rounding” of the absorbance trace as the flow stops. Successive pushes with the same solutions were very reproducible and we did not have to discard any of them when the system had been pre-evacuated and the solutions degassed to prevent bubble formation. The flow system has been used a t temperatures up to 40 “C with good results and no detectable thermal artifacts. Quantitative Tests. One of the systems used to test the performance of the instrument was the production of peroxychromic acid from hydrogen peroxide and acidified solutions of dichromate according to the reaction: HCr04- 2H202 H+ 9 CrOs 3H20 This reaction was shown by Wilkins et al. (16) to be thirdorder overall, first-order in each reactant. The decay of the absorbance of HCr04- can be followed a t 354 nm and the growth of Cr05 a t 600 nm with a clean isosbestic point a t 480 nm. At high acid concentrations, a slow decay of the Cr05 occurs (16). Figure 5 shows the spectral changes which accompany this reaction. Only selected spectra are displayed to show the decay a t -350 nm, growth a t -600 nm, and isosbestic point a t -480 nm. Figure 5 also shows the effect of the av-
+
+
ANALYTICAL CHEMISTRY, VOL. 47,
+
NO. 9,
AUGUST 1975
1647
I
:..
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00
I
................
I
386
348
0
592
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WAVELENGTH (nm)
Figure 5. Characteristic spectra taken at 75 scans per second during the reaction of HCr04- with H202 and H+ Starting from the top of the decay at 350 nm or the growth at 600 nm, each successive spectrum is the average of 1, 2, 4, 8, 16 consecutive spectra
356
nm
A
360 mm
0
376 nm
0 0
580 nm
510 nm
05
010
10
15
TIME (sed
-
eraging scheme in smoothing the data. Starting from the top (of the decay), each spectrum is the average of 1 , 2 , 4 , 8 , and 16 adjacent 13.3-msec spectra, respectively. A simple test for an “uncomplicated” reaction in which both reactant and product absorb, can be made by plotting lnlA(t) A(tm)lvs. time a t different wavelengths. If the reaction is “uncomplicated”, the curves must have the same slope a t any time regardless of the order of the reaction. Of course, for a first-order or pseudo first-order reaction, straight lines are obtained. Figure 6 shows such plots for the pseudo first-order reaction of 0.5mM HCr04- with 4.54mM H202 and 10.0mM Hf.The parallel straight lines show that the stoichiometry of the reaction is “clean”. In addition, the range of observation over which the data are usable (about four half-lives shown) dramatically illustrates the improvement in S/Nwhich is provided by our averaging scheme. The dead-time of a stopped-flow apparatus is the time required to transfer the solutions from the mixing chamber to the observation point and bring them to a complete stop. If the stopping time is short and mixing is efficient, then the dead time can be estimated from the dead volume and the flow velocity. For our 2-mm diameter flow cell we have VI = volume from the first to the second mixer (-0.015
ml); V2 = volume of the second mixer (-0.024 ml); V3 = volume from the second mixer to the end of the short-path window (-0.009 ml); and Vq = volume from short-path length cell to center of long-path length cell (-0.073 ml). With a typical flow velocity of 18 ml sec-I, we obtain dead times of 2.7 and 6.7 msec, respectively, for the two path lengths. The dead time was also computed from data obtained with the peroxychromic acid system. A semi-log plot of A ( t m )- A ( t ) vs. time (measured from the time of stopping) gives a straight line under pseudo first-order conditions. Extrapolation to the value that this function would have if no reaction had occurred gives the dead time as the difference between our time zero and the true zero of time for the reaction. This method yielded dead times of 2.5 and 5.5 msec, respectively, for the two path lengths. The quantitative tests with the peroxychromic acid system (made a t 30 “C) are summarized in Table I. Comparison can be made with the results of Wilkins et al. (16) who
Table I. Observed R a t e Constants for the Reaction: HCr04-
+ 2H202 + H +
h‘avelength i n nm
340 374 405 354 360 376 376“ 590 580 540 580 608 580 580 580
Figure 6. Plots of In1A Ami vs. time at various wavelengths for the reaction of 0.5mMHCr04- with 4.54mM H202and 10.0mM H+ The top three lines are decays and the bottom two are growths
[HAl 3
[E3202 1
LHCIC4- 1
N u m b e r of
mM
m If
Ir .!“i
pushes
10.0 10.0 10.0 10.o 10 .o 10 .o 20.0 10.o 10 .o 10.o 10 .o 10 .o 20 .o 22.5 20 .o
4.54 4.54 4.54 4.54 4.54 4.54 9.085 4.54 4.54 4.54 4.54 4.54 9.085 44.97 89.94
0.5 0.5 0.5 0.5 0.5 0.5
2 2 2 3
1.o
3 2
0.5 0.5 0.5 0.5 0.5
1 46
4b
2 2
5 .O
2 4b 2b, d
10 .o
Ib,d
1.o
k x
k ps sec‘l
M - 2 rec-1
1.61(8)a 1.70 (7) 1.70(6) 1.53(16) 1.40 1.47(18) 5.09(11) 1.50(1) 1.90(3) 1.76(13) 1.76(11) 1.74(13) 5.71(51) 30.5(11) 42.5
Standard deviations of the last digit given were computed for multiple pushes from the equation: u =
3.54(18)“ 3.74(16) 3.74(13) 3.37(35) 3.08 3.24(40) 2.80(10) 3.30(2) 4.19(8) 3.88(29) 3.88(25) 3.84(28) 3.14(28) 3.01(11) 2.37
1 % [-A ( k , - )2]1 -
The
1=1
estimated standard deviations of kpi’ obtained by fitting individual curves is generally much smaller. All experiments were carried out a t an ionic strength of 0.1M adjusted with K N 0 3 . The H202 solutions were titrated with a standardized solution of K M n 0 4 . Fixed wavelength pushes: others were obtained from scanning data obtained a t 7 5 spectra per second. < Initial deviation from pseudo first-order decay. P a t h length 0.200 cm: others used a path length of 1.85 e m .
1648
ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
studied this reaction as a function of temperature and obtained for the third-order rate constant k = 107.6 o.2 exp(-4500 f 200/RT)M-2 sec-l At 30 OC, this yields a value of 2.26 X lo4 M-2 sec-' with error limits of 1.5 x I O 4 and 3.8 x IO4 M-2 sec-l. We obtained the data in Table I by fitting the observed data to either a growth or a decay pseudo first-order expression by means of a nonlinear least-squares program (17). At concentrations of HCr04- greater than 0.5mM, we observed the same initial deviation from pseudo first-order decay as had been previously reported (16). We used the reaction of aqueous NaOH with 2,4-dinitrophenyl acetate (DNPA) to test the effect of various averaging schemes on the rate constant. This was a pseudo-first order, fixed wavelength experiment a t 400 nm. From the results, we conclude that the rate constant is remarkably insensitive to the averaging scheme used. Only for a grouping factor of four is there any indication that excessive early averaging might cause problems. A remaining problem with this system, caused by the need to use four valves between the syringes and the mixing chamber and to employ strain relief spirals, is the relatively large hold-up volume. The total hold-up volume of -2 ml is detrimental when working with limited amounts of reactants. In addition, some back diffusion from the mixing chamber can occur, depending upon the solution densities and the time between successive pushes. This problem has been overcome by making two pushes in quick succession and discarding the first one. Good quantitative performance under actual operating conditions a t three different temperatures has also been demonstrated in the study of several enzyme reactions. These studies showed the ability of the stopped-flow system to give reproducible rate constants, enhanced S/N and to detect subtle changes in the absorption spectrum during reaction.
ACKNOWLEDGMENT We thank E. Mei and G. Ho for assistance with some of the measurements, C. Suelter and his students for collaboration in the study of enzyme reactions, and A. Seer of the University Glassblowing Shop for his skill in constructing flow cells.
LITERATURE CITED R. R. Dewald, J. L. Dye, M. Eigen. and L. deMaeyer, J. Chem. Phys., 39, 2388 (1963). L. H. Feldman, R. R. Dewald, and J. L. Dye, in "Solvated Electron", Adv. Chern. Ser., 50, 163 (1964). J. L. Dye and L. H. Feldman, Rev. Sci. Instrum., 37, 154 (1966). J. L. Dye, Acc. Chem. Res., 1, 306 (1968). M. G. DeBackerand J. L. Dye, J. Phys. Chem., 75, 3092 (1971). E. R . Minnich, L. D. Long, J. M. Ceraso, and J. L. Dye, J. Am. Chem. Soc., 95, 1061 (1973). E. M. Hansen, P h D Thesis, Michigan State University, East Lansing, 1970. R . E. Coolen. N. Papadakis, J. Avery, C. G. Enke, and J. L. Dye, Anal. Chern., 47, 1649 (1975). R . K. Chattopadhyay and J. F. Coatzee, Anal. Chern., 44,2117 (1972). R. R. Dewald and J. M. Brooks, Rev. Sci. hstrum., 41, 1612 (1970). The reader is referred to the PhD. theses of N. Papadakis and R . B. Coolen, Michigan State University, 1974, for additional details. J. M. Sturtevant in "Rapid Mixing and Sampling Techniques in Biochemistry", B. Chance, R. H. Eisenhardt, 0. H. Gibson, and K. K. LonbergHolm, Ed., Academic Press, New York, 1964, p 97. M. Kasha, J. Opt. SOC.Am., 38, 929 (1948). F. J. W. Roughton and B. Chance in "Technique of Organic Chemistry", S. L. Friess. E. S.Lewis and A. Weissberger, Ed., Interscience Publishers, Inc., New York, 1963, Vol. 8, p 715. T. Nakamura, J. Biochem., 70, 691 (1971). P. Moore, S. F. A. Kettle, and R. G. Wiikins, lnorg. Chem., 5, 466 (1966). J. L. Dye and V. A. Nicely, J. Chem. Educ., 48, 443 (1971).
RECEIVEDfor review November 21, 19'74. Accepted March 21, 1975. This research was supported in part by the U S . Atomic Energy Commission under Contract No. AT(111)-958 and the National Science Foundation under Grant No. GB25116.
Computer-Interactive System for Stopped-Flow Kinetics with Rapid Scanning Molecular Absorption Spectrometry Richard B. Coolen,' Nicholas Papadakis,2 James A ~ e r y C. , ~ G. Enke, and James L. Dye4 Department of Chemistry, Michigan State University, East Lansing, MI 48824
A rapid scan stopped-flow apparatus was interfaced to a remote PDP-8/1 computer. The data transmission system permits high frequency (up to 10 M H r ) parallel digital data transfer over several hundred feet. A phase-locked loop frequency multiplication system allows sampling to occur at a constant computer-limited rate which is independent of scan speed and is synchronized with the wavelength drive of the monochromator. The data acquisition software establishes a digital averaging procedure which produces realtime signal-to-noise ratio (SIN) enhancement. This averaging varies with time in such a way that adequate spectral and temporal resolution are maintalned while storage requirements are minimal. Present address, E r i e County Laboratories, 462 Grider Street, Buffalo, NY 14215. Present address, D e p a r t m e n t of Chemistry, Cornel1 University, Ithaca, NY 14850. Present address, School o f Chemical Sciences, U n i v e r s i t y o f 11linois, Urbana, IL 61801. A u t h o r t o w h o m correspondence should h e addressed.
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The use of computers for real time data acquisition has become commonplace (1-3). Computer-coupled stoppedflow instruments have been reported (4-6), and a system with rapid scanning absorption spectrophotometric capabilities has been described ( 7 , 8 ) . The present work describes a computer-interactive system for acquiring and processing data from a rapid scanning stopped-flow apparatus. A description of the stopped-flow system is presented in a companion paper (9). The interface system makes use of typical components which are not described in detail here; however, several unique features of the interface are discussed at length in the hope that they might find general applicability. Briefly, the purpose of the interface system is not only to optimize the aicuiacy and efficiency of data collection and processing, but also to permit maximum interaction between the experimenter and the experiment. Principal features of the system include: processing of large quantities of rapidly changing three-dimensional data by a remote minicomputer; a phase-locked loop frequency multiplicaANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
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