The second coming of flow-injection analysis

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Analytrca Chuntca Acta, 2610992) 3-10 Elsevler Science Publishers B V , Amsterdam

The second coming of flow-injection analysis Jaromlr RiElEka Department of Chemtstry, BG-IO, Umversrty of Washmgton, Seattle, WA 98195 (USA)

(Received 29th July 1991)

Abstract

The state of art of the flow-inJectIon techmque IS reviewed and Its future development IS outhned by emphaslzlng the importance of stopped-flow and sequential InJection methodologies and by dlscussmg common aspects of flow mJectlona6a chromatographlc techmques Keywords Flow System, Review

Flow inJection belongs to a family of methods, based on sample inJection into a flowmg stream, which carries the analyte through a chemical modulator into a detector (Fig 1) Thus broad group of methods e races besldes flow mjectlon (FI) or flow-m_leef”” ion analysis (FIA) and also chromatography, electrophoresls and field flow fractlonatlon The mdlvldual members of this family differ m one fundamental respect, VU, the function of the chemical modulator, which alters the orlgmal square-wave input, provided by sample mJectlon, mto a chromatogram, electrophero-

PUMP

IMPULSE

DETECTOR

-

RESPONSE

Fig 1 Flow scheme (top) and concept (below) of analytIca techmques based on mjectlon of analyte mto a carrier stream

gram, fractogram of flagram This 1s why the function of the chemical modulator, or forces within it, are associated with the name of mdrvldual techmques such as chromatography (column), electrophoresls (electric field), field flow fractlonatlon (external force) and flow mjectlon (reactor) Whereas chromatographlc techniques aun at a high resolution obtamed through megarepetltlve mteractlons which modulate the mlgratlon veloclties of analytes through the system, flow mjectlon exploits chemical reactions to transform analytes into species that can be selectively quantified by a detector Although FI 1s the youngest of the flow-based techniques [l], Its apphcatlons have encompassed a full range of reagent assays from morgamc to enzymatrc, from ions to protems and from traces to highly concentrated analytes m aqueous or non-aqueous media Industrial, chmcal, research-oriented, environmental and blotechnologlcal apphcatlons of FIA have been descrrbed m over 4000 papers and SIX monographs [2-71 and discussed at numerous meetings, of which “Flow Analysis” 1s the most recognlzed mtematlonal venue It 1s well recogruzed that m the flow-mnJectlon apparatus (Fig 2) two processes take place sunultaneously physlcal dlsperslon of the sample zone

ooO3-2670/92/$05 00 0 1992 - Elsevler Science Pubhshers B V All rights resexved

.I R&?&I /Anal Chrm Acta 261 (1992) 3-10

Fig 2 Scheme ot a flow-mjectlon system (top) and of the readout obtamed S, Sample mJect]on valve, C, carrier stream, R, reagent stream, W, waste, H, peak height, I, mlectlon pomt Various delay times mdlcate readout pomts as explotted by gradlent techmques

wlthm the carrier stream of the reagent and a chemical reaction between the analyte molecules and reagent molecules thus supplied As the detector 1s tuned to sense species produced by this chemical derlvatlzatlon, the readout has the form of a peak mto which the original square-wave input (as provided by mJectlon) has been transformed As this process can be repeated very reproducibly time after time, the FI apparatus can be used for serial assays with a relative standard deviation (R S D 1 typically better than 1% Low consumption of sample (mlcrohtres) and of reagents (sub-mdhhtres) per assay and a high sampling frequency (2 mm-‘), are the hallmarks of FIA, together with its computer compatlblhty and versatlhty, which 1s due to a vast number of useful combmatlons of reagent selection and reactor designs In its simplest form the reactor 1~ d tube that has been tightly coded to promote radial mwng of sample and reagent If several reagents must be added m succession, additional streams are confluenced and coils are added Amongst the first procedures adapted to FIA were classical colorlmetrlc methods (e g , for ammoma, phosphate, glucose and ethanol), where chart recorders were used to obtain a flagram Over the last 15 years the range of detectors has grown and so has the variety of reactor designs, to accommo-

date solvent extraction 181,gas diffusion [9], photodegradatlon, coulometrlc reagent generatlon and titrations [2] In addition, mmlaturlzed packed reactors (typically 15 mm 1d , 30 mm long), contaming sohd reagents [lo], reductants [21, lmmoblhzed enzymes [ill, ion exchangers 1121or slhcaor polymer-based C,, materials 1131,were mtroduced to convert, catalyse or preconcentrate analyte molecules Not surpnsmgly, some of these separations, performed on a mlcrocolumn, may be viewed as “single plate” or affinity chromatography, thus supportmg the notion of family ties of FI and flow-based separation techniques [14] Most of the above methodologies were proposed during the fn-st 10 years of the exrstence of FIA which was a period of discovery and also of competltlon with the established an-segmented contmuous-flow analysis (AutoAnalyzer) Although crucial for a wider acceptance of FIA, seen m retrospect, focusing towards a high-speed serial assay, while relying on the contmuous-flow approach, was counterproductme, as It emulated contmuous-flow analysis, albeit without air segmentation Only when FIA research became onented towards the exploltatlon of concentration gradients formed by the dispersion process [HI were new techniques using stopped flow, reversed flow, smusoldal flow, reagent mjectlon, sequential mjectlon and single solution cahbratlon developed Of these techniques, stopped-flow mjectlon will become the cornerstone of future developments as it has numerlous practical advantages, allows the lmplementatlon of sequential mjectlon (SI) and will lead to the final transition of FIA from a mere tool for serial assays mto flow injectlon (FI), which 1s here being viewed as a unmersal methodology for the enhancement of chemical sensors and analytical mstrumentatlon

STOPPED-FLOW TECHNIQUE

INJECYION-AN

UNDER-UTILIZED

Optnmzation of a flow-injection system is a balancing act, where dispersion of the sample zone and Its mwng with a reagent must be weighed agamst the tnne required to achieve a

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recorded durmg the stopped-flow interval (Fig 3), which allows the analyte to be quantified This temporal mformatlon can be further enhanced yla multi-wavelength detection, which results m two orthogonal degrees of mformatlon, leadmg to a higher selectlvlty [ 171 Numerous applications of FIA [M] especially m the life sciences and biotechnology [16,18-201 can be slgmficantly enhanced and sunphfled as reactlon rate-based measurements offer a unique way to determme the actlvlty of enzymes, to assay substrates selectlvely m complex matnces or to speed up unmunoassays time Rg 3 Stopped-flow mjectlon for reactlon rate measurement S, Sample mJectlon valve, C, carrier stream, R, reagent stream, W, waste, H, peak height, I, mlectlon pomt Two reactlon rate curves are shown, recorded on top of each other, from the same startmg pomt (I) for low and high analyte concentratlons Note that an Increase m delay hme for stopped-flow measurement will result m changes m the analyte/reagent ratio as Indicated m Fig 2

desired chemical conversion of an analyte mto a detectable species This 1s so because the parabolic profrle estabhshed durmg the mjectlon process expands further during sample zone transport through an open-tubular channel, ultlmately leading to an undesired zone broademng and consequent loss of sensltlvity and sampling frequency It 1s unfortunate that most FI procedures published so far utilize contmuous flow, because such a format 1s not advantageous and 1s difficult to optimize Indeed, by stopping the flow (Fig 3), when the mixture of sample with the reagent reaches the flow cell, many advantages are gamed [2,16] First, as drsperslon ceases while chemical reaction continues, the sensltrvlty of determmatlon increases because the reactants and the product are no longer bemg diluted Second, as neither carrier nor reagent solution 1s pumped during the stopped-flow period, the consumption of solutions (and waste generation) 1s much reduced Third, a selected section of the dispersed sample zone 1s trapped wlthm the flow cell, makmg reaction rate measurements possible, whereby it IS the slope of the reaction rate curve, as

GRADIENT TECHNIQUES-THE SECOND GENERATION OF FLOW-INJEmON TECHNOLOGY

It has been well established that a dispersed sample zone, while travellmg through a detector, offers an mfmlte number of choices of sample/ reagent ratios, the analyte zone dispersing mto a well defined background of a carrier stream and/ or of a reagent [2,15] In Its simplest from, gradlent dilution has been used to accommodate highly concentrated samples wlthm the dynamic range of a detector This has been done by selecting a readout collected with a tnne delay t,, or t, at a peak tall (Fig 2) rather than simply reading peak height In this way, dllutlons up to 20000-fold have been obtamed [21] for process control apphcations Seen m retrospect, selecting the peak top as a point of readout was only a matter of convenience when a chart recorder 1s used as the means of data collection Computerlzed data collection systems now allow us to select either maximum sensitivity through peak height (or peak area) measurement, or to perform “electronic ddutlon” through the choice of t, Going one step further, one may select an mfmlte number of readouts from a single analyte zone By vlewmg the dispersed zone as a matrix of concentrations versus tnne, mto which the original square-wave impulse has been transformed, the original mput can be seen as dlgltlzed mto an infinite number elements of fluid, each of them representing a progressively more diluted calibrator standard [2,15] This concept 1s

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Chnn Acta 261 (1992)3-10

now the basis of a single solution cahbratlon method, recently commercrahzed m the FIAAS instrument by Perkm-Elmer and mgemously expanded mto a novel approach for the ldentlflcatlon and ehmmatlon of interferences m spectroscoplc measurements [22,23] Optimization of reaction rate measurements, as required for enzymatic assays by MlchaehsMenten formalism, 1s done by capturmg a sultable section of the analyte/reagent concentration gradient m the detector and by stopping the flow whde performing the reaction rate measurement (Fig 3) This tunability of analyte/reagent ratio via a dispersion process, and subsequent selection of the readout through the choice of the delay tune t,, 1s also a convenient way of determmmg reaction rate constants or K, values by scanning reaction rates at different ratio of reactants 12,241 Its full potential for umnunoassays and cell studies (see flow-mJectlon cytometry) has yet to be realized

SEQUENTIAL INJECTION-NEW PRINCIPLES?

MECHANICS, OLD

The next logical step m the development of flow-mjectlon methodology 1sbased on dispersion and mutual penetration of sample (9 and reagent (R) zones Sequential mjectlon ‘$1) [25] uses a selector (rather than mjectlon) valve (Fig 4A), through which precisely measured volumes of carrier solution, sample solution and reagent solution are aspirated mto a holding co11(HC) by means of a pump which 1s capable of a precisely controlled stop-go-forward-reverse movement Although m prmclple a penstaltlc or piston pump can be used equally well, so far only a piston pump has been used for this purpose Following the first step of zone sequencing, during which the sample and reagent zones are stacked m the holding conduit adjacent to each other (Fig 4B), the valve 1sswitched mto the detector position In the next step, the flow 1s reversed so that the stacked zones are propelled through the valve and the reactor mto the detector, while they mutually mterdlsperse Durmg this movement the flow reversal creates a complex concentration

Fig 4 Prmclple of sequential lnJectlon (A) Flow scheme, (B) structure of stacked and Injected zones, (C) concentration profiles as seen by the detector HC, Holdmg cod, D, detector, S, sample, R, reagent, H, peak height, I, point of mjecbon Note the smularltles and the progrewvely increasing requirement for precise selectlon of the delay time by comparmg Figs 2, 3 and 4

gradient (Fig 40, m which the sample and reagent zones mutually penetrate, forming a composite region wlthm which the analyte is bemg transformed mto a detectable species The peak observed at contmuous flow will be that shown as a shaded area, as only when R and S penetrate can the detectable product be formed For stopped-flow reaction rate measurement the choice of the delay time td allows optmuzatlon of the analyte/reagent ratio, slmdarly to that m the conventional stopped-flow mjectlon technique Note that the fundamental reqmrement for SI to succeed 1s to achieve maximum zone penetration through a deliberate mcrease m axial dlsperaon, obtained by means of the flow

J REcka /Anal Chun Acta 261 (1992) 3-10

reversal and channel design [24-261 SI 1s mechanically far snnpler than conventIona FI, as it uses only a smgle pump, single valve and single channel The flow path does not have to be reconfigured if the mjectlon volumes, reactlon times or mutual zone dlsperslon are to be modlfled, as these parameters can be altered by changmg the stroke volumes, flow-rates, stopped-flow period and flow reversals via computer control As additional reagents, reactors and detectors can be clustered around the selector valve (the apparent hmlt being only the number of available ports), multi-reagent chemlstnes and multl-detector assays can m prmclple be carried out m a single SI system An example of a flow scheme which accommodates multiple detectlon and two different reagent assays 1s shown m Fig 5A In fact, the majority of solution-handling operations which have been automated by FIA can m prmclple be accomodated by SI Dilution of concentrated analytes, so useful m process control apphcatlons, can be performed with the aid of reverse flow m the system shown m Fig 5B The same flow scheme 1s currently being examined as a means of automated cahbratlon usmg a single standard solution If an mJectlon valve 1s clustered with the sequential valve (Fig 5C) and a column 1s placed m the loop conduit, preconcentratlon and matrix removal for atormc absorption spectrometry (AAS), mductlvely coupled plasmas (ICPs) or mass spectrometry (MS) can be performed The same scheme wdl be useful for interfacing SI systems with a detector whenever the mam stream carrying the analyte IS mcompatlble with the detector performance (water interference m Fourier transform measurements, flow-rates mcompatrblhty for FLMS or FI cytometry Integration of the reactor with the detector, using the well recognized optosensmg scheme [2], 1s well suited for the SI conflguratlon (Fig 5D) Using a large reservoir of acceptor reagent (100 ml) and a reactor/detector with a very small internal volume (50 ~0, the amount of reagent spent and Its contribution to the baseline increase will be negligible when the lower piston durmg its forward and reverse movement replenishes the acceptor within the detection chamber Stopped-flow oper-

light

detector

Rg 5 Sequential mjectlon mamfolds deslgned to accommodate (A) hvo different assays, (B) sample and standard ddutlon, (Cl analyte preconcentratlon and matrur removal and (D) gas d&won and optosensmg

atlon and the absence of product dllutron will compensate for the short optical path (2 mm) through which reflected light 1s travellmg Undoubtedly many other schemes are conceivable and future research will show how many are practical The examples dlscussed here are meant to inspire such thoughts Indeed, m this period of enthusiasm, it appears that m order to interface any solution-based chemistry with a sensor or a complex Instrument, all that 1s needed are sultable hardware components, a computer, customized software, a knowledge of reagent chemistry and lmagmatlon Of these, wrttmg flexible, user-friendly software is the frustrating task As sample injection, controlled dlsperslon and reproducible tmung are the cornerstones of both FI and SI, much of what has been learned from

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the classlcal flow schemes can be applied to zone sequencmg Although the rnechamcs of SI are much simpler than that of FI, the complexity of gradients formed by zone penetration with reversed flow stdl has to be unraveiled The greatest challenge is the theory of flow dynamics, which will one day lead to optimization of flow systems based on flow stopping and reversal The classical approach of contmuous flow, recently rigorously treated for FIA condltlons [271, wdl not yield an answer It is hoped that the use of Impulse/response functions, which was shown to be a much more flexible approach [28], ~111be applied by the same authors to sequential mjectlon Until then, the fundamentals of sequential rnJectlon wdl have to be explored experunentally and summarized m empirical rules 1261

THE SECOND COMING OF FLOW INJECTION-THE ENHANCEMENT OF INSTRUMENTS AND OF CHEMICAL SENSORS

The attributes of FIA, speed, solution contamment, capablhty of numatunzatlon, automated standardlzatlon, sample dilution, matrix removal, analyte preconcentratlon and the ablhty to control the tlme/concentratlon domain of any solution chemistry under mvestlgatlon, are now well recognized and exploited by the worldwide community of flow-mjectlon enthusiasts At long last the next stage 1s being reached a more general recognition of the ablhty of FI to upgrade the performance of analytical mstruments Indeed, FI has been interfaced with a much wider range of detectors than liquid chromatography, which 1s not surprlsmg, as FI m contrast to LC thrives on non-selective and selective detectors alike With selective detectors such as AAS instruments, or blosensors, FI serves as a means of sample pretreatment operations (filtration, dilution, preconcentratlon, matrix removal) based on the physics associated with the flow methodology Less selective detectors, on the other hand, are coupled with reagent chemistries that yield specific products (e g , enzyme assays or immunoassays) Therefore, ultimately, flow-mJectlon systems, most likely m SI configurations, will be recog-

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mzed as universal mlets to both spectroscopic and electroanalytlcal instruments, and wdl also, after suitable mmlaturization, become integral parts of chemical sensors There 1s some evidence that this process has already started, as seen from the followmg examples FlOW-inJeCtiOn atomic absorption spectrometry (FIAAS) was proposed m the late 1970s as a means of interfacing a sample tray with the nebulizer of an AAS instrument [29,30] Serving mainly as a means of sample transport, this configuration did not attract much attention Yet interest m FIAAS grew when it was shown that hydnde generation can be coupled with AAS via FI [31] Other chemistries followed, such as analyte preconcentrations by ion exchange [2,5,12], sorbent extraction [13], analyte conversion and analyte speclatlon [5,321,together with the use of gradlent dllutlon and automated calibration [33,34] Reviews appeared, followed by a monograph coauthored by an international team [61 Most recently the first commercial instrument (FIAAS by Perkm-Elmer) has been successfully launched Since AAS, ICPs and MS share the same “Achilles heel” when being interfaced with solution chemistries, there seems to be an ample scope for the proliferation of FI systems m atomic spectroscopy Interfacing an instrument with a chenucal process, with the auu of continuous morutormg, 1s not a trivial task The urgent need to gain chemlcal mformatlon m a tnnely fashion 1s driven both by economy and by environmental concerns Although unknown m bloprocess momtormg only 7 years ago, when the first truly pioneering work on the use of FI for the control of mdustrlal ferrnentatlon and downstream processmg was published 1351,today the method 1s used for on-line measurements of nutrients and metabohtes [16,181 The blologlcal actlvlty of these products, and the enzymatic assay of many substrates are monitored by stopped-flow mJectlon using UV-visible spectrophotometry Blosensors [361 and mass spectrometers [37] have been interfaced by means of FI with fermenters Most recently a special issue of the Journal of Bwtechnology was devoted to “FIA m the Life Sciences” [lg] Flow mjectlon 1s now a recogxuzed tool for the continuous moru-

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tormg of chemical processes m the petroleum and chemical mdustnes with regular symposia dealing with this t0plc [FACETSXVII (1990), FACSS XVIII (19911, ANABIOTEC (199011 Flow-injectIon cytometry (FIG) is an ongoing project most recently supported by NIH m this laboratory Its aim 1s to perform cytochemlcal reactlons under kmetlc control provided by injectmg zones of reagents onto a cell culture, while momtormg the change m fluorescence or colour caused by interaction of cell components with reporter molecules (fluorophore- or chromophore-tagged reagents) While cell suspensions will be momtored by flow cytometry, munoblhzed cells will be probed by fluorescent microscopy The rationale for mtroducmg FI IS to obtain kmetlc mformatlon and to avoid non-selective rnteractlons of reporter molecules with other than target sites, as such non-selective mteractlons are likely to occur during the prolonged contact of reactants that takes place during conventional perfusion stammg Again, stopped-flow reaction rate measurements are pivotal to this type of research Concluswns

The notion of the second coming of flow-mjectlon analysis 1s based on the observation that this techmque has been transformed from a mere tool for serial assay as orlgmally proposed [ll mto a more general impulse response technique [2], which 1s now becoming a powerful methodology for the enhancement of instrumental analysis However, m Its “teens”, FI 1s still an orphan seekmg admlsslon mto a larger family of flowbased methodologies to which it conceptually belongs [14] The reasons for its exclusion are many and the research fields remam separated, even after chromatography and flow mJectlon have been discussed at a highly successful mtematlonal meeting [38] For theoretlclans it does not fit mto the accomphshed framework of chromatographic techniques Its purpose and means, I e , automation of chemical reactions, rather than of separations, the use of stopped and reversed flow, exploitation of concentration gradients, and on occasion even a deliberate promotion of sample zone broadening, are alien to chromatographlc

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concepts However, both chromatographlc techniques and FI rely on the success of a slmllar balancing act, performed with the understanding of the dynamics of flow Also, FIA may for a long time have appeared to be a sunple post-column derlvatlzatlon system, or a tool for serial assays, not worthy of much conslderatlon Few expenmentahsts pursue both flow mjectlon and chromatography, although many components of these two techniques are similar and some experience can be transferred from one field to the other The value of flow mjectlon as a general tool for solution handling will eventually be recognized as widely as the value of chromatography for separations 1s recognized There is, of course, neither contest nor conflict of interests between LC and FI, as their functions are not competitive, but complementary Chromatography has a tremendous resolvmg power, whereas FI offers selectwlty and reaction rate measurement on a single analyte m a timely fashion It 1s therefore the time to review finally all flow-based Impulse/ response techniques from a umfymg standpoint, as much can be gamed by doing so Not only materials and components but also concepts and approaches can be borrowed and interchanged between FI and chromatography By domg so, novel combmatlons readily come to mind, such as SI chromatography, where SI methodology with suitably time-spaced zone mjectlon could be used to separate analytes by gradient elutlon provided by sequential mJectlon of the sample followed by appropriately selected eluents For FI to be truly muuaturlzed and integrated with chemical sensors, novel low flow-rate generatmg pumps are badly needed Osmosis, electrophoresq electroosmotlc prmclples or hquld drive are the prmclples worthy of conslderatlon Pumps currently m use are reviewed m this issue [161 Whereas practical aspects of FI are thriving, the theory of flow injection lags behind, and will do so even more as the use of flow programming grows, thus dlfferentlatmg FI from the constant monotonous flow mode used m chromatography and chemical engineering In spite of this, or rather because of this development, what 1s truly needed 1s a umfymg theoretical background and a comprehensive way of teaching of all methods

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basedon the physrco-chemical modulation of an

Impulse provided by inJection of analytes (and reagents) into a flowing stream Only then will slmdarltles and differences between chromatography and flow-mjectlon techniques be ldentlfled, correlated and exploited wlthm the framework of a broder dlsclphne which one day will be recognized as ffow analysis

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