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Exploiting the hydrodynamic aspects of continuous-flow systems
Takmta, Vol. 38, No. 12, pp. 13594368, 1991 Printed in Great Britain. All rights reserved
0039-9140/91 $5.00 + 0.00 Copyright 0 1991 Pergamon Press plc
EXPLOITING THE HYDRODYNAMIC ASPECTS OF CONTINUOUS-FLOW SYSTEMS ANGEL Rfos and MIGUEL VALC~CEL Department of Analytical Chemistry, Faculty of Sciences, University of Grdoba,
Ckloba,
Spain
(Received 20 April 1991. Revised 27 June 1991. Accepted 28 June 1991)
Summary-An overview of the analytical potential of the hydrodynamic aspects of unsegmented flow systems is presented. Different approaches involving flow manipulation are described: stoppedflow methodologies, intermittent pumping, selecting-diverting carrier (reagent) streams, open-closed flow systems, flow reversal and flow gradient.
The flow-rate (q) is one of the major variables in flow manifolds. Every method developed is optimized for a characteristic flow rate, which is kept constant throughout analyses through the different available propulsion elements (usually peristaltic pumps). Few methods use variable flow-rates; the best known are probably those based on stopping a pump of the system. The stopped-flow and intermittent pumping techniques are the most representative in this respect. *J However, other methods based on manipulation of the flow-rate during the analysis step also offer major analytical possibilities as shown below. The manipulation of hydrodynamic aspects in flow systems allows interesting analytical applications to be developed and a high degree of automation to be accomplished. Several of such applications were recently reported and could strongly influence the future of unsegmented flow analysers. This paper offers a systematic overview of the different modes involving manipulation of hydrodynamic features of flow systems, their applications and analytical potential. The flow is usually manipulated in its pattern, direction or intensity; thus, pumps and valves are the two key devices allowing the hydrodynamic conditions of flow systems to be suited to one’s requirements. Methodologies developed in this way involve stopping the flow, altering the flow pattern without changing the flow-rate, reversing the flow direction or establishing flow rate-gradients (Fig. 1). Thus, to date, constant flow-rates have dominated the applications of continuous flow systems [Fig. l(a)]: q$ =
constant
where to is the time in which the analysis starts and t,, is the time in which the analysis finishes. Time stopped-flow modes have been used, alternating go/stop steps [Fig. l(b)]: q:;,
cl:;, * * *
3
qt
= q =
constant
but
Flow reversal methodology are based on an analogue flow pattern, but in this case [Fig. l(c)]:
and
ai;,qi;,. . .&,=
-4
Flow rate-gradients [Fig. l(d)] are characterized by the continuous change of the flow-rate according to a linear function: q =
a*? + b(t = time; a = slope; b = intercept)
or a non-linear function: q =
F(t)(polynomic,
exponential, etc.)
This variability is associated with constant flow gradients for the linear functions: Flow-gradient = Q = dq/dt = a = constant and non-constant flow-gradients for the other function, in general:
Q = 4Wllld~ It is also possible to combine two or more of these methodologies by using different mathematical functions [Fig. l(e)]. All these altematives are discussed below.
1359
1360
ANGEL
Rfos
and MIGUEL VALC%RCEL
Stopped-flow modes The flow can be stopped either in the entire system or only in part of it. These two possibilities are related to stops-flow methods and intermittent pumping, respectively. Stopped-flow methods’ involve an abrupt change in the flow-rate (q) from a q-value to zero. During the period over which q # 0 the sample plug is mixed with the reagent and transported to the detector [Fig. 2(a)& When the ilow is stopped, the chemical reaction on which the determination is based develops and the detector continuously monitors the signaltime curve. The slopes of the curves obtained are proportional to the analyte ~on~ntration. Experimentally, stopped-flow methods can be implemented by synchronizing injection with the pump operation, or by using a three-way valve between the injection unit and the detector. In both cases a timer or microcomputer is used to control and synchronize the operation of the valves and the pump. One of the most outstanding practical advantages of stoppedflow methods is the elimination of background signals from the blank or matrix as they are based on measurements of the signal change during the stop period. In this way, RtiZka and Hansen have developed interesting applications for the determination of sulphur dioxide in wine,3 alcohol in blood or serum without sample and glucose in serurn5 The pretreatment, reduction in the number and level of interfering species is another asset of the stopped-flow methods,6 as shown by L&zaro et al. with their catalytic-fluorimetric determination of trace copper.’ An alternative conception of the stopped-flow technique involves stopping the flow not as the sample reaches the detector, but in the tubing zone (reactor) [Fig. 2(b)], in order to increase the residence time and avoid the dispersion of the injected plug. A significant increase in sensitivity is obtained as a result of the longer reaction time without the dilution of the plug that would ensue with ordinary FIA systems. Several methods have been developed by using this methodology.&” The term “parallel analysis” used by RtiiEka and Hansen’ also allows the reaction time to be increased by using two or more reaction coils in parallel, but decreasing the dispersion of the sample; the sample is sequentially stored in parallel coils before reaching the detector. The same authors explained a parallel FIA analyser with eight 80+1 storage
0.3
.qL
7
q&_______._-___--_-- ---- _..--- .-----. ---q-i
-
1 TIME
Fig. 1. Characteristic flow rate-time profiles for different methodologies: (a) anstant flow-rate modes; (b) stoppedflow; (c) flow reversal; (d) flow-gradient and (e) flowgradient/flow reversal coupling.
tubes in which several sample zones are stored in parallel (in a rotating drum) for a desired period of time. An interesting assembly for simultaneous multidete~nations by the stopped-flow technique was recently applied to the determination of free and bound sulphur dioxide in wines.” It uses a doubly stopped-flow manifold and can be considered to be a combination of the two above approaches [Fig. 2(c)]. Free sulphur dioxide directly reacts with fo~~dehyde and para-rosaniline to form a coloured compound which is monitored photometrically, whereas bound sulphur dioxide requires a prior hydrolytic release. Two sample plugs are injected simultaneously. Plug-l merges with the puraro~~~ne~fo~aldehyde mixture. The resulting merging stream is stopped when it reaches the detector, where the indicator reaction is monitored (determination of free sulphur dioxide). Simultaneously, plug-2 is stopped in a previous reactor coil, where its alkaline hydrolysis takes place. Then, on starting the pump, plug-l is flushed out of the system and plug-2 is driven to the detector after merging with the reacting mixture. The second halting is effected as plug-2 reaches the detector and is followed by the
1361
Exploiting the hydrodynamic aspects of continuous-flow systems
monitoring of the signal change with time, which is now made up of the contribution of free and bound sulphur dioxide. On starting the pump again, plug-2 is driven to waste. Intermittent pumping involves stopping and starting one or several pumps from injection and the consequent temporary halting of one or several streams in the course of a determination. According to Rf&ka and Hansen,12 two pumps can be incorporated into a manifold in a variety of combinations, especially if one considers different timings of their stop-go intervals, but, to date, little work has been done in this respect. The simplest application of a two-pump FIA system is to increase the sampling frequency by increasing the washout speed from the coils and from the flow-cell. Thus, the Brazilian FIA group, using commutation units, developed an improved procedure for the determination of nitrite in waters,13 and the turbidimetric determination of sulphate in natural waters and plant digestsI
Constantflow rate methodologies FIA modes involving a constant flow-rate allow one to manipulate hydrodynamic aspects through selecting/diverting valves. These valves arc of great interest to unsegmented flow methods” The six-way rotary valves usually employed in injection systems, converted to selectingdiverting valves by an alternative connection of different channels, probably represent the most useful alternative in this context. Olsen et aZ.16 employed a flow system incorporating three of these valves to preconcentrate heavy metals in a Chelex-100 minicolumn in a first step, followed by an elution step (in a reverse direction) to bring the eluted metal to a flame atomicabsorption detector. These readapted valves can also be used to co-ordinate two FIA subsystems by selecting the stream coming from one of them from a location prior to the detector” [Fig. 3(a)]. In other applications, they perform selecting-diverting operations on flow streams.
a) BUFFERREAGENT-
lN;ECTlON SAMPLE
bl BUFFER
J
-
LA-
s
(3
REAGENT--
iii
WASTE
I
I
STOP
TIME
INJECTION
INJECTION
Fig. 2. Different approaches to the implementation of the stopped-flow technique: (a) Stopping the flow as the reacting plug reaches the detector. (b) Stopping the tlow while the reacting plug is in the tubing zone (reactor). (c) Doubly stopped flow: fkst stop when plug-l (m) reaches the detector and plug-2 (a) is stopped in the reactor; second stop when plug-2 reachea the detector. [m and H indicate the position of the plug at the stop times].
ANGEL RfiXi
1362
and b&XEL
One of the streams reaching the valve is selected
at its exit; the other is driven to waste or allowed to reach a given point in the FIA configuration [Fig. 3(b)]. One such assembly was employed in speciation studies of chro~~.18 The main channel, which contains sulphuric acid as carrier and into which the sample is injected, is merged with channel C from the selectingdiverting valve. The valve allows the selected stream [l,Pdiphenylcarbaxide (DPC) or oxidant] to reach the merging point. Initia~y, the oxidant stream (2) is fed to waste by the valve, whilst DPC (1) flows through channel C, so that the indicator reaction [DPC selectively reacts with Q(W)] can take place in the subsequent reactors. When valve selects the oxidant stream, DPC is driven to the second reactor. Thus, Cr(III) is oxided to Cr(VI) in the first reactor, the indicator reaction takes place in the last reactor and total chromium is determined as a result. Figure 3(c) shows au alternative possibility recently proposed for simultaueous kinetic determina~ons.19 Fe(II1) and Co(B) form ~olourless
vALCk.CEL
complexes with EGTA [reagent 1 in Figure 3(c)], which react at a different rate with PAR (reagent 2) in l&and-displacement reactions, to yield coloured complexes that are monitory photomet~lly at the same wavelength. In one position, the valve Figure 3(c)], selects the sample-EGTA mixture and leads the PAR stream to point B (in this case the reaction only occurs along the last reactor). In the other position, the valve selects the PAR stream and leads the sampl~EGTA mixture to point A. The reaction occurs now along the two reactors before detection, The two continuous signals obtained correspond to different reaction times and allow one to determine the Fe@) and Co(B) present in the sample. Six-way rotary vaives adapted as selectingdiverting valves are a key to designing openclosed systems,2o in which multidetection is carried out by repeatedly passing the reacting plug through a single detector. In this case, one of the streams reaching the valve is the carrier solution and the other is that coming from the detector (Fig. 4). In the open position [Fig. 4(a)],
a) FIA SUBSYSTEM 0
{
FIA SUBSYSTEM 0
bl
C
when
C=@
REAGENT@ CARRIER SAt&‘LE
SAMPLE REAGENT0 REAGENT@
Fig. 3, Different uses of selecting-diverting valves to control the direction of one or two flow streams (see text). C = selected stream; W = waste. @ selecting-diverting valve. 0 injection valve.
Exploiting the hydrodynamic aspects of continuous-flow systems
1363
bl
REACTOR Fig. 4. Role of the selecting~iverting valve (S) in establishing open-closed flow systems. (a) Open-system position; (b) Closed-system position. The signal-time graph shows the response obtained by the detector in the open position configuration (m) (single FIA peak) and in the closed-system configuration (-) (multipeak recording), assuming no chemical reaction takes place (injection of a dye).
the valve selects the carrier solution and drives it to the detector, after which it goes to waste. In the closed-system position [Fig. 4(b)], a closed circuit is established as a result of the selecting-diverting valve selecting the channel coming from the detector. In this latter position the physical and chemical evolution of a sample injected into the flow can be monitored. The analytical potential of open-closed flow systems has been clearly shown in the last few years. Their most remarkable applications are kinetiP*22 and speciamplification methods, 20~21 ation studies, determination of reaction stoiand viscosity,2s simultaneous chiometries” kinetic determinations,26 and determinations of enzymatic activities.27*28 Flow reversal methodology
This methodology is based on the change of flow direction from +q to -q, which establishes constant or non-constant time cycles. As pointed out by Betteridge et aZ.,29flow reversals can be implemented in three different ways (Fig. 5). One involves using two pumps [Fig. 5(a), pump A propels the carrier stream, pump B is unused. Then, the sample is injected and allowed to travel downstream into the reaction coil for a set time. Pump A is then stopped and pump B is started at the same
flow-rate to pull the sample plug back and through the detector and a second coil, and then out to waste. Therefore, this procedure requires two pumps. The second procedure [Fig. 5(b)] uses a single pump and two valves, one for injecting the sample and the other as a selecting-diverting valve, which is the key to this procedure. First, the sample is injected into a carrier stream flowing at +q. After the entire plug has passed through the detector, the flow is reversed (at flow-rate -4) by switching the selecting-diverting valve. The peak is passed back through the detector, thereby establishing a cycle reversal. The simplest procedure to perform flow reversal FIA involves using a single pump and changing the direction of the drum rotation [Fig. 5(c)]. This requires electroni@’ or computer” control. The reversal of the flow direction in continuous automatic analysers was first achieved by the air-segmented mode proposed by Technicon. In the last few years this approach has been used in unsegmented continuous flow analysis (e.g., to control the dispersion and automatic variation of reaction coil length).29 The reaction coil length plays a major role in determining the characteristics of any FIA system and signiflcantly interacts with other factors. Other FIA variables (flow-rate, inner tubing diameter, etc.)
ANGELR~OSand
1364
MIGUEL
play their part in determining the amount of dispersion that a sample plug undergoes as it traverses a flow-injection manifold. Flow reversal may be used in conjunction with choice of flow-rate and sample size to determine the amount of dispersion that a sample undergoes. The flow can be reversed not only by allowing the entire plug to pass through the detector but also by “sampling” a preset zone of the plug, so that the reversal cycles all take place within one FIA peak. 3o In a way, this mode can be considered a peculiar variant of the sampling zone mode proposed by Krug et a1.,32but intended to obtain a multipeak recording that defines kinetic profiles (Fig. 6) that one can readily manipulate through the flow-rate, sample zone and cycle time. Toei used flow reversal FIA for the determination of glucose in clinical samples with a
a)
VALC~CEL
single-33and double-pump system.” The specific enzymatic reaction of glucose oxidase with /3-Dglucose was exploited, but straight calibration graphs could not be obtained when the reaction time was short or when using a stopped-flow FIA procedure. A long period (5 min) was needed for the reaction with end-point rate assay. However, in a conventional single-line manifold with sufhciently long tubing to obtain the required delay, the peak analysis time was rather long because of the large dispersion of the samples in the long reaction tubing. The kinetic FIA procedure in which two peaks are obtained by flow reversal offers better results, probably as a consequence of more efficient mixing that in turn results in a higher reaction rate. Moreover, by measuring the difference between the two peaks for a sample, the blank signal of the samples can also be eliminated. Recently, Wang
SAMPLE
‘-‘CARRIER
--WASTE PUMP
WASTE
B
@ SAMPLE
CARRl
when carrier is driveng by (iJ
SAMPLE
PUMP
Fig. 5. Different procedures for applying the flow-reversal methodology: (a) by means of two pumps, (b) a selecting-diverting valve or (c) a single pump reversing the direction of the drum.
Exploiting the hydrodynamic aspects of continuous-flow systems
et al. also showed the use of repeated reversals of the flow direction to significantly improve the sensitivity of flow-injection stripping voltammetric measurements.” The plating efficiency and the resulting stripping peak currents were greatly enhanced thanks to the repeated passage of the same sample plug over the working electrode, which addressed the time restriction of conventional flow-injection stripping measurements. The direct analysis of gas samples by flow injection is another interesting approach afforded by flow reversal methods implemented without a debubbler. 36 The sample is injected into a liquid carrier-reagent stream and the repeated passage of the liquid zone close to the gas-liquid interface nearest to the detector is monitored to obtain a signal-time multipeak recording. The method lends itself readily to automation and speeds up the analytical process as no prior collection in an absorbent solution or on a filter is required. Flow reversal is also the foundation of a new methodology for performing liquid-liquid extraction processes in automatic unsegmented flow systems based on simplified configurations with no segmenter or separation units.37 The key to this methodology is the placement of the detector in the loop of an injection valve, which is filled with the organic phase, whereas the aqueous phase containing the analyte makes the carrier. The process (reversal cycles as the two liquid-liquid interfaces are created by switching
1365
the injection valve) is repeated as many times as required to achieve a suitable solute transfer between the two liquid-liquid interfaces; and is favoured by the formation of a thin layer of organic phase on the Teflon coil. The phases are never segmented or separated, and the organic phase plug is continuously monitored. Therefore, the solute transfer between the two phases, and the dispersion of the solute into the organic phase, can be continuously monitored. Some kinetic aspects of extraction in continuous flow systems and the behaviour of different ion pairs have been studied.38 Simultaneous kinetic determinations by flow reversal/liquid-liquid extraction have also been reported.3’*38 Clark et al. have recently used flow-reversal flow-injection analysis for FIA titrations.3g They employed a single-line manifold for the titration of strong acids (HCl, H2S0, and H,PO,). The main advantages were the flexibility in operation and the increase in sensitivity. Recently, RdiiEka and Marshall used the movement of the piston of a syringe-type pump coupled with an eight-port valve to generate flow reversal steps and allowing sequential injections in the flow system.40 The synchronization between the movement of the piston and the valve allowed sample zone injection, reagent addition, mixing, measurement, and ejection of the reacted mixture took place by a combination of forward and reversed flow steps. The concept of sequential injection analysis, based on the mixing of the sample with a reagent in order to
TIME
Fig. 6. Multipeak recordings obtained by the flow reversal methodology showing the kinetic profiles defined by their maxima and minima: (a) without chemical reaction; (b) with chemical reaction, by monitoring the formation of a product, or (c) the disappearance of a reactant.
1366
ANGEL
Rfos
and MIGUELVAICARCEL
produce a measurable response, was explained through the random walk model, being defined as a new concept for chemical sensors, process analysis and laboratory assays by the authors. Flow-gradient technique
A flow rate-gradient (flow-gradient) can be defined as the variation of the flow-rate over a given period of time. It has been scantly used in FIA,4’*42but is well-known and widely employed in HPLC to increase resolution. Two FIA references describe the effect of flow-gradients on the more characteristic FIA parameters. In one, the flow-gradient was created by means of a hydrostatic head in a single channel.4’ The flow-rate range obtained was limited to 0.7-2.4 ml/min. In later work, a multifunction pump delivery system was used to establish several flow patterns.” The effects of these flow patterns on systems with and without chemical reaction were evaluated. One of the most important applications in this context is the washout effect (Fig. 7), whereby a higher sampling frequency is obtained. Thus, if a positive linear flow-gradient is established as the sample is injected, the peak-width can be dramatically decreased, the baseline restoration time shortened, the time lapse between consecutive injections minimized and the peak-height increased. Agudo et al. recently obtained similar results by using some non-constant flow rate-gradients.43 Similar results can be obtained by operating with intermittent pumping, as was reported by RdiiEka and HansenI and then reviewed by Krug et a1.,U but in this mode it is necessary to use
two peristaltic pumps (one to propel the reagent stream and another to flush the system with the wash solution). Different types of gradients were obtained by using a microcomputer which controlled (through an interface) the motion of the peristaltic pump drum.45 These flow-gradients can be used to establish concentration-gradients in an easy, fast and accurate way (e.g., to create pH-gradients allowing the photometric determination of acidity constants of compounds active in the UV-visible spectral region with no pH measurements:’ and carry out acid-base titrations&). Other linear concentration gradients can be created in flow systems for several analytical purposes. The stoichiometries of coloured complexes can be determined and complexometric titrations carried out by establishing ligand concentration gradients.43 An interesting flow pattern combining variable flow-rates and flow reversal modes has been proposed by RtEiEka et aZ.47establishing sinusoidal flow functions by a cam-driven computercontrolled piston pump. This system controls two syringes (one for the sample and the other for the reagent) and an eight-port valve, generating alternate load and measurement cycles. The authors give an important number of advantages of the sinusoidal flow pump, its main disadvantages are low sampling frequency and the device being less flexible than a peristaltic pump. The establishment of this and other similar flow patterns through a conventional peristaltic pump controlled by a computer may provide higher flexibility, avoiding the refilling reservoir requirements.
Ibl
(a)
D
TIME
TIME
Fig. 7. Washing effect obtained by using positive linear flow-gradients applied at the residence time of FIA peak (a), and starting simultaneously with the injection of the sample (b). The flow-gradient increases from A to D in both cases.
Exploiting the hydride
aspects of ~ntinuo~-flow
systems
1367
Table 1. Analytical applications of methodologies based on hydrodynamic aspects of unsegmented flow systems
Me~~olo~
Key element to perform the methodology
stopped-flow
Peristaltic pump (valves)
Kinetic studies Blank correction Reduction of interferences Increase of sensitivity Simultaneous determinations
Intermittent pumping
Peristaltic pump
Increase of sample frequency
Open-closed flow systems
~l~ting~ive~ing
valve
Flow reversal
Peristaltic pump Selecting-diverting
valve
Flow-gradients
Analytical applications
Peristaltic pump
Another interesting and useful application of non-constant flow patterns was proposed by RiiiiZka and Flossdorf by using variable forward flow with immobilized enzymes.” They employed designs with stopped flow, flow reversals and variable flow for the spectrophotometric determination of glucose with glucose oxidase and peroxidase ~mobili~d on controlled-pore glass in the same reactor. The variable flow pattern was fast/slow/fast cycles to increase both the contact time and contact volume between the sample zone and the immobilized enzymes without increasing the zone dispersion or Aow resistance. CONCLUSIONS
The analytical potential of methodologies based on hydrodynamic features of flow systems offers interesting possibi~ti~ briefly discussed in this paper and summarized in Table 1. Rumps and valves are the key elements to these methodologies; they can be controlled by electronic timers or microcomputers (through active interfaces) in order to accomplish the maximum possible degree of automation. In addition to the dynamic foundation of these methodologies, most of them involve kinetic aspects that can be exploited for analytical purposes. Other interesting possibilities are the manipulation of dis-
Improvement of sensitivity Kinetic studies Determination of viscosity Determination of stoichiometries Simultaneous determinations Speciation studies Enzymatic activities Dispersion ma~p~ation Improvement of sensitivity Kinetic studies Simultaneous determinations Direct gaseous sample analysis Liquid-liquid extraction Increase of sample frequency ~~blis~ent of inanition-orients Determination of acidity constants Determination of stoichiometries
persion (flow reversal) and sampling frequency (inte~ittent pumping and flow-gradient techniques). The flow reversal mode has introduced a new conception in flow manipulation with major repercussions on the automation of analytical processes. Thus, direct gas sample analyses and liquid-liquid extraction with no segmenter or separation units are two recent examples in this context. The flow-reversal/ flow-gradient coupling is another potentially interesting approach that will offer important applications.
1. J. RGiiEka and E. H. Hansen, Flow Injection Anafysis, 2nd Ed., Wiley, New York, 1988. 2. M. Valclrcel and M. D. Luque de Castro, Flow Injection Analysis. Principles and Applications, Ellis Horwood, Chichester, 1987. 3. J. RfiZiEka and E. H. Hansen, Anal. Chim. Acta, 1980, 114, 19. 4. P. J. Worsfold, J. RtiiEka and E. H. Hansen, Analyst, 1981, 106, 1309. 5. Z&m, Anal. Chim. Acta, 1979, 106, 207. 6. M. Val&cel, Analyst, 1987, 112,729. 7. F. L&aro, M. D. Luque de Castro and M. Valdrcel, Anal. Chim. Acta, 1984, liiS, 177. 8. T. Yamane, A&. C&z. Acta, 1980, 130,65. 9. C. S. kn, J. N. Miller and J. W. Bridges, ibid., 1980, 114, 183. 10. P. Linares, M. D. Luque de Castro and M. Valcircel, ibid., 1984, 161, 257.
1368
ANGELRfos and MIGUELVALC~CEL
11. F. L&zaro, M. D. Luque de Castro and M. Valcarcel, Anal. Chem., 1987, 59, 950. 12. J. Rtidlca and E. H. Hansen, Flow Injection Analysis, 2nd Ed., p. 172. Wiley, New York, 1988. 13. E. A. G. Zagatto, A. 0. Jacintho, J. Mortatti and H. Bergamin FP, Anal. Chim. Acta, 1980, 120, 399. 14. F. J. Krug, E. A. G. Zagatto, B. F. Reis, 0. Bahia, A. 0. Jacintho and S. S. Jorgensen, ibid., 1983, 145, 179.
15. A. Rios, M. D. Luque de Castro and M. Valcarcel, J. Automatic Chem., 1987, 9, 30. 16. S. Olsen, L. C. R. Pessenda, J. REEka and E. H. Hansen, Analyst, 1983, 108, 905. 17. A. Rios, M. D. Luque de Castro and M. ValcPrce.1, ibid., 1985, 110, 277. 18. J. Ruz, A. Rios, M. D. Luque de Castro and M. Valdrcel, Anal. Chim. Acta, 1986, 186, 139. 19. M. Romero-Saldaiia, A. Rios, M. D. Luque de Castro and M. Valcarcel, Talanta, 1991, 38, 291. 20. A. Rios, M. D. Luque de Castro and M. Valclrcel, Anal. Chem., 1985, 57, 1803. 21. A. Rios, F. L&raro, M. D. Luque de Castro and M. Valclrcel, Anal. Chtm. Acta, 1987, 199, 15. 22. S. D. Kolev, A. Rios, M. D. Luque de Castro and M. Valcarcel, Talanta, 1991, 38, 125. 23. J. Ruz, A. Rios, M. D. Luque de Castro and M. Valcarcel, ibid., 1986, 33, 199. 24. A. Rios, M. D. Luque de Castro and M. Valcarcel, J. Chem. Education, 1986, 63, 552. 25. I&m, Talanta, 1987, 34, 915. 26. Idem, Anal. Chim. Acta, 1986, 179, 463. 27. J. M. Femandez-Romero, M. D. Luque de Castro and M. Valcarcel, ibid., 1989, 219, 191. 28. I&m, J. Biotechnology, 1990, 14, 43.
29. D. Betteridge, P. B. Oates and A. P. Wade, Anal. Chem., 1987, 59, 1236. 30. A. Rios, M. D. Luque de Castro and M. Valclrcel, ibid., 1988, 60, 1540. 31. L. E. Leon, A. Rios, M. D. Luque de Castro and M. Valcarcel, J. Laboratory and Robotics, 1989, 1, 295. 32. A. 0. Jacintho, E. A. G. Zagatto, B. F. Reis, L. C. R. Pessenda and F. J. Krug, Anal. Chim. Acta, 1981, 130, 361. 33. J. Toei, Analyst, 1988, 113, 475. 34. I&m, Talanta, 1989, 36, 1233. 35. J. Wang, H. Huiliang and W. Kubiak, Electroanalysis, 1990, 2, 127. 36. F. Cafiete, A. Rios, M. D. Luque de Castro and M. Valdrcel, Anal. Chim. Acta, 1989, 224, 127. 37. Idem, Anal. Chem., 1988, 60, 2354. 38. Ia’em, Anal. Chins. Acta, 1989, 224, 169. 39. G. D. Clark, J. Zable, J. RUiEka and G. D. Christian, Talanta, 1991, 38, 119. 40. J. Rticka and G. D. Marshall, Anal. Chim. Acta, 1990, 237, 329. 41. A. Rios, M. D. Luque de Castro and M. Valclrcel, Talanta, 1985, 32, 845. 42. J. Toei, ibid., 1988, 35, 425. 43. M. Agudo, J. Marcos, A. Rios and M. Valcarcel, Anal. Chim. Acta, 1990, 239, 211. 44. F. J. Krug, H. Bergamin F* and A. E. G. Zagatto, ibid., 1983, 179, 103. 45. J. Marcos, A. Rios and M. Valcarcel, Anal. Chem., 1990, 62, 2237. 46. J. Marcos, Private communication. 47. J. Rfisicka, G. D. Marshall and G. D. Christian, Anal. Chem., 1990, 62, 1861. 48. J. Rt%cka and J. Flossdorf, Anal. Chtm. Acta, 1989, 218, 291.
0039-9140/91 $5.00 + 0.00 Copyright 0 1991 Pergamon Press plc
EXPLOITING THE HYDRODYNAMIC ASPECTS OF CONTINUOUS-FLOW SYSTEMS ANGEL Rfos and MIGUEL VALC~CEL Department of Analytical Chemistry, Faculty of Sciences, University of Grdoba,
Ckloba,
Spain
(Received 20 April 1991. Revised 27 June 1991. Accepted 28 June 1991)
Summary-An overview of the analytical potential of the hydrodynamic aspects of unsegmented flow systems is presented. Different approaches involving flow manipulation are described: stoppedflow methodologies, intermittent pumping, selecting-diverting carrier (reagent) streams, open-closed flow systems, flow reversal and flow gradient.
The flow-rate (q) is one of the major variables in flow manifolds. Every method developed is optimized for a characteristic flow rate, which is kept constant throughout analyses through the different available propulsion elements (usually peristaltic pumps). Few methods use variable flow-rates; the best known are probably those based on stopping a pump of the system. The stopped-flow and intermittent pumping techniques are the most representative in this respect. *J However, other methods based on manipulation of the flow-rate during the analysis step also offer major analytical possibilities as shown below. The manipulation of hydrodynamic aspects in flow systems allows interesting analytical applications to be developed and a high degree of automation to be accomplished. Several of such applications were recently reported and could strongly influence the future of unsegmented flow analysers. This paper offers a systematic overview of the different modes involving manipulation of hydrodynamic features of flow systems, their applications and analytical potential. The flow is usually manipulated in its pattern, direction or intensity; thus, pumps and valves are the two key devices allowing the hydrodynamic conditions of flow systems to be suited to one’s requirements. Methodologies developed in this way involve stopping the flow, altering the flow pattern without changing the flow-rate, reversing the flow direction or establishing flow rate-gradients (Fig. 1). Thus, to date, constant flow-rates have dominated the applications of continuous flow systems [Fig. l(a)]: q$ =
constant
where to is the time in which the analysis starts and t,, is the time in which the analysis finishes. Time stopped-flow modes have been used, alternating go/stop steps [Fig. l(b)]: q:;,
cl:;, * * *
3
qt
= q =
constant
but
Flow reversal methodology are based on an analogue flow pattern, but in this case [Fig. l(c)]:
and
ai;,qi;,. . .&,=
-4
Flow rate-gradients [Fig. l(d)] are characterized by the continuous change of the flow-rate according to a linear function: q =
a*? + b(t = time; a = slope; b = intercept)
or a non-linear function: q =
F(t)(polynomic,
exponential, etc.)
This variability is associated with constant flow gradients for the linear functions: Flow-gradient = Q = dq/dt = a = constant and non-constant flow-gradients for the other function, in general:
Q = 4Wllld~ It is also possible to combine two or more of these methodologies by using different mathematical functions [Fig. l(e)]. All these altematives are discussed below.
1359
1360
ANGEL
Rfos
and MIGUEL VALC%RCEL
Stopped-flow modes The flow can be stopped either in the entire system or only in part of it. These two possibilities are related to stops-flow methods and intermittent pumping, respectively. Stopped-flow methods’ involve an abrupt change in the flow-rate (q) from a q-value to zero. During the period over which q # 0 the sample plug is mixed with the reagent and transported to the detector [Fig. 2(a)& When the ilow is stopped, the chemical reaction on which the determination is based develops and the detector continuously monitors the signaltime curve. The slopes of the curves obtained are proportional to the analyte ~on~ntration. Experimentally, stopped-flow methods can be implemented by synchronizing injection with the pump operation, or by using a three-way valve between the injection unit and the detector. In both cases a timer or microcomputer is used to control and synchronize the operation of the valves and the pump. One of the most outstanding practical advantages of stoppedflow methods is the elimination of background signals from the blank or matrix as they are based on measurements of the signal change during the stop period. In this way, RtiZka and Hansen have developed interesting applications for the determination of sulphur dioxide in wine,3 alcohol in blood or serum without sample and glucose in serurn5 The pretreatment, reduction in the number and level of interfering species is another asset of the stopped-flow methods,6 as shown by L&zaro et al. with their catalytic-fluorimetric determination of trace copper.’ An alternative conception of the stopped-flow technique involves stopping the flow not as the sample reaches the detector, but in the tubing zone (reactor) [Fig. 2(b)], in order to increase the residence time and avoid the dispersion of the injected plug. A significant increase in sensitivity is obtained as a result of the longer reaction time without the dilution of the plug that would ensue with ordinary FIA systems. Several methods have been developed by using this methodology.&” The term “parallel analysis” used by RtiiEka and Hansen’ also allows the reaction time to be increased by using two or more reaction coils in parallel, but decreasing the dispersion of the sample; the sample is sequentially stored in parallel coils before reaching the detector. The same authors explained a parallel FIA analyser with eight 80+1 storage
0.3
.qL
7
q&_______._-___--_-- ---- _..--- .-----. ---q-i
-
1 TIME
Fig. 1. Characteristic flow rate-time profiles for different methodologies: (a) anstant flow-rate modes; (b) stoppedflow; (c) flow reversal; (d) flow-gradient and (e) flowgradient/flow reversal coupling.
tubes in which several sample zones are stored in parallel (in a rotating drum) for a desired period of time. An interesting assembly for simultaneous multidete~nations by the stopped-flow technique was recently applied to the determination of free and bound sulphur dioxide in wines.” It uses a doubly stopped-flow manifold and can be considered to be a combination of the two above approaches [Fig. 2(c)]. Free sulphur dioxide directly reacts with fo~~dehyde and para-rosaniline to form a coloured compound which is monitored photometrically, whereas bound sulphur dioxide requires a prior hydrolytic release. Two sample plugs are injected simultaneously. Plug-l merges with the puraro~~~ne~fo~aldehyde mixture. The resulting merging stream is stopped when it reaches the detector, where the indicator reaction is monitored (determination of free sulphur dioxide). Simultaneously, plug-2 is stopped in a previous reactor coil, where its alkaline hydrolysis takes place. Then, on starting the pump, plug-l is flushed out of the system and plug-2 is driven to the detector after merging with the reacting mixture. The second halting is effected as plug-2 reaches the detector and is followed by the
1361
Exploiting the hydrodynamic aspects of continuous-flow systems
monitoring of the signal change with time, which is now made up of the contribution of free and bound sulphur dioxide. On starting the pump again, plug-2 is driven to waste. Intermittent pumping involves stopping and starting one or several pumps from injection and the consequent temporary halting of one or several streams in the course of a determination. According to Rf&ka and Hansen,12 two pumps can be incorporated into a manifold in a variety of combinations, especially if one considers different timings of their stop-go intervals, but, to date, little work has been done in this respect. The simplest application of a two-pump FIA system is to increase the sampling frequency by increasing the washout speed from the coils and from the flow-cell. Thus, the Brazilian FIA group, using commutation units, developed an improved procedure for the determination of nitrite in waters,13 and the turbidimetric determination of sulphate in natural waters and plant digestsI
Constantflow rate methodologies FIA modes involving a constant flow-rate allow one to manipulate hydrodynamic aspects through selecting/diverting valves. These valves arc of great interest to unsegmented flow methods” The six-way rotary valves usually employed in injection systems, converted to selectingdiverting valves by an alternative connection of different channels, probably represent the most useful alternative in this context. Olsen et aZ.16 employed a flow system incorporating three of these valves to preconcentrate heavy metals in a Chelex-100 minicolumn in a first step, followed by an elution step (in a reverse direction) to bring the eluted metal to a flame atomicabsorption detector. These readapted valves can also be used to co-ordinate two FIA subsystems by selecting the stream coming from one of them from a location prior to the detector” [Fig. 3(a)]. In other applications, they perform selecting-diverting operations on flow streams.
a) BUFFERREAGENT-
lN;ECTlON SAMPLE
bl BUFFER
J
-
LA-
s
(3
REAGENT--
iii
WASTE
I
I
STOP
TIME
INJECTION
INJECTION
Fig. 2. Different approaches to the implementation of the stopped-flow technique: (a) Stopping the flow as the reacting plug reaches the detector. (b) Stopping the tlow while the reacting plug is in the tubing zone (reactor). (c) Doubly stopped flow: fkst stop when plug-l (m) reaches the detector and plug-2 (a) is stopped in the reactor; second stop when plug-2 reachea the detector. [m and H indicate the position of the plug at the stop times].
ANGEL RfiXi
1362
and b&XEL
One of the streams reaching the valve is selected
at its exit; the other is driven to waste or allowed to reach a given point in the FIA configuration [Fig. 3(b)]. One such assembly was employed in speciation studies of chro~~.18 The main channel, which contains sulphuric acid as carrier and into which the sample is injected, is merged with channel C from the selectingdiverting valve. The valve allows the selected stream [l,Pdiphenylcarbaxide (DPC) or oxidant] to reach the merging point. Initia~y, the oxidant stream (2) is fed to waste by the valve, whilst DPC (1) flows through channel C, so that the indicator reaction [DPC selectively reacts with Q(W)] can take place in the subsequent reactors. When valve selects the oxidant stream, DPC is driven to the second reactor. Thus, Cr(III) is oxided to Cr(VI) in the first reactor, the indicator reaction takes place in the last reactor and total chromium is determined as a result. Figure 3(c) shows au alternative possibility recently proposed for simultaueous kinetic determina~ons.19 Fe(II1) and Co(B) form ~olourless
vALCk.CEL
complexes with EGTA [reagent 1 in Figure 3(c)], which react at a different rate with PAR (reagent 2) in l&and-displacement reactions, to yield coloured complexes that are monitory photomet~lly at the same wavelength. In one position, the valve Figure 3(c)], selects the sample-EGTA mixture and leads the PAR stream to point B (in this case the reaction only occurs along the last reactor). In the other position, the valve selects the PAR stream and leads the sampl~EGTA mixture to point A. The reaction occurs now along the two reactors before detection, The two continuous signals obtained correspond to different reaction times and allow one to determine the Fe@) and Co(B) present in the sample. Six-way rotary vaives adapted as selectingdiverting valves are a key to designing openclosed systems,2o in which multidetection is carried out by repeatedly passing the reacting plug through a single detector. In this case, one of the streams reaching the valve is the carrier solution and the other is that coming from the detector (Fig. 4). In the open position [Fig. 4(a)],
a) FIA SUBSYSTEM 0
{
FIA SUBSYSTEM 0
bl
C
when
C=@
REAGENT@ CARRIER SAt&‘LE
SAMPLE REAGENT0 REAGENT@
Fig. 3, Different uses of selecting-diverting valves to control the direction of one or two flow streams (see text). C = selected stream; W = waste. @ selecting-diverting valve. 0 injection valve.
Exploiting the hydrodynamic aspects of continuous-flow systems
1363
bl
REACTOR Fig. 4. Role of the selecting~iverting valve (S) in establishing open-closed flow systems. (a) Open-system position; (b) Closed-system position. The signal-time graph shows the response obtained by the detector in the open position configuration (m) (single FIA peak) and in the closed-system configuration (-) (multipeak recording), assuming no chemical reaction takes place (injection of a dye).
the valve selects the carrier solution and drives it to the detector, after which it goes to waste. In the closed-system position [Fig. 4(b)], a closed circuit is established as a result of the selecting-diverting valve selecting the channel coming from the detector. In this latter position the physical and chemical evolution of a sample injected into the flow can be monitored. The analytical potential of open-closed flow systems has been clearly shown in the last few years. Their most remarkable applications are kinetiP*22 and speciamplification methods, 20~21 ation studies, determination of reaction stoiand viscosity,2s simultaneous chiometries” kinetic determinations,26 and determinations of enzymatic activities.27*28 Flow reversal methodology
This methodology is based on the change of flow direction from +q to -q, which establishes constant or non-constant time cycles. As pointed out by Betteridge et aZ.,29flow reversals can be implemented in three different ways (Fig. 5). One involves using two pumps [Fig. 5(a), pump A propels the carrier stream, pump B is unused. Then, the sample is injected and allowed to travel downstream into the reaction coil for a set time. Pump A is then stopped and pump B is started at the same
flow-rate to pull the sample plug back and through the detector and a second coil, and then out to waste. Therefore, this procedure requires two pumps. The second procedure [Fig. 5(b)] uses a single pump and two valves, one for injecting the sample and the other as a selecting-diverting valve, which is the key to this procedure. First, the sample is injected into a carrier stream flowing at +q. After the entire plug has passed through the detector, the flow is reversed (at flow-rate -4) by switching the selecting-diverting valve. The peak is passed back through the detector, thereby establishing a cycle reversal. The simplest procedure to perform flow reversal FIA involves using a single pump and changing the direction of the drum rotation [Fig. 5(c)]. This requires electroni@’ or computer” control. The reversal of the flow direction in continuous automatic analysers was first achieved by the air-segmented mode proposed by Technicon. In the last few years this approach has been used in unsegmented continuous flow analysis (e.g., to control the dispersion and automatic variation of reaction coil length).29 The reaction coil length plays a major role in determining the characteristics of any FIA system and signiflcantly interacts with other factors. Other FIA variables (flow-rate, inner tubing diameter, etc.)
ANGELR~OSand
1364
MIGUEL
play their part in determining the amount of dispersion that a sample plug undergoes as it traverses a flow-injection manifold. Flow reversal may be used in conjunction with choice of flow-rate and sample size to determine the amount of dispersion that a sample undergoes. The flow can be reversed not only by allowing the entire plug to pass through the detector but also by “sampling” a preset zone of the plug, so that the reversal cycles all take place within one FIA peak. 3o In a way, this mode can be considered a peculiar variant of the sampling zone mode proposed by Krug et a1.,32but intended to obtain a multipeak recording that defines kinetic profiles (Fig. 6) that one can readily manipulate through the flow-rate, sample zone and cycle time. Toei used flow reversal FIA for the determination of glucose in clinical samples with a
a)
VALC~CEL
single-33and double-pump system.” The specific enzymatic reaction of glucose oxidase with /3-Dglucose was exploited, but straight calibration graphs could not be obtained when the reaction time was short or when using a stopped-flow FIA procedure. A long period (5 min) was needed for the reaction with end-point rate assay. However, in a conventional single-line manifold with sufhciently long tubing to obtain the required delay, the peak analysis time was rather long because of the large dispersion of the samples in the long reaction tubing. The kinetic FIA procedure in which two peaks are obtained by flow reversal offers better results, probably as a consequence of more efficient mixing that in turn results in a higher reaction rate. Moreover, by measuring the difference between the two peaks for a sample, the blank signal of the samples can also be eliminated. Recently, Wang
SAMPLE
‘-‘CARRIER
--WASTE PUMP
WASTE
B
@ SAMPLE
CARRl
when carrier is driveng by (iJ
SAMPLE
PUMP
Fig. 5. Different procedures for applying the flow-reversal methodology: (a) by means of two pumps, (b) a selecting-diverting valve or (c) a single pump reversing the direction of the drum.
Exploiting the hydrodynamic aspects of continuous-flow systems
et al. also showed the use of repeated reversals of the flow direction to significantly improve the sensitivity of flow-injection stripping voltammetric measurements.” The plating efficiency and the resulting stripping peak currents were greatly enhanced thanks to the repeated passage of the same sample plug over the working electrode, which addressed the time restriction of conventional flow-injection stripping measurements. The direct analysis of gas samples by flow injection is another interesting approach afforded by flow reversal methods implemented without a debubbler. 36 The sample is injected into a liquid carrier-reagent stream and the repeated passage of the liquid zone close to the gas-liquid interface nearest to the detector is monitored to obtain a signal-time multipeak recording. The method lends itself readily to automation and speeds up the analytical process as no prior collection in an absorbent solution or on a filter is required. Flow reversal is also the foundation of a new methodology for performing liquid-liquid extraction processes in automatic unsegmented flow systems based on simplified configurations with no segmenter or separation units.37 The key to this methodology is the placement of the detector in the loop of an injection valve, which is filled with the organic phase, whereas the aqueous phase containing the analyte makes the carrier. The process (reversal cycles as the two liquid-liquid interfaces are created by switching
1365
the injection valve) is repeated as many times as required to achieve a suitable solute transfer between the two liquid-liquid interfaces; and is favoured by the formation of a thin layer of organic phase on the Teflon coil. The phases are never segmented or separated, and the organic phase plug is continuously monitored. Therefore, the solute transfer between the two phases, and the dispersion of the solute into the organic phase, can be continuously monitored. Some kinetic aspects of extraction in continuous flow systems and the behaviour of different ion pairs have been studied.38 Simultaneous kinetic determinations by flow reversal/liquid-liquid extraction have also been reported.3’*38 Clark et al. have recently used flow-reversal flow-injection analysis for FIA titrations.3g They employed a single-line manifold for the titration of strong acids (HCl, H2S0, and H,PO,). The main advantages were the flexibility in operation and the increase in sensitivity. Recently, RdiiEka and Marshall used the movement of the piston of a syringe-type pump coupled with an eight-port valve to generate flow reversal steps and allowing sequential injections in the flow system.40 The synchronization between the movement of the piston and the valve allowed sample zone injection, reagent addition, mixing, measurement, and ejection of the reacted mixture took place by a combination of forward and reversed flow steps. The concept of sequential injection analysis, based on the mixing of the sample with a reagent in order to
TIME
Fig. 6. Multipeak recordings obtained by the flow reversal methodology showing the kinetic profiles defined by their maxima and minima: (a) without chemical reaction; (b) with chemical reaction, by monitoring the formation of a product, or (c) the disappearance of a reactant.
1366
ANGEL
Rfos
and MIGUELVAICARCEL
produce a measurable response, was explained through the random walk model, being defined as a new concept for chemical sensors, process analysis and laboratory assays by the authors. Flow-gradient technique
A flow rate-gradient (flow-gradient) can be defined as the variation of the flow-rate over a given period of time. It has been scantly used in FIA,4’*42but is well-known and widely employed in HPLC to increase resolution. Two FIA references describe the effect of flow-gradients on the more characteristic FIA parameters. In one, the flow-gradient was created by means of a hydrostatic head in a single channel.4’ The flow-rate range obtained was limited to 0.7-2.4 ml/min. In later work, a multifunction pump delivery system was used to establish several flow patterns.” The effects of these flow patterns on systems with and without chemical reaction were evaluated. One of the most important applications in this context is the washout effect (Fig. 7), whereby a higher sampling frequency is obtained. Thus, if a positive linear flow-gradient is established as the sample is injected, the peak-width can be dramatically decreased, the baseline restoration time shortened, the time lapse between consecutive injections minimized and the peak-height increased. Agudo et al. recently obtained similar results by using some non-constant flow rate-gradients.43 Similar results can be obtained by operating with intermittent pumping, as was reported by RdiiEka and HansenI and then reviewed by Krug et a1.,U but in this mode it is necessary to use
two peristaltic pumps (one to propel the reagent stream and another to flush the system with the wash solution). Different types of gradients were obtained by using a microcomputer which controlled (through an interface) the motion of the peristaltic pump drum.45 These flow-gradients can be used to establish concentration-gradients in an easy, fast and accurate way (e.g., to create pH-gradients allowing the photometric determination of acidity constants of compounds active in the UV-visible spectral region with no pH measurements:’ and carry out acid-base titrations&). Other linear concentration gradients can be created in flow systems for several analytical purposes. The stoichiometries of coloured complexes can be determined and complexometric titrations carried out by establishing ligand concentration gradients.43 An interesting flow pattern combining variable flow-rates and flow reversal modes has been proposed by RtEiEka et aZ.47establishing sinusoidal flow functions by a cam-driven computercontrolled piston pump. This system controls two syringes (one for the sample and the other for the reagent) and an eight-port valve, generating alternate load and measurement cycles. The authors give an important number of advantages of the sinusoidal flow pump, its main disadvantages are low sampling frequency and the device being less flexible than a peristaltic pump. The establishment of this and other similar flow patterns through a conventional peristaltic pump controlled by a computer may provide higher flexibility, avoiding the refilling reservoir requirements.
Ibl
(a)
D
TIME
TIME
Fig. 7. Washing effect obtained by using positive linear flow-gradients applied at the residence time of FIA peak (a), and starting simultaneously with the injection of the sample (b). The flow-gradient increases from A to D in both cases.
Exploiting the hydride
aspects of ~ntinuo~-flow
systems
1367
Table 1. Analytical applications of methodologies based on hydrodynamic aspects of unsegmented flow systems
Me~~olo~
Key element to perform the methodology
stopped-flow
Peristaltic pump (valves)
Kinetic studies Blank correction Reduction of interferences Increase of sensitivity Simultaneous determinations
Intermittent pumping
Peristaltic pump
Increase of sample frequency
Open-closed flow systems
~l~ting~ive~ing
valve
Flow reversal
Peristaltic pump Selecting-diverting
valve
Flow-gradients
Analytical applications
Peristaltic pump
Another interesting and useful application of non-constant flow patterns was proposed by RiiiiZka and Flossdorf by using variable forward flow with immobilized enzymes.” They employed designs with stopped flow, flow reversals and variable flow for the spectrophotometric determination of glucose with glucose oxidase and peroxidase ~mobili~d on controlled-pore glass in the same reactor. The variable flow pattern was fast/slow/fast cycles to increase both the contact time and contact volume between the sample zone and the immobilized enzymes without increasing the zone dispersion or Aow resistance. CONCLUSIONS
The analytical potential of methodologies based on hydrodynamic features of flow systems offers interesting possibi~ti~ briefly discussed in this paper and summarized in Table 1. Rumps and valves are the key elements to these methodologies; they can be controlled by electronic timers or microcomputers (through active interfaces) in order to accomplish the maximum possible degree of automation. In addition to the dynamic foundation of these methodologies, most of them involve kinetic aspects that can be exploited for analytical purposes. Other interesting possibilities are the manipulation of dis-
Improvement of sensitivity Kinetic studies Determination of viscosity Determination of stoichiometries Simultaneous determinations Speciation studies Enzymatic activities Dispersion ma~p~ation Improvement of sensitivity Kinetic studies Simultaneous determinations Direct gaseous sample analysis Liquid-liquid extraction Increase of sample frequency ~~blis~ent of inanition-orients Determination of acidity constants Determination of stoichiometries
persion (flow reversal) and sampling frequency (inte~ittent pumping and flow-gradient techniques). The flow reversal mode has introduced a new conception in flow manipulation with major repercussions on the automation of analytical processes. Thus, direct gas sample analyses and liquid-liquid extraction with no segmenter or separation units are two recent examples in this context. The flow-reversal/ flow-gradient coupling is another potentially interesting approach that will offer important applications.
1. J. RGiiEka and E. H. Hansen, Flow Injection Anafysis, 2nd Ed., Wiley, New York, 1988. 2. M. Valclrcel and M. D. Luque de Castro, Flow Injection Analysis. Principles and Applications, Ellis Horwood, Chichester, 1987. 3. J. RfiZiEka and E. H. Hansen, Anal. Chim. Acta, 1980, 114, 19. 4. P. J. Worsfold, J. RtiiEka and E. H. Hansen, Analyst, 1981, 106, 1309. 5. Z&m, Anal. Chim. Acta, 1979, 106, 207. 6. M. Val&cel, Analyst, 1987, 112,729. 7. F. L&aro, M. D. Luque de Castro and M. Valdrcel, Anal. Chim. Acta, 1984, liiS, 177. 8. T. Yamane, A&. C&z. Acta, 1980, 130,65. 9. C. S. kn, J. N. Miller and J. W. Bridges, ibid., 1980, 114, 183. 10. P. Linares, M. D. Luque de Castro and M. Valcircel, ibid., 1984, 161, 257.
1368
ANGELRfos and MIGUELVALC~CEL
11. F. L&zaro, M. D. Luque de Castro and M. Valcarcel, Anal. Chem., 1987, 59, 950. 12. J. Rtidlca and E. H. Hansen, Flow Injection Analysis, 2nd Ed., p. 172. Wiley, New York, 1988. 13. E. A. G. Zagatto, A. 0. Jacintho, J. Mortatti and H. Bergamin FP, Anal. Chim. Acta, 1980, 120, 399. 14. F. J. Krug, E. A. G. Zagatto, B. F. Reis, 0. Bahia, A. 0. Jacintho and S. S. Jorgensen, ibid., 1983, 145, 179.
15. A. Rios, M. D. Luque de Castro and M. Valcarcel, J. Automatic Chem., 1987, 9, 30. 16. S. Olsen, L. C. R. Pessenda, J. REEka and E. H. Hansen, Analyst, 1983, 108, 905. 17. A. Rios, M. D. Luque de Castro and M. ValcPrce.1, ibid., 1985, 110, 277. 18. J. Ruz, A. Rios, M. D. Luque de Castro and M. Valdrcel, Anal. Chim. Acta, 1986, 186, 139. 19. M. Romero-Saldaiia, A. Rios, M. D. Luque de Castro and M. Valcarcel, Talanta, 1991, 38, 291. 20. A. Rios, M. D. Luque de Castro and M. Valclrcel, Anal. Chem., 1985, 57, 1803. 21. A. Rios, F. L&raro, M. D. Luque de Castro and M. Valclrcel, Anal. Chtm. Acta, 1987, 199, 15. 22. S. D. Kolev, A. Rios, M. D. Luque de Castro and M. Valcarcel, Talanta, 1991, 38, 125. 23. J. Ruz, A. Rios, M. D. Luque de Castro and M. Valcarcel, ibid., 1986, 33, 199. 24. A. Rios, M. D. Luque de Castro and M. Valcarcel, J. Chem. Education, 1986, 63, 552. 25. I&m, Talanta, 1987, 34, 915. 26. Idem, Anal. Chim. Acta, 1986, 179, 463. 27. J. M. Femandez-Romero, M. D. Luque de Castro and M. Valcarcel, ibid., 1989, 219, 191. 28. I&m, J. Biotechnology, 1990, 14, 43.
29. D. Betteridge, P. B. Oates and A. P. Wade, Anal. Chem., 1987, 59, 1236. 30. A. Rios, M. D. Luque de Castro and M. Valclrcel, ibid., 1988, 60, 1540. 31. L. E. Leon, A. Rios, M. D. Luque de Castro and M. Valcarcel, J. Laboratory and Robotics, 1989, 1, 295. 32. A. 0. Jacintho, E. A. G. Zagatto, B. F. Reis, L. C. R. Pessenda and F. J. Krug, Anal. Chim. Acta, 1981, 130, 361. 33. J. Toei, Analyst, 1988, 113, 475. 34. I&m, Talanta, 1989, 36, 1233. 35. J. Wang, H. Huiliang and W. Kubiak, Electroanalysis, 1990, 2, 127. 36. F. Cafiete, A. Rios, M. D. Luque de Castro and M. Valdrcel, Anal. Chim. Acta, 1989, 224, 127. 37. Idem, Anal. Chem., 1988, 60, 2354. 38. Ia’em, Anal. Chins. Acta, 1989, 224, 169. 39. G. D. Clark, J. Zable, J. RUiEka and G. D. Christian, Talanta, 1991, 38, 119. 40. J. Rticka and G. D. Marshall, Anal. Chim. Acta, 1990, 237, 329. 41. A. Rios, M. D. Luque de Castro and M. Valclrcel, Talanta, 1985, 32, 845. 42. J. Toei, ibid., 1988, 35, 425. 43. M. Agudo, J. Marcos, A. Rios and M. Valcarcel, Anal. Chim. Acta, 1990, 239, 211. 44. F. J. Krug, H. Bergamin F* and A. E. G. Zagatto, ibid., 1983, 179, 103. 45. J. Marcos, A. Rios and M. Valcarcel, Anal. Chem., 1990, 62, 2237. 46. J. Marcos, Private communication. 47. J. Rfisicka, G. D. Marshall and G. D. Christian, Anal. Chem., 1990, 62, 1861. 48. J. Rt%cka and J. Flossdorf, Anal. Chtm. Acta, 1989, 218, 291.