1540
Anal. Chem. 1988, 6 0 , 1540-1545
Analytical Potential of Flow-Reversal Injection Analysis Angel Rios, M. D. Luque d e Castro, a n d Miguel Valcircel* Department of Analytical Chemistry, Faculty of Sciences, University of CQdoba, CBrdoba, Spain
The repetltive reversal of the flow dlrectlon In unsegmented flow systems can be accomplished by suitable programmlng of the electronic control of a perlstaltlc pump. At sufflclently short reversal cycle tlmes, a preselected zone of the flow injection analysis peak can be sampled and Its evolution monltored. This multldetectlon mode with a single detector allows one to manipulate the dlsperslon of the process over a wide range and to accommodate any signal wtthln the dynamic detection range. The behavior of a dye (dilution process) and processes involving chemlcai reactlons monltored through the dlsappearance of a reagent or the formation of a product are studied.
The information provided by a single FIA (flow injection analysis) peak is insufficient for some purposes. So much so that several procedures have been designed and different detection techniques have been used to increase the information obtained from one sample injection (multidetection). The simplest, though also the most expensive way to perform multidetection in FIA, is the use of several detectors of the same ( 1 ) or different (2) nature, located in series (3) or in parallel ( 4 ) ,or the use of image detectors (5). The cheapest alternative is the manipulation of the FIA configuration for multidetection purposes, which can be accomplished in two ways: Design the configuration so as to obtain a dual peak. This is of special use in simultaneous determinations based on the injection of an anomalously large sample volume (6), the split of the sample plug and the subsequent confluence of the split streams prior to the detector (7),and the use of two injection valves located in series (8) or in parallel (9) or coupled internally (10). Use configurations that allow several peaks per injection. This alternative, the one providing the more information, has two variants that can be used with continuous analyzers ( l l ) , namely open and closed systems. The reversal of the flow direction in continuous automatic analyzers was first reported with the air-segmented mode proposed by Technicon. In the last two years this approach has been used in unsegmented continuous flow analysis (11-14). This paper is a part of our ongoing research in multidetection with a single detector (15-18), though applied to open systems. The iterative passage of the sample plug through the detector in an open flow system is carried out by repeated reversals of the flow under the control of an electronic system acting on the pump. The reversal of the flow is carried out not by allowing the entire plug to pass through the detector but by “sampling” a preselected zone of the plug, so that the inversion of the cycles (up to 22 in number) all take place within one FIA peak. In a way, this mode can be considered a peculiar variant of the sampling zone mode proposed by Krug et al. (19) and applied here to obtain a multipeak recording that defines kinetic profiles easy to manipulate through the flow rate, sampled zone, and cycle time. Flow-reversal FIA has much in common with stopped-flow FIA. Instrumentally, both require synchronization of injection and the pump motion (the latter must be programmed).
Analytically, the information offered by both techniques (kinetic in nature) is quite similar. Nevertheless, there are major differences between both; namely, the pump is never stopped during a flow reversal experiment (only its direction is changed), so that the signal is continuously affected by dispersion as well as by the chemical kinetics (in stopped-flow, however, the dispersion is virtually unaffected during the pump stop). As a result, the signals obtained are rather different. This paper intents not only to show the advantages and disadvantages of reversal flow versus stopped-flow but also to show the potential of the new FIA mode, especially the possibility of manipulating the dispersion and, hence, the reaction kinetics. This offers interesting possibilities for simultaneous determinations, currently under study by our team. EXPERIMENTAL SECTION Reagents. Bromcresol green stock solution was prepared by dissolving 0.100 g of the dye in 6.25 mL of ethanol and diluting to 25 mL with M borax solution. N,N-Dimethylphenylene-1,4-diamine solution was prepared by dissolving 0.5 g of this reagent in 40 mL of concentrated HCl and diluting to 250 mL with distilled water. Iron(II1) solution was prepared by dissolving 1.75 g or iron ammonia sulfate in 20 mL of concentrated HC1 and increasing the volume to 250 mL with distilled water. Aqueous 1.022 g/L sulfide solution was prepared from sodium M was prepared in sulfide. Potassium permanganate (2 X M oxalic acid in distilled a phosphate buffer of pH 6.5 and 1X water. Apparatus. A single-beam Pye Unicam SP-500 spectrophotometer equipped with a Hellma 178.12QS flow cell (inner volume 18 pL) and a Gilson Minipuls-2 peristaltic pump commanded by a home-made timer allowing the drum rotation rate, start of the reversal cycles (tJ,number of cycles (a),and cycle duration (At) to be programmed was used. A Rheodyne 5041 injection valve and a Tecator TM I11 chemifold were also used. Configuration. The general scheme for the three chemical systems is shown in Figure 1. The long reactors on both sides of the injection valve-detector unit avoid the loss of part of the sample plug in long cycles. The drain is driven through the pump to ensure constancy in the flow rate in both directions of the flow. RESULTS AND DISCUSSION The potential of this multidetection tool was assessed with three chemical systems, namely, (A) without chemical reaction (Bromcresol/borax system) and (B) with chemical reaction, by monitoring (Bl) the disappearance of a reagent (permanganate/oxalic acid system (20))or (B2) the formation 01 a product (S2-/N,N-dimethylphenylene1,4-diamine system (21)).
There are two basic ways to perform these studies: First, change the direction of the flow once the entire FIA plug has passed through the detector. In this case, almost Gaussian peaks subsequent to the first one are obtained (Figure 2). As dispersion and hence the peak width at the base line increase abruptly, the time for each reversal cycle is variable and dramatically lengthened by the successive passage of the sample plug through the detector. Second, establish constant time (of the order of a few seconds) for the reversal cycles and apply them to a preselected peak zone. In this case, the kinetics of the process can be
0003-2700/88/0360-1540$01.50/00 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1, 1988
1541
PUMP
SAMPLE CARRIER
PHOTOMETER
Flgure 1. General iterative reversal flow injection analysis configguration.
2 min
A-
Flgure 3. Influence of the sampled zone on the shape of the recordings.
Table I. Values of the Constants and Initial Rate Dilution for Different Sampled Zones’
From Maxima
-t
Flgure 2. Iterative flow reversal once the entire sample plug has passed through the detector (injection of 6.0 pg/mL of Bromcresol Green in lo-* M borax in the conflgtration in Figure 1 (4= 1.1 mL/min).
manipulated through the flow rate, sampled zone, start time (t, is start time for the cycles from the injection instant, to), and cycle duration, (At). These possibilities are unaffordable by the first mode. The proposed mode provides two types of information: Signal from the different maxima and minima of the recording and typical parameters such as v and k (process rate and rate constant) of the processes, obtained from the kinetic profiles of the maxima and minima. The process rate is obtained from the initial linear portions (AAlAt) of these curves, while k is calculated from log ( A , - A,) = log ( A , A,) - ktl2.303, A, being the initial absorbance, A, the absorbance at time t, and A , the final absorbance. As A , tends to cancel in an open flow system, it is taken as the maximum absorbance of the kinetic curve. Without Chemical Reaction. A solution of bromcresol green in lo-’ M borax solution was injected into a M borax carrier (Figure 1)to avoid changes in the pH of the sample carrier. The only process occurring in the system was the dilution of the dye in the carrier, which was monitored photometrically at 617 nm. Figure 3 illustrates the influence of the sample zone and summarizes the experiments performed after different times from injection, namely, on injection (to),coinciding with travel time ( t a ) ,between travel time and the residence time ( t r ) at , t,, and at different times longer than t, (for a flow rate of 1.8 mL/min and a constant cycle duration of 10 s). A total of 22 reversals were performed in all cases. The shape of the recording was different for each sampled zone. As the reactor between the injection and detection systems is relatively short, a cycle start time shorter than t, or equal to to could be selected, as long as suitable flow rates and cycle times were also used. In this case, the peaks were obtained only when
tats
Ai
4
A3
u, s-1
0.0 4.0 9.0 11.0 14.5 17.0 22.0 28.0 35.0
0.000 0.000 0.038 0.375 0.655 0.653 0.658 0.657 0.656
0.000 0.008 0.183 0.349 0.495 0.651 0.522 0.315 0.100
0.004 0.040 0.211 0.315 0.412 0.513 0.521 0.299 0.110
2.0 x 10-4 1.6 x 10-3 7.3 x 10-3 -1.3 x 10-3 -8.0 x 10-3 -7.0 x 10-3 -6.8 X -8 X 5 x 10-4
k, s-l 1.4 X 1.3 X 8.1 X -8.6 X -2.9 x -1.7 X -1.8 X -6.4 x 1.6 X
10-2 ?O-2
lo-’ 10-3
From Minima
a
t,, s
Ai’
Ai
Ai
u , s-1
0.0 4.0 9.0 11.0 14.5 17.0 22.0 28.0 35.0
0.000 0.000 0.000 0.000 0.030 0.141 0.141 0.054 0.026
0.000 0.000 0.003 0.013 0.078 0.204 0.201 0.070 0.028
0.001 0.002 0.008 0.034 0.115 0.226 0.212 0.080 0.030
2 x 10-4 3 x 10-4 6 X lo4 1.0 x 10-3 2.4 x 10-3 7.8 x 10-3 1.1 x 10-2 2.9 x 10-3 1.2 x 10-3
k, s-l 8.7 x 6.5 x 9.3 x 1.1x 2.1 x 5.5 x 5.9 x 1.2 x 5.2 x
10-3 10-3 10-3 10-2 10-2 10-2 10-2
10-2 10-3
Working conditions: q = 1.8 mL/min, t , = 10 s, t, = 9 s, and T
= 14.5 s.
the dispersion of the sample plug was large enough to allow its arrival at the detector; subsequently, some information was lost compared to cycles longer than t,. Each recording in Figure 3-obtained for the same dye concentration-consists of two dispersion profiles: one obtained from the envelope of the maxima and another defined by the envelope of the minima. These profiles cover a wide range of constants and initial rates of dilution (Figure 4), which varied over ranges shown in Table I. The dilution process must conform to a first-order kinetics with a general equation u = const[dye], influenced by the variables t,, At, and q (flow rate).
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1, 1988
Table 11. Influence of the q , t , , and t oon v and k (Bromcresol Green System)
From Maxima t, = T
t, = t , q,
mL/min
t,,
0.8
u,
8
4.1 x 3.3 x -1.7 x 7.3 x -5.0 x -1.4 x 5.8 x 1.1 x -1.2 x
10 30 60 10 30 60 5 10 30
1.8 3.0
k , s-l
s-1
10-3 10-3 10-4 10-3 10-4
1.6 X 3.2 x -2.2 x 8.1 X -3.9 x -4.5 x 3.0 X 1.0 x -7.0 x
10-3 10-3 10-2 10-3
10-3 10-3 10-3 10-1
10-3
u, s-1
2.1 x 3.8 x -2.9 x -8.0 x -1.4 X -7.0 x -1.2 x -1.4 X -6.4 x
10-3 10-3 10-3 10-3 lo-' 10-3 10-2 lo-' 10-3
k , s-l -9.5 -7.6 -3.8 -2.9 -1.7 -2.1 -2.2 -2.2 -8.3
x 10-3 x 10-3 x 10-3 X lo-' X lo-'
x 10-2 x 10-2
x 10-2 x 10-3
From Minima q, mL/min
t,, s
0.8
10 30 60 10 30 60 5 10 30
1.8 3.0
a)
k , s'l
u, s-1
3.8 x 3.5 x 1.2 x 6.3 x 2.5 X 1.0 x 1.5 x 8.7 x 2.1 x
10-4 10-4 10-4 10-4 10" 10-4 10-3 10-4 10-4
8.4 x 3.1 x 1.6 x 9.3 x 2.9 x 2.0 x 1.1 x 9.0 x 7.5 x
10-3 10-3 10-3 10-3 10-3 10-3 10-2 10-3 10-3
u, s-1
7.8 X 6.1 x 1.7 X 2.4 x 3.4 x 8.5 x 6.1 x 1.7 x 2.5 X
lo4 10-4 lo4 10-3 10-4 10-5 10-3 10-3 lo4
k , ssl 1.0 x 7.0 x 2.0 x 2.1 x 5.0 x 2.7 x 4.4 x 1.9 x 6.5 x
10-2 10-3 10-3 10-2 10-3 10-3 10-2 10-2
10-3
s, t , = 9 s, and t, = 14.5 s). Differences were established
I
E
20 5 17 5 15s
22 s
13 S
25s
11s
9s
7s 4s
2s os
.-
26 5
31 s
35s 40 S
Figure 4. Kinetlc profiies from the recordings obtained in experiments performed at different start times of inversion cycles: (a) from the maxima of the recordings; (b) from the minima.
From the data in Table I was studied the influence of t , on the kinetics of the process (for q = 1.8 mL/min, At = 10
between the kinetic profiies of the maxima and minima. There was no clear-cut variation in the maximum absorbance value with t,; neither was there one in the parameters u and k which, as shown in Figure 5a, can take positive or negative values (the negative sign is related to a decrease in the absorbance). There are zones of maximum positive (at t, = At) and negative (at t, = t,) rate. When kinetic profiles defined by the minima were used, u and k were always positive and their changes with t, followed similar trends, the dilution process being faster when the reversal cycles started about 22 s after injection (Figure 5a'). The influence of at on u and k is shown in parts b and b' of Figure 5 for q = 1.8 mL/min and three characteristic t , values (equal to t , and t,' and larger than t,). With regards to the kinetic profiles of the maxima (Figure 5b), the absolute values of u and k , their variation with At was similar and depended on t,. The maxima of these plots were always obtained for At values between 10 and 30 s. As to the kinetic profiles of the minima (Figure 5b9, the values of u and k were always positive, with the same shape for the different t, values and with a maximum at At = 10 s. The influence of q on u and k is difficult to generalize as it is different for the maxima and minima of the flow reversal (fr) FIA recordings and is also dependent on t, and At. In Table II are summarized the u and k values calculated at three different flow rates (low, medium, and high), variable cycle times and start of the cycles, and t, and t,. The fastest kinetics corresponded to high flow rates and short cycles ( A t 5 or 10 S) *
Influence of the Dye Concentration. In addition to the large amount of information on the dispersion process, the proposed configuration offers the possibility of manipulating data from the recording to broaden the determination range of the analyte. Thus, the unknown concentration can be obtained for any of the different parameters of the multipeak recording as far as its magnitude is within the suitable working range. Table I11 illustrates some of these possibilities, which correspond to linear equations with different slopes. As with closed systems (15),a possible application of the configuration is the implementation of dilution and amplification methods. In the present case, t , determines to a great extent the con-
ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1, 1988
I =9s O I
20 30 40
so 60
o io
Ir(d
io io
40
i o do
1543
1
I.(s)
Flgure 5. Influence of various parameters on the rate and constants of the dispersion process, obtained from the maxima (a, b) and minlma (a', b') of the recordlngs: (a, a') start cycle time, t,; (b, b') cycle duration, t,. Blank
Table 111. Normal, Dilution, and Amplification Methods for Bromcresol Green equation For t, = t, A, = -0.007 + (1.66 X 104)[C]a A2 = -0.006 + (0.3 X 104)[C] A3 = -0.003 + (0.50 X 104)[C] E:Ai = -0.006 + (0.69 X 104)[C] E f A i -0.024 + (1.71 X 104)[C] El'OAi = -0.124 + (6.21 X 104)[C] u,
= (8.35 X
urnin =
lo4)
-(1.16 X lo4)
+ 305.0[C]
+ 53.75[C]
For t, = t, A, = -0.007 + (1.66 X 104)[C] A2 = -0.014 + (2.64 X 104)[C] A3 = -0.007 + (1.53 X 104)[C] E:Ai = -0.002 + (4.65 X 104)[C] E t A j = -0.208 + (9.35 X 104)[C] El'OAi = -0.280 + 16.18[C] urn, = (-6.30 X lo4) + 171.O[C] urnin = (2.96 X lo-)' + 54.44[C]
regression coeff 0.999 0.995 1.000 0.998 0.999 0.999 0.999 0.998 0.999 0.999 0.999 0.999 0.999 0.998 0.999 0.994
[Cl = concentration, M.
ditions of one of the other types of method. Thus, for t, < t,, dilution methods are to be preferred; on the contrary, t, 3 t , is suitable for application of amplification methods (see Table 111). For the dilution process, rate measurements offer no additional information (Table 111) and their equations are very similar to one another. These measurements are of great interest when a chemical reaction further to physical dispersion occurs. With Chemical Reaction. When a chemical reaction occurs in the system, the dispersion of the sample plug plays a secondary role subordinated to the chemical process, the process of actual interest for analytical purposes. The hydrodynamic characteristics of the proposed configuration result in effective reagent-analyte "mixing" from the iterative reversal of the flow faciliting the reaction.
2ppm
5ppm
10 ppm
Flgure 6. Shape of the recordings provided by the different chemical systems: (a) monitoring of the disappearance of a reagent (different t , and tc):(b) monitoring of the formation of a product (different analyte concentrations).
Mpnitoring of the Disappearance of a Reagent. If a chemical reaction takes place in the system, the process most closely resembling the dilution of a dye is the monitoring of the disappearance of a reagent, as both physicaldispersion-and chemical-reaction-aspects act equally on the signal. The classical reaction between permanganate (sample in Figure 1) and oxalic acid (carrier) was chosen for this study. The carrier contained Mn(I1) traces for catalyzing the reaction, which was monitored at 525 nm. As the reaction development could be manipulated through the oxalic acid concentration, this was set to M, suitable for easy evaluation of the chemical contribution. Kinetics of the Process. As with the above-described system, the dispersion of the system could be manipulated through the parameters t,, At, and q. As can be seen in Figure 6a, when the disappearance of a reagent is monitored, the kinetic profiles obtained from the peak maxima offer more information more closely related to the evolution of the chemical process. They were thus used in this study. The influence oft, was critical for fixed chemical M oxalic acid, 8 X 10" M potassium permanganate, and pH 2.1) and hydrodynamic (q = 1.8 mL/min) conditions of the fr-FIA system. For t, 6 t,, u tended to zero, and increased somewhat at values above t,, attaining a maximum that was closer to t, as At increased (t, = t, for d t 3 15 s, t, = 18 s for At = 10 s, and t, = 25 s when At = 5 s). The influence of the flow rate was also decisive. Taking t , = t , as reference, i.e. a reversal cycle always starting at the maximum of the FIA peak the increase in u with the low rate was almost linear. In general, an increase in u of 60-70% resulted from doubling of the flow rate. The influence of t,, At, and q on k was negligible. Determination of Permanganate. The influence of t,, At, and q on the kinetic of the process can be exploited for analytical purposes. Thus, depending on the analyte concentration, the values of these parameters are chosen to
1544
ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1, 1988
Table IV. Features of Determinations of Permanganate by Multidetection fr-FIA (working conditions: 5
q = 3.0
mL/min, t , =
9)
measured parametera
equation A,(t, T )= -0.076 + 858.3[C] A&, = T ) = -0.092 + 565.5[C] A3(t, = T ) = -0.070 + 381.2[C] Az(t, = t,) = -0.013 + 24.3[C] A7(t, = t o ) = -0.002 + 5.11[C] C13Ai(t,= T )= -0.158 + 1633.4[C] E16Ai(t, = t,) = -0.043 + 192.3[C] u ( T ) = (1.63 X lo-') + 24.58[C] u(t,) = -(9.2 x 10-4) + i.g5[c] u ( t o )= -(5.1 x 105) + 0.042[c]
a
Absorbance of peak i (Ai); u in s-l.
determination range, Fg/mL 1.3 x 2.5 x 3.0 X 2.0 x 5.0 x 1.2 x 4.0 x 7.5 x 9.5 x 2.0 x
104to 1.2 x 10-4to 2.0 x lo4 to 2.6 X 10-3 to 3.4 x 10-3 to 1.0x 10-4 to 8.0 x 10-4 to 5.0 x 10-5 to 1.3 x 10-4 to 5.0 x 10-3 to 5.0 x
C = permanganate concentration (MI. for n = 11.
10-3 10-3
io-* 10-1
10-4 10-3 10-3 10-3 10-2
equationb
f0.56 10.68 f0.64 11.12 f1.63 f0.95 f1.44 10.61 f1.02 f1.33
concn,d pg/mL 8X
8X 8X 5x 5 x Bx 5x Bx 5x 5x
10-3 10-3 10-4 10-3 10-4 10-3 10-3
Concentration for RSD.
Table V. Features of Determinations of Sulfide by Multidetection fr-FIA (working conditions: measured parametern
% RSDc
q = 2.2
mL/min, t , = 10 s)
determination range, pg/mL
% RSD'
concn,d pg/mL
0.3-3.0 0.2-2.2 2.0-50.0 8.0-150 2.0-40.0 0.1-1.5 0.1-1.0 0.2-2.5 1.O-40.0 20-500
f0.88 f0.92 f1.34 11.02 f1.89 f2.03 f2.19 f1.26 11.76 f1.52
2.0 2.0 2.0 25.0 2.0 1.0 1.0 2.0 2.0 25.0
"Absorbance of peak i (Ai), u in s-l. b C = sulfide concentration (pg/mL). CForn = 11. dConcentration for RSD.
achieve values of the signal within the useful working range, and normal (based on measurements of the maxima absorbance of the peaks), amplification, and dilution methods can be applied as for the bromcresol green system. At high and@ concentrations, t, values smaller than t, (even t, = to)can be used; on the contrary, at low concentrations, the t,, A t , and q values must be those resulting in the maximum rate. Table IV shows the features of the determination of permanganate based on the measurement of different parameters. The normal FIA method, involving the measurement of the maximum absorbance of a single peak, corresponds to A , (t, 3 t,). From the equation in Table IV were obtained a series of slopes (sensitivities). For equations based on absorbance measurements, the sensitivity varied between 5.11 and 1633.4 M-' (maximum ratio 319.6), while for those using rate measurements, the sensitivity varied between 0.042 and 24.58 M-' (maximum ratio 585.2). Most of the methods based on absorbance measurements and the monitoring of the disappearance of a reagent were dilution methods (slopes less than that of A , (t,3 t,)),except for those using the s u m maximum absorbances of several peaks for t, = t, (for example, E13A,(t, = t,) in Table IV), because the first peak in this sum is Al ( t , = t,). In short, a wide concentration range is covered with good precision (lower determination limit for u(t,)). Monitoring of the Formation of a Product. The reaction used in this case was that between sulfide and N,N-dimethylphenylene-1,4-diamine in the presence of iron(II1) to form Methylene Blue. The carrier in Figure 1 is an Fe(II1) aqueous solution and the sample (sulfide) merges with the reagent prior to injection (sample flow rate 0.5 mL/min and reagent 0.3 mL/min). The reaction was monitored photometrically at 662 nm. In this system, the chemical reaction and physical dispersion of the formed dye act contrarily on the monitored signal. Kinetics of the Process. The recordings obtained in this case under different experimental conditions (Figure 6b) are in contrast with those corresponding to the systems described above. In this case, the kinetic profiies of maxima and minima provide enough analytical information, although the former
are more interesting for low analyte concentrations and the latter for high concentrations. The profiles defining the minima have the advantage, from a kinetic point of view, of closer correspondence with the evolution of the process (formation and dilution of the product) and were thus used in this study. Increasing t, values (at constant At and q ) resulted in increased u and k , similar to the bromcresol system (Figure 5 ) and with maximum t, values slightly larger than the residence time. At these t , values, the maximum of the kinetic curve was attained quickly and was followed by a decrease indicating the prevalence of the dispersion of the product formed. The influence of At on u and k was analogous to that of t , and was maximum a t 10 < A t C 15 s. The parameters u and k increased exponentially with the flow rate, the effect being a maximum below 1.5 mL/min and minimum above 2 mL/min. Determination of Sulfide. Similar to the determination of permanganate, Table V summarizes the features of the determination of sulfide based on the use of different parameters for the measurements. The slopes of the calibration curves also change over a wide range. Considering the equations obtained from absorbance measurements and taking into account that A , (t, 2 t,) corresponds to the normal FIA method, there are several parameters resulting in increased sensitivity ( A , ( t , = t,), C:Ai (t, = t,), and A , (t, = t,) and others decreasing it ( A , (t, = t,), A, (t, = to),and A6 (t, = to)). The maximum slope ratio for equations based on the absorbance is 191.5 and for those implemented by rate measurements is 33.3 (smaller manipulation capacity). The overall determination range is between 0.1 and 500 pg/mL of sulfide, with an average relative standard deviation of about 1.5%.
CONCLUSIONS The features of flow-reversal systems are similar to those of multidetection in closed systems (15). The proposed configuration, though, has distinct features. First, the volume in which the sample plug is diluted can be indefinitely large and the final equilibrium reached in closed systems is unattainable here.
Anal. Chem. 1988, 60, 1545-1548
Second, multidetection is performed on a small zone of the sample plug (whose magnitude is determined by At and q ) ; to that an effective sample concentration is set by choosing t,. Third, the multidetection frequency (the chief weakness of closed systems) is considerably increased in fr-FIA. Finally, reproducibility, expressed as relative standard deviation, is similar for both types of configurations. These advantages features are of special interest to the application of this type of multidetection to simultaneous determinations by introducing new elements in the configuration and using the kinetic information provided.
NOMENCLATURE to = sample injection time t , = travel time (time elapsed between injection and the rise of the FIA peak) t , = time at which the reverse cycles start At = duration of flow reversal cycles t , = residence time (time elapsed between injection and the appearance of the FIA peak) n = number of the flow reversal cycles q = flow rate u = reaction time k = rate constant Ai= maximum absorbance of peak number i among the peaks obtained in a complete flow-reversal FIA experiment Ai’=minimum absorbance of peak number i among the peaks obtained in a complete flow-reversal FIA experiment LITERATURE CITED (1) Hooley, D. J.; Dessy, R. E. Anal. Chem. 1983, 55. 313. (2) Zagatto, E. A. G.; Jacinth, A. 0.; Pessenda, L. C. R.; Krug, F. J.; Reis, B. F.; Bergamin, F. H. Anal. Chlm. Acta 1981, 125, 37.
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Virtanen, R. Anal. Chem. Symp. Ser. 1981, 8 , 375. Basson, W. D.; Van Staden, J. F. Fresenlus’ 2. Anal. Chem. 1980, 302, 370. Uzaro, R.; Rios, A.; Luque de Castro, M. D.; Valcircel, M. Analusis, 1988, 14, 378. Fernindez, A.; Luque de Castro, M. D.; Valclrcel, M. Anal. Chim. Acta 1987, 193, 107. Ferndndez, A,; Luque de Castro, M. D.; Valcircel, M. Anal. Chem. 1984, 5 6 , 1146. Fern&dez, A.; Luque de Castro, M. D.; Valdrcel, M. Anakst (London) 1987, 112, 803. Ruz, J.; Rios, A.; Luque de Castro, M. D.; Valclrcel, M. Fresenius’ 2. Anal. Chem. 1985, 322, 499. Rjos, A.; Luque de Castro, M. D.; ValcBrcel, M. Anal. Chem. 1988, 5 8 , 863. Valdrcel, M.; Luque de Castro, M. D.; Rios, A,, Spanish Patent, No. 554 725, May 1986. Wade, A. P.; Betteridge, D.; Crouch, S. R. “Flow Reversal FIA for Rapid Speclation and Optimization Studles”. Presented at the FACSS XI11 Conference, St. Louis, MO, Oct 2, 1986. Wade, A. P.; Stub, C. L. M.; Crouch, S. R. “Flow Reversal FIAMuses on Uses”. Presented at the FACSS XIV Conference, Detroit, MI, 1987. Betteridge, D.; Oates, P. 0.; Wade, A. P. Anal. Chem. 1987, 5 9 , 1238. Rios, A.; Luque de Castro, M. D.; Valcircel, M. Anal. Chem. 1985, 5 7 , 1803. RIos, A.; Luque de Castro, M. D.; Valcircel, M. Anal. Chim. Acta 1986, 179, 463. Rios, A.; Luque de Castre, M. D.; Valcircel, M. J. Chem. Educ. 1986, 6 3 , 552. Rjos, A.; Luque de Castro, M. D.; Valcircel, M. Talanta 1987, 34, 915. Jacintho, A. 0.; Zagatto, E. A. G.; Reis, 8. F.; Pessenda, L. C. R.; Krug, F. J. Anal. Cb/m. Acta 1981, 130, 361. Day, R. A,, Jr.; Underwood, A. L. Quantitative Analysis, 4th ed.; Prentice-Hall: London, 1980. Leggett, D. J.; Chen, N. H.; Mahadevappa, D. S. Anal. Chim. Acta 1981, 128, 163.
RECEIVED for review June 19,1987. Accepted March 25,1988. The CICYT is thanked for financial support (Grant No. PA86-0146).
Mixed Plant Tissue-Carbon Paste Bioelectrode Joseph Wang* and Meng Shan Lin Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003
A new approach for preparlng electrochemkal blosensors, based on a “mlxed plant tlssue-carbon paste electrode” Is described. The bloelectrode Is made .from carbon paste which Is doped during preparatlon wRh the blocatalyst. The elknlnatlon of the long dlffuslon path, characterlzlng conventlonal tissue electrodes, allows rapid response to changes In the substrate concentration. Response t h e ( t o 5 % )as low as 12 s has been determlned. Hence, rapid detectlon In dynamk flow systems Is feaslble. At least 60 assays per hour can be made. The selectlve detection of dopamine at a “mlxed banana-carbon paste electrode” Is used to Illustrate the new concept. Flow lnjectlon measurements ylelded a detectlon llmit of 1.3 X lod M (20 ng) dopamine, wlth an upper linear range of 9 X lod M and relatlve standard devlatlon of 0.8 %. The response is characterlzed also with respect to paste composttion, canvectlve mass transport, voltammetrlc waveform, and possible Interferences. selectlve measurements of dopamine In the presence of ascorblc acid are Illustrated. The modified carbon paste can be easily Incorporated In varlous sensor conflgurations (mlcro, flow, etc.) relevant to cllnkal analysis. This method of biosensor construction should be widely applicable to other blocataiysts generatlng (or consuming) an electroactlve specles. 0003-2700/88/0380-1545$01.50/0
One of the most interesting developments in the field of electrochemical biosensors in recent years is the use of biocatalysts such as animal or plant tissues and microbial cells, instead of isolated enzymes (1, 2). In particular, the employment of tissue slices as catalytic layers is very attractive because of their high stability, high level of activity, and low cost compared to the use of isolated enzymes. Usually, tissue-based electrodes are prepared by physically retaining the biocatalyst with a support membrane (i.e., spatially separated catalytic and detection sites). The optimum thickness of the tissue slice reflects a compromise between mechanical stability and response time. Nevertheless, the low speed of response remains a severe limitation of these bioelectrodes. The response time tends to be slow because of the long diffusion path between the test solution and the inner detector surface. Initially, typical response times were in the 10-20-min range (3-5). By elimination of the additional support membranes, the response time was decreased to 3 min (5). For clinically relevant applications (e.g., flow or in vivo analyses), response times shorter than 30 s are desirable. This paper describes a new approach €or the construction of tissue-based amperometric bioelectrodes, based upon incorporating the biocatalyst into a carbon paste matrix. Sensor fabrication is accomplished simply by mixing the desired 0 1988 American Chemical Soclety
