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ANALYTICAL CHEMISTRY, VOL. 51,
NO. 12,
OCTOBER 1979
Automatic Sampling and Monitoring of Potentiometric Electrodes: Steady-State Response by First and Second Derivative Techniques Dave Skogberg" and Tom Richardson Department of Food Science, University of Wisconsin, Madison, Wisconsin 53706
Tony Blasczyk Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706
In the past few years, a great deal of attention has been given to ion selective membrane electrodes and biomembrane electrodes for quantitative analysis, as evident by the large number of review articles (1-8). Potentiometric electrodes of this type develop an electrical potential as a result of a difference in chemical potential inside and outside the electrode membrane. Development of this potential depends upon the electrode reaching a steady-state equilibrium with the solution to which it is exposed. One of the primary limitations of such electrodes is the long response time caused by rate-limiting diffusion across the membrane barriers (9). Response times of 2-10 min are not uncommon, particularly for immobilized enzyme electrodes where the enzyme layer acts as an additional barrier to the diffusion (10-11). It would be advantageous with such electrodes to use an automated slope analyzer to allow the continuous monitoring of the slope of the response as an indication of when the probe has reached steady state with the sample. Such a system used with a strip chart recorder allows monitoring of the entire response and recovery curves of the electrode. An automated slope analyzer for measuring the first and second time derivatives of the mV response of the probe enable the user to predetermine the maximum allowable slope criterion for steady state of the probe with the sample. This system also allows the user to predetermine a starting point for the response curves which does not require that the probe return to the response of the blank solution. This allows faster recovery times by not requiring that the probe recover fully to the zero point of the blank solution, but merely to a predetermined value which is less than the steady-state response of the lowest concentration solution to be analyzed. The primary advantages of this system is in saving time and in characterizing new probes when the operating parameters are not already known.
INSTRUMENTATION The block diagram in Figure 1shows the major components of the automated slope analyzer. Each component has been numbered and labeled for ease of reference in the following discussion. The input signal received from the specific ion meter is first fed into a low-pass active filter [l].The function of this stage is to filter out high-frequency noise, in particular, 60 Hz picked up by the probe and specific ion meter. The filter used is a three-pole Buttenvorth low-pass filter with a cut-off frequency of 3 Hz. No signal amplification occurs in this stage; however, placement of the filter before the dc amplifier [2] provides a high input impedance to prevent loading of the specific ion meter. The filtered input signal is next fed into a selectable-gain dc amplifier [2]. This amplifier scales the input to workable levels for the four input modes of operation. These four input modes are: (a) mV mode (exact dc voltage from the probe); (b) pIon mode (100 mV/pIon); (c) concentration mode (10 V to 1 mV for 4 decades); and (d) 10 mV/pIon mode. Modes 2 and 3 are needed for the particular specific ion meter used in this experiment. The amplifier [2] also inverts the input 0003-2700/79/0351-2054$01 .OO/O
signal in input modes 2 and 4 so that the signal to the differentiator [ 31 is always positive-going for increasing concentrations of sample. The amplified input signal is applied to the input of the differentiator [3]. This stage consists of two sample and hold stages and a differentiator-input to differential-output amplifier. The differentiator requires two timing signals, C$l and &, which are two repetitive pulse waveforms with pulse widths of 100 ms and a pulse spacing called the Sample Period or AT. Six different sample periods 8, 4, 2, 1,0.5, and 0.25 s are built in and are selectable by a rotary switch on the control panel. The operation of the differentiator can best be understood by examining Figure 2. At time 1 sample and hold A samples the probe signal and stores its value on the hold capacitor. Similarly, at time 3 sample and hold B samples the probe signal. The difference between time 1 and time 3 is the sampling period, AT. At time 4, the signal between the two outputs of the differential amplifier is stable and is proportional to the first derivative of the input signal. Discrete differentiation was chosen over continuous differentiation because it is less susceptible to noise and can be built with more readily available electronic components. The sample and hold circuits used are composed of a bilateral switch IC (CD4066A, RCA, Harrington, N.J.) and a 4.7-pF hold capacitor. Droop of the sampled signal was not a significant factor even with the long sampling period of 8 s. The outputs of the differentiator are input into an electronic reversing switch [4] controlled by the counter logic [8]. Its purpose is to change the sign of the first derivative such that it is always positive-going with respect to the zero-slope comparator inputs. This is required since the first derivative is positive for the response curve and negative for the recovery curve. The zero-slope comparator [5] receives the differentiator output and compares it with a user selectable voltage, AV, called the Slope Window. If the differentiator output voltage is less than the AV selected, the output of the comparator goes to logic state 1 (+5 V) and thus indicates that the zero-slope criterion has been satisfied. If the differentiator output is greater than the AV selected, the output of the comparator goes to logic state 0 (-5 V) and a nonzero slope is detected. A V is made adjustable from 0 to 10 mV by means of a ten-turn dial on the control panel. It must be remembered that both AV and AT (specifically the ratio AV to AT) are needed to set the zero-slope criterion. A flip-flop [6] stores the output state (logic 1or logic 0 ) of the zero-slope comparator when clocked by the pulse 43. Therefore, the status of the zero-slope condition is updated after each sampling interval. The timing logic [7] generates the necessary timing signals C$*, and C$3 for the system. This circuit is a four-phase pulse generator (four nonoverlapping, equal-time spaced outputs) with the second phase unused. Figure 2 illustrates the relative timing of the three outputs. The sampling period, AT, controls the time interval between C#J~ and &. The counter logic block [SI has the function of deciding whether or not the first derivative is indeed stable (a second 0 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 12, OCTOBER 1979
2055
Figure 1. Block diagram of electronics
2
3
;
2
3
L 7-
Figure 2. Timing signals 4 ,, 4 2, and 4 derivative test). The circuit counts sample intervals (2 x AT) t o a preset number (called Count), that is selectable by the Count control switch t o a value of 30,60, or 90. The counting commences upon receiving the zero-slope condition (a logic state 1 from the zero-slope flip-flop [6].When the desired total count (set by Count) is reached, indicating that probe steady state has been reached, the sample control flip-flop [ 101 is set and probe goes t o t h e wash solution. The total steady-state counting period equals 2 X AT x Count seconds. However, if a period of nonzero slope condition occurs for an interval of greater than or equal t o A T X 20 s, the zero-slope count is reset to zero. Therefore, the first derivative is being sampled a t this larger interval ( A T X 20) to ensure that it is indeed stable. The counter logic [a] also provides the reset signal to the sample control flip-flop [ 101 in the Slope mode when probe steady state has been achieved during the recovery portion of the curve. T h e output voltage of the dc amplifier [2] is applied to the negative input of the reset comparator 191. This stage compares the dc level of the signal input, be it probe voltage, pIon, or concentration, with a specific voltage set by the Reset Point control. When the output of the comparator goes t o logic state 1 ( + 5 V), the counting logic [8] is enabled and the
sample control flip-flop and relay [ l o , 111 are reset (Fixed Point mode of operation). The sample control flip-flop [lo] activates a relay [ l l ] to control the sample arm. The flip-flop is set to logic state 1 (indicating go to wash solution) upon receiving a signal from the counter logic that the desired steady-state condition has been maintained. The flip-flop is reset to logic 0 (indicating go to next sample) by the reset comparator [9] in Fixed Point mode, or by the counter logic [8] in Slope mode. In Slope mode, the reset signal is received when recovery steady state has been reached. The steady-state condition for recovery is the same as the steady-state condition for response. Other useful features built into the automated slope analyzer includes indicator lights (LEDs) to indicate the following conditions: (a) zero slope; (b) a signal level which is at or below the reset point; and (c) recovery period. A chart drive control to conserve paper is also included with three possible modes of operation: (a) Continuous (chart drive on continuously); (b) Response (chart drive on only during the response period); and (c) Zero Slope (chart drive on only during steady-state conditions). Complete details of operation and schematic diagrams are available from the authors upon request.
EXPERIMENTAL A L-lysine enzyme electrode (22) was used l o evaluate the automated slope analyzer. This probe has a response time of 6-8 min and a recovery time of 8-12 mln over thv range of concentrations used in this evaluation. The sensitivity of the analysis is determined by setting the AV and AT dials to the appropriate slope criterion for "zero" as shown in Table I. A setting of 1 mV/min at a 1-s sampling period was used with the L-lysine probe. The Count switch sets the number of counts needed to ensure the slope criterion will be maintained for a sufficient time to allow a satisfactory approximation of steady state (Count x 2 X AT = number of seconds). In the case of the L-lysine probe, a zero slope was maintained for 3 min (90 counts X 2 X 1 s = 180 s = 3 min) before allowing the autosampler to move the probe to the wash solution for rinsing. The reset point was set 5-8 mV below the steady-state value (in mV) of the sample of lowest concentration to be analyzed. Shown in Figure 3 are typical response curves obtained when the Chart Drive is in Continuous, Response, and Zero Slope modes. The spikes on the curve occur from movement of the probe
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 12, OCTOBER 1979
~ _ _ _ _ _ _ _ _ - _ _ _ ~ Table I. Slope Analyzer Control Settings for Four Input Modes"
a
input mode
zero-slope criterion re1 to input
mV
slope = 0.6-
pIon
slope = 0.01-
mV
max slope setting
ilV
mV
__
24-
min A T pIon A V
mm pIon
4 :0 .
~
min A T concn AV
concn
slope = 1.0--
10 mV/pIon
slope = 0.01-
40-
mm A T pIon a V min
mm concn
AT
min pIon
0.4-
mm
min slope setting (practical) mV
0.5-
mm
reset point range (re1 to input) 300 t o
t
300 mV
0.008e
10 decades
0.9-
concn = 100.0-0.01
mm concn
min pIon
0.0087 mm
10 decades
A T= 0.25, 0.5, 1, 2, 4, o r 8 . concn (relative to input voltage) = 10.0 x input voltage). Therefore, 10.0 x 1 0 V = 100.0, concn = 10.0 x 1 V = 10.0, etc. -
A V = 0.00-10.00.
concn
=
Figure 3. Response diagram of L-lysine electrode: continuous, response, and zero slope
through the air from sample to wash solution and back to the following sample. The Chart Drive selector allows a saving of paper when steady-state data are all that are required. The Zero Slope mode advances the paper only after the zero-slope criterion has been satisfied. The Response mode records any positive or zero slope, but does not advance the paper during the negative slope or recovery curve. Continuous mode advances the paper continuously and allows evaluation of the entire response and recovery of the probe, which is particularly useful for the evaluation of the kinetic parameters of a new electrode. The L-lysine electrode used to evaluate the slope analyzer consists of a carbon dioxide gas permeable membrane electrode with a layer of L-lysine decarboxylase physically entrapped at the surface of the electrode. When the electrode is placed in an aqueous solution of L-lysine the following reaction occurs: L-lysine
Llysme decarboxylase
cadaverine (l$-diaminopentane)
+ COP
The sample is dissolved in a 100 mM phosphate buffer at pH 6.0. However, during the course of the reaction, it is thought that the localized emf (electromotive force) of the enzyme solution at the tip of the electrode may be affected substantially by the formation of 1,5-diaminopentane as a product of the decarboxylation. Therefore, the slope of the L-lysine electrode deviates substantially from the 59.16 mV predicted by the Nernst equation. This electrode is discussed more fully in reference 12. Standard solutions for the L-lysine probe were prepared in 0.01 M pyridoxal 5M phosphate buffer, pH 6.0, containing phosphate. The wash solution for the electrode was the same buffer containing no L-lysine. Difficulties in maintaining the analytical concentrations of the standard solutions were encountered and attributed to bacterial growth. Inhibition of bacterial growth by mercury salts and
Figure 4. Standard curve for L-lysine electrode
pentachloroacetate resulted in inactivation of the enzyme. Sodium azide stopped the bacterial growth but interfered with the response of the probe. The buffer for the standard solutions was autoclaved in an attempt to reduce the initial bacterial load and the standard solutions were poured into analysis vials and kept frozen until just prior to their analysis. This resulted in more stable standards. Preparation of the standards 1-2 h before use also proved a satisfactory method of assuring accurate standards.
RESULTS AND DISCUSSION The standard curve of the 1,-lysine electrode is shown in Figure 4. An evaluation of the slope analyzer by using the L-lysine probe indicates that the precision of the analysis is a function of the precision of the probe. A precision ramp generator was used to evaluate the sensitivity and accuracy of the slope analyzer. This demonstrated that t h e slope analyzer can detect a minimum slope of 0.5 mV/min and resolve a set slope to better than f0.2 mV/min, which is equal to the maximum sensitivity (drift and noise) usually attributed to most potentiometric electrodes. One of the major advantages of the slope analyzer is its speed of analysis. Instantaneous feedback to the user is provided by the panel lights which indicate when a zero slope is detected, when the counting mechanism is reset, and when the probe is in the blank or wash solution. The zero slope indicator light allows the operator to detect when the zero slope criterion is satisfied. The reset in Fixed Point mode also allows the probe to move quickly from the wash solution back to the next sample by requiring only that the probe recover
ANALYTICAL CHEMISTRY, VOL. 51, NO. 12, OCTOBER 1979
to a voltage below that of the most dilute sample solution. This represents a substantial saving in the recovery time compared with requiring the probe output to recover to steady state with the blank solution after each sample. This reset function also permits the probe to begin a t the same output voltage for each sample and aids in achieving reproducibility of the analysis. Reproducibility is also aided by using the slope analyzer to detect the probe steady state, rather than manually evaluating the slope of the curve by measuring the tangent to the curve with a straight edge. This eliminates the operator bias inherent in such physical manipulations of data. Technician time is reduced by allowing the autosampler to manipulate the probe from the sample to blank to sample. Sample preparation represents the majority of the labor, as the data acquisition and probe handling are done by the recorder and sampler. Data evaluation is simpler because the data can be displayed either in its entirety, as the response curve only, or as a series of bar graphs showing only the data after the final slope is reached a t the probe steady state. The initial capital investment for equipment is low and savings result from reduced labor in this system. Savings also result from the use of an immobilized enzyme electrode, because the enzyme is used over and not discarded with the analyzed sample. Mechanical repairs and maintenance are minimal except from the upkeep on the recorder and autosampler, and routine preparation of the probe. A slope analyzer of this type could easily be modified for use in kinetic evaluation of reactions by using other types of
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equipment as the voltage source rather than the pIon meter. The first derivative can be taken directly from the slope analyzer with a few minor circuit additions. Use of the slope analyzer is an effective way of automonitoring and autosampling the output signal of potentiometric electrodes. This technique represents an efficient method of routine analysis using potentiometric devices with slow response. Savings in labor and analysis cost as well as a high degree of reproducibility make the automated slope analyzer a valuable tool to the analytical chemist.
ACKNOWLEDGMENT The author thanks Paul Rosen for his technical assistance and the Pillsbury Foundation for the financial support.
LITERATURE CITED Ferris, C. D.; "Introduction to Bioprobes"; Plenum: New York, 1974. Kessler, M.; Clark, L. C., Jr.; Lubbers, D. W.; Silvers, I. A,; Simon, W. "Electrodes in Biology and Medicine", 1st ed.; University Park Press: Baltimore, Md., 1976. De Clercq, H. farm. Tijdschr. Belg. 1973, 5 0 , 291. Gough, D. A.; Andrade, J. D. Science 1973, 180, 380. Guilbault. G. B. Biotechnol. Bioeng. Symp. 1972, 3 361 Richnitz, G. A. Chem. Eng. News 1975, 53, 29. Trauberman, L. Food Eng. 1975, 4 7 , 58. Guilbault, G. G. Bull. Soc. Cbim. Belg. 1975, 84, 679. Linek, V.; Benes, P. Biotechnoi. Bioeng. 1977, 19, 741. Blaedel, W. J.; Kissel, T. R.; Bogusiaski, R. C. Anal. (>hem. 1972, 4 4 , 2030. Bowers, L. D.; Carr, P. W. Anal. Chem. 1976, 4 8 , 545A. Skogberg, D.; Richardson, T. Cereal Chem. 1979, 5 6 , 147.
RECEIVED for review April 23, 1979. Accepted July 12, 1979.
Trace Metal Ion Filtration Losses at pH 5 and 7 Ralph E. Truitt and James H. Weber* Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824
Environmental water systems exemplify complex matrices with trace levels of metal species. Pre-analysis manipulations of trace metal ion samples threaten their integrity. Metal ion contaminations and sorption losses can occur from contact with materials encountered during dilution, filtration, storage, etc. Microfiltration (ca. 0.4-pm pore size) is particularly troublesome in metal ion speciation studies of lake water (I), wastewater ( 2 ) ,estuarine water ( 3 ) ,seawater ( 4 ) ,river water ( 5 ) ,and model natural waters (6),where the sample cannot be acidified prior to filtration. The authors of these studies did not establish that the filtration does not significantly alter the metal ion equilibria under study. Other groups acknowledged the problem but offered no solution ( 7 , 8). Some authors considered microfiltration metal contaminations and losses in more detail. Nurnberg et al. (9) reported 70% Cd2+and 8870 Pb2+losses from approximately 0.1 ng/mL metal ion solutions when unconditioned filter membranes are used. Losses of Cd'+ (10-12), Pb2+(13), and other elements (14, 15) are greater for glass than polyethylene or polypropylene, and increase a t higher pH. In addition, a study by Bene5 et al. demonstrated that the extent of sorption losses varies from ion to ion (16). Several groups recommend the use of acid washed vessels, nonsorbing materials, and the fewest possible sample manipulations to minimize losses and contaminations in trace metal ion analyses (17-19). In preliminary experiments we compared Cd2+filtration losses using a variety of filter media (20). We found that filtration losses from 125 ng/mL pH 8 Cd2+solutions were 29% with Whatman =2 paper filters, 35% with Millipore cellulose acetate filters, and 1670 with Nuclepore poly0003-2700/79/0351-2057$01 .OO/O
carbonate filters. Also, medium (10-15 pm) and fine (4-5.5 pm) porosity fritted 40-mm glass filters had 60% filtration losses with 99 ng/mL pH 8 Cd2+solutions. These preliminary tests demonstrated that metal ion filtration losses can be great, and they can vary considerably with different filter materials. The purpose of this note is to point out changes in metal ion concentrations that occur from filtering solutions with pH and metal ion levels similar to many natural water systems. In this study we compare Cu2+filtration losses with glass and polycarbonate filter supports, with two different 0.4-pm porosity filter membranes, and with pH 5 and 7 solutions.
EXPERIMENTAL Reagents and Materials. Analytical grade reagents were used as purchased. Deionized-distilled water was used for rinsing and dilution operations. Glassware and plastic vessels were soaked in 1 M "OB for 12 h, then rinsed and dried prior to use. Equipment. Filtrations were performed using a glass filter assembly or a polycarbonate-Tygon tubing assembly. The plastic assembly (22) consists of a Nuclepore Filter Funnel Assembly (Nuclepore Corp., Pleasanton, Calif.) and a 25-cm tall wide-mouth bottle that is capped with a two-hole 214 rubber stopper. The filter funnel passes through one hole of the stopper and empties into a 200-mL polypropylene collection bottle through a short length of Tygon tubing. The other stopper hole contains a glass tube through which the wide-mouth bottle is evacuated with an aspirator. The bottom of the rubber stopper was wrapped in Parafilm to prevent rubber debris from falling into the collection bottle. The glass assembly consists of a Millipore Pyrex Filter Holder (Millipore Filter Corp., Bedford, Mass.) emptying into a 125-mL Pyrex filter flask. Metal ion determinations were made by DPASV/HMDE using 0 1979 American Chemical Society
