Basilio Morelll University of Bari 70126 Bari, Italy
A Kinetic Experiment Using a Spring Powered, Stopped-Flow Apparatus
The most widely used technique for studying fast reactions is the "Stopped-Flow" method (1-2), whose essential components include two syringes containing reactants, which flow a t high velocity through a mixing chamber into an observation tube where the flow is abruptly stopped. The freshly mixed reactants follow then the course of their chemical nature and the reaction is observed by various methods (3-6). A stopped-flow apparatus is characterized by the so-called "dead-time," that is the time during which the instrument is unable to furnish correct results. This dead-time depends on several factors like the flow velocity (which depends on the chemical and physical characteristics of the-liquid and on the driving system) and the mixer configuration. In order to decrease the dead-time, more and more elahorate mixers have been designed: 2,4,8, or more jet mixers (4. . . 7). and also multi-mixers (8-10). As far as the driving. system is concerned, pneumatic, hydraulic and, more recently, electromagnetic (11-12) techniques were used. We think it should he very instructive to place a stopped-flow apparatus a t the disposal of undergraduates in a chemistry laboratory. In fact, especially if photometric techniques are used, the progress of the reaction in time can be visually followed by each student by means of an oscilloscope; moreover the transmittance data of an oscillographic picture can be manipulated in a simple way yielding interesting information relative to the reaction studied. These experiments could be performed during a normal
Figure 1. The driving system. Is)spring. ( b ) iron rod. ( c ) driving block. (0imn guides. (e) ring. (0pawl. (g)driving syringes. ( h ) release wheel. ( 8 mechanical brass bar. flow stop. (4 microswitch. (m)
laboratory time, so that the treatment of "chemical kinetics" could become a useful and exciting practical exercise. Unfortunately the high cost of the commercially available instruments usually prevents laboratories from using such equipment for teaching purposes. In this paper we suggest a very inexpensive and easy to build stopped-flow apparatus that could be conveniently used to study kinetics of very fast reactions, and profitably utilized by undergraduates or even by research laboratories with particular needs. Experimental Apparatus
Driving System. One of the principal features of the proposed apparatus is represented by the driving system that utilizes the expansion power of a previously compressed C,,VL,S -y....
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A sufficiently detailed diagram of the experimental driving system is illustrated in Figure 1 and clearly explains howthe anoaratusworks. A spring (a), mounted in a hrass containing cylinder, surrounds an iron rod ( b ) ,sealed to the brass driving block (c). The mechanical characteristics of the used spring are: length 120 mm; number of coils 20; pitch 4 mm; diameter of the wire 2 mm; diameter of a coil 16 mm. These dimensions were not alwavs strictlv but thev a " resnected. . . reoresent . good compromise between two needs, i.e., to realize a sufficientlv hiah flow velocitv and. a t the same time. not to have much-difficulty in the manual compression of the spring. However, if necessary, in order to decrease the dead-time as much as possible, a higher flow velocity can be obtained by using a considerably thicker and longer wire in the construction of the spring, without any problem for the driving system to get started. The driving block runs along two iron guides ( d ) by means of axial ball hearings (not shown in the fig.). By operating a traction on the ring ( e ) ,the driving hlock is raised and the spring is compressed; the driving hlock is then maintained in this position by a pawl ( f ) that fits in a groove of the iron rod. At this point a few ml of each reaeent are loaded into the drivine svrinees (g) (bv . . brindna tieir pistons into contact with thk driving block) and s;the system is ready to start. By rotating the small release wheel (h), the pawl is disconnected, the spring is released and the driving hlock rapidly pushes the syringes' pistons: the solutions are then injected through the mixer and the observation tube. The flow is abruptly stopped (after reaching full flow velocity) when the driving bldck impacts on the brass bar (i); a microswitch (0, mounted on the face of this mechanical stop, signals the flow stop to the detection device. An adjusting screw (not shown) is used for a fine regulation of the microswitch position, to make the flow stop as much as possible coincident with the oscillographic trace start. Another brass bar ( m ) permits adjustment of the initial spring compression before the run: The brass bar position along the iron guides can be easily adjusted by two screws and analogously for the stopping bar position. In this way it
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This work was supported by the "Centro di Studio sulla Chimica Analitiea Strumentale di Bari, C.N.R. (National Council of Re-
searches)." Volume 53,Number 2, February 1976 / 119
tomultiplier, whose signal is taken to the recording device. In our experiments available instrumentation was used, i.e. an old Zeiss Opton M4Q monochromator and photomultiplier; a voltage stabilizer for television (250 EFU Type, VA 200, Hz 50, CITE, made in Italy) to stabilize the power supply of a 45-W tungsten source; a Tektronix type 502 A-Dual Beam Oscilloscope, as recording device, with a polaroid camera to photograph the reaction traces. Reaction
Figure 2. Exploded view ol the mixer absewation tube unit. (a)mixer. ( b ) internal view of the mixing chamber. ( c ) observation tube. (d-0 O-rings. (e) quartz slides. (g) ferrule.
is possible to vary t o some extent both the speed of the liquid and the amount of solutions for each run (the latter is very important when one is dealing with expensive or difficult to synthesize reagents). The driving syringes are 5- or 10-ml glass commercial syringes (Inco Record, made in Italy). These are fitted into two Tygon or, more suitably, stainless-steel tuhings i.d. 2 mm (those used for gas chromatography act very well). A little 3-way metal stopcock on these tuhings, permits filling of the syringes from the reagents' reservoir^.^ Each tubing is sealed with two tuhings, i.d. 1 mm, afferent into mixing chamber. Mixinp Chamber and Observation Tube. In Figure 2 an exploded view of the mixer and observation tuhe assembly is presented. Both mixing chamber (a-b) and ohservation tube ( c ) are made of stainless-steel coated with a polymer (14) to make it inert to many reagents. Also other materials, easier to work with, as blackened Plexiglass or brass, could eventually he used with appropriate reagents. The mixer is a 4-iet . cvlindrical chamber. Verv-i m ~.o r t a n t in the mixer construction are (1) the mixing chamber dimensions. (2) the disnosition and the diameter of the inlet and outl~tholes,in order to obtain a good mixing and a t the same time to avoid cavitation. The best arrangement found, resulted in four tangential i.d. 1 mm opposite jets, a pair from one side of the mixer (for the reactant A) and a pair from the opposite side (for the reactant B). In this way the reagents impact frontally and strongly, improving the mixing efficiency; then the reaction mixture passes in the ohservation tube, through a l-mm i.d. hole. The cylindrical mixing chamber dimensions were i.d. 7 mm, height 2 mm. Mixer and observation tube are built in two separate parts sealed together by screws and an O-ring (d) to facilitate components disassembly and cleaning. This arrangement also permits substitution, without any difficulty, of the mixer here illustrated with the multiple mixer which has been described elsewhere (12): The latter can be used when it is necessary to obtain a very high mixing efficiency (very dense and viscous reagents). The observation tube has a square section with a 4-mm optical path to provide a good sensitivity for spectrophotometric recording. Two quartz slides (e) fixed in the ohservation area by an O-ring ( f ) and a threaded brass ferrule (g),permit measurements in the ultraviolet and visihle region of the spectrum. The ohservation area is about 5 mm above the reagent's inlet. Two flexible light-guides (not shown) are inserted in the ferrules to bring the monochromatic light beam from the monochromator-lamp assembly to the ohservation area and the light transmitted t o a pho-
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120 / Journal of ChemicalEducation
T o test the performances of the proposed apparatus the well known reaction between the dichloronhenol indonheno1 and the sodium ascorhate a t p H 7.5 proceeding with a reduction and disappearance of the blue color of the dichlorophenol indophenol, has been examined (1). In our experiments the concentrations used were: 8 X M for the sodium ascorhate, 5 X M for the dichlorophenol indophenol. Both the reactants were in 0.1 phosphate buffer. In these conditions the reaction follows a first-order kinetic with a law
where c(t) and c(o) are, respectively, the actual and initial concentrations of the dichlorophenol indophenol. Referring to the transmittance signal a t time t as h ( t ) and to the 100% transmittance signal as h(100), eqn. (1) hecomes
In Figure 3 is shown a typical oscillographic picture transmittance versus time recorded a t h = 620 A series of experiments a t different temperatures has Also 3-way solenoid valves (K273-ITT Standard) were tested, hut they sometimes gave rise to phenomena such as bubble formation, probably due to their large inlet hole with respect to the diameter of the tubings. The picture presents a little electrical noise due to the imperfect old detector conditions. This noise probably will he eliminated when a phototransistors' (BPX 25) detector, that we are building, is ready.
Figure 3. Typical oscillographic picture for the reduction of 5 X lo-< M d'+ chiorophenal indophenol by 8 X 1 0 P M sodium ascorbate: both lhe reactants were in 0.1 M phosphate buffer, pH = 7.5. T = 26'C. h = 620mp. Horizontal traces: highest 100% transmittance, lowest 0 % transmitlance. 5 0 mslcm time base.
Effect of the Temperature on the Dead-Time and on the Kinetic Constant of the Reduction of Dichlorophenol Indophenol bv Sodium Arcorbate Total
Temperawre
lDcl
First-order kinetic conrtantn
Is-') 1%)
dead-time
t o - t," fmrl
"Mean o f five valuer.
Figure 4. Dependence of the kinetic constant on the temperature in accordance wim Me Arrhenius equation.
been performed by placing flow system and reagents' reservoirs in an air thermostated chamber. The relevant values of the first-order kinetic constants are reported in the table. In the temperature range studied, the linear dependence of the kinetic constants on the temperature has been very well verified, in accordance with the Arrhenius equation 1
.
E
-+ const 2.303 RT
log k = --
(3)
(E = activation energy, T = Temperature (OK),R = gas constant), as can he seen from the plot of Figure 4. This linear dependence is a real demonstration of the efficiency of the proposed apparatus, as proved also by the good reproducibility of the observed kinetic constants: they are all within f3%. From the plot of Figure 4 it has been possible to obtain the activation energy for the process studied, i.e., E = -19385 callmole. Dead-nme of the Apparatus
T o calculate the dead-time we can consider the difference in ms between the time a t which the flow is stopped (recorded by means of the microswitch) and the time t~ corresponding t o the first experimental point exactly fitting the straight line drawn according to eqn. (2). Unfortunately a certain inaccuracy in the determination of the effective initial time is always possible, due to some mechanical inertia of the microswitch orland to imperfect alignment of this on the stopping bar. T o overcome this difficulty we considered that a variation of reagents' temperature will result in a change of the reaction velocity, but not of the initial absorbance value of the reaction mixture. So extrapolating a certain number of experimental straight lines representative of eqn. (2) and relative to different temperatures, their intersection point will correspond to the effective initial time to of the reaction. Only two experimental straight lines can he sufficient
Figure 5. Data treatment for transmittaneeltime curves: (a) 24%. (b) 26°C. (c) 27% ( b corresponds to Me curve of Fig. 3).
to obtain the real initial absorbance value of the reaction mixture (and so the time to) for a certain reaction; then this can be always used to evaluate the dead-time pertinent t o the same reaction, hut t o different temperature values. This method is illustrated in Figure 5: The treatment of the data for three curves similar t o that of Figure 3, a t 24'C (a), 26OC ( b ) , 27'C ( c ) , respectively, are reported (the straight line ( b ) corresponds to the picture of Figure 3). In this way we have calculated, for the reaction and the apparatus described, an average dead-time to - t l (see fig.) of about 4 ms; thus i t is possible to observe reaction halftimes smaller than 1ms (16). The complete results are reported in the table. A progressive decrease in dead-time can be noted as the temperature increases. I t was outside our purpose to overcome the difficulties connected with a complete explanation of this effect. Some hypotheses can nevertheless be made: (1) the reagents' viscosities decrease with the temperature, (2) the friction of syringes' pistons can vary with the temperature and similarly the flow velocity, (3) the slope of the transmittanceltime plot increases with the temperature. A sum of these opposite factors can probably explain the results of the table. The method, illustrated here to calculate the dead-time in the experimental conditions, seems to have some advantages with respect to other extrapolation methods, provided a temperature control can be achieved. In fact, this way i t is possible to eliminate the typical uncertainty of the transmittanceltime curves extrapolation, due to the nonVolume 53.Number 2,February 1978 / 121
linear dependence of the transmittance upon the concentration, and of the latter upon the time. Our method is sometimes preferable also to methods based on one straight line log ahsorbaneeltime extrapolation to the initial absorbance value (that is possible if the latter is known): actually the effective initial to time can be obtained by intersection of whatever number of straight lines, so the error probability is considerably limited. Conclusions
The apparatus we have described has shown a very good efficiency considering the low cost of the equipment (less than $100, excluding the photometric and electronic components, which are always available in a normal research laboratory). The spring powered driving system seems to have many advantages because it furnishes results comparable to those obtained by more complicated hydraulic or pneumatic instruments and i t is so simple to build that mechanical work, with a little experience, can he done, perhaps, by the students. Another advantaee with resoect to the hvdraulic and pneumatic systems is that successive runs at a distance of onlv a few seconds ran he ~erformed. This is verv"imoor. tant in routine analyses. When necessary the observed dead-time can be further lowered using different springs, as previously mentioned.
122 / Journal of Chemical Educatbn
It can he profitably utilized for an undergraduate laboratory studying a large number of reactions falling in the stopped-flow range, among which, particularly interesting for analytical purposes, are biochemical and trace catalyzed reactions. Acknowledgment
The author thanks Mr. V. Sacchetti for his invaluable help in the mechanical set up of the apparatus. Literature Cited (11 Chance, B.. Eismhardf, R. H.. Gibson, Q. H..and Lanberg-Holm. K. K.. (Editors). "Rapid Mixing and Sampling Techniques in Biochemisuv? Aesdemic Pr-. Ine.. New York~hndon.1964. (21 Caldin. E. F., "Fast keactions in Solution? Blackwell Sdcntiflc Publiestions, &ford (Englandl, 1964. I31 Balko,B..Berger,R.L.,snd Friauf, W . , A n o l Chem., 41.1506 (19691. (41 Sirs, J. A , Tram. Foradoy Soc, 54.201 (1958). (51 Prince. R. H.. Trans.Pornday Sor., 54.838 (19581. (6) Berger, R. L., BaLo, B., Barchordt, W., and Frisuf, W., Rou. Sci. InrLr., 39. 486
