Flow Injection Analysis From Test Tube to Integrated Microconduits
A modern analytical laboratory con tains a formidable array of advanced and ancient tools. While microproces sors are among the most recent acqui sitions, volumetric glassware has been around for more than 200 years (Fig ure 1). Thus, we use sophisticated de tectors and electronics at the same time we use solution-metering and -handling techniques designed at the time when gravimetry and titration were the only known methods of quantitative analysis. Amazingly, even automated methods of analysis have been designed merely to mechanize the manual approach to solution han dling, and may therefore be viewed as "assembly lines" constructed to per form the required operations. Flow injection analysis (FIA) has won much recognition since the REPORT by Betteridge was published in this J O U R N A L (1). A monograph (2), several significant reviews—one of them on the use of FIA in clinical chemistry (3), and over 400 papers have been published on FIA thus far. While opponents of FIA still draw at tention to the drawbacks of the meth od (4-5), a large number of FIA-based assays are being performed routinely (6, 7). It would be a pity, however, if FIA were viewed only as a tool for speeding up routine assays. It is the purpose of this R E P O R T to describe
the unique features of FIA, which will change the concept of solution han dling in the chemical laboratory and make chemical analyses truly compat ible with tools of the computer age by enabling us to design microchemielectronic devices. Conventional FIA System
Flow injection analysis is based on injection of a liquid sample into a moving nonsegmented carrier stream of a reagent. The injected sample forms a zone that disperses on its way to a detector. The simplest FIA ana lyzer (Figure 2a) consists of a pump (P) that propels the carrier stream (R); an injection port (S), by which a well-defined volume of sample solu tion is injected into a carrier stream; and a coil in which the sample zone disperses and reacts with the compo nents of the carrier stream, forming a species (e.g., colored) to be sensed by a flow-through detector (D). A typical recording has the form of a sharp peak (Figure 2b), the height of which is re lated to the concentration of analyte. A simple and practical method for designing an FIA system is based on the concept of dispersion. Dispersion is classified according to its magnitude as limited (D = 1-3), medium (D = 3-15), and large (D > 15). The disper sion coefficient D is the ratio of the
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concentration of sample solution be fore (C°) and after (C max ) the disper sion process has taken place (D = C°/C m a x ). A set of rules (2) has been established to design an FIA system for pH and ion-selective electrode measurements, for colorimetry, for atomic absorption, and for other ana lytical techniques. The dispersion coefficient D has been related to the dimensions of the tubular reactor, flow velocity, axial dispersion coeffi cient, and sample volume (8). A well-designed FIA analyzer yields the readout within 15 s (T) (Figure 2b) from the moment of sample injec tion (S). Approximately the same amount of time is required for the dis persed sample zone to clear out of the detector so that the next sample may be injected. With a simple experimen tal set-up, most analyses can be per formed at a rate of 120 samples/h or more, using 30 μΊ-, of sample solution and less than 1 mL of reagent per analysis. Compared with other automated analyzers requiring a large amount of bench space, the second generation of commercial FIA instruments (FIA 5020, Tecator, U.S.; Bifok, Sweden) is as small as a typewriter, yet incorpo rates an injection valve, pumps, coils, thermostat, detector, and a micropro cessor controlling all functions as well 0003-2700/83/A351-1040 $01.50/0 © 1983 American Chemical Society
Report Jaromir Růžička Chemistry Department A The Technical University of Denmark 2800 Lyngby, Denmark
as d a t a collection and processing. A number of other manufacturers fol lowed Bifok's early lead (American Research Products Corporation, Fiatron Systems, Lachat Chemicals, Mark I n s t r u m e n t Company, Hitachi) so t h a t the m a r k e t today resembles t h a t of personal computers several years ago. As the recent advances are being incorporated into the newest FIA analyzers, the previous generation of instruments becomes obsolete. Integrated M i c r o c o n d u i t s
Miniaturization of the injection a n d detection functions of t h e flow system is the most recent step on the way from the test t u b e to t h e integrated microchemielectronic devices. T h e va riety of detectors and techniques com patible with FIA requires a corre sponding variety of flow systems. I t is a common practice to construct such manifolds from pieces of plastic t u b ing t h a t are used to wind coils a n d t o interconnect p u m p s with t h e injection valve, coils, columns, diffusion mod ules, and detectors. T h e result is t h a t inside a neat box with controls and electronic display is a spaghettilike heap of tubing with the potential for leaks and bad connections. I t was therefore logical to design t h e mani fold as a system of integrated conduits situated in a p e r m a n e n t rigid a n d pla-
Figure 1 . Front cover of a monograph written by E. Rancke Madsen, a Danish his torian of chemistry ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983 · 1041 A
Figure 2. (a) The components of the simplest FIA system: R, carrier stream of re agent; P, pump; S, injection port; D, detector; and W, waste, (b) Typical recording showing one sampling cycle, commencing at the point of injection (S) when valve has been turned and terminating when sample zone has passed into the waste (W). The readout, peak height (H), is available at the latest within 30 s (T) after sample has been injected (S)
nar structure (9). The grooves, form ing the flow channels, may be imprint ed or engraved into a transparent plate and then closed by a flat layer, thus forming a structure of conduits with a hemicircular cross section. The rigidity of such a structure ensures perfect repeatability of dispersion of the sample zone, and the small dimen sions of the integrated microconduits allow further miniaturization of the FIA system and reduction of sample reagent consumption to the microliter level. The method of fabrication, engrav ing or imprinting of channels, and lamination of layers ensures perfect uniformity of units even when they are produced on a large scale. The ma terials—plastics such as PVC—are in expensive and can handle the same so
lutions as the PVC pump tubing. This versatile combination of laminated parallel layers, interconnected by per pendicular channels (9), and various construction materials provides the means for integrating a number of so lution-handling tasks. Simplest is the sequential reagent addition module. A more complex module has been de signed for preconeentration of lead and cadmium using a microcolumn packed with Chelex-100. Use of this microconduit (Figure 3) has resulted in a 40-fold increase in sensitivity in the flame atomic absorption determi nation of heavy metals in seawater by an on-line FIA method (10). A further step has been to integrate sample injection, solution handling, and the detector into one microcon duit thus forming a microchemielec-
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tronic device. Optical fibers have been used to interface the microFIA system with a conventional spectrophotome ter (Figure 4). Sample is aspirated from the sample cup (S) by a peristal tic pump (Ρχ) so that a well-defined portion of a sample solution is arrest ed in the loop (L). This sample zone is then merged with reagent stream and swept into the flow cell by the second peristaltic pump (P 2 ). The inflowing streams [carrier (C) and reagent (R), propelled into the microFIA system by P2] have to be pumped by exactly the same rate as the outflowing streams (C + R also by P 2 ), (11,12). Even more compact are the inte grated microchemielectronic devices with ion-selective electrodes as detec tors. The coated-wire approach (13) and PVC membranes (14,15) are fully compatible with microconduit tech nology. Multielectrode systems for si multaneous pH and potassium mea surement using a common reference electrode have been developed in our laboratory. Also, microdiffusion units for C 0 2 and NH 3 assays have been de signed and integrated into these mi crochemielectronic devices. A logical extension of the microFIA systems with flow-through electrodes sensitive to ammonia will be integrated microconduits for enzyme assay using the zone penetration technique. The repeatability of measurement with integrated microconduits is supe rior to that obtainable in FIA systems built from flexible tubing. For exam ple, pH has been measured with a rel ative standard deviation of ±0.002 for samples of pH 6.8 to 7.8 and ±0.01 for samples of pH 4.0 to 10.0, and colorimetric determination of chloride has been performed with RSD better than ±0.3%. The most important feature of all integrated microconduits is their small and uniform size: These devices are about one-third smaller than a cig arette pack and about one-third as thick. The controlling servicing units (microprocessor, pumps, and detector electronics), like those in Bifok's FIA 5020, are in a typewriter-size unit, but will easily be accommodated within a box the size of a portable tape record er into which the integrated microcon duit will fit very much like a tape cas sette. Such a miniaturized analyzer will be suitable as a monitor for clini cal assays, as an industrial monitor (16), or even as a portable microlaboratory. It is beyond the scope of this work to elaborate on the merits of FIA pro-
cess control monitoring except to point out t h a t a number of industrial processes in Europe and the U.S. are already being controlled by first-gen eration FIA monitors. T h e integrated microFIA systems will allow the un precedented multiparameter monitor ing needed in fermentation, biotech nology, pharmaceuticals, dye manu facturing, photography, and in the oil industry. N e w Concept of Solution Handling
What we have learned so far is that FIA is based on a combination of three principles: sample injection, sample zone dispersion, and reproducible timing of the movement of the inject ed zone from the injection port into the detector. Because of the very short sampling cycle there is only one sam ple zone in the FIA flow channel at any one time. Furthermore, while in all previously developed automated systems the steady state or plateau atop the response curve is used for readout (see Figure 10), the readout in FIA has the form of a sharp peak. There is now sufficient experimental evidence t h a t a reproducible and reli able readout can be obtained from such a transient signal. Yet the con ceptual implications of this observa tion became clear only recently, when variations of the FIA method gave rise to a family of entirely new approaches to automation of quantitative assays (12,17). To understand the underlying prin ciple of FIA gradient techniques, let us consider a simple experiment. A colored solution, say of a dye, con tained within the cavity of an injection valve prior to injection, may be viewed as being homogeneous (Figure 5, left), and thus if it could be scanned by a colorimeter it would be recorded as a square signal, the height of which would be proportional to the concen tration of the dye (C°). When the valve is turned, the dye follows the movement of the carrier stream, form ing a dispersed zone, the shape of which depends on the geometry of the flow channel and the flow velocity. Therefore, the response curve has the shape of a peak reflecting a contin uum of concentrations. In contrast to all previous methods of automated assay, there is no single element of fluid that has the same concentration of dye (sample) as the neighboring one (Figure 5, right). In the majority of FIA methods de veloped so far, the top of the peak is chosen as a reference point from
Figure 3. Preconcentration of lead from seawater on a microcolumn of Chelex-100 This flame atomic absorption method is automated by an on-line FIA system with two pumps, timer (T), and the integrated microconduit (shown on top), which incorporates all components within the area en circled by the broken line on the flow diagram (bottom). A large sample volume (S = 2 mL) is injected into a carrier stream (C) and then neutralized by a buffer (B) while water is supplied into the flame; all of this is accomplished by a pump on the left. During this cycle, traces of lead are adsorbed on the col umn while proper balancing of inflowing streams (C + Β = W, where W is effluent solution to waste) ensures that no sample solution enters the detector (D). Acid (E) is used to elute lead from the top of the column in the countercurrent mode so that lead enters the flame (D) in the next cycle when the pump on the left is stopped while the pump on top is operating. Countercurrent operation of the microcolumn avoids clogging. The adhesive tape used to close the column (insert, top left) is elastic; therefore, con siderable swelling of Chelex-100 has no adverse effect on the column operation
Figure 4. Integrated microconduit for FIA The sample is aspirated into the sample loop L (volume 20 μΐ) by pump (P,) while columns of solutions in the rest of the system—part of another closed loop operated by a second pump (P2)—are kept still. As the sum of incoming streams (P2 streams, carrier C and reagent R) equals the combined outgoing streams (P2, C + R) the system is in hydrodynamic balance, and the sample zone aspirated by Pi and injected by P2 moves through channels where it disperses and reacts so that the resulting color can be measured in a flow cell (D) through which the light is transmitted by means of optical fibers
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Figure 5. FIA gradient t e c h n i q u e s While the sample zone prior to injection (left) is homogeneous and will therefore yield a square readout, the dispersed zone (right) is composed of a continuum of changing concentrations that can be employed in a number of FIA gradient techniques
which readout, that is, peak heights, is obtained (Figure 2b, H). For the same reason the dispersion coefficient has been defined as D = C°/Cma*. Yet closer examination reveals that this is just a convenient approach, as we could choose any other point on the response curve to construct the calibration curve (Figure 6), provided that the readout is always taken from the corresponding section of the dispersed sample zone. In other words, while in conventional FIA the original sample concentration (C°) is always related to peak height by taking the readout at the peak maximum (C m a x ), in gradient calibration, C° is related to a concentration at any part of the dispersed zone. This is done simply by taking the readout for a calibration curve after a certain delay time has elapsed from the moment of injection (S) (Figure 6) rather than taking the readout at the time corresponding to the peak maximum. For readouts taken at increasing delay times, e.g., 12, 14, and 16 s, the slopes of the calibration curves thus obtained will decrease as shown in Figure 6, right. The reproducibility of readouts obtained by this approach is excellent, because the zone disperses in space and time in an analogous manner each time, and since the nonsegmented carrier stream is noncompressible, the timing of arrival of the corresponding elements of the dispersed zone at the detector is exactly reproduced within each injection cycle. The practical application of the gradient technique is obvious: Should the concentration of
an unknown sample be too high to be accommodated by the detector (or by the recorder) the readout can be taken at a suitable delay and matched with the appropriate calibration curve. With the aid of a microprocessor, such data collection and processing are easy tasks. In contrast, the conventional method requires that the "out-ofrange" sample be further diluted manually and assayed once again. The next variation on the theme of continuous concentration gradient involves the use of a detector that measures not just a single characteristic (wavelength, potential, etc.), but scans a whole spectrum (Figure 7). This allows closer examination of the components, present or forming, along the decreasing concentration gradient. Besides optical spectra (absorbance vs. wavelength) electrochemical "spectra" (current vs. potential) can also be recorded on a dispersed sample zone: The three-dimensional scan (Figure 7) of a current-voltage-concentration gradient has been obtained on the tail section of a dispersed sample zone containing copper(II) acetate using a stationary mercury electrode (18). With conventional voltammetry each scan would have to be recorded in a separate solution, prepared by serial dilution. Here the scan (lasting 2 s) was repeated 50 times while a single injected zone was moving through the detector. The recorded contour map reflects the decreasing concentration of reducible Cu(II) species on the trailing edge of the peak while the "ridge" is situated on the potential of
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the copper peak. Complex formation may be studied by injecting samples into carrier streams containing complexants (19) or by letting the injected zone be penetrated by a zone of complexing agent (see below). Optimization of conditions for a chemical assay requires investigation of a number of parameters, including the ratio of reagent and sample concentration. Enzymatic assays and immunoassays can be performed successfully in a relatively narrow sample:reagent concentration ratio, and therefore it is always necessary to find, by manual serial predilution, the level at which an unknown sample may be assayed successfully. This occurs because only within a certain relatively narrow range of concentration will the analyte yield a linear calibration curve. Furthermore, in enzymatic assays the reaction rate is measured since it reflects the biochemically important catalytic activity of an enzyme. Lactate dehydrogenase is a case in point. This enzyme is present in human serum at very high levels following heart attack, but certain liver disorders cause a low level of this enzyme to be present. Thus for each assay a suitable sample dilution has to be found. Here again the dispersed sample zone offers a continuum of concentrations ideally suited for selection of the proper concentration range by simply performing reaction rate measurement in a selected section of the dispersed sample zone. This is yet another variation on the main theme of continuous concentration gradient, the gradient reaction rate method. If we inject a sample containing lactate dehydrogenase into a carrier stream containing a suitable reagent (lactate mixed with NADH), the sample zone will disperse and we can, after a certain delay time, stop the forward movement of the carrier stream by switching off the pump (2, 20). Since the dispersed zone is about 10 times longer than the path of the optical detector, it is possible to choose a small section of the dispersed zone for reaction rate measurement. As the solution is arrested within the cell, the detector measures the change of signal due to the chemical reaction only. This change with time is the reaction rate, which is in turn proportional to the catalytic activity of LDH. By repeatedly injecting solutions containing the same activity of LDH and by recording the reaction rate curves from consecutive segments of the dispersed
Figure 6. Gradient calibration is based on extracting readouts from several sections of dispersed sample zone (left) A series of delay times "slicing" through the maximum and tailing concentration gradient is shown (right) with corresponding series of calibration curves. Peaks were recorded with four concentrations of analyte labeled 25, 50, 75, and 100%
Figure 7. Gradient scanning allows a series of spectra to be recorded on a dispersed sample zone (left) The three-dimensional recording (right) shows a voltammogram of copper(ll) being reduced on a mercury electrode. The peak (forming a ridge) is located at a potential (U) at which maximum current intensity (i) is observed due to reduction of Cu(ll). Fifty scans (each lasting 2 s) were recorded on the tailing section of the dispersed zone
Figure 8. Gradient reaction rate measurement As the sample:reagent ratio decreases along the dispersed sample zone (left) the slopes of the reaction rate curve also decrease (solid arrows). By stopping the carrier stream with increasing delay, curves with decreasing slope (a-d), but improving linearity, are recorded when assaying activity of lactate dehydrogenase (LDH) (20)
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Figure 9 . Gradient penetration (a) Two valves are used to simultaneously inject equal volumes of two solutions into the carrier stream. The dispersing zones penetrate each other (b), while their individual profiles S 1 and S 2 are composites of individual peaks Si and S 2 . All curves are recorded from the same starting point
zone (Figure 8, a-d) one can easily identify the optimum delay time at which the slope of the response curve is sufficiently steep, but still linear. In other words this optimization procedure requires only "electronic" manipulation (of the delay time) whereas conventional methods require tedious identification of the optimum substrate:enzyme ratio by manual prediction. We may visualize this approach as an overlapping variation of the gradient calibration technique and the gradient reaction rate techniques: First a standard containing high LDH activity is used to identify the optimum delay/stop interval in the way described above. This allows us to measure the reaction rate as a change in the readout caused by the chemical reaction within a chosen section of the dispersed sample zone arrested in the flow cell. Finally a calibration is performed by using a set of enzyme standards at two delay times for low and high LDH activity ranges. So far we have toyed with only one zone, which was injected into the stream and allowed to flow. But what if two zones are injected and allowed to merge? The variations on this theme are many (2, 21), so let us in-
vestigate the simplest and most practical case (22). The mechanics of the experiment are straightforward; two zones are injected simultaneously and close to each other into the same carrier stream (Figure 9a). As the central stream line in a tubular channel moves at twice the mean flow velocity, the front section of the upstream zone (S2) soon catches up with the tail of the downstream zone (Si). The zone overlap will increase with the distance traveled and will also depend on the distance between the injection ports and on the injected volumes. If we inject equal volumes of the same dyed solution into a carrier stream the colorimetric detector will yield a signal, the recording of which is shown in Figure 9b. As one would expect, the combined double peak is a straightforward composite of the individual peaks recorded at separate injections of the same solution. In a real assay, when chemical reactions are involved, identical sample zone solutions will behave in a similar way and the individual readouts (Sx and S2) will be used as a reference. If we then inject different solutions by each valve we will, by comparison with our reference response,
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be able to investigate the mutual interaction of the species contained in zone 1 and zone 2 throughout a wide concentration range (22). This is indeed an entirely new approach for the study of chemical assays in particular and chemical reactions in general. The analyst's panacea would be a method which, besides being precise and sensitive, is also specific—or at least selective. The reality is far from this dream, and except for ion-selective electrodes very few attempts have been made to describe selectivity of chemical assays in exact terms. Consequently the selectivity of a chemical assay must be tested by a procedure that requires mixing of potential interferents at various concentration levels with a known concentration of the species to be determined and then performing the assay. Only in this way may one reliably find whether a positive or negative deviation from the readout obtained with the pure compound occurs. Here again the FIA gradient penetration technique is a unique tool for performing this task. If we inject a solution of a known standard by the downstream valve (Si) and a solution of a known interfèrent by the upstream valve (S2), then the peak of zone 1 will, if there is no interference, remain undistorted because zone 2 will yield no response. Yet, if the tested compound is to interfere, then distortion of the tail section of the first peak will occur because the second peak, corresponding to zone 2, will appear. That this interfering effect can be quantitatively evaluated follows from the realization that the interfering species, which would exactly mimic the one we wish to determine, will yield exactly the same double peak as if we injected the solution to be determined by both valves. In other words, the vertical displacement of point M becomes a quantitative measure of an interfering (positive or negative) effect (22). At this point a landscape of variations opens before us. The quantitative aspect of partially overlapping zones will allow us to extract a multitude of quantitative information from one sampling cycle by taking readouts at selected sections of the compounded concentration gradient. Thus if a standard solution and a real sample to be assayed are injected by the two valves, a new variant of the standard addition technique (which allows corrections to be made for unknown interferents in the assayed sample) will be developed. In fact, such a conve-
Figure 10. Automation of wet chemical assays follows the pattern of manual assays (top) in that exactly measured volumes of sample and reagent solutions are homogeneously mixed and then allowed to rest prior to measurement The same applies for air segmented continuous flow analysis (bottom), in which homogeneous mixing within individual liquid segments (insert) is effected by bolus flow. The layer of solution, which has been absorbed on the wall, slips behind, and as it is collected by the oncoming slug, causes a carryover from one segment to the next
nient experimental approach for collecting readouts from a number of solutions containing precisely varied sample:standard solution ratios is exactly what the proponents of chemometrics are looking for when searching for the instrumental tool to implement the generalized standard addition method.
Biochemical assays often use the most selective reagents available, the enzymes, but the complexity of the sample material often requires collection of additional readouts for subtraction of separate reagent and sample blanks and for checking whether interaction occurs. The gradient penetration technique using simultaneous
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injection of substrate and enzyme by valves 1 and 2, combined with the stopped-flow approach, will allow collection of all this information by taking readouts at several time delays after the moment of injection. There are other variations on the theme of dispersed sample zone [we did not discuss FIA titrations (2-8),
which also rely on the concept of dispersed sample zone], but by this time we have reviewed a sufficient number of ideas to demonstrate that they are all based on one underlying concept, and it is time to define the difference between the "old" and "new" approach to automation of chemical assays. The "Assembly Line" Approach and Flow Injection Technique
Any wet chemical assay (Figure 10) requires mixing of a precisely metered volume of a sample solution (S) with a precisely metered volume of a reagent (R), followed by a time period necessary to form a measurable product (P). Obviously, all operations must be precisely repeated each time; otherwise the conditions under which the standards and unknown have been processed will not match, and the assays will yield erroneous results. It is the advantage of automation that a machine can perform these mundane tasks tirelessly and with superhuman precision. For most assays the simple scheme outlined in Figure 10 is not sufficient, because a number of reagents usually must be used sequentially (e.g., S + Ri — Pi + R 2 — P 2 ) to produce a detected species (P2)In the past the mechanization of these operations advanced along predictable lines. The small test tubes, to which sample (or standard) solutions were mechanically pipetted, were moved by a miniconveyor belt through sequentially arranged stations where reagent(s) were added and contents mixed (by shaking or stirring) and finally transferred into a detector for measurement. The concept of the assembly line has, indeed, found application in the analytical laboratory. Variations on this theme are ingenious and numerous. Thus small test tubes have been replaced by small plastic bags, in which prepackaged reagents are stored in molded compartments (ACA, Du Pont). The linear movements of sample containers were replaced by circular motion, and even centrifugal forces were employed to achieve homogeneous mixing in socalled centrifugal analysis (Gemsac, U.S.; Cobas, Switzerland). Continuous flow analyzers differ visibly from the above-mentioned batch analyzers in that all samples move sequentially through the same tubing (Figure 10). Samples from individual cups, placed in a carousel, are aspirated by a peristaltic pump. The moving stream is segmented by air
(A), and the reagent streams join the segmented sample stream at strategic points along the main channel. Sample and reagent solutions are homogeneously mixed by multiple successful inversion of the solution segments as they pass through the mixing coils. Air segmentation (Figure 10) was used to prevent carryover of material from one sample to the next, and there has been a great deal of effort to make the air segmentation as effective as possible. Owing to the versatility of the continuous flow approach the air segmented continuous flow analyzer, designed by Skeggs more than 25 years ago (23), became the most successful tool of automation of serial assays (Technicon AutoAnalyzer, U.S.). However, if we examine closely the principles of Skeggs's system, the air segmentation and the readout of the signal at the so-called steady state, we find, to our surprise, that the idea of continuous flow analysis is a highly sophisticated variation of the assembly line concept! The conveyor belt, carrying test tubes, has been replaced by a long piece of tubing along which reagent solutions are added and homogenization performed. Each segment serves as an individual container (with walls formed by air bubbles) within which homogeneous mixing and chemical reactions take place. The imperfection of air segmentation (caused by adhesion of liquid to the surface of the tubing) has always been viewed as a flaw, as it allowed carryover of material from one segment to the next. In conclusion, it is correct to say that all automated analyzers are designed along the assembly line concept but this observation does not lead us anywhere since FIA can also be described by the same analogy. We need to look at the way solutions are being "assembled." In the design of all automated analyzers prior to FIA a conceptual dilemma had to be resolved: on one hand the desire to mix the sample homogeneously with the reagent contained in the carrier stream and on the other hand the need to prevent the resulting loss of sampling frequency due to intermixing of adjacent samples. Yet it is only the manual handling that requires sample and reagent solutions to be mixed homogeneously. There is no way of shaking a test tube to produce exactly the same concentration gradient each time, and this is why the "end stage," that of a homogeneous mixture, is the only one defined and useful for manual and batch assays. This is the crucial differ-
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ence between the old and new concept: homogeneous mixing vs. the controlled dispersion of solutions. Whenever reacting solutions are brought together two processes, both kinetic in nature, take place—mixing and subsequent chemical reaction(s). By replacing the concept of homogeneous mixing by the concept of dispersion of a zone into a well-controlled concentration gradient, an entirely new approach to design and construction of automated analyzers was discovered. It is only now obvious that the manual approach was so firmly entrenched that much effort has been made to make all automated analyzers function in the test-tube-like fashion. Besides serial assays, there are many areas of research in which interaction between solutions (and solid or liquid surfaces) have to be carefully controlled and monitored. Here also the flow injection technique may be applied. Thus diffusion coefficients have been measured (24) by the method based on peak broadening (25). Response time of ion-selective electrodes and/or CHEMFETs (26), diffusion of ions or gases through membranes, or critical micelle concentration are parameters that can be conveniently measured by flow injection, which thus achieves a significance broader than that of a simple analytical tool. It would be tempting to continue speculations on the future use of FIA and to manufacture variations on the theme of future use of concentration gradients, but we would soon stray too far into the imaginary without experimental evidence, which up to this point has guided us to what is real and practical. It is therefore time to conclude the story of FIA by a comment on invention of the theme—or of flow injection in this case. It is true to say that the roots of what we have named FIA can be traced far back to the use of nonsegmented streams (27), to the use of sample injection (28), or even to the continuous flow approach of Skeggs. Yet in retrospect we recognize that this new technique emerged only when its three features—sample injection, controlled dispersion, and exact timing—were combined in a unified experimental approach. The flow injection technique has truly been discovered only now that its underlying concept has been identified and its variations have led us to design of gradient methods. Neither the abacus nor the test tube was capable of performing the job in a dynamic fashion. Yet here we are within the
r e a c h of " t h e chemical c o m p u t e r " b a s e d on t h e microchemielectronic d e vices, which i n t e g r a t e s a m p l e injection, solution-handling t e c h n i q u e s , a n d sensors coupled t o microelectronics. T o h a n d l e gases, m u c h n a r r o w e r c h a n n e l s will be needed, a n d different c o n s t r u c t i o n materials a n d sensors will have t o be applied. T h e ingenious m i c r o gas c h r o m a t o g r a p h developed a t S t a n f o r d University (29) is a p r o t o t y p e of m a n y future microchemielect r o n i c devices for h a n d l i n g a n d m o n i toring of gases. T h e r e , also, principles of F I A will find application.
Acknowledgment I wish t o express m y g r a t i t u d e t o Elo H. H a n s e n w i t h o u t whose inspirat i o n a n d assistance very little would h a v e been accomplished. W e b o t h owe m u c h t o our Swedish colleagues, B o K a r l b e r g , T o r b j ô r n Anfàlt, R u n e L u n din, a n d Stig E k l u n d , for m a k i n g F I A accessible t o t h e public a n d t o our American colleagues, Joel H a r r i s a n d J i r i J a n a t a from t h e University of U t a h , for their c o n t r i b u t i o n s a n d expertise in microdetector technology. W e also t h a n k Don Olson from Shell C o m p a n y , H o u s t o n , Tex., for t u r n i n g our a t t e n t i o n t o aspects of i n d u s t r i a l monitoring. A n d in D e n m a r k t h a n k s a r e d u e t o our co-workers a t t h e C h e m i s t r y D e p a r t m e n t A for friendly discussions, t o Broo S o r e n s e n for p r e paring t h e illustrations, a n d t o t h e D a n i s h N a t i o n a l Council for Scientific a n d I n d u s t r i a l R e s e a r c h for financial assistance.
(12) Rùziëka, J.; Hansen, E.H.H. Anal. Chim. Acta 1983,145, 1. (13) Cattrall, R. W.; Freiser, H. Anal. Chem. 1971, 44, 586; and Freiser, H. In "Ion-Selective Electrodes in Analytical Chemistry"; Plenum Press: New York, N.Y., 1980; Vol. 2, Chapter 9. (14) Moody, G. J.; Oke, R. B.; Thomas, J.D.R. Analyst 1970, 95, 910. (15) Moody, G. J.; Thomas, J.D.R. In "IonSelective Electrodes in Analytical Chemistry"; Freiser, H., Ed.; Plenum Press: New York, N.Y., 1978; Vol. 1. (16) Haagensen, P.; Rûzicka, J.; Sçjborg, H., work in preparation. (17) Rùziëka, J. Phil. Trans. R. Soc. London A, 1982,305, 645. (18) Janata, J.; Rùziëka, J. Anal. Chim. Acta 1982,139,105. (19) Betteridge, D.; Fields, B. Anal. Chem. 1978,50, 654. (20) Olsen, S.; Rùziëka, J.; Hansen, E.H.H. Anal. Chim. Acta 1982,136,101. (21) Bergamin, F. H.; Zagatto, E.A.G.; Krug, F. J.; Reis, B. F. Anal. Chim. Acta 1978,101,11. (22) Hansen, E.H.H.; Rùziëka, J.; Krug, F. J.; Zagatto, E.A.G. Anal. Chim. Acta 1983,148, 111. (23) Skeggs, L. T. Am. J. Clin. Path. 1957, i3,451. (24) Gerhardt, G.; Adams, R. N. Anal. Chem. 1982,54,2618. (25) Vanderslice, J. F.; Stewart, Κ. Κ.; Higgs, D. J. Talanta 1981, 28, 11. (26) Hammerli, Α.; Janata, J. J.; Brown, Η. Μ. Anal. Chim. Acta 1982,144,115. (27) Mottola, H. A. Anal. Chem. 1981,53, 1313 A. (28) Stewart, Κ. Κ. Talanta 1981,28, 789. (29) Agnell, J. B.; Terry, C. S.; Barth, P. W. Sci. Am. April 1983,248, 44-55.
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References (1) Betteridge, D. Anal. Chem. 1978, 50, 832 A. (2) Rûzicka, J.; Hansen, E.H.H. "Flow Injection Analysis"; J. Wiley and Sons: New York, N.Y., 1981. (3) Rocks, B; Riley, C. Clin. Chem. 1982, 28, 409. (4) Holy, H. W. J. Autom. Chem. 1982, 4, 111. (5) Margoshes, M. Anal. Chem. 1981,53, 1313 A. (6) Hansen, E.H.H.; Rùziëka, J. Trends Anal. Chem. 1983,2 (V). (7) Riley C ; Rocks, B. F. J. Autom. Chem. 1983,5,1. (8) Ramsing, Α.; Rùziëka, J.; Hansen, E.H.H. Anal. Chim. Acta 1981,129,1. (9) Rùziëka, J.; Hansen, E.H.H.; Janata, J. Danish Patent Appl. No. 4296/82; U. S. Patent Appl. No. 478 227. (10) Olsen, S.; Pessenda, L.C.R.; Rûzicka, J.; Hansen, E.H.H. Analyst, August 1983. (11) Rùziëka, J.; Hansen, E.H.H. Danish Patent Appl. No. 81.5148,1981, and subsequent U. S. Patent Appl. No. 385 049.
"Robotics Update"
Jaromir Rûzicka holds a chair in analytical chemistry at the Technical University of Denmark and is an adjunct professor in the Department of Chemistry at the University of Utah. He is a graduate of the Charles University, Prague, and holds PhD and Doctor of Natural Science degrees. He is a past president of the Danish Society for Analytical Chemistry and a member of the Danish Academy of Technical Sciences. His research interests are trace analysis, electrochemical detectors, and automation of instrumental analysis with an emphasis on FIA.
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983 · 1053 A
