Novel kinetic approaches to flow injection analysis

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Novel kinetic approaches to flow injection analysis M. D. Luque de Castro and M. Valcarcel Cordoba,Spain Flow injection analysis is an automatic technique applicable to kinetic analyses based on reaction rate measurements by the use of simple instrumentation. Recent approaches to the implementation of this type of analysis are discussed and their advantages over conventional arrangements are emphasized.

The fundamental characteristic of flow injection analysis (FIA) is its intrinsically kinetic nature. From a physical point of view, FIA measurements are always performed under non-equilibrium conditions because, only exceptionally (use of mixing minichambers’ and open-closed systems2*3), is the injected plug homogenised with the carrier solution and measurements performed under total mixing conditions are very rarely exploited. From the chemical point of view, the measurement time is always shorter than the time to reach equilibrium, so that the chemical system is undergoing constant change. Therefore, FIA has a kinetic nature which is physical when the method used involves no derivatizing chemical reaction (use of electroanalytical or atomic optical techniques as a rule) or only chemica11-3; in most cases the method has a dual physical and chemical character. This makes it mandatory to perform measurements at a constant time interval after injection. Ordinary FIA methods are thus implicitly fixed-time kinetic methods of analysis, mostly involving exclusively physical kinetics. The kinetic character of FIA results in a series of positive and negative aspects characteristic of the technique, the most important of which are those related to sensitivity, selectivity and rapidity. The kinetic aspect of the technique can be further utilized by exploiting the kinetics of other controllable processes (catalysis, diffusion of ions or gases through membranes, adsorption, and desorption), incorporated into the system in addition to the reaction and/or dispersion rate. The simultaneous use of several of these effects allows one to dramatically increase the differences in the behaviour of species with otherwise similar characteristics, giving rise to ‘kinetic discrimination’ which in turn results in improved selectivity. 01659936/89/$03.00.

The sampling frequencies of FIA methods are substantially higher than those of equilibrium methods as a result of not having to wait until equilibrium has been attained. As FIA has an inherently kinetic character, only those methods based on reaction-rate measurements are regarded as ‘kinetic’. Fig. 1 summarizes the general types of FIA methods. Fixed-time methods include ordinary FIA methods and those based on measurement of signal increments over a given interval. Variable-time methods rely on measurements of the peak width at a preselected height (FIA titrations4). Initial-rate methods involve halting the flow at the detector and measuring the slope of the linear portion of the recording of the course of the reaction during the halt and those other methods in which reaction and detection are simultaneous because the reagent is located in the flow-cell so that the rising portion of the reaction curve can be measured. In differential methods, two or more analytes having different reaction rates with the same or a different reagent are involved. Finally, FIA configurations providing multipeak recordings yield substantially increased kinetic information and allow the development of initial-rate (using the envelope of maxima or minima), fixed-time, variable-time or differential methods, in addition to the calculation of physical parameters. The ‘conventional’ kinetic FIA methods are briefly discussed below. The advantages of the new approaches with respect to conventional arrangements are also discussed. Conventional arrangements The’ kinetic FIA methods reported so far are limited to the conventional stopped-flow technique - a single halting of the flow per injection, allowing the kinetic determination of a species. The chemical system may be arranged to allow the simultaneous determination of two species5 or to obtain two signals (peaks) at two reaction times by: (a) using a single detector and parallel dual in’ectio#, a single injection and splitting of the flow 4, or a dual-beam spectrophotometer’; (b) two serial detectors and a single injection, or two parallel detectors and dual injection, or even a single injection with splitting of the flow9. The methods developed using both approaches are mainly aimed at differential kinetic de0

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TYPE OF KINETIC METHOD

FIA

METHOD

FIAGRAM

ordinary

FIXED

TIME

multipeaks

VARIABLE

-TIME

time based “titrations”

stopped - flow

S

A La

I I I

I I

STOP

INITIAL-RATE

integrated detect ion

t

S

react ion

a

k%t

s

multipeak

recordings lif!!!L

Fig. 1. Types of FIA methods and recordings obtained in each case.

termination but have also been used for the kinetic determination of a single specieslo. There is only one precedent in the earlier literature on the use of a home-made multidetector based on LEDs for obtaining a large number of absorbance-time data which can be subjected to the proportional equation method, and to logarithmic extrapolation’r. Simple manifolds The simplest configurations proposed for obtaining two measurements at two different reaction times involve a single-channel system and a conven-

t

tional single-beam photometric detector. These units yield dual peaks because of the abnormally large sample loop placed in the injection system, which gives rise to two reacting zones, one at the head and the other at the tail of the injected sample, with a central zone with little or no reaction12. This effect is also possible by using two serial valves, the connection between them acting as a delay coil fixing the time interval between measurements. Doubly stopped-flow The use of an electronic

system to initiate two

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. Additional reagent

n

Fig. 2. Location of the sample plugs in a doubly stopped-flow manifold and type of recordings obtained in the determination of free and initiallybound compound.

halts in the flow per sampling operation allows the determination of two different analytes or two different states of the same analyte and this is extremely useful where one of the species requires a step prior to the derivatization reaction. Fig. 2 illustrates this approach. A dual injection is performed into two different channels which merge close to the detection point. One of the injected plugs merges rapidly with the derivatizing reagents and reaches the detector, whereupon the first stopping of the flow takes place. Meanwhile, the other plug, mixed with the reagent for the preliminary reaction, is stopped at the reactor. Restarting the flow flushes the first plug from the manifold, gives rise to the merging of the second plug with the derivatizing reagents and drives the reaction mixture to the detector. The flow is then halted for the second time, enabling the progress of the reaction to be measured. The overall recording has two linear portions whose slopes are proportional to the concentrations of the analytes. This approach has been applied to the determination of bound and free sulfur dioxide in wine13, and used to implement non-kinetic and kinetic stopped-flow to the same sample. FIA methods based on the speed of the physicochemical processes The rapidity of the processes occurring in flow injection manifolds allows the development of interesting analytical methods, which are very difficult to implement in any other way. Adsorptive pre-concentration on active surfaces is one practical application of this feature. Two active agents have been used for the adsorptive pre-concentration of an analyte in FIA: activated alumi-

na14y15and electrode surfaces’6-18 (carbon paste or platinum). Another electrochemical kinetic effect of great interest is the application of enzyme membrane electrodes as amperometric detectors for diffusion-limited phenomenalg. The implementation of precipitation-filtration-dissolution processes in FIA manifolds is based on the rapid dissolution of the precipitate due to the short interval of time between precipitation and dissolution2’. The determination of chloride and iodide mixtures in foodstuffs21 and the preconcentration and determination of traces of lead at the ng/ml level” are representative examples of this technique. The calculation of acid-base equilibrium constants of some compounds can be masked by side reactions, for instance by hydrolysis at low or high pH, if the conventional manual procedure is applied. Since neutralization is faster than hydrolysis, absorbance measurements made immediatey after mixing will have virtually no contributions from the side processes. Flow injection assemblies are particularly suitable for fast, practically simultaneous measurements. In such systems, the sample is injected and mixed with a carrier to alter the pH. A combined glass-calomel flow-through electrode is placed close to the flow-cell accommodated in the spectrophotometer. The residence time must be as short as possible. The determination of the acidity constant of a pyridinic acyclic amine by manual methods gives three constants (three jumps) whereas the determination by FIA only gives one. The manual methods give rise to ‘false’ constants at extreme pH values, which can be attributed to hydrolysis of the compound, catalysed by hydrogen or hydroxyl ions. Kinetic measurements offer a means of avoiding the incorrect assignation of pK values by overcoming the perturbations from side reactions23. In the spectrophotometric determination of total cyanide in waste waters in flow-injection system with gas-diffusion separation and preconcentration, Zhu and Fang have used an unstable red intermediate product of the reaction of cyanide with isonicotinic acid and 3-methyl-1-phenyl-2-pyrazolin-5one (instead of the conventional blue final product), to improve efficiency24. Coupled reaction-detection Integrated ’ reaction-electrochemical detection has been used for a long time in FIA both in potentiometric25*26 and amperometric27-30 techniques. This has been done by using enzymes immobilized on the surface electrode and by reagent located at the measurement zone cou led to a chemiluminescence31 or bioluminescence !2 reaction. Non-kinetic

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measurements have also been used in FIA for several years. Novel approaches in this area involve the use of photometric detectors whose flow cells can hold various ion-exchange systems or reagents immobilized on suitable supports. The ion exchangers typically used within the cells are cationic (retention of the ligand, PAN, and formation of a corn lex with the analyte which involves a colour change 3p) and anionic resins (determination of iron by formation of a complex with thiocyanate34). There are many possibilities including the use of alumina and the many different chromatographic column packings used in various modes (ion-exchange, ligand exchange, ion-pair, etc.). In addition to kinetic measurements on the rising part of the recording, this approach allows the measurement of kinetic concentrations steps. This involves the recording of the initial part of the concentration curve thus overcoming the chief problem of the preconcentration technique which is its slowness. The immobilization of reagents in the optical flowcell is also carried out in a variety of modes. There have been attempts to immobilize enzymes, the sole catalysts used to date, on the cell walls after treating them to increase their surface area. So far this has been insufficient to retain the necessary amount of enzyme. Another strategy is to introduce the support containing the immobilized enzyme into the cell. The main problem in this case is the size of the supporting material, as the small glass beads used result in a very small sample volume in the interstices. The absorbance from larger beads on the other hand saturates the photometric detector. The most suitable procedure for this purpose is the adhesion of controlled-pore glass to the walls of the flow cell and the subsequent immobilization of the enzyme on the porous glass. This apgoach permits kinetic measurements to be made , which were previously impossible. The large variety of chromogenic reagents that can be immobilized in the flow-cell of a photometric detector is of great potential and is currently being examined in our laboratory36. Repeated passage of the plug through the detector There are two very simple methods allowing the repeated flow of the injected plug past a single detection point, thus allowing the changes taking place in this plug to be measured as many times as required. (a) The use of open-closed systems (Fig. 3A) in which a switching valve can trap the injected plug into a closed loop accommodating the detector flowcell. The number of signals obtained is equal to the number of cycles performed by the plug, which con-

A

PUMP SAMPLE

CARRIER

‘I

‘\ ‘*. \

(t,) CYCLE PROGRAMMER

B

Fig. 3. Open-closed (A) and repeatedly reversedflow (B) configurations and recordings obtained in each case (for details, see text).

tinue until complete homogenisation with the carrier solution within the circuit has been accomplished2p3. (b) The repeated change of the flow direction by changing the direction of rotation of the drum of the peristaltic pump (Fig. 3B)37. In both cases a multipeak recording (Fig. l), providing a number of kinetic data of theoretical and practical interest, is obtained. The number of these approaches (kinetic and nonkinetic individual and simultaneous determinations2Y3Y37, calculation of stoichiometries, liquid-liquid extraction processes without phase separation, gas-liquid separations, lixiviation, etc.) is large and this list is far from exhaustive. Fast detectors The use of a fast detector does not strictly imply the existence of kinetic methods; nevertheless, these instruments endow methods with a kinetic character which is absent from conventional approaches. This arises from the simultaneous control of different values of the instrumental variables, such as different wavelengths in diode array detectors, in addition to conventional signal-time monitoring. It can also arise from the fast change of potential in voltammetric detectors. On the other hand the amount of data generated is so much greater that that from ordinary FIA that the use of a computerized data acquisition system is essential. The large quantity of data also requires computerized treatment. In this respect,

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Fourier transform this group.

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IR detection

can be included in

Amplification methods The wide definition of the term ‘kinetic’ permits amplification methods to be included. They involve obtaining a fan-shaped beam of calibration curves with different sensitivity and concentration ranges, which allow analysis without preconcentration or dilution of an analyte whose concentration varies over a very wide range. This is done by selecting the most suitable of the calibration curves obtained. A kinetic aspect additional to those inherent in FIA is used in each of the three approaches38. (a) The simplest approach is based on measurements on the FIA recording at various intervals after the injection, i.e., at different extents of reaction development. Times far from the maximum are taken for dilution methods, while the sum of the analytical signals at the maximum and at values close to it are used for concentration methods. (b) The repeated passage of the plug through the detector provides a multipeak recording on which measurements of a minimum (dilution methods) and the sum of the signals of the maxima or minima (concentration methods) can be performed. (c) A fast detector (e.g. a diode array detector) allows measurements to be performed at the maximum of the conventional FIA peak at different wavelengths in the absorption spectrum of the monitored substance. Individual measurements performed at wavelengths other than that of maximum absorption are used in dilution methods. The sum of the absorbances at the maximum and at wavelengths on both sides of this are employed in concentration methods. In all cases, there is clearly an additional kinetic character involved in the analysis. Analytical implications Kinetic methods present a number of advantages over conventional FIA methods, advantages which are further enhanced by the use of the novel approaches described. Among the advantages are the following. Sensitivity

In general, kinetic methods have a somewhat lower sensitivity than equilibrium methods as a result of reaction incompleteness. In FIA methods, where chemical equilibrium is not attained, the kinetic method may or may not improve sensitivity with respect to ordinary methods depending on a variety of factors (dispersion, time required for reaction completion, measurement time, etc.). The new ap-

proaches improve sensitivity in most cases by obtaining a number of signals that can be summed as required (systems with repeated passage of the plug through the detector), through a simultaneous analyte concentration process (coupled reaction-detection), or by the use of fast detectors (concentration methods). This increase in sensitivity is not a disadvantage for concentrated samples since these can be measured by a dilution method. Selectivity

This is the parameter that benefits most from the use of kinetic FIA methods. Whilst conventional FIA is more selective than equilibrium methods as a result of eliminating the contributions from side reactions, kinetic methods also eliminate the sample matrix contribution. In this respect, the contribution of these novel approaches is related to fast detectors, in which measurements performed at different values of the instrumental variable allow one to select a value for that variable at which the undesirable contributions are minimum or zero. Speed

The use of a system giving multiple peaks involves decreased sampling frequencies as a result of being unable to inject a fresh plug until the previous one has been flushed out. Fast detectors provide a large amount of information from quite a short interval, much shorter than usually required for data treatment, and hence give noticeably improved sampling rates. The kinetic concentration step performed with coupled reaction-detection is also much faster than with conventional preconcentration FIA methods. Scope of application

The novel approaches given in this article have a number of analytical implications that are not limited to kinetic determinations and which represent improvements over earlier kinetic FIA methods. In addition: (i) They allow simultaneous determinations (speciation, impossible by conventional approaches) by the use of doubly stopped-flow. (ii) They allow for in situ concentration in the detection system, with the resultant shortening of the time elapsed in this step (coupled reaction-detection) .

(iii) Kinetic measurements using immobilized enzymes are also possible. This approach, which is cheap owing to the small amount of the catalyst used, allows the elimination of the sample matrix which is important in areas such as clinical chemistry (coupled reaction-detection). (iv) Calculation of stoichiometries, reaction and

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rate constants, etc. from multipeak recordings from the data provided by fast detectors.

and

Acknowledgement The CICyT is thanked for financial support (Grant No. PA86-0146).

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M. D. Luque de Castro and M. Varcarcel are at the Department of Analytical Chemistry, Faculty of Science, University of Cordoba, 14004 Cordoba, Spain.

Analysis of eumelanin-related indolic compounds S. Pave1 Amsterdam, The Netherlands Eumelanin-related indolie compounds are excreted in the urine of patients with generalized malignant melanoma. Paper and thin-layer chromatography have helped to clarify the structure of some of the metabolites, but are inadequate to identify the complete structure of the indolic conjugates known as Thormiihlen-positive compounds. These substances can be separated from urine by anion-exchange chromatography on DEAE-cellulose. Separation followed 01659936/89/$03.00.

by enzymatic hydrolysis and gas chromatographic analysis allows the elucidation of their complete structures. The gas chromatographic-mass spectrometric determination of eumelanin-related indolic compounds with deuterium-labelled analogues as internal standards is a reliable, but expensive procedure. High-performance liquid chromatography is a cheaper alternative, but this method does not seem to be useful for the routine measurement of all eumelanin-related compounds. Nevertheless, HPLC will undoubtedly find its place in studies dealing with skin pigmentation and malig nant melanoma. OElsevier

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