A Rapid Sampler for Intermediate Speed Reactions H a r r y C. Froede, John Cowan, Ted W. Reid, and Irwin B. Wilson Department of Chemistry, University of Colorado, Boulder, Colo. 80302
A rapid sampler for intermediate speed reactions is described. This sampler delivers 1 to 13 aiiquots of reaction solution to a quenching solution at preset time intervals of from 0.5 to 50 seconds. The sarnR1.e size is 0.100 ml but can be changed by changing nyringes. The apparatus was tested by studying the hydroxide ion catalyzed hydrolysis of p-nitrophenyl acetate under conditions where the half time was about 5 sec. The second order rate constant was 10.1 + 0.11 mole-’ sec-I compared to a value of 10.4 f 0.1 obtained using a spectrophotometer at lower NaQH concentrations where the reaction w p one tenth as fast. The value in the presence of 1M NaCl was 8.3 += 0.1 and 8.6 i 0.1, respectively. THEUSE OF RAPID MIXING and sampling techniques for kinetic investigations has been extensively studied and is discussed in the literature ( I ) . These techniques allow one t o obtain samples at intervals from 5 to 500 milliseconds. The shortest interval possible for sampling reaction mixtures accurately by hand is about 15 seconds. Therefore, it is possible t o sample solutions for reactions that are very fast or fairly slow. Techniques are needed for sampling at “intermediate” time intervals of0.5 to 15 seconds. Thd modification of existing rapid sampling apparatus (I) for use in the “intermediate” range would require too large a sample. Some techniques for sampling in the “intermediate” time range include the following: aspirator sampling (2), single drop sampling (3), pulse sampling (4), and mechanical pipetting (5). The purpose of this paper is to describe a new device for rapid mixing and “intermediate” time sampling. With this sampling apparatus, time intervals of 0.5 second to 50 secoty+ can be selected and the number of samples can be preset from 1 to 99. Solutions can be changed easily, mixing and quenching are rapid; the sample size can be changed, many points can be obtained from a single run, and sample size is reproducible t o 1%. The apparatus (Figure 1)consists of a hydraulic piston that at zero time rapidly drives two reactant solutions through a AxT ing chamber and into a long wire which serves as the reaction vessel. After the wire is filled, a preset number of aliquots of a driving solution of the same composition as the rgactian solution are forced into the wire at preselected time intervals. Jn this way aliquots of reaction solution are rapidly fQrced out of the wire and into the quenching solution. EXPERIMENTAL
The basic design for the apparatus is shown in Figure 1. The apparatus includes two reservoir syringes (D)which are standard syringes of any suitable size (20 ml) and they are used for both flushing out the system and fqr filling the two (1) “Rapid Mixing and Sampling Techniques in Biochemistry,” B. Chance, R. Eisenhardt, Q. Gibson, and K. K. Lonberg-Holm, Ed., Academic Press, New York, N. Y . , 1964. (2) K. K. Lonberg-Holm, Biochim. Biophys. Acra, 35, 464 (1959). (3) R. H. Eisenhardt and 0. Rosenthal, Science, 143, 476 (1964). (4) J. K. Mietinen, Int. Conf. Radioisotopes in Sci. Res., UNESCO/ NS/RIC/NO 146, Paris, 1957. ( 5 ) K. Chmutev, M. Klientovskaya, and V. Avgul, Zh. Fiz. Khim., 39, 1276 (1965). 1204
reactant syringes (G). To avoid breaking the glass tips on standard syringes, we used luer-lock syringes with the lock removed, as reservoirs. The two reactant syringes (G) are 2.5-ml Hamilton gas tight syringes. The filling of the reactant syringes, the flushing of the system, and the discharge of reactant solutions through the mixing chamber (B) are controlled by three-way Hamilton valves (C). The two valves (one right-handed and one left-handed) are fitted with one male and two female luer connectors each. After flushing the mixing chamber ( B ) and reaction vessel ( A ) , with one of the solutions, the two reactant syringes are filled from the reservoir syringes by drawing back the plungers until they are stopped by the tip of the hydraulic piston (F). The reaction is started by driving the hydraulic ram (Armstrong, Beverly, England) forward by means of a hand-driven lever (not shown). The hydrolic ram is set to force approximately 1.5 ml of solution from each syringe through the mixing chamber (B). This volume serves t o initially flush out the reaction vessel and mixing chamber (volume = 1.51 ml) and refill it with fresh reactants. A cut-away of the mixing chamber is shown in Figure 2. It is made from a piece of lucite into which are drilled three holes meeting in the center of the block a t right angles as shown in the drawiqg. Stainless steel tubes are inserted into the holes and held in place by means of screw fittings with O-ring seals. The reaction vessel is a length of stainless steel tubing (61 cm X 1.80 mm i.d.) fitted with a stainless steel reducing plug at the exit which forces the reactant solution to pass through a small orifice prior to quenching. This allows for better mixing of the reactant solution with the quenching solution. Shorter reaction vessels are easier to flush Qut but then fewer aliquots can be drawn. At the moment the hydrolytic ram stops, the timing system (see Figure 3) which is connected to the sampling apparatus (Figure 1) by means of plug ( J ) is activated by hand. At regular preselected time intervals the timing system activates a Ledex rotary solenoid (S-8215.021) (I)to which is attached a hammer ( H ) that strikes the Hamilton double syringe repeating dispenser (E). The repeating dispenser forces of the volume of the two reactant syringes into the reaction vessel and concomitantly forces 0.100 i: 0.48 ml or about 1/15 of the volume of the reaction vessel into the quenching solution. The tip of the reaction vessel is inserted below the surface of the quenching solution. Quenching vessels are changed by hand between each timed pulse. This can be easily done at 1-sec intervals if the tubes are lined up in a rqck. Intervals of 0.5 sec Can be used but in this case there is not sufficient time to easily position the tip of the reaction vessel beneath the surface of the quenching solution. Instead, we have positioned the exit port of the reaction vessel in the air allowing about 2 inches of empty test tube above the surface of the quenching solution, t o accommodate the liquid surge that attends the quenching. The hydroxide ion catalyzed hydrolysis of p-nitrophenyl acetate was used to test the sampler. The rapidity of this reaction can be altered by changing the concentration of NaOH. Thus, the results of a relatively slow reaction run in a continuously recording spectrophotometer can be compared with the faster reaction run with the sampler. Experiment with Sampler, The p-nitrophenyl acetate solution was prepared by dissolving the solid in 1 ml of acetonitrile and diluting to 100 ml with water (2M in NaCl for some experiments). The reaction was started by mixing
ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970
MIXING CHAMBER D I A . = 2.41 mm
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Figure 1. The rapid sampler
Figure 2. Mixing chamber; front view
0.62mMp-nitrophenyl acetate with an equal volume of 0.025M NaOH and was quenched in 3 ml of 0.1M acetate buffer (pH 5.5). For the reaction in the presence of 1M NaCl, 0.010M NaOH was used with 1.24mM p-nitrophenyl acetate. The absorbance of the solution at 400 nm was obtained by adding 0.5 mi of 2.OM Tris buffer (pH 8.0) just prior to obtaining the reading. Spectrophotometer Experiment. The p-nitrophenyl acetate solution (O.lmM) was 0.5% in acetonitrile and 1.OM in NaCl for some experiments. The reaction was started by adding 50 pl of 0.1M NaOH to 3.0 ml of the p-nitrophenyl acetate solution and was monitored by following the absorbance of 400 nm with a recorder. Timing System. The electronics used to drive the rotary solenoid are shown in block diagram form in Figure 3. From the 60-Hz line, we derive a 2-Hz positive rectangular pulse signal using frequency division circuits. This sequence of pulses is passed through the gate when the gate control signal is “low.” We now have a sequence of narrow pulses at the input of the triggered pulse generator. The triggered pulse generator is a variable frequency divider such that the 1, 1 I/z, 2l/2 . . . .49 seconds output pulses have a period of according to the selection of the operator. This circuit also expands the pulse width to a value that is appropriate for the solenoid. This pulse is used to control a power amplifier which drives the solenoid load. The output of the power amplifier is a sequence of pulses whose leading edges are spaced in multiples of precise ‘/z second. Each pulse powers the solenoid and sharply advances the syringe plunger. A preset counter circuit counts the number of pulses sent to the power amplifier and sends a signal to turn off the gate when the preset number have occurred. All the circuits are then reset and waiting for a new start signal. Example: Consider that we want 3 pulses, each 2 seconds apart. We set the dials labeled “time between pulses in l/? seconds” to “04” and then we set the dials labeled “number
of pulses” to “03.” We push the start button. This sets up the start circuits such that the next trailing edge of the 2-Hz clock pulse actually turns on the gate (synchronized starting). The 2-Hz pulses now begin to go to the triggered pulse generator which is set to “04,” therefore when each fourth pulse comes in, a new wide output pulse is generated and sent along to the power amplifier and the pulse counter. Now when the third wide pulse (12 narrow pulses through the gate) is counted, a signal is sent to close the gate, and reset the circuits. Circuit Details. This instrument uses six modules called Decade Counting Units (DCU) which are modified versions of a circuit described in “Electronic Experimenter’s Handbook,” Winter, 1969 (see Figure 4). Southwest Technical Products Corporation of San Antonio, Texas, offers the complete kit of parts including the printed circuit board. Our modifications include the addition of a 3 3 0 4 base resistor for Q4, Qs, and Q6, and the addition of a connector with at least 11 contacts. Then we brought out to the connector the input, outputs, reset, and power supply leads. A description of the operation of these circuits may be found in the article noted above. Here we note that the output frequency is one tenth the input frequency, and that the condition of the six outputs indicates a count of the input pulses. Each of the DCU’s is constructed such that they are mechanically and electronically interchangeable. The instrument also uses four decoding circuits that are mechanically and electronically interchangeable (Figure 5). This circuit uses logic circuit elements to convert the information at the outputs of the DCU from its “Bi-quinary” form to a “ten line decimal” form. The logic circuits are commercially available resistor-transistor-logic integrated circuits which are made by such firms as Fairchild or Motorola. Here, we used three types of Fairchild devices:
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ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970
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More complete information may be secured from the manufacturer. Now let us consider the parts of the block diagram starting with the 2-Hz clock. (See Figures 6 and 7.) This includes logic blocks (LB) 1, 2, and 3, and two DCU's. LB1 is connected to form a Schmitt trigger which takes a part of the steep sides are necessary for proper operation of the DCU. This 60-Hz square wave is divided down to 6 Hz by DCU No. 1. To get 2 Hz we need to divide by 3, but our DCU only divides by 10, therefore, LB?, LB,, LB4 are used to sense three input pulses and reset DCU No. 2. Result: DCU No. 2 starts from zero, counts two input pulses, and on the third is reset back to zero. The reset pulse has a frequency of precisely one-third the input or 2 Hz. The next block just consists of LB5, one half of which is connected as a n inverter, and the second half is used as a gate. The signal at pin 5 (see Figure 6) is a series of 2-Hz negative going pulses from about 1 volt to about 0 volts.
At pin 3 of the gate, the signal is “High” which means output (pin 6) is “Low” or gate is closed. T o open the gate, the signal at pin 3 is dropped to “Low.” Then we see at pin 6 a sequence of 2-Hz pulses going positive from 0 to 1 volt which is the inverse of the input at pin 5. The next block in our diagram consists of the “stop,” “start,” and “reset” switches, LBs, LB7, and LB8. LB7 is connected as a setlreset flip flop with pin 7 “High” in the reset or stop condition. The start switch discharges a charged capacitor into the set input (pin 1) which lowers the signal out at pin 7 down to “Low.” The stop switch discharges a charged capacitor into pin 5 of LB7, and also into pin 6 of the LBs and sets it for the stop condition. The stop condition for LBBis pin 5 = pin 3 = pin 1 = “High.” Reference to the pL923 specification will show that the condition, pins 1 and 3 = “High” results in no change of outputs with a clock input. The output (pin 5) will go to “Low,” however, if pin 1 = “High,” pin 3 = “Low,” and a clock pulse is applied to pin 2. The change occurs at the trailing edge of the clock pulse. This circuit opens the gate synchronized with the clock signal even with random operation of the start button. The diode D1in the stop circuit keeps the stop signal from resetting the other circuits. Logic Block No. 8 along with a reset switch is used to reset the unit manually. Each of the manually controlled switches, Stop, Start, and Reset, may be remotely located. The start switch in particular might be activated by some other part of the system. The next block (triggered pulse generator) consists of DCU3, DCU4, Decoder,, Decoder4, LBg, LBll, and LBn. DCU3 and DCU4 make up a 2-decade counter, each with a decoder unit to give us ten lines for the ten position selector switches. The diagram (Figure 7) shows the switches set for 73. This means that on the 73rd input pulse both of the switch common circuits will be “High” which is inverted by the two halves of LBlz giving both inputs to the first half of LBl1 a “Low” signal. This results in pin 7 of LBll = High. With the switches set as they are, this “High” at pin 7 can only occur on the 73rd pulse in from the “gate” section. This signal is sent to the pulse count section and to a “oneshot” multivibrator consisting of LBg and the second half of LBll. The Resistor and Capacitor values are chosen to change the pulse width to a value required for good operation IO K
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Figure 7. One half of the wiring diagram for the timing system (See Figure 6 also) The power amplifier (Figure 8) is built around a pair of 2N1016B transistors connected in parallel as an emitter follower. These are driven by the voltage amplifier consisting of the 2N699 and the 2N1041. By varying the value of R1 from 2 M to 6808, this amplifier may be used with power supply voltages from 25 volts to 65 volts. The last block diagram is for the power supplies, which at 3 A., 6.0 volt =klxat 0.5 A, and are f3.6 volt =tO.lz about 50 volt at 6 A. Power supply modules from such firms as Technipower, Acopian, or Lambda are recommended for the two low voltage cases. For the 50-volt case a variable
tap primary type transformer such as Stancor No. RT-408 feeding a full wave bridge rectifier and filter capacitor may be used. The primary taps are chosen for about 35 volts ac out to the rectifiers. RESULTS AND DISCUSSION
Characteristics of the Equipment. If the driving solution were to act as a piston, no driving solution would emerge from the reaction vessel until all the reaction solution had been discharged. With the length of wire described, this
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ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970
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Figure 9. A pseudo first order plot of the data obtained with the rapid sampler using 2-sec, 0,and 2.5-sec, 0 , sampling intervals, for the reaction between 0.005M NaOH and p nitrophenyl acetate in 1 M NaCl at room temperature, 25OC. The last 3 points 0 and U correspond to pulses 14,15, and 16 and are expected to be in error as discussed in the text
would occur on the sixteenth pulse. Actually we have to expect that the driving solution will mix with the reaction solution ahead of it and will emerge from the reaction vessel at an earlier time. In order to see how much mixing occurred, the following experiment was carried out. One reactant syringe and its wire leading to the mixing chamber were filled with a concentrated solution of p-nitrophenol in 0.1M PO4 buffer, pH 8.0. The other reactant syringe and the reaction vessel were filled with 0.1M POc buffer, pH 8.0. Thus the driving solution was “colored.” The timing mechanism was then activated, the solution driven from the wire was collected, and the absorbance at 400 mp of each ejected portion of solution was measured after dilution to a known volume. This experiment showed that no driving solution appeared in the reaction solution until the twelth pulse which contained 2 %. This means that for the 1.51-ml reaction vessel used, the first 1.10 ml is free of driving solution. The thirteenth aliquot contained 10% of driving solution, the fourteenth 25 %, the fifteenth 45 %, and the sixteenth 60%. I n the case of a kinetic experiment, the effective error is very much smaller than the per cent of driving solution in the aliquot because the driving solution has almost the same composition as the reaction solution. The driving solution has the same initial composition as the reaction solution but has a slightly different composition at the time they both emerge from the reaction vessel because it has reacted for a somewhat lesser time. For example, aliquot fourteen has 25% driving solution, almost all of which has come from the first driving pulse; it is therefore one pulse or 7% too young. The net error for aliquot fourteen is therefore approximately 2 Z on the time scale. Therefore it should be quite safe to use twelve or thirteen pulses, and this is borne out in the actual experimental results (Figures 9 and 10). The extent of replacing the “old” solution in the reaction vessel with fresh solution forced in at zero time by the hydraulic ram was checked in the following way: the reaction vessel was washed and filled with 0 . 1 M P 0 4 buffer pH 8.0 from one of the reactant syringes. Then both reactant syringes were filled to their maximum capacity, one with the concentrated p-nitrophenyl solution described above, the other with
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Table I. Rate Constants for the NaOH-Catalyzed Hydrolysis of p-Nitrophenyl Acetate NaCl Spectrophotometric Sampler 0 10.4 & 0.1 10.1 rk 0 . 1 1.OM 8 . 6 f. 0.1 8 . 3 & 0.1 Units are liters mole-’ sec-l. Data are the average values for four runs.
the buffer. The ram was forced forward rapidly. The repeating dispenser was activated by the manual switch on the timing device and the absorbance of the discharged aliquot was compared to the absorbance of a 50:50 mixture of the two solutions after proper dilutions. We found that with the 61cm wire, 4% of the old solution (Le., buffer) remained and was discharged in the first aliquot. With a shorter wire, none remains. The first aliquot is discarded because the time and size is uncertain. The size of the first aliquot is uncertain because the hydraulic ram stops at an undetermined position which will generally position the rack so that less than a full aliquot will be delivered on the first pulse. Neglect of the first aliquot makes no difference for a first order reaction. The apparatus currently is being modified so that the timer is activated by a microswitch that is closed by the ram when it comes to a stop. If a longer time for quenching is no problem, a collecting system such as that designed by LonbergHolm can be used ( I , 2). In order to test the performance of the sampling apparatus, a reaction was chosen which could also be followed more slowly. The hydroxide ion catalyzed hydrolysis of p-nitrophenyl acetate was measured using 0.0125M NaOH and 0.005M NaOH for the fast reaction and 0.0016M NaOH for the slower reaction. The experimental data obtained with the sampler can be seen in Figures 9 and 10. These data show both the reproducibility obtained and the use of different sampling times. The slower reaction was followed spectrophotometrically with a recorder. In Table I, the results from the two different techniques are compared, and it can be seen that good agreement is obtained for the rate constant using the different methods.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970
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The sampler can, of course, be used for following reactions that do not produce a suitable change in absorption provided the composition does not change rapidly in the quenching solution, Samples are taken from the quenching solution and analyzed by any suitable method (i.e., radioactive counting). The composition of the quenching solution that is selected will depend upon the reaction as will the time that the products can be left in the quenching solution before analysis.
ACKNOWLEDGMENT
The authors thank Robert Goukas of the chemistry department shop for building the mechanical apparatus. RECEIVED for review May 5, 1970. Accepted July 9, 1970. This work was supported by Grant NB 07156, National Institutes of Health and Grant GB 7904, National Science Foundation.
Determination of Submicrogram Amounts of Chromiurn by Coulometric Titrimetry C. E. Champion,l G. Marinenko, and J. K. Taylor Institute for Materials Research, National Bureau of Standards, Washington, D. C. 20234
W. E. Schmidt Chemistry Department, George Washington University, Washington, D. C. 20006 An improved method has been developed for the determination of micro- and nanogram amounts of chromium by coulometric titrimetry. Samples containing 30 bg to 15 ng of chromium may be analyzed with an uncertainty limit of 1 ng. The method is biasfree within the uncertainty of the measurement due to random errors, and the end-point determination is the limiting factor for further improvements of the method.
THECOULOMETRIC DETERMINATION of microgram amounts of chromium has attracted the attention of several investigators. Cooke and Furman (1) developed a sensitive potentiometric indicator method for locating the end point and used it in a coulometric titration of 100 pg of chromium with a standard deviation of about 1%. Meites (2) reported a similar constant-current method with a more sensitive indicator system and determined 0.1 fig of chromium with a standard deviation of about 9%. Meites (3) also determined chromium by controlled potential coulometry, involving the quantitative electroreduction of chromium(II1) to chromium(I1) and integration of charge required to oxidize it back to chromium(II1). The standard deviation obtained with 5 pg of chromium was on the order of 1%. Several investigators (4-6) have reported precise coulometric methods for the determination of macro amounts of chromium with relative standard deviations on the order of 0.003 %. The large difference in precision between the macro and micro methods encouraged the present investigation of the effects of the various titration parameters on the coulometric determination of submicrogram amounts of chromium and the consequent development of a sensitive micromethod. The successful development at this level resulted from the 1 Present address, Methods Research and Technical Service Division, Eastrnan Kodak, Rochester, N. Y.,14603.
(1) W. D. Cooke and N. H. Furman, ANAL.CHEM., 22,896 (1950). (2) L. Meites, ibid., 24, 1057 (1952). (3) L. Meites, Anal. Chim. Acta, 18, 364 (1958). (4) G.Marinenko and J. K. Taylor, J. Res. Nat. Bur. Stand., Sect. A , 67 ( 5 ) , 453 (1963). ( 5 ) R. G. Monk and G. C . Goode, Talanta, 10,51 (1962). (6) J. Knoeck and H. Diehl, ibid., 16, 181 (1969).
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construction of a small titration cell and an improvement in the amperometric sensing system. EXPERIMENTAL
Current-Time. A commercial coulometric analyzer which is equipped with a synchronous timing motor and digital impulse counter was used as the power source. The output, reading directly in microequivalents, was calibrated by measurement of the potential drop across a standard resistor calibrated at NBS and by comparison with a quartz crystal controlled time interval meter. Other experiments in this laboratory have confirmed a long-term stability performance of this apparatus to at least 1 part in 10,000. Coulometric Cell. The cell development to increase the sensitivity of the system is shown in Figure 1. The anode, cathode, and reference electrode compartments, each with a maximum capacity of 10 cm*, are separated horizontally by intermediate compartments to facilitate filling or emptying by suction or pressure. The cathode compartment has two 1-cm* platinum electrodes [4 and 51 sealed in opposite sides to serve as generator cathode and indicator electrode, respectively. A cylindrical Teflon (Du Pont) encapsulated magnetic stirrer [lo] further reduces the volume in the cell, agitates the solution, and provides a stable diffusion layer at the indicator electrode. A nitrogen inlet-outlet tube [2] and a tube for sample delivery [3] are inserted in the Teflon cover of the compartment. A 2.5-cm2 syringe may be attached to the latter tube which may be raised and lowered to alternately fill and eject catholyte to rinse the walls of the compartment. The platinum anode [8] is confined in a tube in the anode compartment and further isolated from it by a porous Vycor rod [7]. The reference compartment has a similar isolation tube [6] in which the saturated calomel electrode is inserted. The cell design provides an increase in the sensitivity of the detection system by virtue of the small size of the cathode compartment. The compartmentation minimizes diffusional losses of catholyte, effectively isolates it from interfering species such as anode reaction products and the chloride ion of the SCE, and isolates the indicator system to permit measurement of indicator currents concurrently with generation. The cell was mounted above a laboratory model magnetic stirrer, No special precautions were taken to ensure syn-
ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970
