A PC-based titrator for flow gradient titrations

This paper describes a PC (personal computer) based titrator which was developed for gradient flow titrations. Concentration gradients were generated electrolytically or volumetrically in small tubes. Complete titration curves can be recorded on-line and evaluated automatically. The titrator can be used with all liquid flow detectors with low axial dispersion. The titrator was evaluated for the titration of thiosulphate with electrogenerated triiodide and for the titration of ammonia with electrogenerated hypobromite after continuous gas dialytic separation of ammonia from the sample solution.


Introduction
Flow titrations combine the advantages of flow-through analysis (tbr example high reproducibility, fast mixing, short analysis times and reduced interference from contaminants) with those of titration (high accuracy, precision and reliability). Flow titrations working according to the principle of flow injection analysis have been widely reported [1][2][3][4]. There are advantages in this technique (for example high sampling rates and low reagent consumption), but the disadvantages should not be ignored--calibration with a series of standard solutions and non-linearly deformed titration curves which make evaluation difficult.
Programmed coulometric flow titration was developed by Nagy et al. in the late 1970s [5][6][7][8]; in this linear concentration gradients of the titration reagent are generated electrolytically. Two gradients form a triangle, which is continuously mixed with the sample solution in a dripping vessel. Miniaturization of the flow channel, replacement of the dripping vessel for a coiled tube, and optimization of the titration parameters enables nearly absolute working gradient titrations to be performed in flow channels [9][10]. Valcarcel el al. [12 and 13] have used flow rate gradients to generate concentration gradients. They combined a fixed flow rate pump with a programmed flow rate pump. Mixing linear flow rate gradients with constantly flowing solutions resulted in non-linear concentration gradients. Fuhrmann and Spohn [11] extended the 'triangle' coulometric titration concept to volumetric titrations, and thus extended the technique to more applications. They also proposed the volumetric double gradient titration technique [11] which had an extended determination range. The concentration gradients were generated with two computer-controlled micropumps; the resulting flow * Correspondence to Dr Spohn. rate in the titration tube reactor and the flow detector was held constant enabling nearly absolute working flow titrations to be performed.
The general manifold tbr triangle programmed gradient flow titrations is shown in figure working with two flow channels, one.channel contains the sample of concentration c flowing with the rate and a triangle mass flow profile of the reagent is propelled through the other channel. The mass flow profile can be described by: After mixing the determination reaction: aS + bR products.
(2) takes place in the coil M. The flow detector records the corresponding pair of mirror symmetric titration curves. The time difference between the two resulting equivalence points, leq., and leq.,2, depends on the sample concentration.
During coulometric titrations, the reagent mass flow profile is generated electrolytically in a miniaturized flow cell with a low degree of back mixing (see figure 10) by a triangle current programme according to: y /max* l/r 0 < < r I(t) During volumetric titrations, the streams of two computercontrolled pumps are mixed. One pump propels a reagent stock solution, at concentration c, with the flow rate programme: The other pump propels a diluent with the inverse flow rate programme: max(/-) <Z < with a resulting constant total flow rate (t) + )o(t)=

Vmax
The object of the work reported here was to develop a fully automated microflow titrator for both coulometric and volumetric titrations. developed following layouts which were designed using a software package called EAGLE [14].

Flexible pump control
The pumps used had to be precise and adjustable; the Dosimat 665 precision piston pumps (Metrohm, Herisau, Switzerland) and TEC-S rotating plunger pumps (Tecuria, Bonaduz, Switzerland) were chosen for the titrator. These pumps can also be used to propel nonaqueous and highly reactive solutions. The rotating plunger pumps have a very small total volume of around 20 lal of the inflow, the pump chamber and the outflow.
Both pump are driven by electric impulses. A well-defined volume V is propelled per impulse; and the desired flow, 1), is obtained by controlling th.e frequency, f, which can be defined as the ratio f V/V. Figure 3 shows the layout of the pump control card (only one of the six identical channels is included). A quartz clock generator delivers a t?equency of MHz and two programmable 16 To program the timer channels, and the PIO, the data and control busses are connected to those of the PC via the address decoder and the bus buffer. The PIO circuit works in the 0 mode with three independent input/output ports which are defined by the code word 82H. Ports A and C are the output ports and port B is the input port.
The timers work in mode 3 as square-wave generators, with binary counting, and enable the gate inputs to be used. The corresponding control word is 36H plus the channel number.
The litralor slot card Figure 4 shows the titrator slot card, which controls the electrolytic generation of the titration reagents and enables the polarization voltage of the amperometric detection to be held constant and preadjusted by an integrated potentiostat; it also controls magnetic valves. The indicator circuits for the amperometric detector are galvanically separated from the electrolysis current circuit by insulation amplifiers (ISO122) [15]. The corresponding electrode pairs can be placed in the same flow channel, the digital control signals for the range selection of the electrolysis current, the input selection and the magnetic valve switching are insulated by reed relays and optocouplers. DC/DC converters HPR 111 [16] were used to insulate the power supply. A 12-bit DAC AD7548 (MAXIM) is directly connected to the address decoder bus buffer for adjustment of the polarization voltage [ 17]. An output voltage in the range between -10V and + 10 V is generated to use the full range of the insulation amplifier ISO122 to establish the highest possible accuracy. After galvanic insulation the range of polarization voltage is reduced to the range from V to + V by an amplification stage of 0" 1. The output of that stage is connected to the indicator electrode. The corresponding reference electrode is connected to the input of a current-to-voltage converter, with an amplification factor 2 V/gA. The signal output is connected to input of the analogue multiplexer (HI509, Burr Brown), which can also switch between four analogue sources--this means that external flow detectors can be used.

Software
The control and evaluation software consists of an assembler program tbr the Z80 processor and a main program tbr the PC.

Assembler program
The assembler program controls the real-time functions of the titrator card and consists of two parts. The first part contains a small operating system and several drivers tbr the hardware components, which are connected to the ZS0 and to the PC for data exchange. This part of the software is stored in the EPROM.
The second part is the firmware, which is started automatically after loading from the PC into the RAM of the Z80 microcomputer. The received parameter block contains the duration and the maximum current of the triangle current profile, the time intervals and the start time of the detector signal recording. The slot card functions can be easily extended and modified by changing the firmware.
The most important tasks of the firmware, which is started from the main program, are: (    As demonstrated earlier [11], the precalculated lines can be used for calibration after adjustment of optimum parameter values. The analytical results, Cs, are calculated from the measured time differences between the two on-line evaluated equivalence points, teq, and teq, 2 (see table 2). The index max signs the parameter values at z. The maximum value of the mass flow hie, is related to the maximum electrolysis current and to the maximum flow rate ,max" Previously saved calibration curves can be loaded in many cases the important parameters, for example the pump rates and the electrolysis current, can be held constant over long time periods. Coulometric titrations and volumetric titrations with stable titration reagents have calibration lines with a high long-term stability.
After calibration, the analysis ofsample solutions is carried out. The user can preselect the number and the time period of the titrations. The results can be printed or stored in an ASCII-file after each titration. Figure 7 shows the calculation of At from the titration curves. First, a regression line for the base-line is calculated; then the signal height of the curve relative to the base-line is determined. The linear segments of both titration curves are automatically selected on the basis of user-defined upper and lower signal levels, with respect to the maximum signal heights. The data points in the resulting signal window are used to calculate regression lines for the ascending and descending parts of the titration curves.  Results and discussion Table 3 shows the flow titrator's working parameters. Sampling rate and sample consumption depend on the firation time 2z.
The electrolysis current source was tested with working resistors in the range between 0.1 and 47k Ohm. The relative deviation of the measured from the preadjusted current was not greater than 0"29/o in the range between 0"05-2 mA, and smaller than 0"5 in the range 0"05-5 gA.
The time interval between two changes of electrolysis current is adapted automatically to the titration time 2z and the maximum electrolysis current /max in the range fi'om 1"25 ms to 216.1"25 ms, so that the maximum number of current steps approximates a linear time thnction of the electrolysis current. 0"5 gA-2 mA in the three/max ranges between 0 and 2 mA, 100 gA and 5 gA, respectively, with a resolution of 12 bit coulometric titrations: 2z > 10 volumetric titrations: 2z > 50 0"05-0"8 ml/min for the TEC-S pumps 0-05-10 ml/min for the Dosimates 665 0"1-1"5 ml/min For the generation of volumetric concentration gradients, the time between two changes, of the flow rate is adapted to 2z. The shortest time step is 0"1 s. The generation of nonlinear concentration gradients can also be programmed.
The flow rates can be adjusted with a relative standard deviation of0"15 (N 5, z 0"05) for the piston pumps and 0"59/0 for the rotating plunger pumps. Since two 16-bit counters are used to generate the frequency f, the digital resolution does not limit the precision of the pumps.
The detector signal is measured 10 times/s. The precision of the equivalence point determination depends on the slopes of the ascending and descending parts of the titration curves. Standard deviations between 0"2 and 0"5 (N-5, 0"05)can be achieved.
The flow titrator can be used for a variety of volumetric and coulometric titrations: table 4 summarizes tested titration procedures.
Comparing the precalculated (11) and the measured (12) working lines" Cs 0"373 mmoll-5"36 gmoll-s-x. At (11) Cs 0"384 mmoll-5"56 lamoll-s-At (12) demonstrates that nearly absolute determinations are possible for thiosulphate concentrations between 0"01 mM and 0.25 mM. Sulphite and sulphide can also be titrated with electrogenerated triiodide in the same titration set-up and under the same conditions, The advantages of the described flow titrator were also demonstrated for the coulometric titration of ammonia, after gasdialytic separation from sample solutions with electrogenerated hypobromite according to: 2NH 3 + 3OBr-N 2 + 3Br-+ 3H20. (13) Figure 9 shows the set up, which consists of a gas dialysis cell, GD; a flow-through electrolysis cell, EC; a biamperometric flow detector, BFD; and a mixing and reaction coil, M. These are connected by Teflon tubes with an inner diameter of 0"5 mm; all solutions are propelled by miniaturized rotating plunger pumps: P1-P4.  Figure 11 shows the gas dialysis cell: this separation cell consists of two mirror symmetric KelF-plates with groove labyrinths (groove width 1"5 mm, depth 0'2 mm).
A microporous teflon membrane, which has a mean pore size of 0"2gm and is 201am thick (from Sartorius, G6ttingen, Germany), is mounted between the two plates. The membrane exchange area is 505 mm2.
To determine the sum of ammonia and ammonium, the sample solution is mixed with a 0"2 M NaOH solution to convert ammonium completely into the volatile ammonia. Between 0"1 mM and 2 mM ammonia, the calibration graph (14) is. almost identical to the preca!culated 2!5  (14) (where n 4, 0"05, r 2 0"9998) cS 3"45 mmoll-0"04 mmol-s-.At. (15) Separation efficiency can be calculated from the slope thctors to be (99'1 +_ 0"72). The ammonia determination is not disturbed by non-volatile interferences, which are oxidized by hypobromite, for example thiosulphate, sulphite, cyanide, urea and glutamine with concentrations not greater than 50 mM. Greater concentrations can influence the activity coefficients of am.monia and ammonium. The working parameters are V 0"05 ml rain-l, r 78"1 and Ima 2"5 mA.
The combination of the coulometric flow titration of ammonia with a complete analyte separation by gas dialysis and with enzyme reactors, which convert all of the analyte, enables nearly absolute determinations of glutamine and urea to be performed [10]. The titrator was used for simultaneous on-line determinations of ammonia and glutamine in animal cell culture media [-19-].
In addition, other flow detectors can be used to extend the application field fo the PC-based flow titrator.