c-ompurcrs & Chm?fi*rry Vol. 8, No 4. pp 309 313. 1984 Printed in the U S.A

APPLICATIONS SECTION

0097-8485/84 53-w + -00 Pergamon Press Ltd.

ON-LINE PROCESSING OF STOPPED-FLOW DATA FOR MULTIPLE KINETIC REACTIONS USING A MICROPROCESSOR-BASED

SYSTEM

T. S. HASSAN,~ A. H. G. EL-DHAHER t, H. MAARAFIE, H. ABOU SOUD and M. S. EL-EZABY Chemistry Department, Kuwait University, P.O. Box 5969, Safat, Kuwait

(Received 21 Jnnunry 1984)

Abstract-A microprocessor-based system designed to log and process data from a stopped-flow apparatus is described. First order rate constants for reaction kinetics with multisteps can be simply analysed by the system using two methods. Interaction with the investigator is achieved through an alphanumeric/graphics terminal. Re-investigation of the reaction kinetics of iron-(III) thiocyanate showed close agreement with previous results. The reaction kinetics of palladium with methionine has also been studied by the system.

INTRODUCTION Stopped-flow systems are frequently used in chemical and biomedical studies. Conventional methods to obtain the data use a storage oscilloscope from which a photograph is taken and analysed later by hand. Recent investigators have applied minicomputers (Notz, 1977) making the system versatile and fast. However,the cost was not justified; this consideration has led to the use of microprocessor-based systems which are less costly yet powerful and versatile. The systems provide rapid acquisition of data (Fomili & Lapis, 1981; Holtzman, 1980; Taylor ef al.. 1978) and analysis and display of results on-line (Bonnell & Befreese, 1980; Clark & Schuster, 1980). Many sys- tems, however, allow for only little interaction with the investigator. The data to be logged in many cases are accompanied by extraneous items that are some- times difficult to exclude unless there is effective interaction between the system and the investigator. The system described in this paper is based on the 8085A microprocessor on a commercially available board (the Intel SO/24 CPU board); it includes a video terminal with a built-in thermal printer.

printer. The terminal’s module is 2623A Hewlett-Packard.

The system has the following specifications: .

from

I .l’he level ok the Input sIgna may range from * 0.5 v to * 10 v. Sampling lime interval may range from 0.5 to 250 ms. Number of logged points may range from 50 to 500 points. No trigger signal is needed to start logging. Maximum number of reac- tions that can be analysed from a single mixing is five.

. Rate constant calculations may be done for either known or unknown infinity values.

l Rate constant calculations are supported statisti- cally by calculating the correlation coefficient and the standard deviation.

l Rate constant values may be stored for up to 10 runs for calculating the average value.

l Graphic plots are prcscntcd [or the overall reac- tion and for each separate reaction.

INTERACTION WITH THE INVESTIGATOR

The system is capable of analysing up to five successive reactions occurring in one mixing, a capa- bility useful in the analysis of multistep reactions.

SYSTEM DESCRII’TION

The system is designed to log signals from photo- multiplier output from a stopped-flow apparatus, and to calcutate the rate constant(s) of the reaction(s). The calculation of the rate constant(s) is based on the assumption that all the reactions follow an ex- ponential rise or fall (Fleck, 1971).

Throughout operation, the investigation has con- trol over input data and choice of option. The investigator does not need any programming knowl- edge to operate the system. Simple questions are presented at the display. Input options include the following:

l A reference field and date field to identify the reaction. This information appears in the output.

The components are based on the single board computer and the Multibus from Intel, thus provid- ing flexibility and compatibility for future expansion. In addition, these boards can be simply reconfigured to perform other tasks. The system consists of the iSBC SO/24 CPU board, the iSBX 331 math. unit board, the iSBX 3 I 1 analog to digital converter board, and the iSBC 660 cage with power supply. The system is also equipped with a graphics/alphanumeric dis- play with a keyboard and with a built-in thermal

l Choice of sampling interval and the number of data points to be logged. These choices determine the total logging time and affect the output resolution. The available time range is from 25 ms to 125 sec. The resolution is affected by the number of points per reaction. Since the screen of the graphic display allows a maximum of 512 horizontal dots, the maximum number of data points is restricted to 500. The selec- tion of sampling time interval may also be used to select a portion of a reaction. For example, if a fast reaction is followed by a slow reaction, selection of small sampling interval will focus on the fast reaction, while a long sampling interval will emphasize the slow reaction.

. l The investigator may start the logging at any TPhysics Department. time. The system will not log data unless there is a

310 T. S. HASSAN et a!.

* Amplitude

L-$7”‘,

v .

Time

(a)

t

Amplitude

(bl

Time

Fig. 1. Reactions with known infinity value. (a) rising; (b) falling.

change in the photomultiplier output. This eliminates the need for a trigger signal, making the system compatible with other stopped-flow systems. Provision is made to allow the investigator to observe any changes in the photomultiplier output.

l The system displays logged data on the graphic display and the investigator may accept the run for further analysis or reject it. If the logged data are accepted, the investigator is asked about type of analy- sis, whether the infinity level is known or not. Reac- tions of known infinity values (Fig. I) are those that reach a steady state levet which can be considered the infinity level of the assumed exponential. It will be shown in the next section that knowing the infinity level or not affects the algorithms of analysing the reactions to calculate the rate constant. For known infinity values the investigator is asked to determine the percentage of the reaction to be included in the analysis (range from 755, to 95%). Parts of the reac-

tion appearing not to fit as an exponentiat can then be easily excluded. For unknown infinity values, 95$, of the defined reaction is always taken.

l The investigator starts by segmenting the reac- tions appearing on the graphic display. Up to five consecutive reactions can be defined. Segmenting is done by moving the graphics curser to the start and end of the reaction and pressing a letter key on the keyboard. When the investigator has finished defining the reactions, the system calculates, for each reaction defined, the total reaction time, the rate constant (RC), standard deviation (SD) and correfation coeffi- cient (CC). Plots of all the defined reactions with the calculated values are then displayed. A hardcopy of the screen contents may be dumped on paper using the built-in printer of the terminal.

l At the end of the calculations, the investigator has several options. Segmentation may be redefined and results recomputed, the calculated values may be

Table 1. Effects of H + and Fe3+ upon apparent first order rate constant at 25°C carried but by the system

0.010 2.54f.014 2.52zt.015 2.51i.002

On-line processing of stopped-flow data

FE (Cl0 4_ /0_0075

t = 000.39smc RC = .02133E+02 cc z .0977sE+OI SD : .OBSSZE+OO

No of rmacrlans : 03 AvmraSm =.02157E +02 S.D_lOf av*rog*J =.it31a3E-o1

_-- Ip ____ ,__. -.--

,..’

,:.

:

FE (Cl0,,,/0.0075 -.- HCIO, /0.375

311

t e 002.10s*c RC = .02172 E +02 CC = .099S9E 4-O I SD = .06391E-01

No Of r*actlon E 03 Avarag4 = .02f80E + 02 S.D.[of q w,raga) =.17808E-Of

Fig. 2. Analysis of Iron(II1) thiocyanate reaction carried out by the system. (a) using the known infinity value; (b) without using the known infinity value.

stored for averaging, or a new reaction may be started.

ALGORHWMS

All the reactions analysed by the system are as- sumed to follow a first order exponential (Fleck, 1971).

Equations 1 and 2 are the standard equations for a first order reaction (Fig. 1). These are suitable for treating reactions in which the infinity value is avail- able. The absolute value of Y, - Yiti is to be used in

Eq. (2) for evaluating the slope k by standard least squares methods.

Yj = Y(inf) + Y(dif) exp (- kt,) (1)

In [Y, - Yii,,,,] = In [Y(dif)] - kt,. (2)

In reactions where Y(inf) is not reached, the. differential form of Eq. (1) can be used as shown in Eqs. (3) and (4). again with the absolute value of Zi.

312 T. S. HASSAN et al.

PD [S.OE- 5) METH (Z.OE-31 pH(l.251

t 1 000.05**c = 000.05 ‘.C

RC. .0716lE +03 = .05361E+OJ cc = .09960E+Ol = .09981 E +01 SD= .12696E+Ol = -07951 E + 01

Fig. 3. Analysis of the first and second rates of the Pd(I1) reaction.

z, = (dyjdt), = ~ ky(dif) exp (- kti) (3)

ln [- Z,] : In [ky(dif)] - kti. (4)

If the reaction signal is noisy, Eq. (4) cannot be used, but with suitable signal smoothing gives satis- factory results. We use a filter that approximates Eq. (5) (Tompkins & Webster, 1981).

u, = (l/35)(- 3Y,_, t 12y,-, + 17Y,

+ 12&+, - 3y,,,) (5)

The smoothed 7, values are used in the finite diFIerence equation, Eq. (6), to get approximations to the Z+ Filtering is applied after the investigator had defined the reactions.

Standard deviation and correlation coefficient are also calculated to judge the goodness of the fit.

RESULTS

To evaluate the system performance, the study of the Fe(II1) thiocyanate reaction {published by Mie- ling & Pardue, 1978) was repeated. Materials were: (1) Potassium thiocyanate, a stock solution of 2.0 x lo-* M prepared in 1.0 M of (NaClO,). (2) Fe(II1) perchlorate, a stock solution of 0.093 M pre- pared in 0.20 M (HCIO,) and standardized poten- tiometrically at pH = 2 with 0.01 M standard EDTA. (3) Perchloric acid, a stock solution of 3.88M pre- pared and standardized against 0.10 M of standard (NaOH). (4) Sodium perchlorate, a stock solution of 3.0 M prepared by diluting 3.88 M of perchlorate acid stock solution with water. The pH of the stock

(NaClO,) solution was adjusted to 7.0 with Na,CO,. The solution was heated to remove dissolved carbon dioxide.

Measurements were carried out using a Durrum stopped-flow apparatus linked to the system de- scribed. The optical path length was 20mm. The mixing syringes and cuvette were thermostatted at 25°C. The observed rate constants were calculated for 90% of the reaction. The ionic strength was kept constant at 1.0 M by adding the appropriate amount of 3 M NaGlO.,. The measurements were made at 450 nm. Calculations of the rate constant were made using the known infinity level method and the un- known infinity method. We used nine different con- centrations of Fe(ClO,), and HClO, selected from those reported by Mieling & Pardue, 1978. Results are shown in Table 1. Figures 2(a) and (b) show the analysis of the reaction using both methods. Each reaction was repeated three times and then averaged. The results show close agreement with the work published.

The reaction of palladium pd(II)] with methionine (Meth) was studied at constant ionic strength (I = 0. I5 M) and at constant temperature of (25°C). The kinetics were monitored at 320 nm in a pH range of 1.0 to 5.50 at various initial concentrations of the ligands and at a constant concentration of Pd(ll) of 5.0 x 10 ‘M.

Three different reaction rates were observed under pseudo first order conditions at various pH values. These steps are dependent on the total concentration Meth; as well as the pH of the medium. Figure 3 shows the analysis of the 1st and 2nd reactions. A 4th reaction much slower than the others (taking 2-3 min) and with a small amplitude also appeared. However. since it was low in amplitude, it was too noisy to be analysed by our methods.

The mechanism of reaction of Pd(I1) with Meth. will be discussed in a later article.

Pure methionine was used and stock solutions of the ligands were kept in a dark cool place. A stock solution of Pd(lI) was prepared in 1 .O M of HCl. The concentration of Pd(II) was checked by gravimetric methods as Pd(dimethylglyoxime)z (Vogel, 1966).

On-line processing of stopped-flow data 313

REFERENCES Mieling, G. E. & Pardue, H. L. (1978), And. Chem. 50, 1333.

Bonnel, I. R. & Befreese, J. D. (1980) Anal. Chem. 52, 139. Notz, P. Disc ~bsstr. ant. B. 38, I 171. Clark, D. D. & Schuster, S. M. (1980), Cornput Chem. 4, 51. Fisher, R. A. (1963) in Statistical Methods for Research

Taylor, D. G., Demas J. N. & Taylor, R. P. (1978). Biophys.

Workers, Edinburgh, Oliver & Boyd. J. 24. 77.

Fleck, G. M. (1971), in Chemical Reaction Mechanism, Tompkins W. J. & Webster, J. G. (1981), in Design of

New York, Hoit, Reinhart & Winston. Microcomputer-Based Medical Instrumentation, En-

Fomili, S. L. & Lapis, M. (1981), J. Whys. E. Sci. Inst. 14, glewood ClifTs, New Jersey, Prentice-Hall.

1394. Vogel A. 1. (1966) in Quantitative Inorganic Analysis. Holtzman, J. L., (1980). Anal. Chem. 52,989. London, Longmans.