Injec Analysis
Jaromir Ruzicka University of Washington
Elo Harald Hansen Technical University- of Den
BROO SORENSEN
A
lthough the analytical research of the 1990s focused on life sciences and pharmacology and that of the 1980s focused on process control, the thrust in the 1970s was toward trace analysis and clinical assays. At that time, atomic absorption (AA) spectroscopy and ion-selective electrodes were the most active research areas, followed by separations. Advanced solution spectroscopies (FT-IR, Raman, and UV–vis scanning) were yet to blossom. The most striking difference between the mid-70s and now is the presence of personal computers in the laboratory. Major, well-equipped universities, such as the newly built facilities at the Technical University of Denmark, where we conceived flow injection analysis (FIA) in the early spring of 1974, had a central computing facility. However, when we tried to purchase a Hewlett-Packard model HP-35 hand-held calculator, we were severely reprimanded by both the chair of the department and the administration for this outrageous expense. We were told either to learn FORTRAN 1 and use the central mainframe computer or to remain satisfied with paper, pen, and
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logarithmic tables. Thus, FIA was born at a time when a chart recorder and a stopwatch were the essential means of recording measurements in real time, when most of the work in an analytical laboratory was done manually “in a beaker”. The only exceptions to this laborious approach were chromatography and the air-segmented continuous flow system (1) (marketed by Technicon as the AutoAnalyzer), which became the workhorses of the clinical laboratories in the West during the 1960s and 1970s.
The first experiment The components of the first FIA apparatus (Figure 1a) reflected the technology of the 1970s—a peristaltic pump, an injector (consisting of a disposable syringe with a hypodermic needle), a flow-through “air gap” ion-selective electrode, a potentiometer, and a chart recorder, which were to become the basic building blocks of all future FIA systems (Figure 1b). The peristaltic pump propelled the carrier stream (light blue) and reagent stream (dark blue) continuously forward, toward a detector. A well-defined
From Beaker to Microflu The co-inventors of flow injection recall the spirit and technology of the early days and - dis because one of us (J.R.) cuss how the technique
volume of sample solution (red) was injected into the carrier stream, which merged with the reagent stream at a confluence point and formed a desired detectable product (yellow) as it passed through the mixing coil. A flowthrough detector monitored the continuously flowing stream, yielding a peak (in height or area) that was proportional to the concentration of the analyte (Figure 2a). This early flow scheme demonstrated the principles of flow injection—sample injection, controlled dispersion of the injected fluids, and repetitive timing of physical and chemical processes taking place in the flowing stream. The experiment also established that continuous flow analysis could be carried out rapidly and reproducibly, without air segmentation. Indeed, repeatability (±3% RSD) and speed of response (up to 4 samples/min) of this rather primitive device convinced us of its usefulness, and we decided to name the method, apply for a patent, and gather material for the first publication (2). Shortly thereafter, we temporarily parted company
had accepted a one-year assignment with the United Nation’s International Atomic Energy Agency to establish an analytical facility at the Centro de Energia Nuclear na Agricultura (CENA) in Brazil. The institution had a vast number of soil, plant, and water samples that needed analysis for trace nutrients, nitrogen, phosphorus, potassium, and macro components, such as aluminum and iron—important elements of Brazilian arable soils. This abundance of samples presented us with the opportunity to transform FIA from an academic exercise into a practical tool. In 1982, more than 300,000 samples had been analyzed by FIA at CENA by using spectrophotometry, AA, and potentiometry (3). Thanks to the ingenious work and prolific publication activity of our colleagues Henrique Bergamin, Elias Zagatto, and Francesco Krug at CENA, the Brazilian FIA
has grown far beyond
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work became famous worldand reagent economy in a system that operates at microliter levels (Figure 3) are all valuable assets that contribute to the wide. Although an FIA Aapplication of FI to real-world assays. Yet, the main assets of page article made the cover FI are the well-defined concentration gradient that forms of Analytical Chemistry (4), by the end of 1978 only 20 papers on FIA had been pubwhen an analyte is injected into the reagent stream (which lished and the first edition of our FIA monograph (5 ) in offers an infinite number of well-reproduced analyte/reagent 1981 appeared with a mere 100 references. In retrospect, ratios) and the exact timing of fluidic manipulations (which what we learned is that beginnings are difficult; that it is provides exquisite control of the reaction conditions). not a device, but a methodology that must be conceived, Controlling the timing (what Gil Pacey calls the developed, and applied; and that a new method must be “kinetic advantage”) allows us to exploit reaction rates of aimed at solving real-world problems. different chemical reactions to increase the selectivity of In the beginning, our problem was that the established the assay. Although FI was originally conceived as a means doctrine of continuous-flow analysis was based on the use of of automation, it became apparent that FI offered opporair segmentation, which was deemed necessary to maintain tunities to perform unique assays that are not feasible the integrity of processed samples. Also, because 15 of the when performed manually. This finding led to the definifirst 20 papers came from our lab, FIA was initially seen as a tion of FI as a means of “information-gathering from a curiosity; skepticism persisted as to its feasibility and usefulconcentration gradient formed from an injected, wellness (6, 7). Therefore, the visionary work of the Brazilian defined zone of a fluid, dispersed into a continuous unseggroup, Bo Karlberg in Sweden, Miguel Valcarcel and mented stream of a carrier” (8). M. Luque de Castro in Indeed, FI gradient Spain, Fang Zhao-Lun in techniques are based on China, and K. Kina and selecting one or several (a) Nobuhiko Ishibashi in sections of the concenJapan became the essentration gradient in which tial elements that paved to take the measurethe way to wider acceptment, either in the conance of the technique. tinuous-flow or stopped(Kina and Ishibashi flow modes. Over the introduced FIA in Japan years numerous gradient and co-founded the techniques have been Japanese Association for developed (8–11), such Flow Injection Analysis, as FI titration, gradient which publishes the dilution and calibration, Journal of Flow Injection selectivity enhancement, Analysis, now in its 16th and, ultimately, sequenyear.) Now, the compiled tial injection (SI) (Figure FI bibliography consists 2b). Stopped-flow gradiSample (b) of 10,000 papers, more ent techniques are ideMixing injector Pump than a dozen monoally suited for enzymatic coil Detector graphs, and close to 150 assays, in which selecting Carrier Waste Ph.D. theses. We hope a proper reagent/analyte that the slow, but evenratio is essential and Reagent tual, recognition of FIA reaction rates are the is encouraging to anyone basis for determining struggling to introduce a substrate concentrations new method! or quantifying enzyme FIGURE 1.The beginning. activities. Assets of FIA An outstanding (a) Apparatus on which the first FIA experiments were performed. The system was designed to monitor ammonium concentrations in effluents via its conversion to ammoAutomated sample proexample of the continunia, which then was determined potentiometrically by means of an air-gap sensor (white cessing, high repeatabilous-flow gradient techcylinder on the left)—a combination glass electrode suspended in a chamber above a ity, adaptability to nique is the recent flowing stream of carrier solution. The carrier solution (NaOH) was pumped by a peristaltic pump (right), while sample solutions (2 mL) were injected into the carrier manually microminiaturization, work of Koupparis (10, by a syringe with a hypodermic needle. (b) The classic FIA flow scheme became a bluecontainment of chemi11) in which the interprint for automating countless reagent-based assays by using a variety of spectroscopies cals, waste reduction, actions between drugs or other instrumental techniques for detection.
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(a) Flow and time
S
(b)
(c)
S
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FIGURE 2.Analyte (red) and reagent (blue) zones as they pass through a detector and the resulting respo (a) In FI mode, the injected analyte zone disperses within the reagent carrier stream, forming a measurable product (yellow). (b) In SI mode, the separately injected analyte and reagent zones merge and the product is formed where the zones overlap. (c) In BI mode, the beads carry the reagent and are held within the detector. The analyte reacts and accumulates on the bead surfaces. Note that in FI mode, the peak is asymmetrical; in SI mode, it’s symmetrical because of the flow reversals used to maximize zone overlap; and in BI mode, the signal steadily increases because of analyte accumulation within the detector.
(“guests”) and synthetic receptors (“hosts”) were investigated. This type of binding study requires that the reactants be mixed in varying well-defined ratios, whereas the kinetics of their interactions are precisely controlled and monitored. Assays of constituents that form metastable compounds are difficult to perform because of the transient nature of the processes involved. However, FI allows capture of the relevant response at an appropriate moment, often immediately following the initial contact of the reactants. This capability is particularly helpful in reactions in which the intermediate and not the end product possesses distinct analytical characteristics (12). Transient light formation, generated by bio- and chemiluminescence, can be related to the analyte concentration only if the mixing of reactants and the timing of all operations are optimized and rigorously repeated (13). In the early 1980s, Astrom had the insight to exploit FIA for automating hydride generation for separating trace toxic metals from complex sample matrices prior to their detection by AA (14). Today, his method is widely accepted and commercialBROO SORENSEN ized as a means to enhance AA spectroscopy and inductively coupled plasma MS (15–17). The interesting aspect of his method is the ability to exploit kinetic discrimination, whereby side reactions known to affect the stability of the hydrides can be eliminated or effectively suppressed. An example of such an approach is the determination of Bi(III) (14) or As(III) (13) in the presence of trace metals such as Cu(II) or Ni(II). We believe, however, that further development of FI atomic spectroscopies will need to adopt the discontinuous flow mode, such as SI, which will especially benefit FI–electrothermal AA (18).
Second-generation FI and microflow analysis The transition from continuous to discontinuous reversedflow mode was facilitated by the proliferation of personal
computers and the availability of automated, high-precision syringe pumps and valves. Thus FIA changed from FI to SI (19) and, most recently, to bead injection (BI) (20). FI has even been applied to biological assays by using live cells as targets for drug discovery and toxicology (20, 21). As FI developed, it assimilated and grew with technological advances—automated valve-based injectors, stepper-motordriven high-precision syringe pumps, and scanning flowthrough detectors. In contrast to the modest beginnings of continuous flow, the advanced SI and BI modes (Figures 2b and 2c) use microfluidic systems performing precisely orchestrated forward- and reversed-stopped flow sequences tailored to the needs of the assay. The heart of the system is the sample-processing unit, which is designed to minimize the volume of the sample path from injector to detector by integrating the flow-through detector into a monolithic structure mounted atop a six-position valve (Figure 3a), resulting in the “lab-on-a-valve” concept (22). To facilitate sample introduction, the sample solution is aspirated through a flow-through port by a peristaltic pump (Figure 3b). The system operates in SI mode by aspirating a sample zone from the flow-through port, then aspirating the appropriate reagents sequentially into the holding coil. Sample and reagent zones are mixed within the holding coil by means of flow reversals and then directed into the flow cell by forward flow. By stopping a selected section of sample–reagent mixture within the flow cell, the reaction rate measurement can be performed. Fiber optics connect the flow cell with the light source and the solid-state scanning spectrophotometer that provides either UV–vis photometry or fluorescence measurements. The microliter volume of the flow path between the injector and the flow cell and the microliter flow rates make the system more economical then traditional milli-
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(a)
(b)
Syringe pump
Holding coil
Beam out
Holding Syringe coil pump
Waste Standard
Carrier
Beam in
Carrier
Reagent #1 Reagent #2
Sample Peri-pump Waste
FIGURE 3.The latest. (a) A mezzo fabricated microfluidic system of flow channels integrated with the flow-through cell mounted atop a multiposition valve. The apparatus consists of fiber optics (blue cables), a scanning spectrophotometer/fluorometer, two syringe pumps, and a computer for control of the fluidics, detector, and data acquisition. This system can function in SI or BI mode. (b) Micro-SI system with two syringe pumps. The central rotating groove (red) of a six-position valve sequentially connects the sample and reagent outlets with the flow cell (yellow box) integrated into the structure mounted atop the valve. Using two pumps allows a wide choice of flow rates and their simultaneous connection to the central port offers a wide variety of sample-processing opportunities, facilitated by flow reversals through this central confluence point (22).
liter-scaled systems and more practical than picoliter- and nanoliter-scaled devices as envisioned by micro-total analysis system technology. The distinct advantage of the micro-SI system is its versatility, which is inherent in its highly asymmetric flow path. Although the injector is connected to the flow-through cell by a small-volume channel, holding coils upstream provide ample volume for sample and reagent dilution and mixing, which relies not only on passive diffusion but is assisted by flow reversals. The large 0.5-mm internal channel diameter is designed to process solutions and bead suspensions (for BI mode) and is therefore suited for processing real-world samples that may contain suspended matter. Today’s microfluidic manipulation of samples, reagents, and bead suspensions is technologically vastly different from the initial FIA setups, yet the principles of the methodology remain the same: sample injection, controlled dispersion of the injected fluids, and exact repetitive timing of events result in precise control of physical and chemical processes on which repeatability of an assay is based. The devices, however important, are not the essence of a technique—rather it is the methodology and underlying conceptual principles. BROO SORENSEN Has FI been an unqualified success? Not entirely. Although numerous papers and meetings convincingly document the versatility and advantages of FI, and although FI has been used extensively in research, routine analytical applications have been accepted at a relatively slow pace. A case in point is the pharmaceutical industry where FI is seldom used or else it is used as a
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cheap substitute for chromatography. This notion is perpetuated by the use of homemade FI systems consisting of odd pieces of chromatographic equipment. The result is poor performance with inherent artifacts (high pressure, pulsation, poor mixing, inability to precisely control the flow, etc.) caused by the piecemeal instrumentation. This situation will change, however, as monographs and papers show that FI is a superb vehicle for drug-release measurements (11, 23), bioligand interaction studies, and functional assays for drug discovery (21). Lack of industryoriented commercial SI instrumentation with fully developed assay protocols has also hindered adoption (in a classic “hen–egg” situation), in which an industrial need is not obvious enough to encourage the necessary investment. An exception is FI-AA instrumentation developed by PerkinElmer’s European division. Much of the credit for this instrumentation goes to Bernhard Welz, who championed this line of research and business.
Where are we heading? So where is FI heading after the first 25 years?— microminiaturization, interfacing SI with electrothermal AA and inductively coupled MS, combining SI with CE, and using beads as reagent carriers. As to these trends, it is fair to note that FI was integrated into microconduits long before the advent of microTAS. Because of its speed and multi-analyte resolution, the combination of FI with CE is a perfect synergy as shown by the elegant works of Karlberg (24) and Fang (23). We believe, however, that CE, combined with SI, will become an ideal marriage. The use of BI in such diverse fields as radiochemistry (25), biosensors (20), and drug discovery (21) indicates its potential. Finally, the design of microfabricated analytical systems is yet another area of research where principles of FI have been applied but
where the advantages of the SI and BI modes still have to be appreciated. The recently developed “labon-a-valve” system is, therefore, in our opinion, a step in the right direction. These developments remind us of the excitement in the early years of FIA, and we hope they inspire a new generation of researchers and provide a focus for future work. It is rewarding to see the growing ranks of participants at FI meetings such as the annual International Conference on Flow Injection Analysis that have been organized by Gary and Sue Christian over the past 10 years, and the triennial flow analysis conferences that were initiated back in 1979. Although it is tempting to speculate on the impact of etechnology on the future of FI, it is prudent to restrain ourselves, knowing that we could never have predicted where FIA would go. It surpassed our expectations, had an impact on analytical chemistry, and became a partner to chromatography.
(14) str m, O. Anal. Chem. 1982,54, 190. (15) Flow Injection Atomic Spectroscopy, Burguera, J. L., Ed.; Marcel Dekker: New York, 1989. (16) Fang, Z. Flow L. Injection Atomic Spectrometry; Wiley: Chichester, England, 1995. (17) Flow Analysis with Atomic Spectrometric Detectors, Sanz-Medel, A., Ed.; Elsevier: Amsterdam, The Netherlands, 1999. (18) Nielsen, S. C.; Hansen,Anal. E.Chim. H. Acta in press. (19) Ruzicka, J.; Marshall,Anal. G.Chim. D. Acta 1990,237, 329. (20) Ruzicka, J.; Scampavia, Anal. Chem. L. 1999,71, 257 A. (21) Hodder, P. S.; Ruzicka, Anal. Chem. J. 1999,71, 1160. (22) Ruzicka, J.; Flow Injection Analysis: Principles, Tutorials and Resources; Self e-published on CD-ROM, August 1999.
[email protected]. (23) Fang, Z. L.; Liu, Z. S.; Anal. Shen, Chim. Q. Acta 1997,346, 135. (24) Kuban, P.; Karlberg, Trends B.Anal. Chem. 1998,17, 34. (25) Grate J. W.; Egorov Anal. O. Chem. B. 1998,70, 779.
Additional resources
Ruzicka, J.; Hansen, Flow E. Injection H. Analysis (Japanese); Kagakudonin: Kyoto, Japan, 1983. We wish to express our appreciation to the following Ueno, K.; Kina,Introduction K. to Flow Injection Analysis. Experiments and organizations for supporting FI research through the Applications (Japanese); Kodansha Scientific: Tokyo, Japan, 1983 years: the Center for Process Analytical Chemistry at the Valcarcel, M.; Luque de Castro, Flow-Injection M. D. Analysis (SpanUniversity of Washington; the National Institutes of ish); Imprenta San Pablo: Cordoba, Spain, 1984. Health; the Danish National Science Foundation; Brd. Hansen, E. Flow H. Injection Analysis; Polyteknisk Forlag: Copen Hartmanns Fond, Ib Henriksens Fond, A. Fiskers Fond, hagen, and Thomas B. Thriges Fond (Denmark). Finally we both Denmark, 1986. wish to extend our most sincere thanks to Alison McDonTrojanowicz, Flow M. Injection Analysis: Instrumentation and Applications; ald, former editor-in-chief of Analytica Chimica Acta, for World Scientific: River Edge, NJ, 1999. encouragement and unyielding trust. Valcarcel, M.; Luque de Castro, Flow-Injection M. D. Analysis. Principles and Applications; Ellis Horwood Ltd.: Chichester, England, 1987. References Ruzicka, J.; Hansen, Flow E. Injection H. Analysis, 2nd ed. (Chinese); (1) Skeggs, L. Anal. T. Chem. 1966,38, 31 A. Beijing University Press: Beijing, People?s Republic of Chi (2) Ruzicka, J.; Hansen, Anal. E. Chim. H. Acta 1975,78, 145. (Dan. Fang, Z. Flow-Injection L. Separation and Preconcentration; VCH Verlags Pat. Appl. No. 846/74, Sept. 1974; subsequent U.S. gesellschaft: Pat. Weinheim, Germany, 1993. No. 4,022,575). Frenzel, W. Flow Injection Analysis: Principles, Techniques and Applications; (3) Hansen, E. H.; Ruzicka, TrendsJ. Anal. Chem. 1983,2, 5. Technical Univ. Berlin: Berlin, Germany, 1993. (4) Ranger, C. Anal. B. Chem. 1981,53, 20 A. Valcarcel, M.; Luque de Castro, Flow-Through M. D. (Bio)Chemical Sen(5) Ruzicka, J.; Hansen, Flow E. Injection H. Analysis; Wiley-Inter sors; Elsevier: Amsterdam, The Netherlands, 1994. science: New York, 1981. Hansen?s FI bibliography available at (6) Margoshes, M. Anal. Chem. 1977,49, 1861. www.flowinjection.com/search.html, accessed on 1/29/00. (7) Snyder, R. Anal. L. Chim. Acta 1980,114, 3. Chalk?s flow analysis database available at (8) Ruzicka, J.; Hansen, Flow E. Injection H. Analysis, 2nd ed.; www.fia.unf.edu/fad/fad.html, accessed on 1/29/00. Wiley-Interscience: New York, 1988. Karlberg, B.; Pacey,Flow G.Injection E. Analysis: A Practical Guide; Else (9) Ruzicka J.; Hansen E. H.; Mosbaek Anal. Chim. H. Acta 1977,92, vier: Amsterdam, The Netherlands, 1989. 235. Nielsen, S.; Sloth, J. J.; Hansen, Talanta 1999,43, E. H.867. (10) Georgiou, M. E.; Georgiou, C. A.; Koupparis, Analyst M. A. Flow Injection Analysis of Pharmaceuticals: Automation in the Laboratory, 1999,124, 391. Calatayud, J. M., Ed.; Taylor & Francis: London, England, 1 (11) Georgiou M. E.; Georgiou C. A.; Koupparis, Anal. Chem. M. A. 1999,71, 2541. Jaromir Ruzicka is a professor at the University of Washington. His research interests include applying FI to process analytical (12) Bendtsen, A. B.; Hansen,Analyst E. H. 1991,116, 647. chemistry, automation of chemical and biological assays, and apply(13) Hansen, E. H.; N¿rgaard, L.; Pedersen, Talanta, 1991,38, M. ing microfluidic manipulation and spectroscopic techniques 275.
Acknowledgments
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