Kent K. Stewart Department of Food Science and Technology Virginia Polytechnic Institute and State University Blacksburg, Va. 24061
Flow InjectionAnu/y~.NewTool for Old h a y s New Approach to Analy
Since its conception in the early 1970s flow injection analysis (FIA) has grown enormously. Today many believe that it has the potential to become the method of choice for many automated and semiautomated assays. A number of reviews (Z4), one textbook (61, and two views of its early history (7,8)have appeared. Papers on all aspects of FIA are appearing with increasing frequency in the literature. In this REPORT the standard uses of FIA will be briefly reviewed, and the unique features of FIA as an analytical measurement system will he discussed. No attempt will be made to provide a comprehensive review of FIA systems; rather the author's goal is to provide a general overview and some discussion and speculation to stimulate further development of FIA systems and their uses.
Standard FIA Systems for Standard Assays In most classical assay systems, the reagents and samples are placed in a test tube, beaker, cuvette, etc., allowed to react for a period of time, and then transferred into some detecting system where a measurement is made. These batch operations have been 0003-2700/831035 1-93 1A$01.50/0 i' 1983 American Chemical Sociew
used for making either kinetic or equilibrium analytical measurements. In the vast majority of the assay systems the analytes, reagents, products, etc., are uniformly distributed throughout the reaction vessel. Virtually all the theoretical considerations are based on uniform distribution. This type of thinking predominates today and influences how most analysts view analytical processes. Even with continuous flow analysis (CFA), analysts are encouraged to view the system as if it were a series of small beakers separated hy air bubbles (9). FIA could be perceived as a CFA system without bubbles and thus as an extension of the beakers-on-a-conveyor-belt concept. This analogy is incorrect, and serious errors can occur if the analyst views FIA in this way without any qualifications. However, many beaker, test tube, and cuvette assays can be adapted to FIA determinations using the beakers-on-a-conveyor-belt analogy if empirical systems are used (i.e., if the concentration of the unknown is determined by comparison with a standard curve prepared by running a series of standards in the same system). There are numerous examples of such FIA assay systems that yield
rapid, precise results. FIA analytical systems that rely on absolute measurements have not been developed. The basic FIA systems for mixing sample and reagents, reacting them, and getting a readout are shown in Figures l a and lb. Figure l a presents the popular European version of a semiautomated FIA system ( 1 0 ) in which a sample is inserted into an unsegmented stream of reagent pumped by a peristaltic pump, the analyte is mixed with the reagent by convective and diffusion forces, and the product is measured as it passes through the detector. Peak height measurements are normally made. Figure l b represents the version first introduced in the U.S. (ZZ) in which the sample is aspirated from a sample cup in a sampler tray into the sample loop of a sample insertion valve. Then the valve is actuated, and the sample is inserted into an unsegmented continuous stream of sample solvent that is mixed with a reagent stream, and the resulting mixture flows on to the detector as before. Depulsed positive displacement pumps are normally used. Peak area and/or peak height measurements can be made. Both systems can perform routine replicate assays
ANALYTICAL CHEMISTRY', VOL. 55. NO. 9, AUGUST 1983
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Figure 1. Basic FIA systems (a) Schematic 01 a semiautomated FIA system. (b) Schematic of an automated FIA System. Reprinted with permission from Referr--- 4 n
t
.
.--re 2. Recorder tracing of an FIA determination of serum albumin with bromcresol green Reprinted with permission from Reference 13
at 100 or more samplesh. In many cases, results for individual samples can be obtained within 15 s after the sample is inserted into the system. Precisions ranging from 0.5 RSD to 2.0 RSD have been reported repeatedly for a wide variety of individual assays. Typical FIA recorder tracings are shown in Figure 2. Critical to the success of the systems is the use of smallbore tubing (commonly 0.5-mm i.d.), the use of precisely controlled flow rates (1-14 mL/min), and minimization of system mixing volumes. These features result in a minimized and controlled sample dispersion, one of the unique aspects of FIA. These simple concepts have proved extremely successful in the development of analytical systems for a wide variety of analytes. FIA has been used with many different types of detectors, including colorimetric, fluorimetric, flame emission, atomic absorption, inductively coupled plasma, refractive index, chemiluminescence, thermochemical, and a variety of electrochemical detectors (2-8). I t is probable that any detector that can be used with HPLC systems can be used with FIA systems. FIA systems are obvious candidates for computer interfacing, and several papers have discussed the combination of computers and FIA systems ( 1 2 , 2 4 ) . FIA assay systems have been described for many different compounds (1-8) for use in wide variety of areas, such as clinical chemistry, agricultural chemistry, environmental chemistry, biochemistry, and immunological chemistry. Enzyme assay systems were some of the earliest FIA systems described (11, 15) and the precision of the FIA assays makes them quite attractive for enzyme determinations. Several workers have described novel FIA enzyme systems including stopped-flow systems and enzyme reactors ( 5 , 6 ) .It is likely that many more FIA assays will be developed in the near future. I predict that most of the assays developed for CFA could be readily adapted to FIA systems. Many of the special techniques used in CFA systems such as dialysis, two-phase systems, and merging zones have already been adapted for FIA systems. At present, the usual requirements for the adaptation of the manual and CFA assays to FIA systems are that the analytes, reagents, and products are soluble in the assay solvent, that sufficient analyte or product be developed within 60 s, and that sample dispersion is rigorously controlled and
1 Figure 3. Schematic of an automated FIA-dilution s! Reprinted with permission from Reference 12
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Figure 4. Concentrations at the detector of several FIA sys (a)Concentration 01 an analyie injected into a standard System (Figures la and lb). (b) Concentration of the reagent if the system in Figure l a is used. (c) Concentration of the reagent if the System in Figure l b is used. (d) Product Concentrationswhen System la is used with excess reagent concentrations (blue)and With inadequate reagent concentrations (red) minimized. (Some special cases where extensive dispersion is useful will be discussed in another sertion.) While the shrlrtw reaction times might appear t n limit the number of CFA ilssays that can be adapted, preliminary studies suggest that although the CFA systems may have reaction times in minutes, the chemirally analogous FIA systems often have reartion times in seconds. This probably occurs because the CFA reactinn times are actually longer than necessary for sufficient product formation and berause of the more limited dispersion of FIA syscems. While there are several differences between the classical CFA system of Skeggs (16) and FIA, the author believes that the two syscems are closely related and are complementary tools for the analyst. The CFA systems appear to he more suitable for assays requiring more than 2 tnin reaction rime andlor that require the sequential addition uf threr or more reagents; FIA systems appear to be more suitable for assays requiring 30 s or less and use unly une or two sequential reagent ad-
ditions. Many assay chemistries can be used with either flow system.
Special Uses for Standard FIA Systems In addition to the systems described above, stopped-flow systems and dilutors for FIA systems are basically extensions of traditional beaker analytical chemical systems. Ruzicka and coworkers have developed FIA stoppedflow systems (17)as an extension of the system shown in Figure 1and Malmstadt et al. have developed an FIA stopped-flow system using syringe pumps (18).Successful stoppedflow assays have been developed with sample throughputs of about 100 samplesh. Stewart et al. (12) have developed an FIA automated dilutor system, shown in Figure 3. The sample dilution is controlled by the sample loop size, the diluent flow rate, and the time between fractions in the fraction collector. The use of standard FIA systems as a means of automating classical assays bas been remarkably successful. The combination of FIA with the classical
procedures has resulted in a marked increase of sample throughput usually accompanied by increased precision and a decrease of required operator skill and time.
FIA As It Really IsChemistry in Flowing Streams Basically all FIA systems fit into Pardue’s classification of a kinetic method of analysis (19). An FIA system is in equilibrium only when there is no sample in the system. Not a very interesting case! While assays based on a theoretical assumption of equilibrium conditions can (and frequently are) used in FIA systems, interpretation of the results requires caution. Empirical methods often work; however, the underlying principles are not always the same. For example, in traditional assays, analytes, reagents, and products are usually uniformly distributed throughout a beaker, test tube, or cuvette; in FIA, the analyte is not evenly distributed throughout the system. Rather the sample bolus is inserted into a moving stream, and its concentration distribution along the
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 9, AUGUST 1983
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bolus into a moving stream inside a segment of narrow-bore tubing and the measurement of the analyte a t some point downstream. The theoretical description of the time-dependent concentration profile of analytes, reagents, and products under these rather simple conditions is currently under debate. While there is general agreement that the work of Taylor (20) is the starting point of the modern theory, there is disagreement as to what is the best theoretical description. One camp prefers the “tanks in series” model (21) in which the dispersion of an injected bolus is estimated by using a classical model of a series of totally mixed tanks. The alternative approach is that of Vanderslice et al. (22) in which the flow is assumed to be completely laminar and the dispersion of a sample bolus is based on numerical solutions of the diffusion-eonvection equations in the regions in which FIA systems are usually operated. The two key parameters predicted by Vanderslice et al. are the time (t,) from injection to the initial appearance of sample bolus at the detector and the baseline to baseline time ( A b ) for each sample bolus a t the detector (see Figure 4a). Equations 1and 2 show the relationship of the crucial parameters needed to predict these two times. The definition of the svmbols is aiven in Table I.
P Wall 2
7
= 0.092
1
ZI 2
3
3
2
1
Tubing Wail
1 Figure 5. Relative concentration gradients inside FIA tubing at different T (reduced time, see Table I) The numbers on Um gradient profilesare normalized values when initial wncentrations of 10 are injected. (a) A convection-conkoiied region (7 = 0.004). (b) A ConVeCliondiffUSionSOntr~ii~ region (r = 0.092). a common region of FiA systems. (cl A diflusion-controiied region ( r = O.W, the Taylor region. This region is m m o n for CFA systems. uncommon for FiA system
tubing is a time-dependent function (See Figure 4). These and other features of FIA require that the serious student of FIA examine the theoretical basis of assays to be performed in FIA systems. For example, there are several means of mixing samples with reagents in FIA. Two common approaches are to insert the sample into the reagent and mix the sample and reagent by convective and diffusion forces (Figure la) or to insert the sample into a carrier stream and then mix the carrier stream with a reagent stream (Figure lb). With the former,
the reagent concentration is not constant along the sample bolus (Figure 4b), and it is possible in reagent-limiting situations to obtain a decrease in the product concentration in the middle of the sample bolus (see Figure 4d). However, if the reagent concentration is constant along the sample bolus (see Figure 4c) the probability of product concentration dips is quite small.
Theoretical Considerations The simplest FIA system can be described as the insertion of a sample
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It is interesting to note that while dispersion is traditionally represented in units of volume, these authors represent it in units of time. In FIA systems time is a common unit and is rather easily measured; volume can be measured diredly but it is rather cumber. some to do so. An examination of the concentration profiles inside a tubing segment (Figure 5) demonstrates that not only does the bolus shape change with the changing parameters but the concentration profile also changes inside the bolus. These changes can have significant implications for those investigating such areas as kinetic measurements in FIA systems (23). Recently Gerhardt and A d a m (24) used Vanderslice’s equations with FIA systems to obtain diffusion constants for a number of compounds. These workers demonstrated that FIA can be effectively used to get accurate diffusion constants with good precisions.
Yo*c"l.r
New tools for Drotein sep'arations.
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670.000 150.000 45.000 77,500 1.355
2
020
-
015
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