Report Kent K. Stewart Department of Food Science and Technology Virginia Polytechnic Institute and State University Blacksburg, Va. 24061
Flow Injection Analysis NewTool for Old Assays New Approach to Analytical Measurements
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 (1-5), one textbook (6), 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 be 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 0003-2700/83/0351 -931 A$01.50/0 © 1983 American Chemical Society
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 by 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 la and lb. Figure la presents the popular European version of a semiautomated FIA system (10) 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. (11) 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 · 931 A
Figure 1. Basic FIA systems (a) Schematic of a semiautomated FIA system, (b) Schematic of an automated FIA system. Reprinted with permission from Reference 12
Figure 2. Recorder tracing of an FIA determination of serum albumin with bromcresol green Reprinted with permission from Reference 13
Figure 3. Schematic of an automated FIA-diiution system Reprinted with permission from Reference 12
932 A · ANALYTICAL CHEMISTRY, VOL. 55, NO. 9, AUGUST 1983
at 100 or more samples/h. 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 (1-8). It 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 (12,14). 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
Figure 4. Concentrations at the detector of several FIA systems (a) Concentration of an analyte injected into a standard system (Figures 1a and 1b). (b) Concentration of the reagent if the system in Figure 1a is used, (c) Concentration of the reagent if the system in Figure 1b is used, (d) Product concentrations when system 1a 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 section.) While the shorter reaction times might appear to limit the number of CFA assays that can be adapted, preliminary studies suggest that although the CFA systems may have reaction times in minutes, the chemically analogous FIA systems often have reaction times in seconds. This probably occurs because the CFA reaction times are actually longer than necessary for sufficient product formation and because of the more limited dispersion of FIA systems. While there are several differences between the classical CFA system of Skeggs (16) and FIA, the author believes that the two systems are closely related and are complementary tools for the analyst. The CFA systems appear to be more suitable for assays requiring more than 2 min reaction time and/or that require the sequential addition of three or more reagents; FIA systems appear to be more suitable for assays requiring 30 s or less and use only one 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 1 and 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 samples/h. 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 has 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 I s — Chemistry 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
ANALYTICAL CHEMISTRY, VOL. 55, NO. 9, AUGUST 1983 · 933 A
bolus into a moving stream inside a segment of narrow-bore tubing and the measurement of the analyte at some point downstream. The theoreti cal description of the time-dependent concentration profile of analytes, re agents, 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 mod ern theory, there is disagreement as to what is the best theoretical descrip tion. One camp prefers the "tanks in series" model (21 ) in which the disper sion 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 numeri cal solutions of the diffusion-convec tion equations in the regions in which FIA systems are usually operated. The two key parameters predicted by Van derslice et al. are the time (t„) from in jection to the initial appearance of sample bolus at the detector and the baseline to baseline time (Aij,) for each sample bolus at the detector (see Figure 4a). Equations 1 and 2 show the relationship of the crucial parame ters needed to predict these two times. The definition of the symbols is given in Table I. /Γ\ΐ.025
ta = 109 α 2 D 0 0 2 5 Δί,,
Figure 5. Relative concentration gradients inside FIA tubing at different τ (reduced time, see Table I) The numbers on the gradient profiles are normalized values when initial concentrations of 10 are inject ed, (a) A convection-controlled region (r = 0.004). (b) A convection-diffusion-controlled region (τ = 0.092), a common region of FIA systems, (c) A diffusion-controlled region (τ = 0.8), the Taylor re gion. This region is common for CFA systems, uncommon for FIA systems
tubing is a time-dependent function (See Figure 4). These and other fea tures of FIA require that the serious student of FIA examine the theoreti cal basis of assays to be performed in FIA systems. For example, there are several means of mixing samples with reagents in FIA. Two common ap proaches 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 sam ple into a carrier stream and then mix the carrier stream with a reagent stream (Figure lb). With the former,
the reagent concentration is not con stant along the sample bolus (Figure 4b), and it is possible in reagent-limit ing situations to obtain a decrease in the product concentration in the mid dle of the sample bolus (see Figure 4d). However, if the reagent concen tration 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 de scribed as the insertion of a sample
934 A · ANALYTICAL CHEMISTRY, VOL. 55, NO. 9, AUGUST 1983
35.4 α5 L\0.64 £)036
(1) (2)
QI
It is interesting to note that while dis persion is traditionally represented in units of volume, these authors repre sent it in units of time. In FIA systems time is a common unit and is rather easily measured; volume can be mea sured directly but it is rather cumber some to do so. An examination of the concentra tion profiles inside a tubing segment (Figure 5) demonstrates that not only does the bolus shape change with the changing parameters but the concen tration profile also changes inside the bolus. These changes can have signifi cant implications for those investigat ing such areas as kinetic measure ments in FIA systems (23). Recently Gerhardt and Adams (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 diffu sion constants with good precisions.
Thus, an elegantly simple new tool has been developed for the measurement of physical constants, and Vanderslice's theoretical view of FIA is inde pendently confirmed. Theory of Pseudotltrations and Exponential Dilution Chambers
A special type of FIA system is shown in Figure 6, the FIA pseudotitration system. This was originally de scribed as an FIA titration system (25). However, as Pardue and Fields (26) have pointed out, titration is a term reserved for analytical systems in which the amounts of titrant and analyte are either equal or integral multi ples of each other. Since in FIA titra tions it is the concentrations of the ti trant and analyte that are equivalent, and not the amounts, these systems have more recently been called pseu dotltrations (27). A number of work ers have described ingenious designs for FIA pseudotltrations (28-35). In the FIA pseudotltrations the sample dispersion is manipulated so as to pro vide an exponential increase in con centration followed by an exponential decrease in concentration. Such dis persion can be obtained with either a stirred mixing chamber or a suitable length of wide- or narrow-bore tubing. The resulting concentration profiles are shown in Figure 7. Under these conditions Equation 3 is applicable. The symbol definitions are given in Table I. Atpq = K4 In
^
as
+ K,
Table 1. Definition of Symbols a ^
Internal radius of tubing in c m as
° reg D K1 and K 2 f fa
Atb Ateq
Original concentration of analyte Original concentration of titrant Diffusion constant of analyte in c m / s Constants of individual FIA systems Time in seconds Time from injection to initial appearance of peak
L
Time from initial appearance of peak to final appearance of peak Equivalence time for pseudotltrations and scale expansion systems Reduced time (equals Dt/a2) Length of reaction tubing in c m
q
Flow rate in mL/min
τ
(3)
The FIA pseudotltrations are a unique type of measurement system in which the measured time is propor tional to the concentration of the ana lyte. There is no unambiguous analo gous measurement system in the tra ditional equilibrium assay systems that the author is aware of and only a few analogous measurement systems in the kinetic assays in stirred con tainers. Pardue and Fields have re cently developed the theoretical basis for these pseudotitration systems (26, 36). It is interesting to note that this work not only explains the obser vations made by Ruzicka et al. (25) on the relationship of the baseline to baseline time to concentration, but also provides the basis for under standing the early observations that peak height was related to concentra tion in such systems (37). Recent work has shown that the time measure ments may also be used under much more general conditions and that stirred mixing chambers may also be
Figure 6. Schematic of an automated FIA system for pseudotltrations
Figure 7. Concentration profiles for FIA pseudotltrations (a) Concentration profiles for three different samples with original concentrations C 1 , C2, and C3 as the effluent emerges from the mixing chamber, if no titrant was present, (b) Concentration profiles of the three samples after reacting with the titrant
936 A
·
ANALYTICAL
CHEMISTRY,
V O L . 5 5 , N O . 9, A U G U S T
1983
u s e d in a scale e x p a n s i o n s y s t e m for m a n y assay s y s t e m s (27). In t h i s t y p e of s y s t e m t h e mixing c h a m b e r is placed i m m e d i a t e l y after t h e s a m p l e i n s e r t i o n valve, a n d t h e r e a g e n t is m i x e d with t h e effluent from t h e mix ing c h a m b e r . U n d e r t h e s e conditions t h e c o n c e n t r a t i o n of t h e s a m p l e s can be empirically d e t e r m i n e d using E q u a t i o n 4.
AtP
Ki In C°as + K2
(4)
T h e general n a t u r e of t h e s y s t e m was d e m o n s t r a t e d by i n c o r p o r a t i o n i n t o F I A s y s t e m s using colorimetric, fluorimetric, c o n d u c t o m e t r i c , a n d flame emission s y s t e m s . Scale e x p a n sions of two- to thirtyfold were o b t a i n e d in t h e s e cases. It is m o s t likely t h a t such a scale e x p a n s i o n s y s t e m can b e i n c o r p o r a t e d into a n y s t a n d a r d F I A system. S u c h scale e x p a n s i o n s y s t e m s could h a v e p r o f o u n d effects on t h e design of f u t u r e F I A s y s t e m s . For e x a m p l e , since t h e p r i m a r y c o n c e n t r a t i o n mea s u r e m e n t is t i m e , t h e d e t e c t o r is n e e d ed only as a trigger a n d t h u s t h e d e t e c t o r r e s p o n s e n e e d n o t be linear w i t h c o n c e n t r a t i o n ; it only n e e d s t o yield r e p r o d u c i b l e t i m i n g p o i n t s . In a similar fashion, t h e d a t a acquisition s y s t e m n e e d be only a relatively sim ple digital clock coupled w i t h a micro processor for e x p o n e n t i a l o p e r a t i o n a n d m u l t i p l i c a t i o n . F u r t h e r m o r e , only sufficient r e a g e n t is n e e d e d to p r o d u c e e n o u g h p r o d u c t t o activate t h e trigger; a d d i t i o n a l r e a g e n t is n o t n e e d e d t o r e a c t w i t h higher c o n c e n t r a t i o n s of t h e a n a l y t e . T h u s t h e r e is a p o t e n t i a l for r e a g e n t cost r e d u c t i o n . It is diffi cult t o visualize w h e r e all t h i s will lead. Certainly it suggests t h a t s i m p l e r and/or miniaturized FIA systems could be developed using t h e scale ex pansion system. R e c e n t s t u d i e s h a v e suggested t h a t a l t e r n a t i v e s to t h e use of n a r r o w - b o r e t u b i n g as a reaction c h a m b e r can be q u i t e useful. S o m e h a v e suggested t h a t tightly coiled t u b e s (38) be used; o t h e r s h a v e suggested t h a t p a c k e d b e d s (39) be used. O n e of t h e m o s t p r o m i s i n g a l t e r n a t i v e s a p p e a r s t o be t h e t h r e a d e d - b e a d r e a c t o r s (40) in which t h e reaction c h a m b e r consists of a l e n g t h of t u b i n g p a c k e d w i t h b e a d s of d i a m e t e r s on t h e o r d e r of 50% of t h a t of t h e t u b i n g . I t is n o t clear w h e t h e r or n o t l a m i n a r flow prevails in such a system. O n e would s u s p e c t not.
Conclusion T h e technically correct view of F I A is t h a t it is a kinetic m e a s u r e m e n t sys
t e m in which t h e s y s t e m is n o r m a l l y not completely mixed and the radial a n d axial s a m p l e c o n c e n t r a t i o n p r o files are t i m e - d e p e n d e n t functions. I believe t h a t t h i s view of F I A will b e very p r o d u c t i v e in t h e long r u n . W h i l e it is difficult t o p r e d i c t t h e o u t c o m e , several exciting o b s e r v a t i o n s a l r e a d y h a v e been m a d e using t h i s view of t h e s y s t e m . T h e m e a s u r e m e n t of diffusion c o n s t a n t s a n d t h e use of t i m e for con c e n t r a t i o n m e a s u r e m e n t s in F I A sys t e m s a r e two e x a m p l e s of t h e p o t e n t i a l of t h e s y s t e m . It is obvious t h a t m u c h r e m a i n s t o be d o n e in t h e develop m e n t of F I A . T h e n e w t h e o r e t i c a l con c e p t s h a v e y e t t o be c o m p l e t e l y ex ploited. N e w r e f i n e m e n t s are being developed a t an a m a z i n g r a t e . As F I A systems become better understood new c o n c e p t s of m e a s u r e m e n t of a n a lyte c o n c e n t r a t i o n will n o d o u b t be possible. C e r t a i n l y t h e possibilities for increased s a m p l e t h r o u g h p u t , s y s t e m o p t i m i z a t i o n , a n d m i n i a t u r i z a t i o n of F I A s y s t e m s have b a r e l y b e e n s c r a t c h e d . O n e can only look t o t h e fu t u r e of t h i s field w i t h great e n t h u siasm.
References (1) Betteridge, D. Anal. Chem. 1978, 50, 832-46 A. (2) Ruzicka, J.; Hansen, E. Chemtech. 1979, 9, 756-64. (3) Ruzicka, J.; Hansen, E. Anal. Chim. Acta 1980,114, 14-19. (4) Ranger, C. B. Anal. Chem. 1981, 53, 20-32 A (5) Rocks, B.; Riley, C. Clin. Chem. 1982, 28, 409-21. (6) Ruzicka, J.; Hansen, E. "Flow Injection Analysis"; Wiley: New York, N.Y., 1981. (7) Mottola, H. A. Anal. Chem. 1981, 53, 1312-16 A. (8) Stewart, Κ. Κ. Talanta 1981, 28, 78997. (9) Snyder, L.; Levine, J.; Stoy, R.; Connetta, A. Anal. Chem. 1976, 48, 942-56 A. (10) Ruzicka, J.; Hansen, E. Anal. Chim. Acta 1975, 78,145-57. (11) Beecher, G. R.; Stewart, Κ. Κ.; Hare, P. E. In "Protein Nutritional Quality of Foods and Feeds," Proceedings of an ACS symposium entitled "Chemical and Biological Methods for Protein Quality"; Friedman, E. M , Ed.; 1975; Pt. 1, pp. 411-21. (12) Stewart, K. K.; Brown, J. F.; Golden, Β. Μ. Anal. Chim. Acta 1980, 114, 11927. (13) Renoe, B. W.; Stewart, Κ. Κ.; Beech er, G. R.; Wills, M. R.; Savory, J. Clin. Chem. 1980, 26, 331-34. ( 14) Slanina, J.; Lingerak, W. Α.; Bakker, F. Anal. Chim. Acta, 1980, 117, 91-98. (15) Bergmeyer, H. U.; Hagen, A. Fresenius Z. Anal. Chem. 1972, 261, 333-36. (16) Skeggs, L. T. Am. J. Clin. Pathol. 1957,28,311-22. (17) Ruzicka, J.; Hansen, E. Anal. Chim. Acta 1979, 106, 207-24. (18) Malmstadt, H. V.; Walczak, Κ. Μ.; Koupparis, M. A. Am. Lab. September 1980, pp. 27-40.
940 A · ANALYTICAL CHEMISTRY, VOL. 55, NO. 9, AUGUST 1983
(19) Pardue, H. L. Clin. Chem. 1977,23, 2189-201. (20) Taylor, G. Proc. Roy. Soc. A. 1953, 219, 186-203. (21) Ruzicka, J.; Hansen, E. Anal. Chim. Acta 1978, 99, 37-76. (22) Vanderslice, J. T.; Stewart, Κ. Κ.; Rosenfeld, A. G.; Higgs, D. J. Talanta 1981,28,11-18. (23) Painton, C. C ; Mottola, H. A. Anal. Chem. 1981,53, 1715-17. (24) Gerhardt, G ; Adams, R. N. Anal. Chem. 1982, 54, 2618-20. (25) Ruzicka, J.; Hansen, E.; Mosbaek, H. Anal. Chim. Acta, 1977,92, 235-49. (26) Pardue, H. L.; Fields, B. Anal. Chim. Acta 1981,124, 39-63. (27) Stewart, K. K.; Rosenfeld, A. G. Anal. Chem. 1982, 54, 2368-72. (28) Nagy, G.; Toth, K.; Pungor, E. Anal. Chim. Acta 1975, 47, 1460-62. (29) Horvai, G.; Toth, K.; Pungor, E. Anal. Chim. Acta 1976, 82, 45-54. (30) Nagy, G.; Feher, Z.; Toth, K.; Pungor, E. Anal. Chim. Acta 1977, 91, 87-96. (31) Nagy, G ; Feher, Z.; Toth, K.; Pungor, E. Anal. Chim. Acta 1977, 91, 97-106. (32) Nagy, G.; Feher, Z.; Toth, K.; Pungor, E. Anal. Chim. Acta 1978,100, 181-91. (33) Astrom, O. Anal. Chim. Acta 1979, 205,67-75. (34) Ramsing, A. U.; Ruzicka, J.; Hansen, Ε. Η. Anal. Chim. Acta 1981,129, 1-17. (35) Stewart, K. K.; Rosenfeld, A. G. J. Automatic Chem. 1981, 3, 30-32. (36) Pardue, H. L.; Fields, B. Anal. Chim. Acta. 1981,224,65-79. (37) Nagy, G.; Feher, Z.; Pungor, E. Anal. Chim. Acta 1970, 52, 47-54. (38) Tijssen, R. Anal. Chim. Acta. 1980, 224,71-89. (39) Van Den Berg, J.H.M.; Deelder, R. S.; Egberink, H.G.M. Anal. Chim. Acta 1981,224,91-104. (40) Reijn, J. M.; Van der Linden, W. E.; Poppe, H. Anal. Chim. Acta 1981, 123, 229-37.
Kent K. Stewart is professor and head of the Department of Food Science and Technology at VPI & SU. He received his AB in chemistry at the University of California at Berkeley and his PhD in chemistry at Florida State University in 1965. His current research interests include flow injection analysis, laboratory automation, and the analytical chem istry of foods. He has been working on the development of flow injection analysis since 1972.
