Flow Injection Analysis

289 Chapter 11

FLOW INJECTION ANALYSIS

M. VALCARCEL Department of Chemistry, Faculty of Sciences, University of Cordoba, 14004 Cordoba (Spain)

11.1

INTRODUCTION

One of the most promising trends in Analytical Chemistry involves the replacement of human intervention in laboratory processes, i.e. the implementation of automatic methods of analysis (ref. 1). There are three major alternatives in this respect:

(1) in discrete or batch methods,

each sample preserves its integrity in a small cup where the different analytical stages, including detection, take place; (2) robotic methods rely on the use of a microprocessorcontrolled minirobot which mimics the actions of a human operator in applying analytical methodology; and (3) in continuous methods, the samples are introduced successively at regular intervals into a channel through which a liquid containing the reagent is circulated, and a flowcell located at the detector permits the acquisition of a transient signal as the analyte or its reaction product passes through it. Automatic continuous methods can be classified into two major groups according to the way in which carry-over between the samples is minimized or avoided. The classical alternatives in this context, represented by commercial instruments (e.g., those from Technicon and Skalar), rely on the use of air bubbles to separate the bubbles in the flow, whereas the more recent approaches make use of air segmentation, thereby resulting in simpler automatic configurations. The most significant variant of automatic unsegmented continuous-flow methods is Flow Injection Analysis (FIA).

Since its advent in the mid-1970's (ref. 2) it has reached a

remarkable development stage (refs. 3,4). Fig. 11.1 shows a schematic diagram of a basic FIA system, which consists of four main parts. The propulsion unit establishes the flow at a rate, as constant as possible for one or several solutions, either containing a dissolved reagent or merely acting as carriers.

This

function can be performed by a peristaltic pump, a gas pressure system or even by gravitation. An inexpensive low-pressure rotary valve allows the reproducible insertion of accurately measured sample volumes without halting the flow as in high performance liquid chromatog­ raphy (HPLC). The transport-reaction system is often, and sometimes improperly called a "reactor."

This can be a straight, a coiled or a knotted tube, a mini-mixing chamber, a tube

packed with a chemically active material (e.g., an oxidant or reductant, an immobilized enzyme)

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291 or a continuous separation device (e.g., a dialyzer, a liquid-liquid extraction assembly). A flowcell accommodated in an optical or electroanalytical detector transduces a given property of the analyte or its reaction product into a continuous signal fed to a recorder or microcomputer. The manual switching of the valve allows the use of a semi-automatic analytical switch of great practical interest, which marks the difference between FIA and other automatic methodologies.

The full automation of the basic FIA system requires the incorporation of a

sampling system, a propulsion unit, an electrically controlled injection valve and a microprocessor furnished with an active interface. The signal obtained in FIA is transient in nature.

It is similar to those provided by

chemiluminescence and electrothermal atomization atomic absorption spectrometric (AAS) methods, and also resembles a chromatographic peak. Analytical measurements on this signal can be based on different parameters such as the peak height or area, like in HPLC (Fig. 11.1). Time-based determinations rely on the relationship between the time elapsed until a preset signal is reached and the logarithm of the analyte concentration. There are other alternatives such as peak-to-peak measurements in stopped-flow reaction-rate determinations and the slope of the rising portion of the signal in integrated reaction-detection systems. Flow injection analysis is thus a major alternative to automatic continuous methods and is characterized by the following basic features:

(a) the flow is not segmented by air bubbles,

which results in clearly simplified configurations; (b) the partial dispersion or dilution of its reaction product can be readily manipulated by controlling the geometric and hydrodynamic characteristics of the manifold; (c) the chemistry and continuous separations involved in the analytical process

are liable to ready automation; (d) neither

physical

equilibrium,

homogenization of the flow, nor chemical equilibrium, and/or reaction completeness has been reached by the time the transient signal is detected. Therefore, FIA methods can be regarded as fixed-time modes of kinetic methods of analysis; and (e) the operational timing must be highly reproducible insofar as measurements are made under non-equilibrium conditions. Fig. 11.2 depicts the most common types of FIA configurations. There are numerous other possibilities not included in this figure. The first three configurations show the ease with which analytical processes involving several coupled reactions can be implemented in FIA systems. The reversed FIA mode features increased sensitivity and is applied when there are no restrictions to the sample volume available.

The merging zones configuration, which incorporates a dual

injection valve intended to insert small volumes of sample and reagent(s), is essential in dealing with expensive or scarce reagents. The stopped-flow mode allows reaction-rate measurements to be made and overcome interferences arising from the sample matrix.

Finally, the

incorporation of a continuous separation unit offers major advantages as regards selectivity (interference removal) and sensitivity (preconcentration).

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Fig. 11.2 Most common FIA configurations. (Ń: pump; C: carrier; AC: additional carrier; R: reagent; S: sample; IV: injection valve; D: detector; W: waste) (1) Single-line manifold. (2,3) Multi-line manifolds. (4) Reversed FIA configuration. (5) Symmetric merging zones configuration. (6) Stopped-flow manifold. (7) Non-chromatographic separation techniques implemented in FIA configurations.

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There are several reasons accounting for the impact and popularity of FIA within the scientific and technical communities, namely:

(a) the utmost simplicity and versatility of

configurations; (b) the low cost of their different components; (c) the drastically reduced sample and reagent consumption; (d) the high sampling rates achieved; (e) the good reproducibility of the results obtained; (f) the enhanced selectivity resulting from kinetic discrimination; (g) the ease of use for continuous separation techniques; (h) their use as a tool for introduction of samples into atomic spectroscopic instruments—which is the focus of this chapter—and for developing chemiluminescence based methods; (i) their convenient use with immobilized reagents or catalysts (enzymes); and (j) there suitability for the resolution of real problems in fields such as environmental (ref. 5), pharmaceutical (ref. 6), clinical (ref. 7), industrial and food chemistry (ref. 8). Although there has been a negative trend in FIA involving the mere adaption of manual analytical procedures without a clear objective, the last few years have seen the development of novel strategies clearly broadening its scope and opening interesting perspectives (ref. 9). As stated above, one of the assets of FIA is its use as a system for sample introduction in atomic spectroscopy (AS). This topic has been covered at length in a monograph (ref. 10) and several literature reviews (refs. 11-13).

This chapter deals with the potential of FIA in this

context. Far from making an exhaustive review of the already vast literature on this topic—over 200 papers—it is concerned with the most relevant aspects. After describing the technological aspects involved in the association of an automatic continuous methodology with the more common atomic spectroscopic instrumentation, the chapter deals with the incorporation of continuous separation techniques. A discussion of the potential of FIA as a sample introduction system in atomic spectroscopy precedes a brief description of the most relevant applications reported to date and a survey of the most promising future trends. Flow injection systems can be regarded as an interface between samples and standards and the instruments providing useful analytical information about the qualitative and quantitative composition of samples. In association with atomic spectroscopic instrumentation, FIA systems can serve four basic types of function (Fig. 11.3), namely:

(1) act as a mere alternative to

manual introduction; (2) function as an automatic system for the introduction and mixing of samples with auxiliary chemicals intended to react with analytes or perform specific functions in the nebulizer or atomize; (3) serve as the means of use for a continuous separation technique (dialysis, gas diffusion, ion exchange, liquid-liquid extraction); and (4) allow the automatic development of chemical reactions coupled to a continuous separation unit. 11.2

TECHNICAL ASPECTS OF THE FIA-AS ASSOCIATION

This section deals with general aspects concerning the on-line coupling of an FIA assembly to an atomic spectroscopic instrument.

On account of their differences, due distinction is made

between atomic absorption (AA) and inductively coupled plasma (ICP) spectrometry.

Fig. 11.3 Roles of FIA as an interface between samples and standards and atomic spectroscopic instrumentation.

294

295

11.2.1

Atomic Absorption Spectroscopy (AAS)

Apart from its destructive nature, the use of an atomic absorption spectrometer as a detector in FIA systems involves some peculiarities.

The nebulizer suction requires the optimum flow

rate of the FIA manifold (usually between 1 and 3 mL/min. ) and that of aspiration of the nebulizer (normally in the range 6-10 mL/min.) to be made compatible. On the other hand, the typical dispersion arising from the FIA system is increased by that originating in the nebulizer, which acts as a well stirred mixing chamber (ref. 13). The flow rate of the carrier stream pumped into the nebulizer should always be greater than the natural aspiration rate of the nebulizer in order to achieve the optimum performance of the FIA-AAS association. The apparent increase in the FIA peaks resulting from increasing sample pumping rates does not arise from increased nebulizer efficiency, but from increased sampling delivery (ref. 14). In fact, it has been demonstrated that a reduction in the sample uptake rate decreases the signal but increases the atomization efficiency.

The net result being a 33 %

decrease in the peak height, but a four-fold increase in the peak area (ref. 15). As can be seen in Fig. 11.4 A, the height of the FIA peaks approaches that of the peak obtained by conventional continuous aspiration of the sample as the pumping rate increases. As shown by Tyson (ref. 13), the variation of the peak height with the flow rate takes the shape of the curve in Fig. 11.4 B. Many authors have found no optimal zone as they have not investigated a sufficiently broad flow rate range. The most straightforward and inexpensive way of establishing the flow in the FIA-AAS association involves using the nebulizer aspiration as the sole propulsion system. However, the sensitivity and precision have been shown to decrease considerably when the flow into the nebulizer is not forced (ref. 16). A number of strategies have been proposed for accomplishing the best compromise between the optimal carrier and nebulizer flow rates. Four of them are worth special note, namely: (a) the use of a long piece of tubing prior to the nebulizer.

This poses the problem arising from

considerably increased dispersion in the injected zone which in turn results in decreased sensitivity; (b) the removal of the nebulizer from its customary location in the instrument, which results in very poor nebulization efficiency; (c) the application of solvent compensation methods. These involve the use of an additional stream merging with the carrier at a point close to the nebulizer input in order to increase the carrier flow rate without disturbing the FIA system.

The sensitivity is thus decreased as a result of dilution, although some authors (ref.

16) have shown such a decrease not to be too significant (only about 10%); and (d) the incorporation of an air stream prior to the nebulizer (air-compensation) through a T-piece which acts as a pre-nebulizer. This approach, originally proposed by Yoza et al. (ref. 17) was widely developed recently by Sanchez-Pedreno et al. (ref. 18). Increased sensitivity is achieved (especially at low pumping flow rates) as a result of more effective liquid fragmentation. This gives rise to signals comparable with those provided by the conventional continuous aspiration method.

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Fig. 11.4 Influence of the carrier flow rate signal on the signal obtained in the FIA-AAS association. (A) comparison between the signals obtained by conventional aspiration and by FIA at different carrier flow rates (q-j > q2 > qß). (Â) relationship between the peak height and the flow rate.

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From the above discussion it follows that the sensitivity of measurements in the FIA-AAS association is a function of:

(a) the aspiration flow rate of the nebulizer (AAS); (b) the carrier

flow rate (FIA); (c) the system used to make both compatible; and (d) the injected sample volume (the higher the volume, the higher the sensitivity). The precision of measurements in the FIA-AAS association depends largely on the constancy of the pumping flow rate and the absence of pulsations. The precision has been shown to increase substantially as a result of the use of a gravity based propulsion system instead of the traditional peristaltic pump to establish the hydrodynamics of the FIA system.

When using a peristaltic

pump, the precision of the FIA-AAS association is not significantly different from that of conventional aspiration (ref. 16).

A recently reported systematic study (ref. 14) showed that

the signal-to-noise ratios of FIA-AAS peak heights and areas approach but never exceed the ratios achieved by conventional nebulization. The use of air as a substitute for liquid carriers in FIA systems coupled on line to AAS nebulizers results in increased sensitivity and sampling frequency (~ 600/hour), and similar precision (ref. 19). The fact that organic solvents burn efficiently in the flame and are more efficiently vaporized on account of their lower viscosity and surface tension has been exploited with the FIA-AAS association by using an organic solvent carrier (usually MIBK) and merging the sample with a water miscible organic solvent (e.g. methanol). This ensures considerably increased sensitivity (refs.

20,21).

The use of a dual injection valve instead of a conventional valve minimizes the errors arising from the so called "nebulizer memory" in analyzing samples with high elemental concentrations (ref.

22).

11.2.2

Inductively Coupled Plasma (ICP) Spectrometry

As stated in a recent paper by McLeod (ref. 23), the FIA-ICP-atomic emission spectrometry (FIA-ICP-AES) association provides transient signals analogous to those of other alternatives such as pulse nebulization and electrothermal vaporization and in contrast to the traditional measurements based on steady state signals. Unlike FIA-AAS, the technical aspects of FIA-ICP-AES assemblies have been the subject of few papers.

The influence of the different variables involved does not differ much in both

associations, with the exception of those related to the differential features between AAS and ICPAES (or ICP-mass spectroscopy [ICP-MS]).

It is interesting to note that the optimal sample

flow rate in ICP-AES depends on the particular element-analyte.

In general, the signal is

scarcely affected by changes in such flow rates in the range 1-5 mL/min. (ref. 24). As a rule, a compromise must be made between analytical sensitivity, plasma stability, and sampling rate. The last two also depend on the sample flow rate with values higher than 5-6 mL/min. causing fluctuations in the reflected power.

298 Fassel et al. (refs. 25,26) have shown that pneumatic nebulization results in low analytical transport efficiency in the FIA-ICP-AES association.

They designed a total injection

microcentric nebulizer that yielded nearly 100% efficiency.

Their system, applicable to

effluents from FIA (or HPLC) manifolds, circulating at rates between 0.1 and 0.2 mL/min., is comparable to or better than conventional pneumatic nebulization in terms of detection limits. This performance contrasts with most other studies, where reduced detection capabilities have been reported. A new interface system consisting of a conventional concentric nebulizer, a heated desolvation chamber and a jet separator was recently developed and applied to sample introduction in ICP spectrometry with special emphasis on continuous flow manifolds (ref. 27). Martin and Ihrig (ref. 28) have proposed the full automation of the FIA-ICP-AES association. Their system includes the following features: (1) automatic analysis of undiluted samples with sample introduction by FIA by means of a sampler with 76 positions and no operator intervention; (2) automatic operator selectable fixed dilution and analysis of samples; (3) automatic analysis of samples by computer guided sequential dilutions to place all elements within the optimal calibration range; (4) use of merging zones mode to accomplish dilutions up to 200-fold; and (5) automatic addition of standards to a sample and subsequent analysis for a standard addition study. Further advantages of the use of FIA in this context include improved plasma stability, accuracy and precision, and lower detection limits for many elements. Ebdon et al. (ref. 29) recently demonstrated that the use of FIA as a sample introduction system in ICP-MS also offers the advantages inherent in the use of FIA coupled to atomic spectroscopy in general. 11.3

CONTINUOUS SEPARATION METHODOLOGIES COUPLED ON-LINE WITH SPECTROSCOPIC INSTRUMENTATION

Automatic continuous configurations based on FIA principles are some of the best alternatives to the use of non-chromatographic continuous separation processes with a variety of interfaces, namely: (1) solid-liquid (precipitation, ion exchange, adsorption, lixivation), (2) liquidliquid (dialysis, extraction), and (3) gas-liquid (gas diffusion, hydride generation) (refs. 30,31).

The broader application of these systems in FIA as compared to air-segmented

automatic methodologies is probably the result of overcoming a series of technical problems arising from the passage of the air-segmented flow through the continuous separation system. This normally results in perturbations decreasing the analytical reproducibility.

This is not

always the case, as among other devices, dialyzers can be used successfully with both types of automatic continuous methodologies. The second class incorporated into FIA set ups, can either be introduced in a continuous way (e.g. in hydride generation, dialysis and liquid-liquid extraction), be generated in-situ (precipitation, distillation) or be a permanent part of the continuous separation device (adsorption, ion exchange). The continuous separation process may involve a chemical change, a physical change or both as a means of facilitating the transfer of matter across the interface.

299 This can be rather

small

(dialysis, gas diffusion, liquid-liquid

extraction)

or

large

(precipitation, ion exchange). A state of the art discussion of non-chromatographic continuous separation techniques coupled on-line with atomic spectroscopic techniques is presented in Chapter 5 of the monograph edited by Burguera (ref. 10). The inherent advantages of FIA as a system for sample introduction in atomic spectroscopy (decreased human intervention, drastically reduced sample and reagent consumption, and increased sampling rate) are supplemented by those arising from the use of an analytical separation

technique,

preconcentration.

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(a)

indirectly

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This involves two negative aspects which contrast with the inherent

advantages of FIA, viz. decreased sampling rate and increased injected volume requirements. If preconcentration is the primary objective, it is preferable to resort to the continuous aspiration of sample (at a fixed flow rate over an accurately controlled interval) rather than inserting it through an injection valve; (b) indirectly enhanced selectivity arising from interference (matrix effect) removal, aided by kinetic discrimination.

The interval during which phases are

kept in contact is somewhat short compared with those typical conventional separation systems; (c) the scope of application of atomic spectroscopic techniques is broadened with the use for the determination of both inorganic and organic anions which are laborious and tedious by conventional procedures; and (d) increased precision compared with that of conventional separation techniques involving the use of an atomic spectroscopic system as a detector. Below are briefly discussed some of the most significant achievements in this field. 11.3.1

Ion Exchange

The use of micro-columns packed with an ion exchanger in FIA systems is one of the best ways whereby a continuous separation can be coupled on-line to an atomic spectroscopic system. The principal objectives of this association are as follows:

preconcentration of trace metals,

elimination of matrix or interfering effects, and implementation of sequential determinations (including speciation). Due to their excellent capability for retention of metal cations, particularly transition metals, commercial chelating

resins (e.g., Dowex A - 1 , Chelex-100, Muromac-1)

and

exchanging materials synthesized in the laboratory (e.g 8-hydroxyquinoline immobilized on controlled pore glass beads (ref. 32)), have been the exchangers most frequently used for preconcentration and determination of metal ions in FIA-AS configurations.

Commercially

available anion exchangers (e.g., amberlite IRA-400, De-Acite FF) are used in this context for interference removal purposes (e.g., the separation of sulfate and phosphate in the determination of calcium (ref. 33).

McLeod et al. (refs. 34,35) used activated alumina in its acidic form for

the preconcentration and removal from the matrix of oxyanions such as Chromate, arsenate, borate, molybdate, phosphate, selenate, and vanadate.

These polyvalant anions are strongly

retained on the resin, and are difficult to remove and require strong bases for removal.

300 Minicolumns used in these systems are generally made of glass, Teflon®, Tygon or PVC tubing with one of the types commonly employed in FIA. They are usually between 1.0 and 10.0 cm in length and 1.5-5 mm inside diameter.

On account of their relatively large particle size,

commercially available exchangers are easy to pack and condition. One of the typical requirements of FIA assemblies accommodating ion-exchange minicolumns is that they must be designed to operate in two stages.

The first is the retention or

preconcentration and the second involves the elution of both the cations-analytes (which are led to the atomic detector) and the interferents isolated.

These are driven to waste in a wash

operation repeated at a frequency which is a function of the concentration of the interferents and the mini-column capacity. There are a variety of FIA set ups incorporating an ion exchange column coupled on-line to an atomic spectroscopic detector. single-line continuous manifold.

Fig. 11.5 depicts the three typical alternatives available for a The simplest of such alternatives (Fig. 11.5 A) involves the

use of a single valve for the sequential injection of the sample and eluant (generally nitric acid). The sampling frequency can be increased by using two serially arranged valves (Fig. 11.5 B), one for the sample and the other for the eluant.

Nevertheless, these configurations are only

intended to overcome matrix effects. The third alternative (Fig. 11.5 C) involves placing the resin mini-column in the loop of the injection valve.

In addition to eliminating matrix effects.

It allows preconcentration to be

accomplished by passing a large sample volume through the loop of the injection valve while in its filling position, and permits the downstream elution of the metal ions retained in the preconcentration step. This avoids problems arising from the resin compactness. Other much more complex manifolds reported in the literature offer no substantial advantages over those depicted in Fig. 11.5. Preconcentration has also been developed by using configurations with continuous aspiration of sample, downstream elution, etc., which require the use of several lines and two or more valves (ref. 36). The purpose of increasing the sampling frequency of trace metal preconcentration used FIA systems designed using two parallel columns working simultaneously.

While the sample is

passed through one of them to effect preconcentration, the other is used for the elution of the analytes, which are driven to the atomic detectors (refs. 37,38). The one advantage of these set-ups, apart from their complexity, lies in the need for both columns to have the same capacity.

IEC Fig. 11.5 Different single-line FIA configurations accommodating an ion exchange mini-column (IEC) coupled on-line to an atomic spectroscopic instrument.

ELUTING SOLUTION

301

302

11.3.2

Precipitation-Dissolution

Not with standing its wide use for quantitative (gravimetric) and qualitative purposes in classical Analytical Chemistry, this technique has so far been scarcely employed with automatic methods of analysis (ref. 1) because of the inherent difficulties involved in the total or partial replacement of human intervention in this process.

Nevertheless, the nature of FIA recently

made possible the development of systems for the continuous precipitation and precipitationfiltration, washing and dissolution.

This offers

substantial advantages over manual batch

precipitation methods applied prior to determination by atomic spectroscopy. A major reason for the good results obtained is the rapidity with which the different stages are implemented, which lessens the shortcomings originating in typical stages of precipitation processes (viz. décantation, precipitate evolution with the potential risk of contamination by different mechanisms, etc.). Every continuous precipitation system includes two essential elements namely:

(a) the

precipitation reactor, usually a helically coiled Teflon® tube of 0.3-0.7 mm i.d. and 100-300 cm in length, and (b) the filters, which were originally designed as cleaning devices for HPLC. The best results were obtained with those of cylindrical shape and large filtration areas (~ 3 cm ), pore sizes between 0.2 and 2 μηι and chamber inner volumes larger than 300 μ ί . 2

The determination of the analyte in these systems is based either on the dissolution of the precipitate or on the increase in the cation-reagent concentration in the reaction zone once the precipitate has been retained on the filter. Fig. 11.6 shows the four types of FIA-AS assemblies involving the precipitation of the analyte. In the simplest of all four configurations (Fig. 11.6 A), which is used to implement the normal FIA mode, the sample containing the analyte is injected into a reagent-cation stream. Precipitation takes place in the coil and the precipitate is retained on the filter.

As the

concentration of the reagent-reaction decreases in the reaction plug zone, a negative peak is obtained upon arrival at the detector after passage through the filter.

An identical volume of

water provides a small FIA peak that is used as a blank. Owing to the need for an excess of precipitating cation which corresponds to a concentration falling outside the detector linear range, a water stream is incorporated into the flow system to dilute the cation prior to the nebulizer. The use of continuous precipitation in a reversed FIA configuration (Fig. 11.6 B) involves the use of a further selecting valve to perform blank measurements. First, the reagent-cation is injected into a water stream and a tall FIA peak is obtained. Then, the selecting valve is switched and the sample is continuously pumped into the system.

Another identical injection of the

reagent causes the analyte to form a precipitate, which is retained on the filter. The FIA peak obtained in this case decreases with increasing analyte concentration in the sample. This type of configuration does not always require the incorporation of the water stream for dilution of the reagent-analyte cation prior to the nebulizer as this is already diluted in the carrier.

303 The three basic operations involved, namely precipitate formation, washing and dissolution can be performed in the configuration depicted in Fig. 11.6 C. This uses three valves, with one for injection and the other two for selecting purposes.

In the precipitation step, the sample is

injected into a reagent-cation stream, the precipitate formed as a result is retained on the filter and the reagent stream is sent to waste through SV2. In the washing step, SV1 allows the introduction of a wash solution stream that is led to the filter through the second selecting valve (SV2).

The analytical signal yielded tend to raise the baseline. In the third step, a dissolving

solution precipitate yields a positive FIA peak, the height of which is proportional to the analyte concentration in the sample. Another configuration allowing continuous precipitation and dissolution without injection developed for the continuous preconcentration of trace metals is shown in Fig. 11.6 D. In the preconcentration step, both the sample and the reagent are continuously pumped into the system and the precipitate thereby formed is retained on the filter. In the dissolution step, the selecting valve is switched to pass a stream of dissolving solution, through the precipitate, which, once dissolved, gives a positive FIA peak proportional to the amount of analyte present in the inserted sample volume. The use of continuous precipitation as a separation technique coupled on-line with AS can have three basic aims: (a) the indirect determination of both inorganic and organic cations (or, in general, any species precipitating with a given reagent-cation); (b) the preconcentration and determination of traces and subtraces of metal ions; and (c) the removal of interferents by precipitation without the concourse of the analyte in the continuous separation. 11.3.3

Liquid-Liquid Extraction

Every continuous liquid-liquid extractor of those typically used in FIA configurations (refs. 3,41) has three essential elements, namely:

(1) Solvent Segmenter, in which the incoming

streams of the two phases involved will merge. It must provide an outgoing stream of alternate and size-controlled segments of both phases.

Despite the availability of very sophisticated

designs, a simple T-, Y-, or W- shaped tube of low, known void volume is adequate for most applications.

The flow rate ratio is comparable to that of the volumes of both phases in the

conventional procedure; (2) Extraction Coil, which receives the segmented flow emerging from the segmenter.

It is here that the transfer of matter at the interface between segments take

place. The length of this process and hence the coil's efficiency depend on its length and the flow rate. The coil is usually made of Teflon® (the organic phase wets its wall while the aqueous phase occurs as bubbles) when the analyte is originally present in the aqueous phase; and (3) Phase Separator, which receives the segmented flow and continuously splits it into two separate streams of each phase. Its efficiency rarely surpasses 85-90%.

As a result, the phase where

detection is to take place (generally the organic one) must be fully free from the other.

The

performance of this device is based both on the relative densities of the phases and on the different wetability of its inner wall by each phase. Phase separators are available in a variety of designs, of which two are preferentially used, namely:

(a) glass T-shaped tubes with an

304 internal Teflon® coat intended to facilitate the separation of the organic phase, and (b) membrane separators resembling dialyzers and gas diffusion cells.

A microporous Teflon®

membrane, for instance, allows only the organic phase to pass through. One of the practical difficulties posed by continuous liquid-liquid extraction lies in the establishment of a uniform flow of the organic phase.

In fact, peristaltic pumps give rise to

major perturbations as a result of the corrosion of flexible tubes. addressed in three ways, namely:

This problem can be

(1) by using flexible tubes of extremely inert materials

resistant to some organic solvents. Unfortunately, most have limited lifetime; (2) by employing displacement flasks (see Fig. 11.7 A), which involves pumping an aqueous stream into a closed container that is filled with the immiscible organic solvent, which in turn is fed at a constant flow rate to the FIA system; and (3) by setting a constant pressure with the aid of an inert gas forcing the extractant to circulate along the FIA manifold. Although there exists a variety of designs of FIA manifolds incorporating a liquid-liquid extractor, two of them are worthy of special note, particularly when coupled to atomic spectroscopic instrumentation. As can be seen in Fig. 11.7, they differ in the relative positions of the injection zone and the continuous extractor. When injection takes place after the separation process (Fig. 11.7 A), the sample is continuously aspirated into the system, where it is or not mixed with a carrier that can be a buffer and/or contain a reagent (e.g., a chelating ligand or a bulky counter-ion intended to form ion pairs) forming an extractable product at reactor L. The streams of both phases merge at the solvent segmentor (SS) prior to entering the continuous extractor. The emerging organic phase stream, which contains the analytes, fills the loop of an ordinary injection valve and its flow rate is controlled by a peristaltic pump.

An

aqueous carrier of high flow rate transports the organic phase plug to the detector without dispersion and a rate compatible with that of aspiration of the nebulizer. This type of manifold utilizes the displacement flasks described above. Although sample consumption is higher than usual for FIA, this configuration is particularly suitable for use with atomic spectroscopic detectors. In the configuration shown in Fig. 11.7 B, the sample is injected in the conventional FIA fashion prior to the extraction process. The organic phase directly enters the nebulizer. The incompatibility between the flow rates can be overcome by using the solvent or the air compensation methods by means of a T-piece placed between the phase separator (PS) and the atomic spectroscopic instrumentation. This configuration utilizes flexible pump tubes which are highly resistant to corrosion. The association of liquid-liquid extraction with FIA and AS can serve a variety of purposes, namely:

(a) indirect determination of non-metal species; (b) elimination of matrix effects

(spectral interferences); and (c) selectivity enhancement through the use of organic solvents, etc. The preconcentration factors achieved are not very large owing to the technical constraints to the range of variation of the flow rate ratio. The manifold depicted in Fig. 11.7 A features an additional advantage where only a small volume of solvent actually reaches the flame, thereby avoiding the toxicity of some vapors, particularly that of chlorinated organic solvents.

Fig.11.6 Continuous precipitation FIA systems coupled on-line to an atomic absorption spectrometer. (1) without precipitate dissolution, in the (A) normal and (B) reversed FIA mode. (2) with precipitate dissolution, (C) and (D) without sample injection.

306

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W(a)

A AS I CΡ

Fig. 11.7 FIA configurations with continuous liquid-liquid extraction coupled on-line to atomic spectroscopic instrumentation. The injection point is located prior to (A) or after (B) the extractor's basic components, namely: solvent segmenter (SS), extraction coil (EC), and phase separator (PS). (IV: injection valve; DF: displacement flask; W: waste; and L: reactors ).

307

11.3.4

Hvdride Generation

The problems posed by a conventional, whether commercially available or not, hydride generation system (viz. large sample consumption, high interference levels) can be minimized if the hydrides of some elements (e.g. arsenic, antimony, bismuth, and selenium) are formed in an FIA system provided with a continuous gas-liquid separator, permitting the gas phase to be driven to the atomic absorption or ICP instrumentation. This also results in very good detection limits (of the order of a few nanograms per milliliter in most cases). Fig. 11.8 shows the two FIA configurations for the continuous generation of volatile hydrides.

In the design in Fig. 11.8 A (ref. 42), a few microliters of the sample are injected

into a hydrochloric acid stream that is merged with another stream of sodium borohydride in a basic medium. The volatile metal hydride is formed in the reactor. Phase separation is aided by means of a stream of inert gas (nitrogen or argon) whose flow rate (100-200 mL/min.) is decisive. A debubbler similar to those used in air-segmented continuous flow analyzers allows the gas phase carrying the analyte to be led, free from the aqueous phase, to the atomic spectroscopic instrumentation. Adequate performance of the separator requires controlling the outgoing waste liquid stream.

In addition, its inner volume should be as small as possible in

order to avoid undesirable phenomena arising from dispersion. Continuous gas liquid separations have also been implemented with other systems such as microporous Teflon® membrane (ref. 43) or a classical gas diffusion cell (ref. 44). In the latter instance (Fig. 11.8 B) advantage is taken of the porosity of a microporous membrane to separate the aqueous liquid stream from a gaseous hydrogen stream which functions both as carrier and as acceptor.

There are no

significant differences between the two FIA manifolds shown in Fig. 11.8. As stated above, one of the most salient features of these continuous coupled assemblies is the reduction of the negative influence of other metal species. This beneficial effect arises from the kinetic discrimination resulting from the short time over which the sample resides in the system, and, according to other authors, from the reduced absolute amounts of interferents. A comprehensive and interesting discussion on this topic is made in Chapter 6 (written by Fang) of the monograph edited by Burgura (ref. 10). 11.3.5

Cold Vapor Methods(for the determination of mercury)

This type of method has been widely used for the AAS determination of mercury on account of the unique properties of this metal. The use of an FIA system for implementing this alternative, offers substantial advantages (e.g. simplicity, ease of automation, low sample consumption, high efficiency, and stability) over conventional procedures applied in a laboratory made or commercially available device. A number of configuration have been used for this purpose. They are not essentially different from those described above for the hydride generation, although the quartz tube is not heated in this instance.

A gas-liquid separator for use in this context was

recently reported (ref. 45). Also of note is the novel and interesting approach by Andrade et al. (ref. 46), in which the membrane gas-liquid separator is housed in the measuring flow cell.

Á

Fig. 11.8 Flow injection continuous hydride generation systems, (a) with a gas liquid separator, and (b) with a gas diffusion cell.

Íá Â Ç

308

309

11.4

FEATURES OF SAMPLE INTRODUCTION BY FIA

The use of FIA systems of different designs (Fig. 11.2) and degrees of automation (Fig. 11.1) coupled on line to atomic spectroscopic instrumentation makes an excellent alternative to the introduction of treated or untreated samples. This section deals with the advantages offered by the use of the FIA-AAS association for such a purpose and with its different possibilities. The main difference between the manual introduction of treated or untreated samples and the use of an FIA manifold lies in the ease with which the latter can be fully automated and the operator only handles the injection valve or changes the aspiration tube from sample to sample (see Fig. 11.1).

This results in a number of advantages inherent in the reduction of human

intervention in laboratory processes (ref. 1), viz faster operation, increased precision, particularly in those cases requiring sample pretreatment, and reduced costs. Each of these and some less obvious assets are commented on separately below. 11.4.1

Sample Throughput

Flow injection analysis allows samples to be introduced into the analytical system at a considerably high rate with sampling frequencies between 50 and 700 per hour are typically achieved depending on whether samples need to be treated or not. The upper end of the above rate range is accomplished when the traditional aqueous solutions used as carriers are replaced with air (ref. 19). Sample carry-over is particularly increased in those instances involving the analysis of high analyte concentrations, which cause "memory effects" in the nebulizer that in turn reduce the sampling frequency. Nevertheless, this problem can be minimized by using a dual valve. The use of a continuous separation technique for increasing sensitivity

through

preconcentration calls for the introduction of larger than usual volumes into the FIA system, which considerably reduces sample throughput (5-25 samples per hour). 11.4.2

Sample Consumption

The use the of an FIA assembly instead of conventional procedures for the introduction of liquid samples into atomic spectroscopic instrumentation results in remarkably consumption of sample (or standard).

lower

This is of special significance in analyzing scant or

valuable samples. Injected volumes in FIA usually range between 30 and 50 μ ί , though they may be as small as 2-10 μL·

The minimum useable injected volume is determined on the one hand by the analyte

concentration (the higher it is the smaller the injected volume required) and on the other hand by the degree of complexity of the FIA manifold (the higher it is, the greater the dispersion or dilution of the sample).

The volumes inserted into those systems involving aspiration rather

than injection through a valve are considerably larger (0.10-10 mL) as the basic aim of the FIA system in such instances is preconcentration.

310 11.4.3

Typical Problems Arising from Sample Introduction

The nature of some liquid or dissolved samples results in some well known problems upon direct introduction by aspiration into an atomic spectroscopic detector (atom reservoir).

The

reduced sample volumes typically used in FIA minimize or in some cases eliminate these problems altogether.

The beneficial effect of FIA in this respect allows a number of

shortcomings to be circumvented. Thus: (a) the nebulizer or burner clogging caused by high dissolved salt content in a sample is readily avoided, which allows the direct introduction of samples containing up to 30% (w/v) of dissolved solid. This is of particular significance when solid samples are to be dissolved in small volumes because the analyte is available at a low concentration or because the sample already has a high salt content (e.g., brine solutions, sea water); (b) the greater the viscosity of a liquid sample, the smaller the atomic spectroscopic signal will be when discrete aspiration is used.

The utilization of an FIA system and large

injected volumes (over 100 μΐ_) and short reactors give rise to the same effect. On the other hand, the use of small injected volumes (10-30 μΐ_) gives rise to steadily constant signals regardless of wide changes in the sample viscosity as a result of the efficient mixing of carrier and sample favored by a low initial viscosity.

This is not of particular significance to the

determination of trace metals in clinical samples, as viscosity differences between standards may give rise to major problems.

Obviously, increased carrier temperatures also have a

beneficial effect (ref. 47); (c) the use of organic solvents in atomic spectroscopy may pose serious health hazards for the operator due to the formation of toxic vapors. This is not the case with FIA systems. The fact that they are closed systems avoids the direct contact of the solvents with the atmosphere.

Moreover, the low solvent volumes typically injected (see Fig. 11.7 A)

result in flame vapors containing minimal amounts of toxic vapors. Chlorinated solvents should be avoided as far as possible for continuous sample introduction into a nebulizer unless special precautions are exercised; and (d) as a rule, the stability of a flame or plasma achieved by using micro-injection is much higher than that accomplished by conventional continuous aspiration as a result of the nebulizer handling an unbroken stream of liquid not interrupted by air.

Sample

introduction by FIA also allows the use of solvents, injected in small volumes which may extinguish a flame or plasma upon continuous aspiration. 11.4.4

Addition of Reagents

The prior mixing of the sample with certain reagents (e.g. lanthanum or strontium ions, EDTA) in order to minimize interferences or supress ionization, can be automatically accomplished in three ways by using FIA manifolds, namely: (a) by using the reagent dissolved in the carrier, which can also be an organic solvent in a single-line FIA configuration (Fig. 11.2.1); (b) by using a second channel of the reagent merging with the main channel where the sample is injected (Fig. 11.2.2); and (c) by using the merging zones approach and a dual injection valve (Fig. 11.2.5). The first of these alternatives is the simplest, but also the most troublesome. The second case results in more efficient mixing of sample and reagent at the merging point and reduces consumption of reagent as this can be circulated at a low flow rate.

311 The third case, which entails the simultaneous injection of very low, identical volumes of sample and reagent that mix at a merging point, dramatically decreases reagent consumption. 11.4.5

Automatic Calibration Procedures

The calibration stage is critical to Instrumental Analysis insofar as it decisively influences the accuracy, precision and speed of the analytical process, the quality of which is adversely affected by the wrong choice of the standards to be used. This is due to the fact that no account is taken of potential matrix effects or because they are cumbersome to prepare.

In this respect,

the use of FIA systems in the development of automatic calibration procedures is one of the most outstanding advantages of their on-line coupling detector.

to an atomic spectroscopic instrument as a

In a recent interesting paper, Tyson (ref. 49) reported a systematic description of

flow injection procedures. The high versatility of FIA has given rise to a number of approaches to calibration. An orderly description of such approaches requires distinguishing between four generic options, namely: (a) the use of different parameters of the transient signal as the basis for the analytical measurement; (b) the utilization of a single or several standards of different concentration; (c) the application of the classical calibration graph method or the standard-addition method; and (d) the use of systems for introduction (injection or aspiration) of samples and standards via the same or different zones, simultaneously or sequentially in the latter case. Fig. 11.1 illustrates the different mechanisms available for obtaining information from FIA peaks.

In addition to the typical way of constructing the calibration graph (i.e., plot of peak

height vs concentration), there are other alternatives which offer some advantages.

Thus,

calibration based on the peak area allows the increased nebulization efficiency obtainable at low flow rates to be exploited.

In addition, peak area measurements give rise to better detection

limits and precision, though they require a microprocessor for data collection, processing and result delivery. The so-called "time based methods" rely on the relationship between the peak width (viz. the time elapsed between two points of the FIA peak at a given preselected signal level) and the logarithm of the analyte concentration. This allows the concentration range of the calibration curve to be expanded by a few orders of magnitude (refs. 50,51). It is also possible to use the rising portion of a peak signal by employing a real mixing chamber to generate an exponentially increasing concentration by continuous dilution of a concentrated standard (ref. 52).

The use of fixed-time points along the falling potion of the signal is another viable

alternative to automatic dilution (ref. 53). Calibration procedures used with the FIA-AS association can be implemented in two ways, namely:

(a) by using a series of standards of different concentrations (most common choice),

and (2) by employing a single standard solution that is automatically diluted in a controlled manner within the FIA system, thereby avoiding the time-consuming manipulations traditionally associated with the manual preparation of a series of standards in volumetric glassware flasks from a concentrated stock solution.

312 Experimentally, FIA-AS calibration procedures can be classified into two large groups according to the way in which a series of standards or only one standard may be introduced to the system.

The two configurations, illustrating the most relevant and representative possibilities

is shown in Fig. 11.9 and Fig. 11.10. The chief differences between the two configurations lie in whether the sample or standards are introduced through the same (Fig. 11.9) or through different places (Fig. 11.10). The most common procedure to implement the determination of a given analyte with the FIAAS association involves the injection of standards of increasing concentration and the subsequent introduction of the samples (Fig. 11.9 A), which results in no significant advancement.

The

possibility of exploiting the controlled dispersion or dilution of a single standard within the FIA manifold avoids preliminary manual dilutions. Figs. 11.9 Β and 11.9 C show two alternatives to the use of reactors of different lengths to achieve the desired extent of dilution of a single standard of high concentration.

In the first instance (Fig. 11.9 B) are used two or more

reactors flanked by a splitting and merging point which divide the injected standard and samples into several subplugs whose volume and extent of dispersion are a function of the geometric and hence hydrodynamic features of each reactor, and whose sequential arrival at the detector gives rise to a multi-peak recording.

The three maxima and two minima obtained with the three

reactors provide dilution factors of 7, 10, 14, and 30 and therefore allow the best suited to each sample to be chosen (ref. 54). The configuration depicted in Fig. 11.9 C is based on the same principle. However, it uses two selecting valves that allow one to choose the appropriate reactor for the required extent of dilution (by factors between 6 and 4) rather than the above mentioned splitting and merging points (ref. 55). The manifolds shown in Fig. 11.9 D and Fig. 11.9 Ε are based on the automatic introduction of a variable, controlled volume of a single standard and the samples.

They require a

microprocessor to time the functioning of some elements in the configuration. Tyson (ref. 49) regards these as "time-based" methods, although they differ from those based on peak-width measurements alluded to above. The so-called "zone sampling mode" (Fig. 11.9 D) uses two injection valves.

The first injects a somewhat large volume that is diluted in a controlled

manner before being passed through the loop of a second injection valve which re-injects part of the dispersed zone. This allows one to choose between the zone of greater (ends) or smaller (center) dilution by controlling the time elapsed between the two injections (ref. 56).

In the

configuration depicted in Fig.11.9 E, the typical injection valve of FIA assemblies has been replaced with an aspiration probe whose motion is automatically controlled and which aspirates the required volume of standard (or sample).

This is accomplished by controlling the time

during which the probe is immersed in the corresponding solution (ref. 57). The pump is kept still while the probe is not immersed, thereby avoiding the introduction of air into the stream unlike the air segmented flow analyzers. The use of a reversed FIA configuration (Fig. 11.10 A) has allowed Tyson (ref. 49) to develop a straightforward, yet ingenious procedure for implementing the standard addition method with the FIA-AS association.

The sample is continuously aspirated into the detector,

313 where it yields a constant signal that can be taken as a baseline. The injection valve introduces standards of increasing concentration that yield positive or negative peaks depending on the particular concentration. By plotting the signal increment (peak maximum) as a function of the standard concentration to obtain a straight line whose intercept (nil signal increment) allows the concentration of analyte in the sample to be determined. The use of a symmetric merging zone configuration (Fig. 11.2.5) is another straightforward approach to the implementation of the standard additions methods in its "generalized" method version which compensates for both matrix and spectral interferences. It involves the simultaneous introduction of samples and standards and has been used with the FIA-ICP-AES association (ref. 58). The configuration depicted in Fig. 11.10 Β illustrates the approach employed by Tyson and Appleton (ref. 52) to carry out calibrations based on the use of a mini-mixing chamber which effects the dilution of a single standard that is introduced into the system by aspiration.

A

microcomputer collects data from the dilution curve and plots the exponential concentration, C, gradient as a function of time, t, according to the equation : Cst = C[1 - exp( - qt/v)]

(11.1)

where Cst is the initial standard concentration, q is the flow rate, and ν the volume of the minimixing chamber. In a second stage, the selecting valve permits the continuous aspiration of the sample, which yields a constant signal almost immediately.

The time tx at which this signal

appears in the dilution curve of the standard is used to calculate the analyte concentration in the sample from equation 11.1. The "zone penetration" method

is another useful alternative to the development of

calibration procedures. Although Fang et al. (ref. 59) use a proportional injector, instead of the usual injection, and selecting valves actuated simultaneously. These have been preserved in the scheme shown in Fig. 11.10 C for consistency with all other configurations described to date. During the load operation, an aqueous carrier arrives continuously at the detector.

The

simultaneous switching of both valves results in the formation an analyte solution zone sandwiched between water (in the front) and the standard (in the rear). Fig. 11.10 C also shows the theoretical profile of standard (dotted line) and sample concentrations (solid curve), as well as the real response of the detector. Times ti and t2 corresponds to the same dispersion in the rising and falling portions, respectively, of the analyte concentration profile. The signal at time ti corresponds to the diluted sample exclusively, while that obtained at t2 corresponds to the sample at an identical concentration (dilution) plus concentration of the standard. The analyte concentration in the sample is determined by relatively straightforward computations. In order to avoid fluctuations in the rate of the flow that enters the nebulizer, the chief source of poor reproducibility in FIA-AS calibration and dilution procedures, Bysouth and Tyson (ref. 60) devised an interesting manifold based on a fixed rate pump acting in conjunction with a computer controlled selecting drive and one other pump.

314

SOLUTION

Fig. 11.9 Calibration in FIA-AS involving the introduction of standards (ST) and samples (S) through the same zone of the flow injection manifold. (C: carrier; R; reagent; P: pump; IV: injection valve; SV: selecting valve; SP: splitting point; CP: confluence point; KL: kinematic linkage; PR: computer controlled moveable probe).

315

Fig. 11.10 Calibration procedures in FIA-AS involving the introduction of standards (ST) and samples (S) through the different mixing zones in the flow injection manifold. (C: carrier; P: pump; MX: mixing chamber: IV: injection valve; SV: selecting valve).

316

11.4.6

Manipulation of Sensitivity

One of the most interesting practical advantages of the FIA-AS association is the ease with which it can be adapted to the concentration level of the analyte in the sample, whether this is too high (e.g. determination of many metals in alloys) or very low (e.g. determination of trace metals in biological fluid) by automatic dilution or preconcentration. 11.4.6 (a) Automatic Dilution Most of the calibration procedures described in the preceding section allow the automatic, controlled dilution of standards and samples, and give dilution factors between 2 and 100. Hence, the dynamic determination range can be expanded by several orders of magnitude to higher concentrations without the expected detriment to precision.

This permits the direct

determination of analytes occurring at high concentrations in the original sample without the need for preliminary manual dilution in order to ensure that the signal yielded falls within the linear range of the calibration curve. This of significance to the routine control of samples in which the analyte concentration may vary over a broad range.

Reis et al (ref. 56) have

developed a method for the AAS determination of potassium in plant digests over the range 0-500 mg/L, with a precision of about 1% by using the flow injection manifold shown in Fig. 11.9 D. Another way of accomplishing automatic dilution involves the use of peak width rather than the peak height (as stated previously, the peak height is proportional to the analyte concentration).

Thus, if the peak corresponding to a very concentrated sample gives a large

signal which goes off-scale on the chart recorder, one can use peak width without the need to dilute the sample in order to perform a new measurement (ref. 50). As a rule, the FIA manifold should result in an analyte dispersion higher than usual (e.g., the use of longer reactors) in order to allow the peaks to approach a Gaussian shape. It is interesting to mention the good results obtained with this "dilution" in the final stage (data collection and processing) of the analytical process, which allows the determination of magnesium at concentrations of 3, 60, 400, and 1000 mg/L with errors of 0.0, -5.0, 0.0, and +3.0%, respectively (ref. 51).

It is

also possible to enact automatic "dilutions" by making measurements at times longer than the peak maximum, i.e. in the tailing portion of the peak (ref. 53).

11.4.6 (b) Automatic Preconcentration The application of a continuous separation technique on-line with atomic spectroscopic instrumentation allows analytes to be preconcentrated to various extents and will result in increased sensitivity.

The concentration range is expanded to smaller values (of the order of

1 0 to 1 0 ) . The widely acknowledged loss of precision derived from the manual application of 9

12

a batch technique is reduced by coupling the continuous separation device on-line with the detector.

Section 11.3 deals with representative examples of this type of FIA configuration

aspect in a general manner.

The preconcentration of analytes requires the introduction of a

317 larger sample volume than is usual for FIA.

Such a volume is normally added by strictly

controlling the time of aspiration at a very constant and reproducible flow rate.

The

configurations most widely employed for this purpose are based on continuous ion exchange (Fig.11.5 C) and precipitation processes (Fig. 11.6 D). The preconcentration factors typically obtained by continuous liquid-liquid extraction (Fig.11.7 A) are much smaller compared to ion exchange methods as they depend on the ratio between the incoming flow rates of the organic and aqueous (sample) phase, which cannot be varied over a wide margin due to technical constraints. A brief description of some representative examples of these configurations will show the potential of these techniques. The use of a micro-column packed with aminoacetate chelating resins allows the FIA-ICP-AES determination of cadmium with a detection limit of 0.05 ng/mL and a relative standard deviation (RSD) of 2.2% for a concentration of 0.1 ng/mL. The sampling frequency was 25 per hour for a volume sample of 5.0 mL (ref. 61). The manifold shown in Fig. 11.6 D has been used for the determination of lead in waste water with a detection limit of 1.0 ng/mL (RSD of 3.0%) by AAS with a hollow cathode lamp (preconcentration factor of 700). The traces of lead present in the aspirated sample are precipitated as a basic salt in the coil upon merging with a stream of ammonia, and the precipitate is retained on a filter. In a second stage, the precipitate is rapidly dissolved by a stream of nitric acid and subsequently driven to the detector (ref. 62). Preconcentration in this type of on-line system poses two main problems.

First, it requires

the use of a large volume of sample, which may be a major problem where the volume of sample is small (e.g. biological fluids).

Nevertheless, by performing injections (1-2 mL) of

hemodialysis fluids through a septum, Sanz-Medal et al. (ref. 63) have succeeded in determining aluminum in the ng per mL range by AAS and ICP-ES, using a mini-column packed with a strongly basic anion-exchange resin in a single-line manifold (Fig. 11.5 A). retained on the resin is eluted by means of an injection of 75

The analyte

of 1 M sodium hydroxide

through a conventional injection valve. The other major problem of these configurations lies in their poor performance as regards sample throughput.

They have sampling frequencies

substantially lower than those typical of conventional FIA configurations as a result of the limitations imposed by the sample aspiration operation. This problem has been addressed by using two parallel columns to perform simultaneous preconcentration and elution (refs. 37,38). There are other ways of increasing the sensitivity of the FIA-AS association which are basically related to the features of the carrier material. Thus the use of air as a carrier for the injected samples results in calibration graphs with slopes which are twice as steep as those typically obtained by using water as the carrier in the AAS determination of zinc (ref.19). Organic solvents such as MIBK also favorably replaces water as a carrier in the determination of some metal ions (refs. 20,21). However, the aqueous samples must be previously mixed with a miscible organic solvent (e.g. methanol, acetone) to avoid the formation of intermediates in the system (ref. 21).

318 11.4.7

Stud y an d Enhancemen t o f Selectivit y

In general , atomi c spectroscopi c technique s ar e mor e selectiv e tha n molecula r spectroscopi c techniques. However , ther e ar e a numbe r o f potentia l problem s i n atomi c spectroscop y which can reduc e th e selectivity . Thes e ar e principall y chemica l an d spectra l an d ca n b e a proble m i n complex matrices . Th e extent t o whic h selectivit y change s wil l var y wit h atomi c spectroscopi c technique (viz . flam e AAS , non-flam e AAS , ICP-AES , ICP-MS , etc.) .

Th e natur e an d

determination o f thes e potentia l perturbation s an d thei r tolerate d leve l wil l allo w th e minimization o f thes e effects . Th e FIA-A S associatio n ha s valuabl e feature s tha t ca n b e exploite d both fo r characterizin g an d fo r enhancin g th e selectivit y o f atomi c spectroscopi c techniques . The systemati c stud y o f th e influenc e o f a larg e numbe r o f specie s (charge d o r uncharged , organic o r inorganic ) i n developin g a n analytica l procedur e i s time-consumin g an d ca n b e tedious a s i t involve s determinin g th e tolerate d leve l o f eac h species . Thi s involve s th e manua l preparation o f a larg e numbe r o f solution s i n whic h th e concentratio n rati o o f foreig n specie s t o analyte mus t var y ove r a wid e range . Th e FI A techniqu e substantiall y reduce s th e tim e require d to determin e th e selectivit y o f a give n analyt e methodolog y involvin g th e us e o f a n atomi c spectroscopic detector . I n addition , th e feature s o f th e transien t signa l typicall y obtaine d b y FI A allows on e t o establis h th e origi n o f th e perturbatio n pose d b y eac h species . Fig . 11.1 1 show s a simple manifol d designe d t o stud y th e interference s wit h th e AA S determinatio n o f calciu m (ref . 47). A strea m o f th e analyt e i s continuousl y introduce d int o th e syste m an d merge d wit h a wate r carrier strea m int o whic h th e potentia l interfèren t i s inserte d vi a a norma l injectio n valve . Th e final concentratio n o f th e interfèren t ca n b e calculate d b y evaluatin g th e coefficien t o f dispersio n of th e manifold . Som e specie s (e.g . aluminum , phosphate ) yiel d negativ e peaks , whil e other s (e.g. organi c solvents , potassium , lanthanum ) give s ris e t o positiv e peak s wit h respect t o th e baseline, whic h correspond s t o th e signa l yielde d b y th e analyt e continuousl y introduce d int o th e nebulizer. Th e firs t pea k correspond s t o a n injectio n o f th e analyt e solutio n an d it s profil e reflects th e actua l concentratio n gradien t establishe d i n th e manifold . Recently, Wad e e t al . (ref . 116 ) develope d a similar , thoug h automate d flo w injectio n approach allow s a n intelligen t choic e o f line s an d experimenta l condition s base d o n th e magnitud e of th e interferin g effect s observe d fo r a wid e rang e o f interferent s an d analyt e concentrations . The FIA-AA S associatio n als o offer s th e possibilit y o f enhancin g th e selectivit y o f a give n determination i n a n automati c fashion , thereb y improvin g th e qualit y o f th e analytica l proces s and broadenin g th e scop e o f applicatio n o f atomi c spectroscopy . Ther e ar e thre e mai n way s o f minimizing o r eliminatin g interferences , namely : (1 ) b y automati c additio n o f reagent s (e.g . lanthanum, EDTA ) whic h ar e mixe d wit h th e sampl e i n th e FI A manifol d t o lesse n chemica l interferences an d avoi d unwante d ionizatio n o f certai n element s i n th e flame ; (2 ) b y applyin g automatic calibratio n procedure s addressin g matri x effects . I n sectio n 11.4. 5 wer e describe d several alternative s t o th e automati c implementatio n o f th e standar d additio n method ; an d (3 ) b y using continuou s separatio n device s couple d on-lin e t o a n atomi c spectroscopi c detector . I n thi s manner, th e analyte(s ) i s (are ) isolate d fro m th e interferent s prio r t o enterin g th e nebulizer . This i s th e mos t efficien t alternativ e o f th e thre e methods .

Fig. 11.11 Manifold used by Tyson et al. to study the selectivity for the determination of calcium by FIA-AAS. Reproduced from (ref. 45) with permission from The Royal Society of Chemistry.

time

+ +

Ca lOOppmJ

319

320 A continuou s separatio n ca n b e aime d a t retainin g (isolating ) th e interferent s an d henc e no t involve th e analyte . Thus , phosphat e i s separate d fro m calciu m (th e analyte ) b y mean s o f a continuous precipitatio n syste m i n th e determinatio n o f th e latter . A colum n packe d wit h a n anion exchange r i s use d fo r th e systemati c remova l o f anioni c interferent s i n th e determinatio n of a variet y o f cationi c analyte s (refs . 33,64) . I n thi s an d othe r simila r methodologies , th e concentration o f interferent s an d th e retentio n capacit y o f th e syste m determin e th e frequenc y with whic h i t mus t b e replace d o r regenerate d (precipitat e dissolution , specie s generation) . This ca n b e don e i n a continuou s fashio n b y usin g additiona l stream s an d selectin g valves , o r i n a discrete manne r b y replacin g th e exhauste d continuou s separato r wit h a regenerate d one . Continuous separatio n system s i n which th e analyt e plays a n activ e role , i t i s transferre d t o a secon d phase whil e th e interferent s remai n i n th e startin g phase , ar e fa r mor e commo n an d interesting. Whe n th e aforesai d secon d phas e i s a soli d (e.g . i n precipitation , io n exchange) , th e analyte mus t b e transferre d i n a secon d stag e t o a liqui d strea m intende d t o driv e i t t o th e detector. I f i t i s a liqui d (liquid-liqui d extraction ) o r ga s phase (hydrid e generation , col d vapo r mercury generation) , i t i s transporte d directl y t o th e nebulizer .

I n thes e instances , th e

enhanced selectivit y achieve d i n th e atomi c spectroscopi c determinatio n arise s fro m tha t o f th e separation process . O n th e othe r hand , interference s no t involve d i n th e transfe r o f matte r because o f thei r natur e (e.g . uncharge d o r non-gaseou s species ) ar e eliminate d mor e o r les s completely.

O n th e othe r hand , th e reductio n wit h respec t t o th e manua l procedur e o f

interferents tha t ma y tak e par t i n o r distur b th e separatio n proces s i s th e resul t o f tw o effects , namely: (a ) th e so-calle d "kineti c discrimination " (ref . 65) , whic h stem s fro m th e fac t tha t the tim e ove r whic h th e transfe r take s plac e i s muc h longe r tha n th e conventiona l procedur e a s equilibrium i s rarel y reached , particularl y i n liquid-liqui d extraction .

A s thi s proces s i s

optimized fo r th e analyte , interferent s ar e onl y partiall y transferred ; an d (b ) th e decrease d injected volume , whic h obviousl y reduce s th e absolut e amoun t o f interferent s presen t i n th e analytical system . The indirec t AA S determinatio n o f chlorid e ion s b y continuou s la p precipitation-filtratio n with silve r io n a s reagen t i s muc h mor e selectiv e tha n it s manua l counterpart . Thus , th e selectivity factor s (i.e . th e ratio s betwee n th e concentration s o f interfèren t tolerate d b y th e FI A and th e manua l method ) fo r iodid e an d bromid e ar e 24 0 an d 70 , respectivel y (ref . 66) . Precipitation wit h silve r als o allow s th e determinatio n o f sulfonamide s i n a variet y o f pharmaceuticals withou t interferenc e fro m an y o f th e usua l excipient s o r diluent s (vanillin , glucose, fructose , sucrose , glycerol , starch , ethyleneglycol , polyviny l pyrrolidone , etc. ) (ref . 67). The continuou s extractio n o f zin c a t concentration s betwee n 0. 5 an d 1. 6 mg/m L a s thiocyanate i n MIB K allow s it s absorbanc e a t a wavelengt h o f 213. 9 n m t o b e measure d fre e fro m the otherwis e majo r interferenc e o f iron , whic h i s no t extracte d i n th e proces s (ref . 69) . A systematic compariso n o f interference s betwee n th e FI A an d manua l (wit h décantatio n funnels ) variants o f th e metho d fo r determinatio n o f anioni c surfactant s base d o n th e formatio n o f ion pairs wit h a cationi c chelat e [1,10-phenanthroline-Cu(ll) ] an d subsequen t extractio n i n MIB K

321 revealed the greater selectivity of the continuous automatic procedure (selectivity factors of 50 for Triton X-100, 5 for Perchlorate, 20 for phthalic, succinic, glutamic, and benzoic acid, 200 for nitrate, 2.5 for iodide, etc.) (ref. 69). By using the manifold shown in Fig. 11.8A, Astrom (ref. 70) showed the determination of bismuth at the nanogram per milliliter level to be feasible even in the presence of interference concentrations of 100 to 1000 times higher than those tolerated by the conventional hydride formation procedure. Similar results were found in the determination of selenium in rocks (ref. 71). 11.5

APPLICATIONS

The earliest use of the FIA-AS association was reported in 1978. In the intervening years there have been reported a number of applications whose foundation and generic features were dealt with in the preceding sections of this chapter.

As a detailed description of all areas is

beyond the scope of this chapter, the interested reader is referred to recent published monographs on this topic (refs. 3,4,10). This section comments only on the most relevant and representative of applications. They are divided according to whether the analyte of interest is determined directly or indirectly and whether it is a metal or non-metal species (organic or inorganic). 11.5.1

Pireçt Determinations In these methods, the analyte is introduced into the atomic spectroscopic detector and the

analytical signal yielded is proportional to the concentration. This type of determination can be further divided according to whether or not the analyte is transformed in the FIA manifold. 11.5.1 (a) Determination without Transformation In the most straightforward configurations used for this purpose, the analyte takes part in no chemical reaction or separation process in the FIA manifold. The sample is merely diluted and suitably mixed with reagents intended to reduce interferences.

The analyte arrives directly at

the detector. Judging by the number of reports for the determination of metals in real samples, particularly in the journal, Clinical Chemistry, this is the most widely and preferred method. The FIA-AAS association has been employed in the determination of copper, zinc, and iron in whole blood (ref. 72); zinc (refs. 73,74), calcium and magnesium (ref. 75), lithium (ref. 76), in serum ; sodium, potassium, calcium, magnesium, iron, copper, and zinc in cerebrospinal fluid (ref. 77); iron and copper in human milk (ref. 78), and lead in human hair (ref. 79). The FIA-ICP-AES association has been used in the development of a rapid and precise method for the simultaneous determination of eight elements in serum (ref. 80).

Other

straightforward FIA manifolds coupled on-line with atomic spectroscopic instrumentation have been used to solve analytical problems in environmental chemistry e.g., the determination of sodium, potassium, magnesium, and calcium in waters (ref. 81), in industrial control e.g., the determination of lead in gasoline (ref. 82), copper in effluents from a zinc plant (ref. 83), and

322 lead, bismuth, antimony, and silver in steels (ref. 82), in food chemistry e.g. the determination of iron and copper in infant powdered milk (ref. 84), and lead and calcium in oyster tissues and bovine liver (ref. 85), and in rock analysis e.g. the determination of calcium (ref. 86) and magnesium (ref. 87). Direct determination of non-metal species by FIA-AS can be used by molecular emission cavity analysis (MECA).

The determination of organophosphorus insecticides (ref. 89) and

sulfur anions such as sulfide, sulfite, and sulfate (ref. 90) has been obtained with MECA. However this approach has not yet been systematically applied to the determination of these analytes in real samples. 11.5.1 (b) Determination with Transformation of the Analvte In this method, the analyte does take part in a chemical process (formation of a chelate either in solution or on a resin, a precipitate or a hydride) generally associated with a continuous separation process (e.g., liquid-liquid extraction, ion exchange, precipitation, gas formation, and separation).

The transformed analyte yields a signal which is proportional to its original

concentration in the sample upon arrival at the detector. The transformation undergone by the analyte is intended to indirectly increase both the selectivity (interference removal) and sensitivity (preconcentration) of the determination. The formation of neutral metal chelates and their continuous extraction into an organic solvent has allowed the determination of zinc in biological and environmental samples (ref. 91) and lead in human urine (ref. 92).

A

continuous precipitation-dissolution system (Fig. 11.6D) has been used to determine lead in waters at the ng/mL level (ref. 62). Olsen et al. (ref. 93) have reported a variety of FIA manifolds using a packed mini-column packed with chelating resin Chelex-100 for the AAS determination of traces of heavy metals (cadmium, copper, lead, and zinc) in sea water. Aluminum has been determined in hemodialysis fluids by using a mini-column packed with an anionic resin (ref. 63) or a synthetic chelating resin (ref. 94). A column packed with activated alumina in its acidic form has been used in FIA-ICP-AES for the determination of sulfate in different types of water (ref. 95). The same type of detector has been used in the development of a method for the rapid sequential determination of chromium (III) and chromium (VI) in waters (ref. 34).

FIA systems involving hydride generation have been advantageously applied in

various areas in the determination of selenium in rocks (ref. 71) and arsenic in glycerin (ref. 96). 11.5.2

Indirect Determination

These are normally based on the measurement of the signal yielded by a species acting as a reactant in the analytical process.

The analytical signal is indirectly related to the analyte

concentration. The use of FIA configurations coupled on-line with atomic spectroscopic detectors is rather advantageous in this context as manual procedures are sluggish, cumbersome and scarcely precise. A distinction should be made from a practical point of view according to whether the

323 analyte to be determined is a metal or non-metal. The above classification based on whether or not the analyte is transformed is less appropriate here. 11.5.2 (a) Determination of Metals Valcarcel et al. have developed indirect methods for the determination of aluminium (ref. 97), uranium (ref. 98), and lanthanum and cerium (ref. 99) based on the enhanced absorption signal obtained in a fuel-rich air-acetylene flame. The FIA manifold used for such a purpose is very simple.

The sample is injected into a carrier containing iron (III) as a masking agent

intended to avoid the precipitation of the reagent cation at the optimum pH. These are sui generis indirect determination as the analyte is not transformed within the FIA system and the reagent exerts its action in the flame. The former is determined through its positive action on the AAS determination of the reagent. 11.5.2 (b) Determination of Non-Metals As a rule, the indirect determination of non metals by FIA-AS are based on a prior reaction between a reagent responsible for the atomic spectroscopic signal used and the analyte in the FIA manifold. A continuous separation technique is used in most cases to avoid or control the arrival of the reagent at the detector.

By using a column packed with CuS, Ruzicka et al. (ref. 100)

determined cyanide. This anion elutes as the reagent-cation by forming cuprocyanide complexes that are driven to the AAS system used.

Indirect determinations involving liquid-liquid

extractions are based on the use of charged metal complexes which form extractable ion-pairs with the analytes. This is a case in the determination of Perchlorate in serum and urine by use of the cuproine-like chelate copper

(l)-6-methylpicolinaldehyde

azine (ref.

101), anionic

surfactants in waters by the use of the copper (ll)-1, 10-phenanthroline complex (ref. 69), cationic surfactants in waters using the

tetracyanatocobaltate (II) complex (ref. 102), and

nitrate and nitrite in meat using the copper (l)-2,9-dimethyl-1, 10-phenanthroline (refs. 103,104).

Continuous precipitation-filtration-dissolution systems used in an FIA manifold

(Fig. 11.6) have been successfully applied to the determination of chloride ion in waters (ref. 66) and of mixtures of chloride and iodide in foodstuff (ref. 105) by using the silver ion as a reagent, sulfate in waters by using lead (II) as precipitant (ref. 106), and sulfonamides (ref. 67) and local anaesthetics (ref. 107) in pharmaceutical preparations by use of silver and copper (II) as précipitants. 11.6

TRENDS

Many authors have acknowledged that one of the most interesting feature of FIA is the possibility of using it as a tool for the introduction of samples in atomic spectroscopy.

As in

other fields, FIA poses some problems regarding the development of truly innovative approaches to its association with atomic spectroscopy.

It facilitates the resolution of real problems

concerned with routine control in a variety of areas such as clinical, environmental, industrial control, pharmaceutical and food chemistry.

It is therefore not surprising that two established

324 commercial manufacturers of atomic spectroscopic instrumentation are currently developing modules for AAS and ICP-AES intended to automate preliminary operations such as calibration, preconcentration, and dilution. This will be a great asset to control laboratories handling large numbers of samples and will no doubt promote atomic spectroscopy as an analytical technique. Continuous sample introduction systems (FIA) coupled on-line with

electrothermal

atomization AAS have not be widely used (refs. 108,109) on account of the intrinsic discreteness of such detection systems. The fact that this atomic spectroscopic alternative is subject to major sources of perturbation makes on-line incorporation of separation techniques for interference removal a promising option in this context. Advancements in various aspects of continuous separation techniques (e.g., the use of new exchange materials, the implementation of liquid-liquid extraction without phase separation, the sequential dissolution of one or several precipitates, etc) will no doubt endow the analytical methodologies involved with greater simplicity, precision, selectivity and rapidity, and expand their scope of application.

It is of interest to note the possibility of using two continuous

separation techniques (e.g., liquid-liquid extraction and hydride generation (ref. 110)), thereby exploiting their complementary advantages. One of the most serious drawbacks of automatic methods of analysis is that automation does not embrace the entire analytical process.

Most "automatic analyzers" require the manual,

discrete collection and preparation of samples. "automatic analyzers."

These are treated and introduced to the

This is the actual "brake" to the process which is frequently passed

over in describing the disadvantages of analyzers. preliminary operations is of practical interest.

The elimination or automation of these

Direct near real-time determination of metals

in the atmosphere by atomic spectroscopic techniques (ref. 111) makes an interesting approach in this context, as does the direct introduction of solid samples into FIA systems. In these cases they are lixiviated or dissolve in a controlled, automatic fashion by using one of the following two approaches, neither of which has been used with atomic spectroscopic detectors, namely: (a) by using additional energy in the form of an electrical discharge (ref. 112) or ultrasounds (ref. 113); (b) by repeatedly passing a lixiviating carrier through an open-closed (ref. 114) or reversed-flow manifold (ref. 115). ACKNOWLEDGEMENT The author wishes to express his gratitude to the Comision Interministerial de Ciencia y Technologia of the Spanish Government for financial support through Grant No PA86-0146 in connection with this work. REFERENCES 1 2 3

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