Chapter III

AUTOMATIC ANALYTICAL METHODS FOR ENVIRONMENTAL MONITORING AND

CONTROL

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III.1. INTRODUCTION

In the last decades can be observed the use on a larger and larger scale of automation in various domains of science and technique, and especially in the domain of environmental quality monitoring. This was possible due to the great progresses in the top technology fields, like micromechanics, microelectronics and especially computer construction.

The automation of some processes or technologies presents great advantages, which will not be mentioned here, allowing the achievement of great performances and low costs for the products obtained, which would otherwise be inaccessible.

Automation found a wide application domain even in the field of analytical chemistry, presently being harder and harder, and in some cases even impossible, to resolve the problems arising for a chemist without using automated analysis methods.

Generally, the automation of the analytical processes can be made in several ways. Thus, automated analyzers for discrete samples were created, together with flow analyzers and robotic analyzers. From these, of extreme importance are the analyzers based on the principle of flow analysis.

In the last decades special attention was paid to the flow analysis methods being applied for the solving of routine or research analytical problems. The common characteristic of these methods is the fact that the measurements are made in an analytical channel through the sample to be analyzed and the reagents circulate. The devices constructed for the applying of these analysis methods are characterized by simplicity from a mechanical point of view and operation safety. In addition, certain important characteristics of the flow analysis methods, like analysis quickness and reproducibility of the determinations, are very high.

The use of computers to command, control and diagnose the equipment used, and for the processing of the obtained experimental data, has increased even further the performances of the flow analyzers, many of them being able to operate in a fully automated regime.

The flow analyzers can be used for the ‘on-line’ or ‘off-line’ analytical determinations.

In the ‘on-line’ version, the flow analyzer is placed near the sampling location. Thus, the samples can be taken (generally automatically) from an industrial flow, a wastewater evacuation channel, etc. The analysis begins immediately after sampling, the result of the analysis being obtained in a short time from a few tens of seconds to at most a few minutes. Proceeding in this manner, the results of the analysis are obtained in ‘real time’ so that efficient actions can be taken, whenever necessary. The ‘on-line’ flow analyzers are generally analyzers dedicated to certain chemical species, for some concentration domains. These analyzers, generally, must

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have a robust construction and this is due to the location in which they operate and where vibrations, large temperature variations, etc. may be present.

In the ‘off-line’ version, the flow analyzer is located at a certain distance from the sampling location, in a laboratory. The samples, collected from various points, are brought to the laboratory for analysis. Between the sampling and the analysis a long time may pass (hours or even days) which is often inconvenient.

Different methods for conducting the flow determination ‘in-line’ also exist. In this case a parameter, or even a species to be analyzed from an industrial flow, is determined with the aid of a sensor introduced in the respective flow.

In the following pages a short presentation of the automated flow analysis methods will be made with applications in the environmental quality domain, together with other automated analysis methods with important applications in the mentioned domain.

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III.2. FLOW ANALYSIS TECHNIQUES Mihaela Carmen CHEREGI, Mihaela BADEA, Andrei Florin DĂNEŢ III.2.1. INTRODUCTION IN CFA, SFA, FIA AND SIA

Automatic flow methods of chemical analysis, unknown for more then a half a century ago are now widespread in most analytical laboratories. Since the original paper published by Skeggs in 1957 on multisegmented continuous flow analysis, many improvements and even simplifications have been made on this field.

The importance and interest of these continuous flow analyses regarding the study of water quality is reflected in numerous reviews which have been carried out within this field either with a general approach or in the case of their application to the determination of certain parameters. The majority of these papers are related to the application of flow injection analysis (FIA) and, in the last years, to sequential flow analysis (SIA). The explanation is the fact that either they are no longer in an widespread use (segmented flow analysis) or they have no fast development (multicommutation flow analysis and multisyringe flow analysis).

III.2.1.1. Continuous Flow Analysis

Continuous flow analysis (CFA) refers to any process in which the concentration of the analyte is measured uninterruptedly in a stream of liquid or gas. The basic principle of continuous flow analysis is to eliminate chemical analysis by hand-mixing of reagents in individual items of glassware and to substitute a continuously flowing stream of liquid reagents circulating through a closed system of tubing. Therefore, in CFA the sample is converted into a flowing stream by a pumping system and the necessary reagent additions are made by continuous pumping and merging of the sample and reagent streams. The mixing and the chemical reactions take place while the sample solution is on its way toward a low-through cuvette, where the analytical signal is continuously monitored and recorded. The principal difficulty to overcome is to prevent intermixing of successive samples during their passage through the analyzer, this intermixing causing overlap and loss of discrimination at the recording stage. In general, to minimize this so-called carry-over, the design of the timing sequence between samples was optimized by reducing the processing rate and by inserting a washing solution (e.g. water) between each sample. The higher the sample throughput the greater is the interaction between samples and this accounts for the restriction on sample processing rate in continuous analyzers.

The diagram of the simplest CFA system is presented in Figure III.2.1.a and it consists of:

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- a pump (P) that is used to propel the carrier stream through a thin tube; - a coil of tubing also named reaction coil (RC) in which the sample zone disperses

and reacts with the components of the carrier, forming species that are detected by a flow-through detector;

- a recorder/personal (PC) computer which registers the CFA typical signals. Continuous analyzers are made with fewer moving parts, because it is the

liquid that is in motion and for this reason they have the merit of being mechanically simpler and easier to construct then the discrete ones. Sample transport and reagent additions require only a suitable pump. Peristaltic pumps are almost always used in commercial continuous analyzers and the readily availability of multichannel peristaltic pumps enables a single device to control the entire sequence of events. This technique requires flexible tubes in order to set up the continuous flow system and these tubes must not be attacked by the materials under examination, and this may place certain limitations on the scope of the method. Certain reactive and corrosive materials cannot be satisfactory pumped although advances have been made in the development of inert plastics and other synthetic materials.

The continuous flow approach is the most flexible way to carry out a number of operations necessary to perform a chemical assay. In addition to sample dilution, heating, mixing, and reagent insertion, operations executed in a discrete analyzer too, the continuous flow mode can perform dialysis, gaseous diffusion, distillation, solvent extraction and other types of chemical pretreatment directly on the sample, during its transport to the detector.

A greater variety of detectors may be applied to a flowing stream, the most common being the photometric ones.

Types of continuous flow methods The continuous flow methods can be classified into three groups:

1. Continuous mixing methods (Figure III.2.1.a) – that involve the sample insertion into the system, mixing it with the carrier or reagent, measuring the reaction plug as it passes through a suitable detector and either sending to the waste (open systems) or recirculating it (closed systems). Also, the sample can be inserted into the system in an intermittent mode, with washing solutions intercalated between samples in order to avoid carry-over.

2. Stopped-flow continuous mixing methods (Figure III.2.1.b) – the flow is stopped at various stages during the process in order to prevent air from entering the system between sample aspiration and reagent aspiration or washing. A kinematically controlled probe aspirates a certain sample volume through a steel tube dipped into the sample solution, after which it is raised and the pump is stopped. The probe is then immersed in the reagent/washing solution reservoir and the pump is restarted. In kinetic methods, the flow is stopped into the detector flow-cell to monitor the evolution of the reaction.

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C WRC

P

B

A

S

DC W

RC

P

B

A

S

D

(a)

CW

RC

P

A

SD

KL

CW

RC

P

A

SD

KL

(b)

WRC

P1

A

SD

P2

ttrttr

Ana

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al si

gnal

time (s)

WRC

P1

A

SD

P2

ttrttrttrttr

Ana

lytic

al si

gnal

time (s)

(c) Figure III.2.1. Diagram of CFA system. (a) continuous mixing method; (b) stopped-flow continuous mixing method – reagent A is continuously aspirated and mixed with the alternating flow sample S and carrier C; (c) continuous-flow titration – P1 – pump of constant speed, P2 – pump of variable speed, controlled by computer. P – pump; RC – reactor (reaction coil); D – detector; KL - kinematically controlled probe; C – carrier; S – sample; A, B – reagents; W – waste; ttr – titration time.

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3. Continuous-flow titrations – the sample is continuously inserted into the system either by keeping its speed constant and changing that of the titrant (Figure III.2.1.c), or both sample and titrant can be kept at a continuous speed and measurements can be made as a function of the analytical signal obtained.

Applications of CFA systems. The continuous monitoring of cyanide anion, which is a highly toxic ion, has

been carried out by using a CFA system (Figure III.2.2) and a classical analytical method with spectrophotometric detection (barbituric acid and chloramines T). As Figure III.2.2 illustrates, the stream of chloramines T is mixed with the sample then merged with a pyridine/barbituric acid stream after the corresponding reaction coil. A second reaction coil allows the colored reaction product to form and be measured at 578 nm. This method, also realized by normal FIA and reverse FIA, permits a comparison between these two modes and CFA.

Figure III.2.2. Diagram of CFA system used for cyanide spectrophotometric determination. P – peristaltic pump; RC – reaction coil; D – detector; W – waste.

Chloraminne TRC1=25 cm

P

Pyridine-Barbituric acid

Reagent

Sample

Buffer (pH=6.3)

D W

RC2=525 cm

35° CChloraminne TRC1=25 cm

P

Pyridine-Barbituric acid

Reagent

Sample

Buffer (pH=6.3)

DD WW

RC2=525 cm

35° C

III.2.1.2. Segmented Flow Analysis

The segmented flow analysis (SFA) was the earliest contribution in the field of automated methods development. It originated from the scientific paper of Dr. L Skeggs (University of California, USA) published in 1957 and has found widespread use in almost every facet of analytical chemistry. Skeggs’s studies were materialized in the design of the first continuous dynamic measuring system with sequential introduction of samples and the use of a flow-cell. Sample carry-over was prevented by segmentation with air bubbles introduced between successively aspirated samples.

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Several international corporations, such as Technicon, Skalar, Burkard, etc. have contributed to the development and commercialization of analyzers and assemblies based on Skeggs’s idea. For many years, these were the only alternative available for the automation of high-throughput control laboratories.

General aspects and instrumentation SFA is characterized by the use of one or several liquid streams where the

samples are introduced by sequential aspiration and separated by means of air bubbles aimed at avoiding the carry-over. Therefore, the final liquid stream is segmented into small discrete liquid slugs by bubbles of air or other gas that entirely fills the stream tubing bore.

The sample and reagents are mixed by passing through glass coils and through a temperature controlled heating coil if heat is required to speed the development of the reaction product before detection. In the initial work, it was found that in some analyses the high molecular weight components contained in samples were interfering with the chemical reactions. This problem was ingeniously overcome by the use of a cellophane dialysis membrane to remove them. Today, the technique has become one of the most reliable and widely used methods for automatic chemical analysis in routine and research analytical laboratories. This technique has the advantage of for measuring large batches of samples for up to 16 analytes simultaneously at speeds of up to 120 samples.

A SFA system is presented in Figure III.2.3 and it comprises a series o modules each performing a specific function, e.g. sampling, propelling device, sample transport, heating, separation unit, detector equipped with a flow-cell, data recording and analysis module, all of them coupled on-line to one another.

Reaction coil

Separation unit

Debubber

Air Sampler

Pump

Diluent

Reagent

Waste

Air

Detector

Flow-cell

Thermostatedbath

Reaction coil

Reaction coil

Separation unit

Debubber

Air Sampler

Pump

Diluent

Reagent

Waste

Air

Detector

Flow-cell

Detector

Flow-cell

Thermostatedbath

Figure III.2.3. Scheme of a SFA system.

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Sampling device – which consists of a sample turntable and moving articulated aspiration probe.

Propelling device – aimed to provide the sample and reagent and air streams. These are generally multi-channel peristaltic pumps (Figure III.2.13) but may also be used piston pumps and the pressure exerted by a gas or gravitational force. The flow-rate of the streams can be adjusted and maintained as constant as possible by using flexible plastic tubes that withstand the mechanical pressure to which they are subjected.

Reaction-mixing coils – sample and reagents merge in the appropriate stages through “T”-connectors and then pumped through the glass, PTFE or polyethylene tubing where the mixing of reactants and the analytical reaction takes place. The tubing is coiled and horizontally mounted. It provides the reactants mixing by repetitive inversions of the liquid phase and its length governs the time over which the reacting mixture ‘resides’ in it and hence the sampling frequency.

Heating device, continuous separation device – are introduced in SFA system, if required, and they are connected in series to the other components of the manifold. For heating, usually thermostated baths or electrical wires wrapping the coils favoring the analytical reactions development are used. For separation, devices such as dialysers, liquid-liquid extractors, sorption or ion-exchange micro-columns, filters are used. These devices are placed before the reaction coils to remove potentially interfering species.

Debubbler – its aim is to remove the introduced air bubbles just before the liquid stream reaches the flow-cell of the detector. The removing is necessary in order to prevent the parasitic signals produced by the air bubbles upon reaching the detector. The debubbler is not normally necessary in the most recent designs as the signals from the detector are handled by a computer capable of discriminating between these undesirable signals and those actually corresponding to the reaction mixture.

Continuous detection system – any optical or electrochemical detector that can be equipped with a flow-cell is used as detection unit in SFA. The air bubbles are efficiently removed from the liquid stream if the waste tubing of the flow-cell is coupled to a channel of the peristaltic pump (Figure III.2.4). The flow rate of the liquid entering the flow-cell must be higher than the flow rate of the liquid drawn through the pump.

Data recording and treatment unit – which should be prepared to operate in continuous mode and be as simple as a typical Y–t recorder or a sophisticated as an advanced microprocessor/computer carrying out both operations or delivering directly the required results. The most modern SFA systems are fully operated by a high-performance computer.

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Light source

Photocell

Liquid drawn through pump

Segmentedstream

Air or same liquidsto waste

Light source

Photocell

Liquid drawn through pump

Segmentedstream

Air or same liquidsto waste

Figure III.2.4. Scheme of a spectrophotometric flow-cell with debubbler. Principles and kinetic aspects of SFA An operational feature of the SFA system is that, in addition to sample and

reagents, air is drawn into the system through one of the pump tubes and it produces segmentation of the liquid stream once it has been merged with it. This segmentation is maintained through the succeeding stages of the analysis up to the detection unit where the air is removed and a continuous solution phase is reformed. The air introduction causes each individual sample to be divided into a number of small discrete liquid slugs and this presents several advantages. First, the air segments are responsible for maintaining a sharp concentration profile at the leading and following edges of each individual sample. Second, the presence of air bubbles promotes the mixing of the phases. Each stream slug can invert efficiently as it rises and falls through each turn of the mixing coil. For maximum mixing efficiency the length of each liquid slug must be less than half the diameter of the coil. In addition, the wiping action of the air along the tube wall prevents the build-up at the surface of residues from the preceding liquid slug.

The measurements carried out in SFA systems are made under physical (homogenization of the sample-reagent slug between two consecutive bubbles) and chemical (analytical reaction reaches the equilibrium state before the reacting slug reaches the detector) equilibrium. Therefore, in these systems the steady-state signals are recorded and hence their design and the operation should achieve these equilibria.

The main advantage of a determination realized in a SFA analyzer, namely precision and rapidity are drastically influenced by operational parameters such as the

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extent of carry-over and mixing reactant, the time spent by reacting mixture in the system, etc.

Analytical signal The performance of a system that processes discrete samples at intervals is

related to the dynamics of the flowing stream. A continuous stream of liquid flowing through tubing exhibits a velocity profile, the flow being faster at the center and slower at the tubing surface where frictional retardations occur (Figure III.2.5.a). If part of the fluid races ahead and part lags behind, this can lead to contamination from one sample to the next. Segmentation of the liquid stream by air-bubbles reduces contamination by providing a barrier to mixing. As shown in Figure III.2.5.b, each liquid slug between two bubbles is well mixed by turbulence due to wall friction, and laminar flow and contamination between samples is prevented by complete separation between each pair of liquid slugs. The bubbles continually clean the system by wiping the walls of the tubing and driving forward any stationary liquid film that might contaminate following samples. However, the air-bubbles do not entirely prevent the sample carry-over, because mixing in the surface layer can still occur.

(a) (b)

Figure III.2.5. (a) Parabolic profile of liquid velocity in narrow tubing. (b) Reactants mixing in liquid slugs.

The standard response of the detector for a SFA system and its characteristic

parameters are shown in Figure III.2.6. It is obtained upon passage of the reacting mixture zone, placed between two reagent or washing solution zones, the air bubbles having been previously removed, through the detector flow-cell. It is composed of three parts: a rising portion, a plateau (steady-state signal) and a falling portion, the inverse of the rising portion, merging again with the baseline. Detailed studies reveal that the rising portion of the SFA signal is exponential, thus the measured concentration C as a function of time, t, is given by the equation:

)( tE CCkdtdC

−=

where Cε is the equilibrium concentration ant Ct that corresponding to a given time, t.

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Ana

lytic

al si

gnal

timp (s)trtin tout

tr

tL

tw1/2

Ana

lytic

al si

gnal

timp (s)trtin tout

tr

tL

tw1/2

Ana

lytic

al si

gnal

timp (s)trtin tout

tr

tL

tw1/2

Figure III.2.6. Standard signal obtained in SFA systems. tr – residence time, delay time between sample start and detection unit; tin – time for which probe is in the sample vial; tout – time for which probe is out of sample vial.

The significance of the signal parameters presented in Figure III.2.6 are: tr –

time elapsed between the start of the sample aspiration and its arrival at the flow-cell, also known as residence time; tin – aspiration time over which the probe is submerged in the sample vial, and tout – interval during which the aspirating probe remains outside the sample vial withdrawing air and washing solution. Additional to these parameters, there are another two of great significance, namely the lag phase (tL) and the half-washing time (tw1/2), which have been demonstrated to be fundamental in calculating the performance characteristics of a SFA system. They afford a correlation between the approach to steady-state, fraction of steady-state reached in a given time and contamination between samples.

A plot of log Ct against time is presented in Figure III.2.7. The lag – phase is related to the first portion of the signal and it is defined as the interval elapsed between the signal start and the obtainment of the signal plateau and is expressed numerically as the value of the intercept of the linear portion on the time axis. The half-washing time is defined as the time required for the signal at a given point to change from its value to half of its value and is calculated directly from the slope of the linear portion of the plot.

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time (s)

log

Ct

-0.5

-1.0

-1.5

-10 0 10 20 30 40 50

tL

(Lag phase)

log 2

log 2

tw1/2 tw1/2

time (s)

log

Ct

-0.5

-1.0

-1.5

-10 0 10 20 30 40 50

tL

(Lag phase)

log 2

log 2

tw1/2 tw1/2

Figure III.2.7. Graphical response of lag phase tL and half-washing time tw1/2. Sample carry-over The sample carry-over occurs mainly in unsegmented streams and in terms of

the standard SFA system design this implies: a. the aspiration device, the probe is contaminated internally and externally by the

previous sample unless a washing mechanism is employed; b. flow system, a static, thin liquid film prevents direct contact of air with the tubing

walls, thereby generating a heading sample residue. Upon arrival at the same point, part of the first sample is mixed with and incorporated into the segment of the second (Figure III.2.8.a).

c. connection between the debubbler and the detector input. It favors mixing through the absence of any physical separation between successive samples. The connection must be as narrow and short as possible in order to avoid axial diffusion

The effect of carry-over on the SFA signals compared with the theoretical situation is presented in Figure III.2.8.b. Substantial carry-over results in strongly overlapped, analytically unusable signals.

Sample contamination (Figure III.2.8) can be determined by sequentially introducing three samples (S1, S2, S3) the first and the last of the same concentration

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and S2 of a much higher concentration. If contamination does not occur, the steady-state signals of S1 and S3 should be identical; if the third is higher then the first, the SFA system is subject to carry-over. The fact that the signal between samples may not reach the baseline is normal in routine determination as long as the signal attains equilibrium, i.e. provides a plateau. A measure of sample carry-over can be quantitatively expressed by using tw1/2 and tL. If the time elapsed between the

aspiration of two successive samples is measured, the value of the equation 2/1w

Lb

ttt −

gives the so-called degree of interaction, Φ ⎟⎟⎠

⎞⎜⎜⎝

⎛ −=Φ

2/1w

Lb

ttt

. Clearly, the smaller the

value of tL and tw1/2 the higher is the performance of the analyzer.

Flow

Tim

e Air Air

Air Air Air

Air

Air

S2 S1

S1S2

Flow

Tim

e Air Air

Air Air Air

Air

Air

S2 S1

S1S2

(a)

(i) (ii) (iii)

(b) Figure III.2.8. Carry-over in SFA systems. (a) Contamination of sample S1 and S2 produced by thin liquid film between air and tubing walls. (b) Effect of carry-over on the SFA signals yielded by three consecutive samples (S1, S2, S3). (i) ideal SFA signals; (ii) SFA signals obtained for a reduced contamination; (iii) SFA signals for a strong contamination.

Ana

lytic

al si

gnal

time (s)

S1

S2

S3S3

Ana

lytic

al si

gnal

time (s)

S1

S2

S3

Ana

lytic

al si

gnal

time (s)

S1

S2

S3

Ana

lytic

al si

gnal

time (s)

S1

S2

S3

Ana

lytic

al si

gnal

time (s)

S1

S2

S3

Ana

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al si

gnal

time (s)

S1

S2

Ana

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al si

gnal

time (s)

S1

S2

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In conclusion, air bubbles are not completely efficient in preventing sample contamination. Taking this into account, TECHNICON introduced an intermediate washing solution (Figure III.2.9), which is aspirated in the system in the following sequence: 1. aspiration of air; 2. aspiration of sample (S1); 3. aspiration of air; 4. aspiration of washing solution; 5. aspiration of air; 6. aspiration of the next sample (S2).

Figure III.2.9. Flow profile after aspiration of a washing solution (WS) between two successive samples (S1, S2)

This cycle is repeated until the last sample has been processed. The role of the

washing solution is to decrease the carry-over in the three parts of the SFA system where it usually appears by:

Flow

Air Air AirS2 WS S1WSAir Air

12345612 Sequence

Flow

Air Air AirS2 WS S1WSAir Air

12345612 Sequence

a. washing the aspirating probe internally and externally; b. removing or substantial dilution of the static, thin liquid film on the tubing inner

walls formed by the small ‘delayed’ amount of the heading sample; c. establishing a liquid barrier between sample zones which drastically reduces the

possibility of reaction zones contamination after the debubbler. Sample throughput The sample throughput is defined as the number of samples processed per

hour and it is one of the features whereby the performance of an SFA analyzer is evaluated. Taking this into account, a determination will be feasible only if the signal reaches the steady-state in a time sufficiently short to permit its recording or acquisition. This means that the tin, sample aspiration time, and tr, interval over which the sample resides in the system, should be minimized. However, they must not be to short in order to prevent physical and chemical equilibrium to be attained.

Carry-over is another factor that influences the sample throughput. The use of a washing solution considerably delays the sampling operation. Also, the long lag phase and half-washing times impose an increase in the sample volume to be aspirated and therefore the sample throughput is reduced.

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Factors affecting SFA signal quality There are three aspects of decisive importance that have repercussions on the

quality of the SFA signals and hence on the analyzer performance. These three aspects are: 1. sample dispersion – the term refers to the spread of an aspirated sample in a

flow system, as a result of the static liquid film formed on the tubing inner wall that reduces the separation efficiency of the liquid slugs with air bubbles. The influence of a series of experimental variables on the dispersion has been studied and these variables were classified as:

a. system variables: tubing inner diameter; flow rate; residence time; segmentation rate (expressed as number of air bubbles circulating per second);

b. sample variables: viscosity; surface tension; molecular or ionic diffusion coefficient.

2. sample-reagent mixing – the liquid slug (containing sample, reagent (diluents)) between two air bubbles must be homogenized. This physical equilibrium is attained during sample zones flow through the manifold to the detector, (tr). There are two parameters that contribute to the slug homogenization:

a. the compressibility of the air bubbles gives rise to a turbulent flow regime which fosters mixing

b. the helically coiled tubing favors the radial diffusion through the centrifugal force additional to the sweeping effect of the flowing stream, which shortens the homogenization time.

The factors that drastically influence efficient mixing in SFA systems are: tubing inner diameter; coil diameter; slug length; flow-rate and the characteristics of the flowing solution: viscosity; density; reactants diffusion coefficient.

3. flow stability – reliable and reproducible results in SFA systems are obtained if the flow profile is stable. This means that the circulating liquid slugs should have the same length. The factors responsible for the slug length irregularities are: flow-rate inconstancy; peristaltic pump pulsations; temperature variation (which affects the compressibility of the air bubbles); impurities in the sample or reagent tubing, etc.

Applications of SFA

The most applications of the TECHNICON SFA analyzers are in the field of various parameters determination in biological fluids, in clinical laboratories. But these analyzers can be easily adapted to other needs in the areas such as: environmental, pharmacology, food, agriculture or chemical industry. Companies such as SKALAR design and make analyzers aimed at non-clinical applications.

The applications of SFA analyzers may be classified according to the type of the detector involved. Thus, 70-75% of all SFA methods used molecular UV –VIS

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absorption, followed by ISE potentiometry (10 -14%) and much less nephelometry, fluorimetry.

Figure III.2.10.a depicts an assembly used for the determination of ammonia in see and tap water. The sample is aspirated (eventually after filtration) in the system and mixed with EDTA (metal ion masker) and then with phenolate and hypochlorite streams to form the dye indophenol blue, whose color is intensified by a nitroprusside stream. After de-aeration, the absorbance is measured at 630 nm. In this manner, nitrogen can be determined over the range of concentration 0.02 - 2 mg/mL at a sample throughput of 60 samples/h.

Sampler

Pump

Waste

Waste (air)

630 nm

Washing solution

Air

EDTA

Sample

Phenolate

Hypochlorite

Nitroprusside

SamplerSampler

Pump

Waste

Waste (air)

630 nm

Washing solution

Air

EDTA

Sample

Phenolate

Hypochlorite

Nitroprusside

(a)

Sampler

Pump

Waste

Waste (air) Washing

solutionAir

Water

Sample

Air

Water

Colourreagent

480 nm

Waste

Dialyzer

SamplerSampler

Pump

Waste

Waste (air) Washing

solutionAir

Water

Sample

Air

Water

Colourreagent

480 nm480 nm

Waste

Dialyzer

(b) Figure III.2.10. (a) SFA manifold for ammonia determination in different types of waters. (b) SFA manifold for chloride determination in waters.

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In Figure III.2.10.b is presented a SFA system for chloride ion determination in waters. It uses a dialysis unit to remove interferences. The reagent is a mixture of Hg(SCN)2 and Fe(NO3)3, which in the presence of analyte, generates a red coloration due to the Fe(SCN)2+ complex as a result of the much more stable HgCl+ complex. The reaction is sensitive enough to determine chloride over the range of concentration 1 – 20 mg/L. III.2.1.3. Flow Injection Analysis

Flow injection analysis (FIA) is a form of flow analysis that does not rely on air segmentation to prevent the interactions between successive samples. Instead, it employs a non-segmented flow under conditions such that sample spreading is minimized and successive samples can be introduced at short time intervals.

Although, non-segmented flow systems have existed for a long time, only in 1975 J. Ruzicka and E. Hansen and K.K. Stewart et al, independently demonstrated that flow injection systems can be used for rapid and precise automated analysis if the proper flow conditions are selected. This approach is known as flow-injection analysis; term coined by J. Ruzicka and E. Hansen.

Since FIA was born, it found many applications both in the laboratory and in process control and became known simply as FI as it was realized that FI is not only a tool for analysis, but also a generally applicable solution handling technique. Its ability to control and monitor kinetic aspects of automated assays has been recognized, and identified as “kinetic advantage”.

By now its scope is broadening into environmental research and into a tool of biotechnology and for the study of the chemistry of life. FI’s versatility, or self-adaptation, and perfect computer compatibility makes it an ideal interface between a computer and a (bio)chemical system.

Principles Flow-injection analysis is based on the insertion/injection of a liquid sample

into a moving non-segmented carrier stream of a suitable liquid. The injected sample forms a zone, which is transported by the carrier through a coil of tubing to a detector. The detector measures a physical parameter of the sample (absorbance, electrode potential, pH, etc.) that changes continuously as a function of time as the sample passes through the flow cell. This means that the concentration of the species being monitored is continuously changing with time. The carrier may contain a reagent that reacts with the analyte to yield a detectable product, or may consist of an inert solution and in this case the carrier serves as a means of transporting the sample to the detector. Thus, the FIA response curve is a result of two processes, both of kinetic nature, the physical process of dispersion of the sample zone within the carrier stream, and the chemical process of the formation of chemical species.

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The diagram of the simplest flow injection system is presented in Figure III.2.11 and it consists of: - a pump (P) that is used to propel the carrier stream through a thin tube; - an injection device (Vi) that introduces into the carrier a well-defined volume of

sample solution (S) in a very reproducible manner; - a coil of tubing also named reaction coil (RC) in which the sample zone disperses

and reacts with the components of the carrier, forming species that are detected by a flow-through detector;

- a recorder which registers the FIA typical signals. A typical recorder output has a form of a peak, its height (H) being related to the analyte concentration.

C

S

Vi

W

RC

D

P

R

C

S

Vi

W

RC

D

P

R

(a)

Ana

lytic

al si

gnal

time (s)

H

T

S

tb

Ana

lytic

al si

gnal

time (s)

H

T

S

tb

(b) Figure III.2.11. (a) Diagram of a FIA system. (b) Typical recorder output. P – pump; V – injection device; RC – reactor (reaction coil); D – detector; C – carrier; S – sample; R – reagent; W – waste; H – peak height; T – residence time; tb – peak width, time that sample zone passes through flow-cell.

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The time span between the sample injection S and the peak maximum, which

yields the analytical output, is the residence time T, during which the chemical reaction occurs. For a well-designed FIA system characterized by a rapid response, this means two samples analyzed per minute, (T + tb) must be smaller then 30 s. The injected sample volumes are usually between 1 and 200 μL that requires a maximum 0.5 mL of reagent per analysis. This makes FIA to be an automated microchemical technique capable of having a sample throughput of 100 determinations per hour, with a minimum consumption of sample and reagents.

In addition to such higher sampling rate and very rapid availability of the analytical response, the most important aspect of the FIA method is the concept of controlled dispersion of the sample zone, an entirely new concept in analytical chemistry at that time, and which allows the design of a FIA system suited to automate a given analytical procedure.

In order to explain the FIA response shape, let us examine the Figure III.2.12. After the sample injection into the carrier stream, the formed zone does not flow down the tube as a compact plug. In a FIA system, the injected sample zone disperses according to the parabolic velocity profile characteristic for laminar flow. This parabolic concentration profile develops because the sample molecules near the walls are retarded by friction while the molecules in the center of the tube are free to move more rapidly. In fact, the solution at the walls of the tube does not move at all whereas the solution in the center of the tube moves at twice the average flow rate.

umax=2u

Injection Laminarflow

Laminar anddiffusion flow

Diffusion flow

time (s)Carrier stream direction

Figure III.2.12. Modes of mass transport through a tube. The development of this parabolic concentration profile is not desirable for

FIA because it dilutes the sample and it spreads it out, causing a decrease in sensitivity. This inconvenient is controlled in FIA by employing conditions that promote radial mass transfer (by diffusion). Molecules left behind along the walls of the tube will tend to diffuse toward the more diluted center of the tube, where it will move more rapidly. Molecules at the leading edge of the parabolic concentration profile tend to diffuse toward the walls, slowing them down. The net effect is to reduce the degree of sample spreading and to cause the carrier and sample to mix.

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Since diffusion in liquids is a slow process, FIA is done at relatively slow rates so that diffusion has time to take place. Also, tubing diameters are small so that the distance from the walls to the center of the tube is small and sample molecules do not have to diffuse very far. Radial mass transfer is also induced by coiling the tubing between the injection device and detector to set up secondary flow patterns. The magnitude of this effect depends on the flow rate, tubing diameter, and the degree of coiling.

The detector signal reflects the degree of spreading and dilution that has occurred during the zone sample transportation through FIA system and its typical shape is generally tailed rather than symmetrical. Thus, the mixing between the carrier and the sample solution is always incomplete, but because the mixing pattern for a given experimental setup is perfectly reproducible, FIA yields reproducible results.

It is important to recognize that FIA methods are dynamic in the sense that neither physical equilibrium (complete mixing) nor chemical equilibrium (the analytical reaction goes to completion) are prerequisite and there are concentration gradients in the system during the sample transportation through the detector.

Dispersion of sample zone Certain analyses in FIA that are used to determine original sample

composition, such as: pH determinations, conductance measurements, atomic absorption determinations, require that the sample solution be transported through the flow-cell as an undiluted zone and in a higher reproducible manner. On the other hand, for the majority FIA systems described in literature, the species being monitored have been generated by on-line chemical reaction. The prerequisite for performing such analysis is that during transport through the system, the sample zone is mixed with reagents and sufficient time is allowed to produce a desired compound in an easily detectable amount. Additionally, sometimes the sample must be diluted, so that the resulting signal can be accommodated within the dynamic range of the detector, or in the case of trace analysis, the excessive dilution must be avoided to obtain a maximum sensitivity. All these diverse requirements can be fulfilled by manipulating the dispersion of the sample: a) how much of the original sample solution is diluted on its way toward the detector, and b) how much time must elapse between sample injection and analytical signal recording.

J. Ruzicka and E. Hansen introduced the concept of dispersion coefficient, D, that has been defined as the ratio of concentrations before and after the dispersion process has taken place in the element of fluid that yields the analytical readout.

D = C0/Cmax = H0/H

where C0 is the initial concentration of the sample before it is diluted, Cmax is the maximum concentration of the sample that flows through the flow-cell. In most FIA methods the analytical readout is based on the measurement of the peak height H, and

307

therefore the dispersion coefficient may be expressed as a ratio between the heights of the initial (H0) and diluted (H) sample. Similarly, the dispersion coefficient for the reagent is:

DR = C0R/Cmax

R

But D may be calculated in any point on the ascendant or descendent part of the FIA signal. Taking into account that the FIA signal is characterized by an infinite number of Cg values, where Cg is the concentration at any point on the gradient, D may be defined as:

Dg = C0/Cg

The values of Dg range from infinity (Cg = 0) to unity (Cg = Cmax). Thus, D = 1

indicates no dilution of sample at the center of the injected sample zone and D = 3 indicated a dilution factor of 3. As a function of D, the FIA systems may be classified in FIA systems using limited dispersion (1<D<3); medium dispersion (3<D<10); and large dispersion (D>10). FIA systems in which the analytical determination is based on chemical reactions must be designed in such manner to provide D of 3 or more, allowing adequate mixing of sample with the carrier reagent with a moderate loss in sensitivity due to the dilution.

There is a tendency to regard D as a characteristic of the manifold, but it should be remembered that factors that affect the diffusion of the injected sample such as its size, viscosity of the carrier stream, etc., will affect the D value, too.

A considerable amount of experimental works summarized that D is influenced by:

1. sample volume. D increases with the volume injected because a large volume of sample leads to a large zone and to a high sensitivity.

2. length of reactor, tubing between injection valve and detector. D increases with the squared root of reactor length. But the reactor must be long enough to allow the reaction product development.

3. flow rate. D increases with the flow rate, but for faster flow rates a loss in sensitivity can occur since there is less time for the chemical reaction to develop. The characteristic parameters for a FIA system with a medium dispersion are

shown in Table III.2.1. Instrumentation The instrumentation for FIA is simple, as it is show in Figure III.2.10. The

requirements for designing a FIA system include a pulse-less, easily controlled flow, a reproducible injection of the sample; a detector equipped with a flow-cell and readout devices.

308

Flow injection analyzers are today commercially available as integrated units comprising valve(s), pump(s), detector, auto-sampler and computer controlled/data station from a number of manufacturers in a number of countries. Their distinguishing feature is their automated sample-handling capacity, hundreds of samples being analyzed for different analytes per unit of time.

Table III.2.1. Typical parameters for a FIA system of medium dispersion.

Parameter Value Injected sample volume (μL) 10 – 200 Carrier flow rate (mL/min) 0.3 – 2.5 Reaction coil length (cm) 10 – 200

Tubing inner diameter (mm) 0.3 – 0.8 Flow-cell volume (μL) 8 – 40 Sample throughput (h-1) 60 – 360

Time for one determination (sec) 10 – 60

Pump The syringe-drive, pressurized-gas, and peristaltic pumps are some means of

propelling the carrier stream(s). The advantage of the syringe-drive pumps is the pulse free flow, but periodic refill is necessary. The most used and suitable device for propelling the liquid flows is the peristaltic pump, because it may accommodate several channels whereby, according to individual tube diameters, equal or different pumping rates may be obtained. A schematic diagram of a peristaltic pump is presented in Figure III.2.13.

1

2

3

4

1

2

3

4

Figure III.2.13. Scheme of a peristaltic pump. 1 – thermoplastic flexible tube; 2 – rotor; 3 – rollers; 4 – stoppers. The arrows indicate the sense of liquid circulation through the pump tube.

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The popularity of the peristaltic pump can be attributed to its design, which uses an electric motor to turn a set of rollers. The rollers compress and release a flexible tube as they pass across the tube. The liquid will follow the rollers until the tube is no longer compressed and by this time a 2nd or even 3rd roller is compressing the tube, preventing flow back, pushing the initial dose of liquid out of the pump. By a repetitive operation as the rollers rotate a pumping movement is created, which has an element of pulsing as a standard. By the squeezing of the tube the rotor creates suction lift and outlet pressure. This squeezing action creates a vacuum, which then draws fluid through the tubing to achieve the pumping action. Because the flexible tubing is the only wetted part, maintenance and cleanup are simple and convenient.

Peristaltic pumps are never completely pulse free. A well-constructed pump must: a) stop and start instantaneously, allowing the precise control of all moving streams for stopped-flow or intermittent pumping functions; b) have many closely spaced rollers that rotates rapidly and thus the tubes are compressed frequently for short periods of time, generating a pulsation of high frequency and a low amplitude. The Gilson Minipulse and the Ismatec Minipump are examples of pumps that allow a stepwise regulation of the flow rates, generate nearby pulse-free flows, start and stop instantly. A new alternative is the use of individual, computer-controlled peristaltic pumps, which make it possible to run each analytical channel under full computer control and to select individual methods to be run on a batch of samples.

Sample injectionIn order to save reagent, to increase the residence time with a minimum

sample dispersion, and to accomplish zone sampling ingenious flow manifolds have been designed with different devices for sample introduction. Such devices have evolved from the first device equipped with a syringe, as described by J. Ruzicka and E. Hansen, to a device equipped with an automatic injection with multiple injection sections, culminating in the widespread employment of six port valves, which are in fact rotary injection valves. The valve injection mode approximates the plug injection and is a more facile way of inserting well-reproducible sample volumes into the carrier without disturbing its motion. Figure III.2.14 shows a rotary injection valve and its operating mode.

The loading and injection steps employed by displacing a movable part between two resting positions are a common feature of these devices. Air bubbles and pressure surges must be avoided during the injection because they will modify the pattern of the flow in FIA system, affecting dispersion and precision. The dimensions of the sampling loops define the volume of the injected sample.

Flow lines, reactors, connectorsPump tubes are available in different materials and have two bridges for fix

them around the mobile part of pump. These bridges are often color-coded to

310

designate the delivery rate. The choice of the peristaltic tube material depends on the nature of the solvent.

The tube placed between the injection valve and the detector represents the dispersion coil or the reaction coil and it has a uniform internal diameter of 0.3 – 2 mm (most frequently used being 0.5 mm). It usually is a Teflon tube tightly wound in the form of a coil to promote mixing. Reactors filled with chemically inactive glass beads have been used to improve the mixing without increasing the dispersion. In these rectors a well-controlled and reproducible dispersion pattern of the sample zone and high sample throughput can be achieved.

S

FT

to RC

W

1

2

3 4

5

6

LOADING STEP

L

INJECTION STEP

S

FT

to RC

W

1

2

3 4

5

6L

Figure III.2.14. Schematic diagram of a FIA four ports injection valve with and its operating mode. S – sample; R – reagent; FT – carrier; RC – reaction coil; L – valve loop; W – waste. When the analytical readout is based on peak width, the case of FIA titrations,

the use of a mixing chamber is necessary. This device has an inner volume (Vm) much greater then the sample injected volume (Vi), and the injected sample zone is homogeneously and instantly mixed with the reagent solution in the chamber. A variety of other units have been reported for specific purposes such as: - dialysers and gas diffusion units (Figure III.2.15.a) in which the separation of

analytes from a donor stream to an acceptor stream is carried out using a permeable membrane that separates the two streams;

- solvent exaction units (Figure III.2.15.b) that contains two special components: - a segmentor that creates a regular pattern of organic and aqueous segments, - a separator for the organic phase.

- packing reactors in which the packing material may be an ion-exchange resin used for analyte pre-concentration or/and for removing the interfering species;

311

immobilized enzymes used for analyte selective degradation; oxidizing or reducing agents that act directly on the analyte or generate reagents “in-situ” that react with the analyte.

The FIA manifold, the flow lines from the injection valve to detector, must be held in a fixed position because movement may affect the flow patterns and influence the distribution of the sample. Also, the fittings should be designed not to impart flow irregularities.

Bottom block

Top block

Bolt holes

Flow channel

Inner view

Side view

Bottom block Donor

outlet

(a) (b) Figure III.2.15. (a) Scheme of a dialyser/gas diffusion unit used in FIA manifold. (b) Scheme of a solvent extraction unit used in FIA manifold (i) organic phase segmentor, (ii) phase separator. Detectors Generally, chromatographic detectors with cell volumes smaller then 20 μL

are well suited for FIA work. On the other hand, conventional detectors may be furnished with flow-through cells provided that the system will accommodate and handle flow cells of sufficiently small holdup volumes and apertures. Two types of flow-cells used for spectrophotometric measurements are shown in Figure III.2.16. Any detection technique that is compatible with a flowing stream is suitable for flow injection.

Spectrophotometry, nephelometry, fluorescence, chemiluminescence, atomic absorption, flame photometry, potentiometry with ion-selective/modified electrodes or field effect transistors, amperometry with sensors and biosesors and voltammetry with wire-type or rotating disk electrodes are the most important detection techniques used in FIA.

Donor inlet

Top block

Membrane

Acceptor inlet

Acceptor outlet

Bottom block

Top block

Bolt holes

Flow channel

Inner view

Side view

Bottom block Donor

outletnor

inlet

Top block

Membrane

Do

Acceptor inlet

Acceptor outlet

Inner view

Side view

Bottom block

Organic phase

Donor outlet

nor inlet

Top block

Membrane

Do

Acceptor inlet

Acceptor outlet

Bottom block Donor

outletnor

inlet

Top block

Membrane

Do

Acceptor inlet

Acceptor outlet

PTFE thread

Waste

Organic phase + analyte

(i) (ii)

Organic phase

Aqueous phase + analyte

PTFE thread

Waste

Organic phase + analyte

(i) (ii)

Aqueous phase + analyte

312

(a) (b)

Figure III.2.16. Spectrophotometric flow-cells. (a) “Z” configuration and (b) “U” configuration.

Kinetic determination The most important feature of FIA that distinguishes it from the other

continuous flow techniques is the well-defined concentration gradient formed when an analyte is injected into the carrier stream. The gradient approach yields the capability to perform procedures not feasible by conventional continuous flow analysis. Many gradient techniques have been developed, including titration, gradient dilution and calibration, gradient scanning, FIA stopped-flow technique and simultaneous injection of two zones, which completely or partially overlap permitting the execution of selectivity studies and standard addition procedures.

A FIA signal represents a variation of concentration from zero to Cmax and there is no single element of fluid with the same concentration as the neighboring one. Any element of fluid along the gradient corresponds to a fixed dispersion (D) and they can be related to a fixed delay time elapsed from the moment of injection. In gradient techniques, one of these elements is selected to take the measurement in continuous or stopped-flow mode.

The principle of gradient dilution is based on selecting for the measurement any point, other than the peak maximum. The advantage of this technique is that of obtaining the readouts after pre-selected delay times (Figure III.2.17.a), manual pre-dilution of the sample can be avoided and a large concentration range can be accommodated within the dynamic range of the detector used.

The gradient calibration technique is an extension of the above described one. Its main goal is to avoid the usual repetitive calibration by means of a series of diluted solutions, as the information sought is in fact already contained within some of the segments of fluid originating from a sample zone of the most concentrated standard sample solution (Figure III.2.17.b).

The FIA stopped-flow approach is based on a combination of stopped-flow measurements and of gradient dilution. It is based on the increase of residence time, keeping the reactor short and decreasing the flow-rate of the carrier stream. Choosing appropriate stop-go time intervals, the reaction time will increase and the reaction rate

hνhν

313

will be proportional to the concentration of the analyte (Figure III.2.17.c), for a pseudo-zero-order reaction. This technique allows a fine adjustment of the reagent/sample ratio by selecting a corresponding delay time.

(a)

0.25 0.50 0.75 1

•

•

•

•

t1t2t3t4

S

Ana

lytic

al r

esp o

nse

Scan

10 s3 min

t0%

a

b

c

d

S

25 s

Ana

lytic

al r

espo

nse

time (s) (b) (c)

Figure III.2.17. Schematic diagrams of FIA gradient techniques. (a) Gradient

dilution. Peaks recorded for different concentrations of a dye, he most concentrate one being 1%; t1…4 – different delay times; calibration curves. (b) Gradient calibration. Different injected concentration of a dye solutions corresponding to different delay times, t1..4, on peak of the most concentrated one. (c) FIA stopped-flow: a – continuous pumping; b, c, d - recorded reaction rate curves for different delay times.

D1

D2

D3

D4S S

t1 t2 t3 t4100%

75 %

50 %

25 %

Ana

lytic

al R

espo

nse

•

•••

•

•

•

•

•

•

•

•

•

•

•

•

t1t2

t3

t4

25 50 75 100 %

Ana

lytic

al R

espo

nse

time (s)

10 s

D1

D2

D3

D4S

D1

D2

D3

D4S S

t1 t2 t3 t4100%

75 %

50 %

25 %

S

t1 t2 t3 t4100%

75 %

50 %

25 %

Ana

lytic

al R

espo

nse

Ana

lytic

al R

espo

nse

•

•••

•

•

•

•

•

•

•

•

•

•

•

•

t1t2

t3

t4

25 50 75 100 %

Ana

lytic

al R

espo

nse

•

•••

•

•

•

•

•

•

•

•

•

•

•

•

t1t2

t3

t4

25 50 75 100 %

•

•••

•

•

•

•

•

•

•

•

•

•

•

•

t1t2

t3

t4

25 50 75 100 %

Ana

lytic

al R

espo

nse

10 s10 s

Ana

lytic

al R

espo

nse

time (s)

314

Multiple-determination and multiple-detection FIA techniques timization of

systems

is injected and two or more signals are recorde

he signals being delayed; measured continuously within a

- he same type displaced in series or in parallel.

obtainin

-determination – of one or more analytes from a sample may be sequent

of these two FIA techniques, it results that the multiple

presented below:

Another trend in FIA development has been the design and op for multiple-determination and multiple-detection. These techniques are used

for kinetic determinations or for simultaneous determinations of two or more components from the same injected sample.

Multiple-detection – a single sampled in different ways:

- using a single detector, t- using a dynamic detector, a physical parameter is

certain range (e.g. absorbance vs. wavelength, current vs. potential, etc., along the dispersed sample zone; using more detectors of t

The multiple-detection (Figure III.2.18) may be successively performed, g more data of a certain measured parameter, or simultaneously, using a single

detector that performs simultaneous measurements at different working parameters of the instrument.

Multipleially realized, performing a number of injections equal with the number of

analytes to be determined or simultaneously, determining more analytes from the same injected sample (Figure III.2.18.b).

Considering the definitions-determination may be realized by the multiple-detection, but the multiple-

detection does not means multiple-determination, strictly. Two examples of simultaneous determinations are (a). Simultaneous determination of Fe(III) and Ti(IV) (Figure III.2.19.a) –

both analytes react with Tiron (R). Two sample volumes are simultaneously injected into 1 M HCl carrier stream by means of two injection valves (Vi1, Vi2). The injected sample zone with Vi1 passes through the reduction column (RedC) where Fe(III) is reduced to Fe(II), which does not react with Tiron. The injected sample zone injected with Vi2 is treated with Tiron and the mixture zone goes to the spectrometer where the absorbance due to Fe(III)+Ti(IV)–complex with Tiron is recorded. For each double injection two peaks are recorded, first corresponding to the complex absorbance of Fe(III)+Ti(IV)–Tiron and the second corresponding to the complex absorbance of Ti(IV)–Tiron, only. On the bases of heights of these two recorded peaks, the concentrations of Fe(III) and Ti(IV) can be calculated by using calibration curves.

(b) Simultaneous determination of nitrite and nitrate (Figure III.2.19.b) – is based on the measuring the absorbance of the reaction product formed between the nitrite and sulfanilamide (R1) and N-(1-naphthyl)-ethylendiamine (R2). The injected sample zone is split into two sub-zones using a “T” connector. One of the sub-zones is treated with R1; the nitrite ion is diazotized with sulfanilamide and then, the diazotization product is coupled with R2 to form a colored azo-dye for which the absorbance is recorded. The other sub-zone passes through a reduction column (RedC)

315

that contains copperized cadmium and here, the nitrate is reduced to the nitrite. The sub-zone is then treated in the same manner as the other sub-zone and the overall mixture passes to the spectrometer where the absorbance due to nitrite and nitrate is measured. The concentrations of nitrite and nitrate are evaluated from the peaks heights by using calibration curves.

P

C

S

Vi

W

RC1

DRC2

RC3

Ana

lytic

al si

gnal

time (s)

S

P1

P2

P3

T1 T2

P

C

S

Vi

W

RC1

DRC2

RC3

P

C

S

Vi

W

RC1

DDRC2

RC3

Ana

lytic

al si

gnal

time (s)

S

P1

P2

P3

T1 T2

Ana

lytic

al si

gnal

time (s)

S

P1

P2

P3

T1 T2

Ana

lytic

al si

gnal

time (s)

S

P1

P2

P3

T1 T2

(a)

(b) Figure 2.1.18. Schemes of FIA systems for successive multidetection technique. (a) separation of the injected zone in three equal sub-zones that reach the detector after different periods of time. Each injection produces 5 calibration values, 3 peaks (P1, P2, P3) and 2 troughs (T1, T2). (b) more detectors displaced in series, their response being computed by a microprocessor. P – peristaltic pump; Vi – injection valve; RC – reaction coil; D – detector; C – carrier; S – sample; R – reagent, W – waste.

C

S

Vi

W

P

D1 D2 D3 D4 D5C

S

Vi

W

P

D1 D2 D3 D4 D5C

S

Vi

W

P

D1 D2 D3 D4 D5

316

(a)

Figure 2.1.19. (a) Scheme of FIA system used for simultaneous determination of Fe(III) and Ti(IV) using Tiron as reagent. (b) Scheme of FIA system used

III.2.1.4

) was first reported in 1990 by J. Ruzicka nd G.D. Marshall at the University of Washington, as an evolution of the flow

analysis

(b)

for simultaneous determination of nitrite and nitrate. P – peristaltic pump; Vi – injection valve; RedC – reduction column; D – detector; C – carrier; S – sample; R – reagent, W – waste (see explanations in text).

. Sequential Injection Analysis

Sequential injection analysis (SIAa

process. This technique, for automatic sample analysis, is based on the same principles as FIA, namely controlled partial dispersion and reproducible sample handling, and it offers different possibilities with a series of advantages and disadvantages in relation with its parent technique. The instrumental simplicity, robustness, ease and efficiency with which hydrodynamic variables can be controlled, and high flexibility and modes maintenance requirements of this modified technique have turned it into a very popular choice with both research and industrial analysis

C W

P

R1

S

Vi

DRedC

R2

C W

P

R1

S

Vi

S

Vi

DDRedCRedC

R2

C W

P

R

S

Vi1

S

Vi2

DRedCC W

P

R

S

Vi1

S

Vi1

S

Vi2

RedCDD

317

laboratories. On the other hand, SIA has proven to be a technique that can be designed to operate in a multi-parametric way, which is of special interest when considering the design of the environmental monitors. Thus, the monitoring of wastewater has been proposed for ammonium, nitrate, nitrite, total nitrogen, orthophosphate, total phosphorus, detergents, etc., and it can be determined in 15 min. However, in spite of these advantages, SIA presents two major disadvantages: the sample throughput is lower then that of the usual flow systems and major difficulties in the mixture of sample and reagents.

SIA is a single-line system, completely microcomputer controlled, that can be configured to perform most operations of conventional FIA, with no or minimal physica

n operational manifold design for SIA is illustrated in Figure III.2.20. The heart of is a multi-port selection valve, used in place of a conventional injectio

g coil by the pump moving in a forward motion. Each measuri

l reconfigurations of the manifold, allowing to perform determinations of different analytes.

Principles A the systemn valve and this is the primary difference between FIA and SIA. This valve

delivers accurately measured volumes of carrier, sample, standards and reagent solution to a holding coil by connecting its common port to a reversible pump featuring a precisely controlled forward-stop-backward motion. The common port can access any of the other ports by electrical rotation of the valve. The holding coil placed between the valve and the pump prevents the aspirated (injected) solutions from entering the pump.

Initially, the system is filled with washing or carrier solution, which is aspirated into the holdin

ng cycle begins by switching the multi-port valve to the sample line and aspirating a precise measured volume (few μL) of sample into the holding coil by the pump moving in a backward motion; the pump is stopped during the rotation of the valve to avoid pressure surges. Next, the valve is switched to the reagent line and a precisely measured volume of reagent is drawn into the holding coil. Thus, the sample and reagent solutions are sequentially injected into the holding coil next to one another, hence the name of this technique. A second reagent may be aspirated on the other side of the sample. Finally, the valve is switched to the detector port and the pump propels the sequenced zones forward through the reaction coil to the detector. The cores of sequenced zones penetrate each other via laminar flow and diffusion. If the radial mixing is promoted by a suitable choice of coil geometry, the analyte and reagent zones mix and produce detectable species and a transient signal as in conventional FIA is recorded. The complex reagent/product/sample zone can be either transported through the detector continuously, or stopped within detector, resulting in measurement of the rate of formation of the reaction product. In this way, kinetic information can be extracted from the SIA signal. There is no limit to how many

318

solutions or devices (reaction coils, mixing chambers and detectors) can be nested around the multi-port valve. A series of standards can be permanently nested around valve, being ready for automated recalibration whenever is necessary.

P

HC W

S

D

St

R

Vp

R

W

R

St

S

P

R

DHC

Vp

W

R

St

S

P

R

D

HC

Vp

Figure III.2.20. Schematic diagram of a SIA system. P – single-line reversible (high-precision bi-directional) pump; HC – holding coil; p – multi-

config of the flow path. The injected sample volume, reaction time, sample ilution, reagent/analyte ratio or system calibration are controlled from a computer

Vport valve; D – detector; S – sample; R – reagent; St – standard solution; W – waste.

The biggest advantage of SIA over FIA is that is not necessary the physical urationre

d

319

keyboar

rm FIA and what are its drawbacks?” The characteristic feature of FIA patterns is that the injected sample zone

flows to treams merge in a continuous fashion and in this ma of the passing carrier s

Figure III.2.21. (a) Formation of sample z – sample line concentration, in FIA mode via a confluence point supplying a steady reagent, R – reagent line concentration. (b) Mutually penetration of

(or ove the relative volumes, in addition to the usual parameter of tubing size and length, reaction coil geometry and

d. Indeed SIA is fully computer-compatible and allows the configuring of the system to perform complex chemistries. In addition, SIA requires very low sample reagent volumes (μL/analysis).

Operational parameters in SIA “How does SIA differ fo

a confluence point where two snner; an equal volume of reagent is added to each element tream. The result is a concentration gradient of an analyte within the constant

background of reagent. In contrast, in SIA no confluence points are used. The multi-port line is used to sequence the zones into a holding coil. This valve does not serve, as a confluence point as it connects only two ports (not three) at a time. The result is a sharp boundary between adjacent zones and only a partial overlap of analyte and reagent picks is possible (Figure III.2.21.b).

R

(a) (b)

one concentration gradient, S

sample, S, and reagent, R, zones in SIA mode. Reaction product - shaded zone. In SIA mode it is needed a computer control otherwise injected zones and their intermixing would be poorly reproduced. Thus, the parameter of prime importance in SIA is the degree of penetration rlay) of the adjacent zones. This is dependent on

time (s)

S

IH

R

time (s)

S

IH

R

S

time (s)

IH

S

IH

R

time (s)

320

the flow

agent) the overlapping decreases, but the disp

raight reactors allow a greater zone penetration through axial dispersion than the

ssure syringe was used as liquid driver that provided a sinusoidal flow as a result of non-uniform motion of the piston in the cam-driven strating that the flow is not constant but reproducible.

flow-cell detector, tubing/reaction coils and connectors (as those used in FIA) and a p

rate delivered by the peristaltic pump. The degree of penetration and the dispersion determine the signal to be recorded:

Figure III.2.21.b shows the mutual zone penetration in SIA. A small zone volume results in increased overlapping, but the dispersion is relatively large. By increasing one or both volumes (sample and re

ersion at the maximum overlapping is less. In general, the injected sample volume should be less than the volume at half-maximum signal and the reagent volume should be at least twice the sample volume. The reaction is instantaneous if the concentration of reagent is higher then analyte concentration, the recorded signal maximum should occur at the isodispersion point (the point of maximum overlapping).

Tube diameters of 0.9 – 1.5 mm decrease the backpressure compared with 0.5 mm, resulting in improved precision without excessive decrease in the zone penetration. St

coiled reactors preferred in FIA. Instrumentation

In previous SIA works a low-pre

piston pump, illuPeristaltic pumps of high precision were tested for propelling the flow and their performances were compared with the above described piston pumps. Thus, their sampling cycle is shorter than that of the piston pumps as the pump needs to be refilled periodically and also peristaltic pumps are much more commonplace in the laboratories than piston pumps. On the other hand, the disadvantage of the peristaltic pump arises from the need of fairly elastic tubes, which have a much shorter life. In order to circumvent this inconvenient, an auto-burette was used to propel the flow in SIA. It has been demonstrated that a small-volume syringe pump provides the highest precision, of 0.3 %, and the peristaltic pumps and the auto-burette provide a precision of 1 %.

In conclusion, a SIA system is assembled using a multi-port (usually 10 ports) electrically activated selection valve, a high-precision peristaltic or syringe pump, a suitable

ersonal computer. Appropriate software must be available to control the flow direction, rate and timing of the pump, the position of the multi-port valve and to collect and process the data. At the present there are several commercialized software for SIA users, including: Atlantis, MATLAB, Microsoft QuicBasic, Labview, FlowTEK, DARRAY, FIALab, etc.

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III.2.1.5. Hyphenated Systems

ifferent continuous flow techniques described above of great interest area. It is well-known the success of

elation between environmental reservation and standard of living has led to the need of vigilance and continuous ontrol

The combination of the dith powerful instrumentation is w

the FIA in conjunction with atomic absorption spectrometry (AAS), which involves flames, electrothermal, or hydride generation schemes, as evidenced by the large number of published papers; works presented at various scientific events or by the book published by J.L Burguera in 1989 and entitled: Flow Injection Atomic Spectrometry. Therefore, the combination of AAS with SIA brings about advances as well as challenges of synchronizing the discontinuous flow mode of SIA with continuous operation of the nebulizers or quartz flow-cells, while the interaction with the discontinuously fed graphite tubes appears as an attractive option. Continuous flow techniques were coupled to different techniques, such as gas chromatography, capillary electrophoresis or mass spectrometry. The results are hybrid systems, which comprise the advantages of both methods and drawbacks of none: multi-component resolution, the high sample throughput and the sample handling of FIA/SIA. Recently, Fourier Transform Infrared Spectrometry (FTIR) combined with FIA/SIA was reported as another example in fashion, systems where enzymatic degradation can provide kinetic information will become a useful tool for bioanalysis. On the horizon another technique appears: Raman spectrometry, which due to its qualities and drawbacks is a suited target for enhancement by flow injection. II.2.2. AUTOMATED FLOW ANALYZERS I

The acceptance of the existence of a corrpc of a large number of environmental parameters. A large number of environmental samples are submitted to routine laboratories every day in order to satisfy these increasing demands. Traditionally analyses have been performed by off-line methods, which mean all the samples must be carried to a centralized laboratory. These off-line measurements may lead to a significant delay between submission of a sample and analysis result, particularly when demands upon staff and instrumentation are great. One of the current trends in environmental parameters analysis involves avoiding the sampling by utilizing automated, unattended, analytical instrumentation, which may not be very selective but allows the detection of alarm situations and the performing of a complete analysis in those samples where analysis is required. With this instrumentation fast process monitoring is achieved, thus facilitating processes regulation and enabling quality assurance and quality control.

322

High quality chemical information attainable in “real-time” requires having a rapid, accurate, reliable, robust, without demanding the continuous presence of the analyst,

d on-line pro

handling of solutions, and provides strict control

ues. New on-line UV

e different in technology, costs and sample types or number.

with a low consumption of reagent and whenever possible multi-parametric system like the continuous flow analyzers available. Certain analyses of environmental samples are difficult to achieve in a completely automated manner, but aqueous samples are especially well adapted to be analyzed by flow techniques.

Among all the continuous flow techniques, flow injection analysis (FIA) is a well-established, powerful, sample handling procedure for laboratory analysis an

cess analytical chemistry, with a wide range of applications in environmental situations. Maybe this is the reason for which FIA enthusiasts agree on: “This is a technique that allows chemists to easily automate and optimize well-developed wet chemical methods for routine laboratory use. You can even program an analyzer to switch from one analyte to another during the analysis of a batch of sample… But FIA was never been a popular product of larger instrument companies, so smaller firms produce most of these analyzers”. In spite of all, the technique is surprisingly under-used by laboratory chemists and this perhaps automation was too expensive or took too long when FIA was introduced in 1975.

FIA offers several advantages over the manual handling of solutions, such as: it is computer-compatible, allows automated

of reaction conditions. Also, because of its versatility in sampling handling, FIA serves as front end to practically all spectrophotometric and electrochemical detectors and to various environmental, clinical and industrial assay. Other applications include “real-time” monitoring of chemical processes, automated renewal of the sensing layer in chemical transducers, and electrochemical methods, such as hydrodynamic voltammetry and ion-selective electrode measurements.

The recent results achieved in computerization, microfluidics, and hardware have facilitated the further development of new flow injection techniq

-digestion technique, combined FIA-sequential injection analysis (SIA) techniques, the incorporation of ion-selective electrodes and improvements of data handling are some highlighting examples. The company Global FIA, Inc. adds that mixed media (beads, bubbles, immiscible liquids) in flow systems have opened new applications. FIA methods are also presently undergoing standardization protocols established by the Organization of International Standards and the U.S. Environmental Protection Agency.

Table III.2.2 lists some commercial continuous flow analyzers and their features, which can b

323

Table. III.2.2. Commercial continuous flow analyzers and their haracteristics.

Produc QuikChem 8000 QuikChem® FIA+

FIAstar 5000 FIAstar5010

CNSolution 3000 Flow Solution IV

SIA2000-S FIAflo2000

0

S

c

t

QuikChem®FIA +®IC QuikChem IC

+ LabTOC2100

Flow Solution 3000

SFA 2000 DUOflo2000

B 200WATERLAALERT 2000 –(industrial process monitor)

Company LACHAT® INSTRUMENTS,

I

TECATOR AB, Box 70, S-263 21

ALPKEM -OI ANALYTICAL HEADQUART151 Graham Road

ERS,

PO Box 9010 College Station, Texas 77842-9010, U.S.A

BURKARD SCIENTIFIC Ltd. PO Box 55, Uxbridge, Middx,

.K. UB8 2RT, U

INC. 6645 W. Mill Road, Milwaukee, W53218 U.S.A.

Hoganas SWEDEN

www.lachatinstrume www.foss.dk www.oico.com www.burkardcientific.Website address nts.com co.uk

Applications Inorganics and total organic carbon in environmental, industrial, agronomy, food and beverage analysis.

Wet chemicalanalysis of

s nutrients and

other parameterin water, soil, meat, food analysis

Environmental, oceanographic, agricultural and industrial analysis.

Water, soil, food and beverage, industrial process,agricultural and biochemical analysis

Sample throughput

samples/h samples/h 6 - 90 samples/h 60 – 180 samples/h

30 – 75 30 – 120

Injected volume L 2 μL – 2 mL 20 – 400 μ 40 – 200 μL Variable loop, minimum 10 μL

Detector Photometric, Iflame

SE,

ic

at e

c,

ded

e photometric, amperometric, pH, fluorimetr

Digital dual-wavelength photometer threduce baselindisturbances for higher accuracy and lower detection limits

AmperometriISE, pH, photometric, tandem detector design, expenrange detection technology

Photometers, fluorescence, flamphotometer, chemiluminescence detector

324

(Table III.2.2. continued)

Product FIMS 100 FIMS 400

stems

EVOLUTION II SINGLE: “one-

A

ASIA Flow Injection analyzer

A)

FIAlab 2500 FIAlab 3000

FIAS Flow Injection SyFI-MH

shut” Analyzer The INTEGRAL

Integral FUTURCHEMLINE

(modular FI

Company

2

es

ISMATEC SA Feldeggstrasse 6,

FIAlab®

INSTRUMENTS The PERKIN ELMER Corporation 761 Main Avenue,

TNorwalk, C06859-001U.S.A

ALLIANCE S INTRUMENT

Zone d’activitles bosquets 4, BP 31, 95540 Mery-sur-Oise, FRANCE

CH-8152 Glattbrugg/Zurich GERMANY

Inc. 1440 Bel-Red Riad, Suite 208 Bellevue WA 98007-3926 U.S.A.

Website address www.perkin-elmer.comwww.inforoute.cgs.fr/a www.ismatec.com www.flowinjection.colliance m

Environmental control, medicine, food, agriculturegeology, industry, research and teaching

, od, ty

nd

y, ng,

s

Water, wine, tobacco, agriculture, fochemical industries analyses

Environmental onitoring, qualim

mental studies,

control of food abeverage, bio- and chemical-process monitoring and control, sensor testing

EnvironApplications

laboratory research in chemistry, biotechnologdrug screeniindustrial procescontrol.

Sample throughput

120 – 180 samples/h

6 – 120 samples/h 30 – 120 samples/h

Injected volume μL < 500 μL 10 μL 20 – 1000 Detector Atomic

absorptionP

, ICP--MS

Photometer with fibers,

, IES, Photometric, uorescence-

OES, ICoptical

Photometerbiosensors fl

microscopy, luminescence,FTIR, MS, electrochemical

325

(Table III.2.2. continued)

n+

Analyzes:

TN

yzer

yzer

PISCESTM

9000 (portable

FloPro 4P FloPro 9P

ASI/Eppendorf Variable

Product Skalar Sa

San++

FormacsLT

cs Forma PrimacsSC

Primacs SN

Robotic Anal SP 100 BODcompact

Fluorecence Analyzer FluoImager Toxicity AnalToxTracer

SIA system) Analyzers

Company

Head Office

CONSTELLATION

.

Global FIA, Inc. PO BOX 480, Sixth

A

AMKO Systems, Inc.,

eaver

SKALAR’s

PO Box 3237, 3800DE Breda, NEATHERLANDS

TECHNOLOGY, 7887 Bryan Dairy Road, Suite 100, Largo, Florida 33777, U.S.A

St, Fox Island, W98333, U.S.A.

250 W. BCreek Road, Unit 6, Richmond Hill,Ontario, CANADA L4B 1C7

www.skalar.com www.constech. www.globalfia.co www.amkosystems.com

Website address com m

Application Waters, detergents, fertilizers,

er, o

rs, nic analytes, es;

;

ental, gy, pharmaceuticals,

soil, plants, bewine, food, tobacc

Environmental wateindustrial process analyses

Inorganic and orgaon-line processindustrial analysislab and field analysis

Chemical, environmbiotechnolopharmaceutical, food and beverage.

Sample throughput

ple 20 – 140 samples/h 1 – 3 min/sam

Injected volume 2.5 μL number and

0 samples/h with 2 – 100 μL

Up to 0.3 mL/min < Variable sample

volume

2

samples Detector Photometer, flame

photometer,

r;

Integrated UV-VIS

tro ter;

ce, fluorimeter, IES, pH-meter, conductivity-meteUV- VIS; IR; chemiluminescence detector

spectrophotometer for absorbance and fluorescence detection.

UV-VIS specphotomechemiluminescenamperometric

Colorimeters, ISEs; conductivity detector;

326

III.2.2.1. Continuous and Discontinuous Systems

nd most of them can be classified : segmented flow analysis (SFA), completely continuous flow analysis (CCFA); and

flow inj

economical in terms of reagent consumption and waste generat

cial Automated Flow Analyzers

continuous or discrete (batch) struments. The continuous system constantly measures some physical or chemical

properti

analysis and sequential

There are many variations of flow analysis a

inection analysis (FIA) with its variants sequential injection analysis (SIA) and

bead injection analysis (BIA). Hundreds of automated methods, such as: standard wet chemical environmental tests, agricultural analysis and process control analysis, are available for each technique.

While the “classic” or continuous flow systems can operate well without the aid of a computer, they are un

ion, because of the continuous pump of all solutions. In contrast, the recently designed variants of FIA, SIA and BIA, are based on discontinuous flow and consumes reagents only when the sample is treated, the reagent being injected not pumped into the carrier stream. Nowadays, the use of personal computers and the availability of dedicated and object-orientated software make SIA and BIA widely accessible, although these new variants impose some constraints on the versatility of operation as compared to FIA. The principles of FIA and SIA are discussed in Chapter III.2.1. BIA combines the advantages of solid-phase chemistry with the novelty of fluidic handling of microcarrier beads (beads diameter of 30 – 150 μm), allowing automated surface renewal and post-analysis manipulations. It operates in SIA mode but instead of reagent solutions, bead suspensions are used as reagent carrier. The injected bead suspension is trapped in a flow-through detector in FIA manifold, where it is subsequently perfused by the analyte solution, buffers, or auxiliary reagents. Chemical reactions occur at the bead surface and can be analyzed in “real-time” directly on the solid phase or within the eluting liquid phase. Monitoring simultaneously the changes in solid and liquid phase, a multi-parameter assay is possible. At the end of an analysis cycle, the beads can be automatically discarded, collected, or rerouted. BIA is applicable in diverse fields of research, including chemical sensors, bioassay, affinity chromatography, spectrophotometry and electrochemistry.

III.2.2.2. Commer

The analysis system may be classified as in

es of the sample and yields a signal that is a continuous function of time. A discrete, or batch system analyzes a discrete or batch- loaded-sample, and information is supplied only in discrete steps. In either case, the information on the measured variable is fed back to the monitoring or control equipment.

Flow-based analytical techniques fall into one of four categories: air-segmented, unsegmented continuous flow; flow injection

327

injectio

s. Automated instruments perform the following operatio

ple dispensing;

velopment; in the detection system;

of the commercial flow analyzers is their ing capability and many analysts and instrument

manufa

new alternative in the use of individual computer-controll

mated flow analyzer: a.

also

b. and precision should be calculated from the

c. gent

n analysis. Multi-commutation and binary sampling can be implemented in both unsegmented and segmented flow systems and potentialities and limitations have been pointed out by B.F. Reis et al.

Automated flow systems are intended to analyze multiple samples, either for a single analyte or for several analyte

ns: 1. Sampling (from a small cup on auto-sampler, or assembly line); 2. Sam3. Dilution and reagents adding; 4. Chemical reaction de5. Directing the reaction product 6. Reading the recorded data; 7. Processing the data.

The distinguishing featureautomated sampling handl

cturers add this feature to larger instrument systems, such as: for automated electrospray MS, ion chromatography, HLPC, or atomic spectroscopy. In addition, flow analysis immunoassay, a relatively new developed hyphenated technique, has shown promise for automated, reproducible immunoassay. The determination is easily optimized during the its run because the analytical protocols and system parameters are controlled by the computer.

In some cases, new pumps are becoming available. The time of multichannel peristaltic pump is gone and a

ed peristaltic pump appears on the horizon. LACHAT Instruments affirms that: “A system can be set up with four chemistries, and you can select any one, two, three or all four to be run on different batches of samples”. Therefore, these new devices make possible to run each analytical channel under full computer control and to select individual methods to be run on a sample batch.

Besides the above-mentioned essential aspects, it is important also to provide the main figures of merit for a proper description of an auto

Sample throughput – the number of samples processed per unit of time should be estimated in association with specified carry-over level. It is recommended to specify the consumption of sample and/or reagents when sample throughput is calculated. Analytical characteristics – accuracy, sensitivity, detection limit, selectivity, dynamic range of concentration data according to the IUPAC regulations. The analyzer should be validated using a standard method with confidence limits and tests of significance. Robustness – is discussed from two points of view: the dependence of the analytical signal on the variation of system parameters (temperature, reaconcentrations, flow rates, etc.) and the instrumental stability, which is manifesting it-self as a shift and/or drift in baseline and/or analytical signal.

328

The stability of the analytical curve, cost and maintenance requirements should be specified. Portability – to another operational environment and consequent suitability for in situ, in vivo or on

d. –site analyses, as process analyzer for on-line monitoring.

to furnihow to

. BURKARD Scientific is an English company. BURKARD Series 2000 automa systems meeting the needs o

ntrol are catered for in singl

s been designed for economy and ease of use to the mod

After the automated flow analyzer has been characterized, it is recommended sh some additional information: zone sample sampling; merging zones and get them; sample electrostatic dissolution, in-line gas sampling; multi-

commutation; adherence to well-established variants of flow analysis, etc. Examples of commercial analyzers and their features 1

tic chemical analyzers are a range of high specificationf today's demanding routine chemical sample processing. The series 2000 analyzer analyses ions, nutrients and metals down to ppb

levels. Both discrete laboratory applications and on-line process coe or multi-channel configurations. The systems are modular allowing the user

to purchase only what they need, from a basic single channel analytical system with manual or automatic sample presentation through to multi-channel systems with computer control and data processing. The analyzers offer a wide range of applications based on continuous flow technologies including segmented flow (SFA), flow injection (FIA) and ion selective electrode (SIA). Techniques include on-line distillation and UV digestion, on-line automatic calibration and standards preparation, over-range dilution and re-analysis.

FIAflo 2000 is a new multi-use high precision flow injection analyzer based on colorimetric and UV detection. It ha

ern laboratory and it offers: fast flexible analysis; 2 to 6 channel instruments; up to 4 simultaneous heated methods; easy interchange of chemistry manifolds; wide choice of detectors for maximum performance, Windows® data processing and control.

The instrument comprises three separate units and these are basic analytical modules, automatic sample presentation and data capture. The analysis can start immediately with the analytical module consisting of a manifold constructed for the plug-in chemistry. Ready assembled units are available for the most commonly used methods and where is possible a single manifold can mount two related chemistries. The pump tube and valve connections are conveniently positioned and colored for identification. It takes only a few minutes to change the analytical method. BURKARD methods combine the latest improvements in performance with low reagent consumption. FIAflo 2000 offers fully automatic sampling with a choice of carousel or XYZ sample changer. Automatic dilution of over-range samples is available as an option on the XYZ. However to alleviate the need for dilution the MicroStream data-processor has an extended concentration range enabling the results to be printed out at up to 10 times the normal calibration standard. When the

329

instrument is not used with a sample changer a low-cost manual injection timer is available to ensure precise loop filling. In addition, each valve has a manual injection button as standard to assist the analyst with setting up of the individual chemistries. The standard high sensitivity colorimetric and UV detectors coupled to FIAflo use filter technology and flow through cells with a rage of path lengths from 3-50 mm. Free standing alternative detectors for Chemiluminescence, Fluorescence, and Ion Selective Electrode interface with the chemistry manifold using the appropriate couplings. FIAflo 2000 comes with MicroStream, a powerful multi-tasking system based on Windows® software. MicroStream will speed up your calculating and reporting. Starting with a low-cost package for single-channel use, this system can be expanded to run up to four independent multi-channel analyzers. New quality control software checks the accuracy of analysis and all results can be downloaded to other software packages and LIMS. MicroStream is compatible with Windows® based networks. FIAflo 2000 analyses nutrients, ions and metals and the most popular applications include: waters; soil samples; landfill samples; fertilizers; food and drinks; chemicals/pharmaceuticals; quality control.

The SIA2000-S automatic chemistry analyzer is a low cost and versatile instrument for a range of applications based on ion selective electrode technology. It is ideal for the laboratory with small sample numbers that arrive on irregular basis. The modular design of SIA2000-S system allows the individual purchase of sampler, analytical chemistry unit, data processor or recorder to better meet the exact analyst requirements. The “add-on” potential means that options may be acquired as the work load increases and the chemistry module interfaces with other manufacturers instruments. The analysis module comprises a multi-channel fixed or variable speed peristaltic pump, an injection valve (valves), heating bath fitted with two PTFE coils and a dual flow cell carrying the ion selective and reference electrodes. An interface is fitted to enable the electrode response to be amplified, linearized and output to a chart recorder or data handling system. Ionic species, both monovalent and divalent, may be determined over a wide concentration range giving an excellent linear calibration. BURKARD SCIENTIFIC method sheets provide information on flow rate, regents, sample loop sizes and manifold configurations for standard or specific determinations. The laboratory based analyzer handles with up to 40 samples per hour. The simple construction enables quick interchange of electrode and flow-cells. When the method incorporates a flow injection valve, reproducible timing of the loop volume can be accomplished using the Burkard BT2000 loop timer with pencil switch.

Originally designed for fluoride determination in water, this technology has been extended to include applications in clinical, agricultural and industrial science. The fol

rs, sea-water, sewage) and effluents.

lowing species are of typical uses for SIA: ammonia, nitrate and nitrite in soil sample; sodium and chloride for clinical methods; fluoride in soil and waters; total and free SO2 in beer and wines; pH and conductivity.

The ALERT 2000-S is a chemical analyzer for continuous on-line monitoring of industrial processes, water (drinking water, rive

330

It offers a flexible system that can be supplied for local measurement of a single chemical, or as a much larger network of multi-channel systems for remote monitoring over a wide geographical area. The analytical unit comprises a multi-port valve precision pump unit, chemistry manifold, a choice of detector and a pre-programmed database providing a range of control options and graphical indicators. The sample is continuously introduced via a diaphragm pump of suitable flow-rate into a weir. At intervals the sample and calibration standards are pumped at selected flow rates into the chemistry stream, where they may be diluted and mixed with reagents to form a colored complex which is measured at either visible or UV wavelengths in a colorimetric detector. The analogue amplifier signal is received by the Data A-D converter, analyzed, and continuously printed out showing sample concentration levels. From the processed data received, if concentration limits are exceeded, corrective measures will be taken by the process pumps to restore acceptable operating conditions. The analyzer has main isolations switches situated inside each cabinet. The chemistry module has a main ON-OFF switch and additional switches for the chemistry pump and sample pump. The pH-meter has a separate power source requiring a low voltage supply. At the heart of all ALERT 2000-S systems is an industrial computer. This is an all-solid state system consisting of a single board computer together with one or more intelligent analogue cards and a mix of other industry standard input/output, control and communication interface cards, depending on the application. Solid state relays and optional opto-isolated digital inputs and outputs allows the computer to operate valves, pumps and power supplied to directly control the analyzer, activate alarms, etc. For controlling industrial processes, three-term or PID functions may be added. Each ALERT 2000-S is designed to satisfy individual requirements and it is available in three configurations: • Monitoring locally with ALERT 2000 having built-in keypad and VDU. • Monitoring locally with connection to Homebase computer via RS232 (single

ALERT 2000 over distances of up 100 meters) or RS422/RS485 (multi-drop over

distances up to 1200 meters). • Monitoring over long distance using single or multiple ALERT 2000 systems

connected to a Homebase via telephone links. The above configurations define how ALERT 2000-S will communicate with

the operator and depends on how close the unit is to the monitoring point. Other options of the system include self-diagnostics tests, reagent level

monitoring and leak detection. On the communications side it offer a radio links to the telephone network. ReMAC allows access into the analyzer's data logs and audit trails. Files of data can be downloaded to Homebase, and graphic displays of past and current test levels may be displayed and plotted. ReMAC also allows complete control of the remote site by re-setting limits, and enabling and disabling of functions etc.

The ALERT 2000-S has a wide range of applications in the field of the on-line monitoring of wastewater; effluents; industrial process monitoring; food and drinks; pharmaceuticals, quality control.

331

All BURKARD analyzers can be fitted with the computer controlled automatic start-up and shut-down. A motorized wash/reagent changeover system ensures that all reagent lines are automatically connected to a water-wash on comple

ven a single sample and to analyze 60-180 samples/h (Figure III.2.22). The standard digital dual-wavelength (DDW) detector used allows

tion of analysis. The computer returns the valve to reagents for restart. Other computer control options are possible, e.g. reagent level sensing, special functions and interfacing to other instruments.

2. The FIAstar 5000 system’s analysis time per sample is 20-60 seconds, which permits the analyst to run e

measurements in a wide dynamic range, from μ-absorbance units to 2.5 absorbance units. This reduces the need of sample dilution into range. The wide dynamic range is in reality limited by the linearity of the chemistry involved (Beer’s Law) and the presence of refractive index (RI) effects. At low absorbance values, the refractive index effect may become significant and cause appreciable error in the analysis. In the FIAstar 5000 system measurements are performed at reference and measuring wavelengths simultaneously and the RI effects are not strongly dependent on wavelength, therefore forming the difference measure-reference can eliminate them. In reality this also reduces or eliminates the effect of air bubbles, reducing the need for checking and re-running of samples or de-gassing of reagents. As the DDW detector basically reduces all effects that are not wavelength-specific, this leads to a much more stable reading and substantially lower detection limits. For many analytes, sub-ppb detection levels can be achieved.

Figure III.2.22. FIAstar 5000 analyzer.

By using a computer and the SoFIA software (Figure III.2.23.) the analyzer may be e to evaluate, print out and store the

utine testing results and to control the entire FIAstar system from the computer set-up into a fully automatic system, abl

ro

332

keyboar

3. ALLIANCE Instruments is a French company providing automatic

analyze tories. The CHEMLINE is the ALLIANCE Instruments’ first on-line monitor and it

gives a on a new

principl

motoriz

d. Calibration and recalibration can be done quickly and easily, and displayed on the screen.

Figure III.2.23. SoFIA software for FIAstar 5000 analyzer.

rs to the industrial market and for routine and research labora

correct answer to the problems of reliability, accuracy and low maintenance. CHEMLINE is unique with its innovative-patented reactor basede of analysis called STEP-CHEM ALLIANCE Instruments. The principle is

the following: samples and reagents are mixed in the reactor equipped with 10ed valves and piston managed by a stepper motor that delivers very precise

volumes (up to 10 μL if required) (Figure III.2.24). a. The sample is admitted into the reactor by opening a specific valve and moving

down the piston. A precise volume is admitted into the reactor by programming and then the valve is closed.

b. The first reagent is admitted into the reactor by opening the second dedicate valve. The piston moves down in order to admit the right volume of reagent then the second valve is closed.

333

c.

334

d.

g.

h. If it is i reaction product by heating,

rations: step number of the motor, opening and closing of the valves ar

The mixing of sample with the reagent is performed by moving the piston and admitting a gas (air or nitrogen) with the third valve. The step can be operated one or more times. The chemical reaction takes place in the reactor for some minutes. During this step all the valves are closed and piston is stopped.

e. If necessary, a second reagent can be added or dilution performed by using the same principle.

f. The last step is to take the measurement by pushing the solution to the detector, which can be a colorimeter, an infrared detector, a UV photometer or selective electrode. Another valve, at the top of the reactor is used. After closing this valve, there are three possibilities:

A new cycle is started; eration is performed; A complete washing op

Calibration is performed development of themportant to accelerate the

to distillate the sample or to use a UV digester, another valve will be selected in order to send the solution to a specific module: heating-bath, distillation unit, UV lamp, etc. The same valve will permit the return to the reactor if another reaction has to be done.

ll the opeAe controlled by a microprocessor, each step is fully programmable. This

patented principle allows a very high accuracy of volume insertion and guarantees very good quality of the analysis. Before or during the analysis, an automatic dilution can be programmed. The level and the factor of dilution are programmable. The analytical section does not require a regular maintenance. Only, some seals and o-rings can be replaced every six month. This new way of mixing samples and reagents without using a peristaltic pump offers a significant advantage to the user: a very low maintenance (no change of tubes, nothing to maintain regularly). It is not required to stop the analyzer the user must only refill the CHEMLINE with reagents.

In most cases, colorimetric detection is performed in the range 400-800 nm. The colorimeter includes a tungsten-halogen lamp, lenses which focus the light in a 10, 30 or 50 mm flow-cell. The detection is dichromatic: the transmitted light is divided in 2 equivalent beams by a prism and analyzed at two different wavelengths. The final measurement is obtained by difference. This type of detection cancels matrix effects like color of the sample especially important when it is changing.

335

Figure III.2.24. Schematic new principle of analysis: STEP-CHEM.

Two software solutions are available: One software allows the analyzer to be a stand-alone unit, its main functions

being: - management of all analytical specifications; - automatic calibration with 1 to 5 points; - archiving and data transmission; - high an low level alarms - auto dilution; - reanalyze in case of problems; - diagnostics.

One remote software under windows for maintenance and operation on compatible computer that allows:

- full remote control; - remote diagnostics; - upload/download of methods, programs and results.

The installation of a CHEMLINE must be as near to the sampling point as possible in order to have a permanent on-line monitoring. The analyzer is used for continuous monitoring of water quality: ammonia; total nitrogen; chloride; cyanide; iron; nitrates; phenols; phosphates; silicates; total organic carbon and for the process control in chemical industry.

The SINGLE had been designed for the control and analysis of a specific parameter important to laboratory or production unit. It comprises a built-in sampling system with probe and wash receptacle (a sampler with 12 cups). One analytical unit includes: a pump; a manifold; one or two heating bath; one colorimeter with a filter in the range 340-880 nm; a compartment for reagents; flasks and wash valves; two standard solutions and micro-electronic valves. A microprocessor controlled unit which comprises a logical board for data processing and system’s control, a touch panel to access all functions, 20 columns impact printer; scrolling menu software. The SINGLE can be used by virtually anyone, with no specialist knowledge equipped for routine use: simply load in the sample and press the “analyze” key.

The calibration is made with the base line (zero) and two standards (3 points). If the correlation coefficient is out of the user defined limits, the operator is warned by an error message and a new calibration must be performed. The result is calculated from the average of 200 measurements and validated only if their coefficient of variation is below 0.5 %.

The SINGLE is mainly used for: phenols, cyanides, total organic carbon in wastewater; volatile acidity, sugars in wine; nitrates in food products.

The EVOLLUTION II distinguish itself by using a modern proportioning pump and an advanced “unique focusing” colorimeter using optical fibre technology and bichromatism. With a capacity to sample of 104 cups, the analyzer can assay 60 tests per hour for up to 8 methods, including methods where distillation is required. The system incorporates computer-generated data reporting, with a real time display

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of measuring signals, zooming function, selective printout and electronic archiving. The EVOLUTION II is mainly used for nutrients in seawater; phenols, cyanides and detergents in wastewater.

The INTEGRALFutura is the new generation in continuous flow analyzers featuring advanced microflow technology, electronic debubbling, modern electronics and comprehensive computer control and data management. Each FUTURA console can be in fact considered as an independent analyzer and it is directly connected to the computer. III.2.2.3. The Future - Microfluidics

New researches in microfluidics influence strongly the continuous flow methodologies. Many manufacturers on flow analysis equipment are developing microfluidic analyzers, known as micrototal analysis system or lab-on-chip devices. In the future, these devices will completely replace the existing macrosystems. They will have incorporated an in-line filtration device to remove the particulates from the sample and to avoid clogging.

LACHAT considers the range of microliters of solutions as a perfect compromise between reducing reagent consumption and accommodating to real-world samples.

FIAlab developed a microfluidic analyzer called “lab-on-valve” (LOV), which operates in SIA mode. The entire analyzer is micromachined within a single monolithic structure and mounted on top of the multi-port valve; a single multi-stepper syringe pump propels microliters of fluids; it is compatible with UV-VIS and fluorescence spectrometry.

Nowadays, the manipulation of samples, reagents and bead suspensions is technologically different from the initial continuous flow systems, although the methodology principles are the same: sample introduction, controlled dispersion, reproducibility of events, which result in a precise control of the physical and chemical parameters.

A recent discovery is the combination of capillary electrophoresis with continuous flow analysis, which offers a perfect synergy of rapid response and multi-analyte resolution. The use of electro-osmotic flow as fluid propulsion in flow analysis systems in a capillary electrophoresis-like configuration can replace the conventional pump and allows it to be employed in vastly miniaturized formats. From this combination other flow methodologies have been born – capillary FIA, SIA or BIA that can be involved in fields as radiochemistry, biosensors, trace analyses, drug discovery.

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REFERENCES 1. J. Ruzicka, E.H. Hansen, Anal. Chim. Acta, 1975, 78, 145. 2. “Metode Automate de Analiză în Flux”, A.F. Dăneţ, Ed. Univ. Bucureşti,

Bucureşti, 1992. 3. “Flow Injection Analysis”, J. Ruzicka, E.H. Hansen, John Wiley & Sons, Inc.,

New York, 1981. 4. “Flow Injection Analysis. Principles and Applications”, M. Valcarcel, M.D.

Luque de Castro, Ellis Horwood Ltd., 1987. 5. “Flow Injection Analysis”, J. Ruzicka, E.H. Hansen, Second Edition, John

Wiley & Sons, Inc., New York, 1988. 6. “Flow Injection Atomic Spectroscopy”, J.L. Burguera (Ed.), Marcel Dekker,

New York, 1989. 7. “Flow Injection Analysis. A Practical Guide”, B. Karlberg, G.E. Pacey,

Elsevier Sci., Publ. Co. Inc. The Neatherlands”, 1989. 8. “Flow Injection Separation and Preconcentration”, Z.L. Fang, VCH, Verlags-

gesellschaft, Weinheim, Germany, 1993. 9. “Flow Injection Analysis. Principles, Techniques and Applications”, W.

Frenzel, Technical Univ. Berlin, Berlin, Germany, 1993. 10. “Flow Analysis with Atomic Spectrometric Detectors”, A. Sanz-Mendel (Ed),

Elsevier, 1999. 11. “Flow Injection Analysis of Pharmaceuticals: Automation in the Laboratory”,

J. Martinez Calatayud (Ed), Taylor,& Francis, London, England, 1997. 12. “Flow Injection Analysis: Instrumentataion and Applications”, M.

Trojanowicz, Word Scientific, River Edge, New York, 1999. 13. J. Ruzicka, E.H. Hansen, Trends Anal. Chem., 1998, 17, 6. 14. E.H. Hansen, J. Ruzicka, Trends Anal. Chem., 1983, 2, 5. 15. R.R. Kowaslki, J. Ruzicka, G.D. Christian, Trends Anal. Chem., 1990, 9, 8. 16. E.A.G. Zagatto, B.F. Reis, C.C. Oliveira, R.P. Sartini, M.A.Z. Arruda, Anal.

Chim. Acta, 1999, 400, 249. 17. T. Gubeli, G.D. Chistian, J. Ruzika, Anal. Chem., 1991, 63, 2407. 18. A.N. Araujo, J.L. Costa Lima, M.L.M.F.S. Saraiva, R.P. Sartini, E.A.G.

Zaggato, J. Flow Injection Anal., 1997, 14, 151. 19. G.D. Chistian, Analysit, 1994, 119, 2309. 20. A. Cladera, E. Gomey, J.M. Estela, V. Cerda, A.Alvarey-Osario, F. Rincon, Int.

J. Environ. Anal. Chem., 1991, 45, 143. 21. B.F. Reis, M.F. Gene, E.A.G. Zagatto, J.L.F.C. Lima, R.A.S. Lapa, Anal. Chim

Acta, 1994, 239, 129 22. C.E. Lenehan, N.W. Barnett, S. Lewis, Analyst, 2001, 127, 997. 23. A. Cladera, C. Tomas, E. Gomey, J.M. Estela, V. Cerda, Anal. Chim. Acta,

1995, 302, 297.

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24. F. Mas, A. Cladera, J.M. Estela, V. Cerda, Analyst, 1998, 302, 297. 25. V.P. Andreev, G.D. Chistian, Anal. Lett., 2001, 34, 1569. 26. G. Chistian, Anal. Chim. Acta, 2003, 499, 5. 27. J.F. Staden, Anal. Chim. Acta, 2002, 467, 61. 28. E.A.G. Zagatto, J.F. van Staden, N. Maniasso, R.I. Stefan, G.D. Marshall, Pure

Appl. Chem., 2002, 74(4), 585 29. www.flowinjection.com 30. www.fia.unf.edu/fad/fad/html 31. www.foss.dk 32. www.perkinelmer.de 33. www.zelana.com/lachat 34. www.fia.unf.edu 35. www.oico.com III.2.3. APPLICATION OF THE FLOW TECHNIQUES OF ANALYSIS IN ENVIRONMENTAL MONITORING AND CONTROL José MARTÍNEZ CALATAYUD, Mónica CATALÁ ICARDO III.2.3.1. Introduction The analytical challenge from environmental monitoring and control means to the availability of portable multi-sensor monitoring rather than analyzers into the chemical laboratory. The emergent advances for monitoring portability (last 10-15 years) is a clear consequence of the recent development of chemical sensors, biochemical’s, gas sensors and disposable strip sensors. The combination of sensors with electronic transducers (electrodes-electrochemical, transistors-optical, semiconductors-impedance, etc.) allows the conversion of the molecular (or ionic) recognition response into an electrical output to establish the analytical concentration of the substance to be tested. A lot of efforts resulted in the existing (commercially available or in the home-made phase) plethora of portable devices developed for such different scientific fields as bio-medical, mining, industrial, oceanographic studies, domestic water production and, in environmental monitoring and control, especially for testing water quality and atmospheric pollution. The future wide use of these portable devices is clearly connected with the manufacturing of robust, compact, low-cost, long-term and easily transportable monitoring devices. At the present, one must recognize we are far away from the ideal.

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Flow procedures when are provided with miniaturized detectors, fit especially well with portable multi-sensor monitoring instrumentation. The low-cost miniaturized flow-cells containing electrochemical sensors (potentiometry, conductimetry, etc.) was the starting point to establish a radical departure from traditional analytical ways (sampling and transport) followed by the LED spectrophotometric detectors. The next natural step was the combination of different cells in the same flow manifold or multi-sensor arrays. Examples are the portable FIA manifolds with three or four ion sensors for calcium, potassium, nitrate and chloride. Gas analyzers based on multi-cell potentiometric sensors have been also proposed and developed for many toxic gases: namely, sulfur dioxide, volatile organic compounds and carbon monoxide or nitrogen oxides. Even some portable devices have been designed to be adapted to liquid or gas samples depending on the input (type of pump) and sensors. A flow system for complete environmental monitoring and control must contain the sampler, the flow assembly and the detector and recording devices. The central part of the system, the flow assembly in different modalities (FIA, SIA, multi-syringe, multi-commutation, etc.) fits well with the rest of the system due to the easiness to conform to the requirements of the “standard recommended procedure”, usually proposed in a batch mode. Flow methods also fit well with any kind of analytical detector with the single requirement to change the batch flow to a flow-cell; and, normally it is not a complicated task, to adjust either sample and reagent concentrations to conform to the batch empirical conditions. An advantage of flow procedures over its batch counterparts is that they improve the precision of the batch method, because of the reproducible timing and controllable sample dispersion. Another interesting point is the capability of flow methods to modify the sample matrix (sample pre-treatment) by integrating different experimental steps like analyte pre-concentration (ion exchange, liquid-liquid extraction, and gas diffusion) or dilution, the filtration of turbid samples or containing suspended solids, etc. These changes in the sample matrix resulted in benefits to improve the detector performance. For instance, when the sample matrix is filtered, some constituents that enhance the base line are removed; then, the filtration improves the detectability of the compound of interest. The pre-concentration of the analyte means lower detection limits. Analytical flow procedures are relatively young; segmented-flow analysis is the “father” of the dynasty; then, the apparition of FIA supposed an explosive and widespread use of the flow methodology. New flow-methodologies are continuously expanding and appearing new modalities: in this order, mono-segmented-FIA, SIA, Multi-syringe, Lab-on a valve, Multi-commutation, etc. And last but not least, the primary advantage of the flow methods over other automatic methodologies is economic; an aspect of paramount relevance when the environmental monitoring and control means an unimaginable number of daily analyses.

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Flowing stream techniques are well adapted to in-situ determination of analytes in water samples (about 2500 references up to 2003) and a lesser extent to air analyses. An overview on the flow analytical procedures gives a clear result; FIA is the preferred and by far, the most used methodology. Different extensive reviews can be found in analytical literature dealing with FIA-water monitoring. And the detector “married” with the FIA is also by far, the UV-VIS absorption spectrophotometer. A different problem is the soil monitoring (nutrients and pollutants) due to non-fluid nature of the matrix, a problem of special interest in regions with intensive agriculture. Most of the problems from polluted soils are quickly passed to the water quality. When thinking about environmental monitoring and control, one should think on the commercially availability of robust, compact, portable (even submersibles) and multi-parametric systems. Most of flow assemblies were originally designed for one or few parameters and for off-line analysis. In addition, the miniaturization of the flow manifolds will result in lesser sample and reagents consumption. The present requirements to be pointed out are the design of miniaturized, multi-parametric systems to fulfill the needs of the on-line complete analysis. The environmental monitoring requires separating water and atmospheric samples; both of them comprising many different matrixes (like drinking water, marine water, wastewater, deep ground waters, etc.) and each matrix includes a large number of analytical parameters; like common sample constituents and pollutants from an external source. A simple classification will divide constituents and pollutants in water samples in anions (mostly of nutrients are included into this group), cations and organic compounds. On the other hand and as above reported, there are different flow methodologies. An overall vision of the complete problem obliges necessarily to select examples to illustrate the present possibilities.

A selection of some examples based on different flow-methodologies and devoted to one kind of the following areas: (a) Water monitoring and control: coastal sea waters, FIA nitrites and nitrates; aqueous sediments, dissolved oxygen; rain water, fog and snow, hydrogen peroxide; and sulfur(IV); and wastewater, SIA multi-parametric. (b) Atmospheric monitoring and control: urban areas, ethanol; workplace, NO2; and general atmospheric ambient, ozone and SO2. (c) Soil pollutants, pesticides. III.2.3.2. Water Monitoring and Control

Sea water monitoring, Nitrogen (Nutrients) The adaptation to flow systems from classical batch procedures means in many occasions to change some chemical steps. The determination of the total nitrogen is performed through nitrate or ammonium, after a digestion step by the

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traditional Kjeldhal; a suitable alternative for a flowing stream method is the on-line photo-degradation with the aid of chemical oxidants. On the other hand, a plethora of flow spectrophotometric procedures for nitrite determination have been published; most of them rely on the modified Griess or Shinn procedures. The nitrate determination is performed by the same nitrite reaction after a prior redox process to convert nitrate into nitrite, being the solid-phase reactor filled with copperized cadmium is the preferred for most of authors. Other alternative fitting best for flow methods are the homogeneous reduction with hydrazine; the photo-reduction by irradiation with a low pressure Hg-lamp; or enzymatic processes. A certain number of FIA assemblies integrated nitrate, nitrite, ammonium and total nitrogen with the aid of a spectrophotometric detector. For more information about the chemistry of these procedures see Section III.3 “Automation of the Spectrophotometric methods”. The speciation of nitrogen, sequential determination of nitrite (Sinn reaction), nitrate (with the copperized-cadmium reactor) and total nitrogen (UV photo-degradation) can be easily integrate in a compact flow-manifold as depicted in figure III.2.25.

Monitoring nitrate in estuarine and coastal waters The main point to support the in-situ monitoring is the changes suffered by

samples during collecting and storage: this analytical requirement obliges to design and prepare field-portable laboratories able to perform in different natural environments and conditions. This has been solved in some less difficult situations like workplace air; rivers, lakes, etc.; in more complicated situations the number of available multi-parametric solutions is certainly rare. Some situations are not clearly solved like sampling deep water in a lake, sea or estuarine; transporting the sample from the original place to the ship board-laboratory increases the solved air (among other inconveniences) with the inherent disadvantages.

A recent and maybe representative example of the efforts to solve this problem is the manifold nitrate determination in estuarine and coastal water samples by a “submersible flow injection analyzer” in which size, weight and low buoyancy [the difference between the upward and downward forces acting on the bottom and the top of the cube, respectively, is called buoyancy] and easy use of the set-up were carefully studied. According to the moment requirements, the instrument can be operated in manual (diagnostic) or automatic (long term monitoring) mode and operated as a bench-top, shipboard or submersible instrument. The instrument has been proved as a shipboard mode for mapping nitrate concentration in North Sea and submersible mode for the Tamar estuary transect.

The chemical procedure is the well known modified Griess procedure for nitrite with the prior reduction of nitrate by the cadmium-copperized solid-phase reactor as depicted in the Figure III.2.25. A sample on-line filtration unit is also included.

342

0.45 μm

Sample

Carrier

Iv Sulphanilamide

NED

P reactor

Cu/Cd Pre-

filter

D

W

Syringe

Figure III.2.25. Flow Injection Analysis manifold for determination of nitrite and nitrate in water samples. Iv: Injection valve with a sample volume of 260 μL; D: Detector 540 nm and 20 mm path length; reactor length, 1 m; P, peristaltic pump; W, waste; flow-rates (in mL min-1): filtered sample 0.80; carrier, 0.32; sulfanilamide, 0.16; and, naphtyl ethylene diamine dihydrochloride, NED, 0.16.

W CNED Sulphanilamide

Standard inlet

Sample inlet

Power connector

“O” ring

Sample loop

P1: reagents

P2: Standard/sample

Distribution manifold

PVC cover plate

SV

FC/SSDIv

Reducing column

Reactor

“T” Conector

W CNED Sulphanilamide

Standard inlet

Sample inlet

Power connector

“O” ring

Sample loop

P1: reagents

P2: Standard/sample

Distribution manifold

PVC cover plate

SV

FC/SSDIv

Reducing column

Reactor

“T” Conector

Figure III.2.26. Block diagram of submersible flow injection analyzer. W: waste; P: pump; C: carrier; SV: 3-way switching valve; FC/SSD: Flow cell/solid-state detector; Iv: injection valve

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The Figure III.2.26 depicts the block diagram of the submersible flow injection analyzer built within a pressure housing engineered for a single book of PVC. The quantification is performed by a flow-through, solid-state detector, incorporating an ultra-bright green light emitting diode (LED) as a light source, and a photodiode. Either the length of the flow-cell and the sample volume can be altered to get a larger dynamic range. From the range 2.8 - 100 μg L-1 N up to 100 - 2000 μg L-1 N; the detection limit is 2.8 μg L-1 N with a light-path 2 mm long and 250 μL sample aliquot.

FIA determination of dissolved oxygen (DO) in sediments by the Winkler method

The Winkler titration is the traditional robust method for the determination of water-dissolved oxygen, DO. In a first step, the oxygen oxidizes the Mn(II) hydroxide block. Addition of sulfuric acid in the presence of an excess potassium iodide dissolves the oxidized manganese hydroxide producing tri-iodide, which is in a final step, titrated by thiosulfate with the aid of starch for clear end-point. The classical procedure seems not suitable for purposes of accurate respirometric measurements. Several suggestions appeared in the analytical literature for automating the DO measurements, some of them into the FIA field. The selected example is based on the spectrophotometric monitoring of released tri-iodide; the assembly is of type of r-FIA (reverse FIA, where the sample is the carrier and reagents are inserted instead of the sample) to avoid blockage of tubing when Mn (II) ion solution merges continuously with the alkaline (sodium hydroxide) stream. The water sample is the carrier where the Mn (II) is inserted. The resulting stream merges with an alkaline-KI mixture, and the reaction product (oxidized manganese hydroxide) is solved by merging with the sulfuric acid solution. Tri-iodide is spectrophotometrically monitored at 440 nm.

Figure III.2.27 depicts the flow assembly that, according to authors, has been

Figure III.2.27. FIA system for determination of the disso

successfully applied in either laboratory and field situations.

lved oxygen ture of KI and

P, peristaltic pump.

1, solution of MnSO4 to inject 50 μl; 2, water sample (as carrier); 3, mixNaOH; 4, acid iodide wash solution; and 5, H2SO4. Flow-rates 1.4 ml min-1.R1, reactor 100 cm long; W, waste, Iv, injection valve; s-v, 3-way solenoid valve; D, detector; and

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III.2.3.3 Control in Rain Water

ative characteristics has relevant

effects on atmospheric chemistry; like the quick conversion of dissolved SO to

ided with aquarium pumps for producing the flow. e ma

nels; one contains the solid-phase reactor to eliminate the ydroge

mberlite (IRA – 743);

at pH 4.8 and at –1.00 V uring 1

mple throughput was 90 h-1 and reproducibility under 1%. The semb

ste.

. Monitoring and

The hydrogen peroxide and due to its oxid2

sulfuric acid the main component on the acid rain; other oxidants present in atmosphere as ozone give the same process but retarded and requiring certain pHs and presence of metallic catalysts. The amperometric determination of hydrogen peroxide in rainwater was performed in a flow assembly provTh nifold is provided with a solid-phase enzymatic reactor filled with catalase immobilized on a resin type Amberlite and the detector is formed by gold electrodes modified by electro-deposition of platinum. The H2O2 is amperometrically monitored at +0.60 V versus the reference electrode Ag/AgCl and a stainless steel tube was used as auxiliary electrode. The sample is inserted into a carrier of electrolyte; this channel splits to form two independent chanh n peroxide. Both channels (with and without reactor) merge in a single channel to lead the sample (treated and untreated) to the electrochemical cell. The outputs difference gives the hydrogen peroxide concentration. The immobilization of the catalase is performed by the usual way of treating the resin with glutaraldehyde to be linked to the amino groups of the Athen is added the enzyme and finally the reactor is washed. The electrochemical cell contains a set of gold microelectrodes. The platinum electro-deposition was made with 2 x10-3 mol L-1 K2PtCl6, d 5 min. The life span was of 15 and 7 days for the reactor and Au-Pt electrodes, respectively. Saas ly was tested with a rainwater sampler placed outside of the lab. The proposed flow-assembly is depicted in the following Figure III.2.28.

AV

Figure III.2.28. FIA system for hydrogen peroxide determination in rainwater E: electrolyte; AP: aquarium air pump: AV: aquarium valve; Iv: injection valve; TR: tubular reactor; D: Potentiostat and electrochemical cell; W: wa

E

E

Iv

AP D TR

W

345

of hydroge

is Ag/AgCl and placed at the p

second, all the SMS is converted in S(IV) by increasi

liary enzymatic pro s

Figure III.2.29. FIA system for determination of H2O2, HSO3 and hydroxyl

ethane sulfonate

HSO3 and HMS respectively; AE: main electrolyte flow,

A former article was also dealing with the amperometric determinationn peroxide and sulfur (IV) in rain, fog, snow and cryo-sampled atmospheric

water vapor. The bisulfite is directly measured; and, the rest of the S(IV), present as hydroxyl methane sulfonate (HMS) or forming other carbonyl adducts, indirectly determined by releasing sulfite with an alkaline treatment.

The electrochemical micro-cell contains two homemade platinum electrodes (indicator and auxiliary electrodes). The reference electrode

ti end of the tubular micro-cell. The working electrode is mounted on a Nafion tube, which is a cationic exchanger with the goal of separating hydrogen peroxide and bi-sulfite (it is only permeable to hydrogen peroxide). The selectivity among both compounds is implemented with a careful selection of the pH. At high pH (alkaline medium) the HO2

- is firstly oxidized (0.30 V) and bisulfite do not interferes the output. At acidic pH and 0.65 V, SO2 is the first to be oxidized with an almost null interference from hydrogen peroxide.

The process for the determination of HMS is based on two steps; first is measured the non protected S(IV); and

ng pH to 11 -12. The assembly is depicted in Figure III.2.29. Other compounds present in the matrix and presenting a low oxidation

potential (versus Ag/AgCl) interferes the measurements. An auxices of destroying hydrogen peroxide (catalase) and sulfite (sulfite oxidase) avoids

interferences from iron, formaldehyde, etc. Sample throughput 30 h-1 and limit of detection 2 10-8 mol L-1.

D P

Iv

Water

AE

ME

-

mME: auxiliary flow, 0.024 to 0.06 mL min-1, 0.1 mol L-1 KOH or 0.1 mol L-1 HClO4 for H2O2 or -

0.075 to 0.18 mL min-1, 0.4 mol L-1 KOH or 0.4 mol L-1 HClO4 for H2O2 or HSO3

- and HMS respectively; Water flow-rate, 0.24 to 0.54 ml min-1; P: peristaltic pump; Iv injection valve (200 μL sample loop); D: electrochemical cell and polarograph.

346

III.2.3. astewater

-procedures is depicted a SIA assembly itable of monitoring different quality parameters using the usual analytical

hemist

onitoring is depicted in Figure III.2.30. It is designed to determine total organic

tmospheric Monitoring and Control

ollution is a challenge for the nalytical monitoring and control; a challenge in which the analyst is “aided” by the

in the pollutan

4. Water Quality, W As a summary of automatic flowsuc ry.

A multi-parametric SIA assembly designed for wastewater quality in-situ and real-time m

carbon (TOC), chemical oxygen demand (COD), biochemical oxygen demand (BOD), total suspended solids (TSS), nitrate, nitrite, ammonium, total nitrogen, phosphate and total phosphorous. Probably and according to many authors, SIA fits better than any other flow methodology with the multi-parametric analytical task. When a special diode array detector, is incorporated in one of the free ports of the second injection valve, additional parameters may also be evaluated in 3 min, like detergents.

III.2.3.5. A

The dramatic increase in the environmental panew and changing legislation rules; in many occasions with significant differences from countries, even countries sharing common borders and the same problems.

In atmospheric monitoring the systems for sampling are formed of different parts; it requires a device for the sample collection; the trapping device to reta

t and, the measuring assembly. The sampling device must be able of an accurate measure of the air volume sampled. The sampling methods frequently used are filtration (passive or active), sedimentation, electrostatic precipitation, centrifugation and impaction; filtration being the most common. The different parts involved in sampling should be constructed in a material not introducing pollution in the sample. Theses considerations should be added to the next step of the analysis; the sample storage; when analysis is not in-line with the sampling.

347

Figure III.2.30. Multi-parametric SIA assembly designed for wastewater

A primary caution in sampling, sample transport and storage should be kept in

mind. S

tion require different sampling procedures according to the

other two parts ample

Monitoring ethanol in ambient air ions which consequence is a change in

atmosph

representative example is the extensive use of ethanol as vehicle fuel (very important

quality in-situ and real-time monitoring.

ampling can disturb the target system and resulting in an unrepresentative final sample. Atmospheric sampling do not alter the original system, it do not causes disturbances in the surrounding ambient. Sample transport and storage do not cause sample deterioration; it is not always an easy task, not all the components remain unchanged after sampling. Studies on air pollutype of ambient to be monitored; a workplace analysis differs from the procedures to be applied for the study of the environment in an industrial area. The study of workplace atmosphere is special based on the level of pollutants to which the workers are exposed; e. g. sometimes a micro-sampling is recommended; the worker carries an adsorptive badge which is send to laboratory at the end of the day. Continuous-flow methods have been connected with the(s r and trap) to configure a complete in-line monitoring system.

There are many anthropogenic acteric chemistry. New compounds were released to the atmosphere in

increasing amounts from relatively recent time. The economic dependence of many countries on imported petrol derived to search for new alternatives to fossil fuels. A

348

in some countries like Brazil), which resulted in an increased concentration of atmospheric unburned alcohol from car exhausts. At the end it resulted in a clear change on the atmospheric composition, to a dramatic extent in urban areas. To known the chemistry interactions at present in atmospheric gasses is required to develop analytical methods for monitoring the ethanol contents in ambient

During air sampling the ethanol reacts with

(a) oil (about 100 cm long) made of borosilicate glass is the sampling

pure water to

t pH 9 with a phosphate buffer.

nm).

s of solution is

e buffer; P:

air. Unburned alcohol is an important ingredient in photo-oxidation or reactions with hydroxyl radicals. The wet chemistry of ethanol is well known contrary to the situation of the atmospheric samples. The following method provides an enzymatic-fluorimetric procedure for monitoring the ethanol in ambient air.alcohol dehydrogenase, ADH, and nicotinamide adenine dinucleotide, NAD+, producing NADH; which is monitored by fluorescence.

The description of the scheme and operating mode has the following steps (see Figure III.2.31):

Air is aspirated through a KI filter to eliminate the ozone influence (b) A scrubber cdevice. This is treated with acetone and 40% HF; then is washed withobtain a hydrophilic surface. The flowing sample bubbles through the absorbing solution; a mixture of 1 mmol L-1 NAD+ and 7.5 U mL-1 ADH a(c) The product of the reaction, the NADH, is lead to the detector flow-cell where fluorescence is measured at 460 nm (excitation wavelength, 360(d) A correction is required to avoid bubbles arriving to detector from the solvent volatility; the flow-rate in channel B is smaller than in A and the exceseliminated through channel C. (f) The flow assembly (detector and pump excluded) fitted into a thermostated box for field experiences.

Figure III.2.31. A: absorbing solution: ADH, NAD+, phosphatperistaltic pump; D: fluorescence detector; V: injection valve. See explanation in the text.

A

C

A

C

Flowmeter, pump

DP V

scrubber-coil Ozone filter

B

air

349

T

QA= 0.2 ; QB= 0.15 mL min ; gas flow rate=1 L min and 70 % relative humidity. Calibration over the range 0 -112 mg mL-1. The dynamic range in not linear as usu n was 6.5 % (c = 44 mg mL-1,

l of analytes should be very low and e sample cannot be analyzed as is it in the environment, which obliges to include an

on-line cedure. The so-

s

topped. A second pump is used to forc from

2.

ctrophotometer.

is o ) with a power source (12 V battery) (for laborato

he system was calibrated with the references: 3 mL min-1 -1 -1

al in enzymatic reactions. The calculated deviation = 5) and the required time per sample 2.3 min.

Determination of NO2 in air with on-line pre-concentration In environmental air monitoring the leveth

sample pre-treatment to obtain a continuous and automated procalled chromato-membrane cell, CMC, has been used in different FIA systems for the on-line sample pre-concentration and separation (gas-liquid, liquid-liquid heterogeneous systems, etc.). It is a continuously working device in which the membrane performs a kind of “chromatographic separation”; when two different phases (polar and organic solvent or air) are forced through the membrane the transfer of analytes between tow phases occurs. This device is included in the FIA assembly. The selected example to illustrate this is the determination of nitrogen dioxide in air so in field as in the laboratory. The analytical method is the “universal” for nitrite; first the nitrogen dioxide should be converted into nitrite. For this conversion iproposed the reported "chromato-membrane cell concentration-distribution device made of a PTFE block with micro- and macropores. The steps of the flow determination are as follows (see Figure III.2.32.): 1. The absorbing solution of triethanolamine is forced to the CMC at a flow-rate of

0.5 mL min-1 up to filling, and then the pump is se the sample air (20 L) into the cell at 7 mL min-1. The NO2 is transferred

the air to the absorbing reagent and transformed into nitrite ions. Turning the valve 2 the resulting solution is inserted into the FIA system and merges with the mixture of sulfanilamide and NED (Saltzman reagent) developing the corresponding color to be monitored in the flow-cell of the spe

3. By starting again in the reverse direction the free NO2 air enters into CMC cell to by renewed and start again the cycle.

The portable set-up is a micro FIA system with PTFE tubing 0.25 mm internal diameter; which is closely tight into a box (box size 16 x 16 x 32 cm) and the detector

f the LED type (light emitting diodery determinations the FIA manifold was formed with PTFE tubing 0.5 mm

internal diameter). The detection limit is 0.9 μg L-1 and the complete cycle is about 5 min.

350

P2

P3

RS

AS

AS

air

Figure III.2.32. Flow system with the chromato-membrane. RS, reagent

lo early recommend a reagent; several were tested. They

tion zone: ) The 200 nm internal

(b) lass surface, 80 x 6 mm, provided with a triangular

(c) VC. The ozone

(d) iluminescent reagent is pumped to the detector device placed at about 20

ith 1 mmol L

solution (sulfanilamide and NED mixture); AS, absorbing solution; SL, standard sample (NaNO2); V, injection valve; P1, double-plunger pump (flow rate 0.05 and 0.25 mL min-1 for μFIA or conventional FIA tubing, respectively); P2, peristaltic pump (0.5 mL min-1); P3, syringe type pump (7 mL min-1); DG, degassing unit; D, detector. CMC = CMC cell.

zone f w-analyzer OThe authors do not cl

prepared an empirical set-up for a hanging droplet and a film-reaction and tested over 70 chemiluminescent reagents for ozone; the azine dyes phenosafranine, methylene blue and safranin O resulted in the higher light emission. The main parts of the ozone analyzer are the detec(a hanging droplet is formed by a PTFE capillary 3 cm long and

diameter. The accurate and reproducible volume of solution is provided by the peristaltic pump; and the droplet is eliminated by the valve which presses briefly the pumping flexible tubing. the film-reagent is a smooth gdistributing device to avoid irregularities of the liquid film over the whole surface. Analytical applications are only studied in the film-reactor mode. The reactor consists of a 35 x 200 mm chamber build in black Pflowing at 2 L min-1 reacts with the suitable reagent (according to the operator choice). The chemmm of the entrance window of the Photon Counting Module.

The flow system was as shown Figure III.2.33. W -1

phenosafranine in ethanol the following analytical figures were obtained: application range, 5.2-330 μg m-3; quantification limit, 2.1 μg m-3 and time per sample 5 s. Comparing results with the UV method this detector present lesser sample

DG

SL

V1

V2

D

CMC

P1

351

consumption and is faster and gives minor results; which can be due to the interferences from aromatic compounds which are not eliminated in the UV detector.

Determination of atmospheric SO2ompounds to be controlled to test the air

quality

ed with on-line sampling and pre

(vinyldene) difluoride,

reagent is inserted by the injection

Figure III.2.33. Flow assembly for ozone determination. RC: Reaction

It is one of the relevant polluting cand many efforts and procedures have been proposed for its determination in

air analyses. Most of these methods lack the required selectivity and suitable sensitivity. A pre-concentration step is the usual sample pre-treatment, which in the next selected example is performed with the aid of a gas permeation device. These devices have been extensively proposed in FIA procedures.

A spectrophotometric monitoring assembly provid-concentration and able to be field applied is reported.

A micro-pore hydrophobic membrane made of polysandwiched between two perspex blocks serves for pre-concentration. Air circulates at one side of the membrane at a flow-rate of 0.9 L min-1; at the other side, the absorbing solution at 0.8 mL min-1 containing 5 10-4 mol L-1 5,5´-dithiobis(2,2´-dinitrobenzoic acid) (DTNB) in 0.025 mol L-1 phosphate buffer. For a 5 – 8 minutes interval the flow is stopped to allow the pre-concentration process. The resulting mixture absorbed SO2 plus valve into the carrier stream formed by the same reagent DNTB at 0.7 mL min-1 and is monitored in the detector flow-cell at 410 nm. A miniaturized optical fibber spectrophotometer is used (Figure III.2.34).

D RC

air

waste air

P V

Reagent

waste solution

chamber; D: photo counting module; V: valve; P: peristaltic pump

352

W

V D P

Sample gas

DTNB

DTNB

W

110 mm

40 mm

20 mm

4 mm

60 mm

10 mm25 mm

Gas inlet

Liquid inlet

Gas outlet

Liquid

Figure III.2.34. FIA assembly (top) and pre-concentration device (lower) for SO2 determination The main interferences of the procedure are the hydrogen sulfide and hydrogen cyanide. The application range and detection limits rely on the pre-concentration time and the reference signal; e. g., for 5 min and reference the carrier DNTB solution, 0-4 mg m-3 and 50 μg m-3, respectively. However, for 8 min of pre-concentration and reference output pure air, the analytical figures of merit are 0-3.2 mg m-3 and 35 μg m-

3. Sample throughput clearly linked to the pre-concentration interval, are 8.5 and 6 h-1

respectively. III.2.3.6. Soil Pollutants Determination of pesticides by a multi-commutation method with photo-chemiluminescence. The pollution of underground waters by the extensive use of pesticides is a new anthropogenic problem affecting the health of the population. Pesticides are in the environment as a consequence of being used in agricultural works but also as being used in other activities as domestic use, airports, golf fields, roadsides, etc. The massive use of pesticides (last two decades) is a new challenge in water treatment plants. Herbicides are the most used pesticides. The manufacturer’s numerical figures give an idea of the size of the problem. In Europe, more than 500

353

tons/year are fabricated of about 40 pesticides; the manufactured amount of pesticides in USA in 1993 is represented in the Table III.2.3. Table III.2.3. Amount of pesticides produced in USA in 1993.

Pesticide Tones Pesticide Tones Atrazine 31,500-33,750 Chlorpyrifos 4,500-6,750 Metalachlor 27,000-29,250 Chlorothalonil 4,500-6,750 Alachlor 20,250-22,500 Propanil 3,150-5,400 Methyl bromide 13,500-15,750 Dicamba 2,700-4,500 Cyanazine 13,500-15,750 Terbufos 22,250-3,600 Dichloropropene 13,500-15,750 Bentazone 1,800-3,150 2,4-D 11,250-13,500 Mancozeb 1,800-3,150 Metam Sodium 11,250-13,500 Parathion 1,800-3,150 Trifluralin 9,000-11,250 Simazine 1,350-2,700 Glyphosate 6,750-9,000 Butylate 1,350-2,700

A pesticide is formulated with additives and co-adjuvant to facilitate its actuation. They are fumigated from air and its concentration in the environment is continuously changing due to dispersion, volatilization, chemical and biological degradation and lixiviation. How these processes are performed is due to the physic-chemical characteristics of each pesticide, but also from the characteristics of waters, soil and atmosphere. There are many flow methods for pesticide determination. The selected example is based on a new strategy by means of the multi-commutation methodology; with the aid of the chemiluminescence detection and photo-degradation to form the detectable compound. The aqueous solution of the pesticide is irradiated with an UV lamp in the suitable medium. The formed photo-fragments merge with the oxidant, potassium permanganate in a sulfuric medium before the chemiluminescence-based detection of the resulting photoproducts. The use of solenoid valves results in substantial reagent savings and constitutes a further extension of clean chemistry procedures.

The pesticide asulam (methyl-4-aminobenzenesulphonyl carbamate), presents a broad spectrum of biological activity. It is used as an insecticide, herbicide and fungicide; and, most often, it is used as a post-emergency herbicide for controlling deciduous and perennial grasses. Asulam acts by stopping cell division and growth of plant tissues. It remains in soil for more than one season. However, it exhibits a high mobility by virtue of the high water solubility of its sodium salt and is therefore a potential water pollutant.

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Monitoring photo-degradation processes has recently proved an effective metho

olenoid valves each o

ion. queous

he optimum insertion profile for each of the two tested oxidant systems is

depict

d for the in situ control of environmental pollutants. The potential of sunlight-based photocatalytic decontamination has lately aroused much interest.

The flow manifold depicted in Figure III.2.35 comprised three sf which acted as a stand-alone commutator with only two positions. Valve

operation was characterized in terms of N*(t1,t2), where t1 and t2 are the intervals during which the valve was ON and OFF, respectively, and N was the number of times of the ON/OFF cycle. The peristaltic pump was placed to aspirate the sample and reagents into the flow-cell behind the reactor; this difference from the usual location of the propulsion system in the FIA-manifolds, was intended to prevent the flow from stopping immediately upon insertion as the valve was actuated.

VV11

Figure III.2.35. Flow assembly optimized for pesticide determinat Q2: photodegradation medium (glycine buffer at pH 8.3); Q1: Asolution of asulam; Q4: Carrier (water); Q3: Oxidant (K3Fe(CN)6 0.1 mol⋅L-1 in NaOH 1 mol⋅L-1 or KMnO4 10-4 mol⋅L-1 in H2SO4 1.2 mol⋅L-1). Flow-rate: 9 and 10 mL⋅min-1 for Fe(CN)6

3- and MnO4- system, respectively. P: peristaltic

pump; PMT: photomultiplier tube; V: Solenoid valve; FC: spiral flow cell; PR: photoreactor consisted of a 173 cm length and 0.8 mm i.d. PTFE tubing helically coiled around a 15 W low-pressure mercury lamp (Sylvania) for germicidal use.

Ted in Figure III.2.36. With the potassium permanganate, an overall of 20

alternate micro-insertions of pesticide and photo-degradation medium were performed. During each micro-insertion, valve V1 was kept ON for 0.3 s to aspirate

VV22 PM

VV33

VV11 QQ11

QQ22

QQ33

QQ44

PPRR

PP

FFCC

ON OFF

VV22 VV33

1100

355

asulam and OFF for 0.1 s to aspirate the buffer used as photo-degradation medium. Valve V2 was kept on to have the peristaltic pump aspirate asulam and the buffered medium throughout the duration of the process (8 s). This loading time allowed the inner walls of the photo-reactor to be efficiently flushed in order to avoid sample carry-over. During the next 90 s, the sample/medium mixture was stopped in the photo-rector for the UV-irradiation. Next, valve V3 was switched ON for 17 s to allow the oxidant to flow and V2 was used to alternately micro-insert photo-degraded

Figure III.2.36. Optimized insertion profile for obtaining a typical transient analytical signal for MnO

pesticide (0.7 s segments) and the oxidant (0.2 s segments) in 17 ON/OFF cycles.

T permanganate and ferrycianide.

ith the selected MnO4 /H2SO4 system, the response was linear up to an asulam conce

h the Fe(CN)6 /NaOH system and the photo-degradation of nit

as in the text)

rs for monitoring of air and water pollution. P. W. Alexander, L. T. Di Benedetto, T. Dimitrakopoulos, D. B. Hibbert, J. C.

2.

4-/H2SO4 systems.

SV1= 0, 20*(0.3,0.1), 0.1

SV2= 97.5, 17

SV3= 0, 8, 90, 17*(0.7,0.2), 0.5

Cycle: 123 20 segments; 8 s 90 seconds 17 segments;

123 seconds

SV1 ON OFF ON

OFF ON

OFF

SV3

SV2

wo oxidant systems were studied: potassium–W

ntration of 5 ppm; the relative standard deviation for the slope of five curves recorded for freshly made solutions on different days was 3.8%. This is a “clean” chemical procedure as the reagent uptake is very low in both cases (only 716 μL for potassium permanganate).

The MnO4–/H2SO4 system proved more selective. Especially strong was the

interference of calcium wit 3-

rate to nitrite which reacted with the permanganate. Copper gave a strong chemiluminescent signal, even at low concentrations. This may require the prior removal of copper from some types of sample with a solid-phase reactor filled with an appropriate ion-exchange resin. The limit of detection was 40 μg L–1 asulam; the equation relating the two was I = 110.42 [mg L–1] – 214.84 (r2 = 0.9926). The overall analysis time was 120 s. REFERENCES (ordered 1. Field-portable flow-injection analyse

Ngila, M. Sequeira and D. Shiels, Talanta, 1996, 43(6), 915 – 925. B. Karlberg, B. Pacey, Flow Injection Analysis, a practical guide, Elsevier, New York, 1989.

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3. Miniature flow injection analyser for laboratory, shipboard and in situ monitoring of nitrate in estuarine and coastal waters. P. C. F. C. Gardolinski, A. R. J. David

4. aKelvie, M. R. Grace, M.

5. of hydrogen peroxide in rain water, R. Camargo Matos, J.J. Pedrotti

6. d D. Klockow, Fresenius'

7. c carbon, nutrients, M. Miró, J. M. Estela

8. w, Fresenius' J. Anal. Chem., 1999,

9. d coupled with a chromatomembrane cell system: application

10.nal. Chim. Acta, 2003, 491(2),

11.ased online sampling and preconcentration. Z.X. Guo, Y.Z. Li, X.X.

12.

and P. J. Worsfold, Talanta, 2002, 58(6), 1015 – 1027. A reverse-flow injection analysis method for the determination of dissolved oxygen in fresh and marine waters. S. Muangkaew, I. D. MRayanakorn, K. Grudpan, J. Jakmunce and D. Nacapricha, Talanta, 2002, 58(6), 1285 – 1291. Flow-injection system with enzyme reactor for differential amperometric determination and L. Angnes, Anal. Chim. Acta, 2001, 441(1), 73-79. Amperometric flow-injection technique for determination of hydrogen peroxide and sulphur(IV) in atmospheric liquid water, I.G.R. Gutz anZ. Anal. Chem. 1989, 335(8), 919-923. Application of flowing stream techniques and related compounds to water analysis. Part I. Ionic species: dissolved inorganiand V. Cerdá, Talanta, 2003, 60(5), 867 – 886 An enzymic-fluorimetric method for monitoring of ethanol in ambient air. M. Schilling, G. Voigt, T. Tavares and D. Klocko364(1-2), 100-105. Absorption, concentration and determination of trace amounts of air pollutants by flow injection methoto nitrogen dioxide determination. Y. L. Wei, M. Oshima, J. Simon, L.N. Moskvin and S. Motomizu, Talanta, 2002, 58(6), 1343-1355. Determination of ozone in ambient air with a chemiluminescence reagent film detector. C. Eipel, P. Jeroschewski and I. Steinke, A145-153. Flow injection determination of gaseous sulfur dioxide with gas permeation denuder-bZhang, W.B. Chang and Y.X. Ci, Anal. Bioanal. Chem., 2002, 374(6), 1141-1146. A new flow-multicommutation method for the photo- chemiluminometric determination of the carbamate pesticide asulam. A. Chivulescu, M. Catalá-Icardo,J. V. García-Mateo and J. Martínez-Calatayud, Anal. Chim.Acta 2004, in print.

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III.3. MODERN TECHNIQUES FOR AIR POLLUTANTS III.3.1. LIDAR (LIGHT DETECTION AND RANGING)

Mihaela BADEA, Mihaela-Carmen CHEREGI, Andrei Florin DĂNEŢ

LIDAR is the optical equivalent of the radar, and so is often referred to as laser radar. In a radar, radio waves are transmitted into the atmosphere, which scatters some of the power back to the radar's receiver. A LIDAR also transmits and receives electromagnetic radiation, but at a higher frequency. LIDARs operate in the ultraviolet, visible and infrared region of the electromagnetic spectrum.

LIDAR is an acronym for light direction and ranging, and is a laser remote sensing technique used in both science and industry. LIDARs are used to precisely measure distances and properties of far-away objects.

III.3.1.1. LIDAR Design A block diagram for a basic LIDAR system is illustrated in Figure III.3.1. A simplified block diagram of a LIDAR contains a transmitter (laser), receiver (an optical telescope) and a detector system.

In LIDAR a powerful laser transmits a short and intense pulse of light. The pulse is expanded to minimize its divergence, and is directed by a mirror into the atmosphere. As the pulse travels upward it is scattered by atmospheric constituents (mostly nitrogen) and aerosol particulates. Light that is backscattered and into the field-of-view of the telescope is collected and channelled toward detectors by an optical fiber or other optics. Filters are used to eliminate light away from the laser's wavelength, and a mechanical shutter blocks the intense low-level returns when required. The amount of light received is measured as a function of time (or distance) using sensitive photo-detectors, and the signals are digitized for storage on a computer's hard drive. LIDARs typically use extremely sensitive detectors called photomultiplier tubes to detect the backscattered light. Photomultiplier tubes convert the individual quanta of light and photons first into electric currents and then into digital photo-counts, which can be stored and processed on a computer. The photo-counts received are recorded for fixed time intervals during the return pulse. In the last years different kinds of lasers were used depending on the power and wavelength required. The lasers may be both cw (continuous wave, on continuous like a light bulb) or pulsed (like a strobe light). Gain mediums for the lasers include,

358

gases (e.g. HeNe = Helium Neon or Xenon Fluoride), solid state diodes, dyes and crystals (e.g. Nd:YAG = Neodymium:Yttrium Aluminum Garnet).

LIDARs are valuable instruments for atmospheric research because they provide an active remote sensing technique that can probe atmospheric regions inaccessible to other instruments, and at high spatial and temporal resolution. The technique has made possible the spatially resolved measurement of atmospheric constituents, as well as various atmospheric parameters, such as temperature, winds, clouds, etc. LIDARs operating from ground and space provide complementary information: where satellite-borne LIDARS provide global coverage but at lower horizontal resolution, ground-based instruments reveal the fine detail required for atmospheric process research.

Figure III.3.1. Scheme of LIDAR system

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LIDAR can exploit different optical techniques as Raleigh and Raman scattering, differential absorption, Doppler and resonance scattering.

In order to obtain information on the concentration of a constituent, two wavelengths must be used. This gives rise to the differential absorption LIDAR, or DIAL, technique often utilized for atmospheric probing. In standard DIAL applications, one wavelength is tuned to a strong absorption line of the species of concern, whilst the other is tuned slightly off the absorption line, allowing the density or concentration of absorbing constituents to be calculated. As the wavelength of the absorption line is specific to each absorbing species, one can isolate the absorption of each constituent. III.3.1.2. Application of LIDAR in Environmental Monitoring

Ozone observation One of the main applications of the LIDAR techniques to environment

monitoring has been in observations of ozone. Ozone absorbs strongly in the ultraviolet part of the spectrum, and furthermore the absorption coefficient varies rapidly with wavelength. These two characteristics make the DIAL method ideal for ozone measurement. The exact choice of wavelengths used in an ozone LIDAR depends on the atmospheric region to be probed: troposphere (< 300 nm) or stratosphere (> 300 nm).

An airborne ozone LIDAR has a lower vertical resolution (a few hundred meters) but is able to measure a curtain of ozone values ~10 km deep.

Pollutants measurement in the atmospheric regions One of the most important applications of LIDAR lies in its ability to make

remote measurements of pollutant emissions from industrial resources. In the modern regulatory environment such a capability is of great commercial value, and many LIDAR systems were developed around the world to provide it. Typically, such a LIDAR is mounted in a truck or van and driven to the perimeter of the site under investigation. The LIDAR performs a two-dimensional scan of the region upwind and downwind of the emission source, mapping the pollutant concentration as a function of height and elevation angle.

Early applications of LIDAR to pollutant monitoring used the Raman technique. The Raman technique has the advantage that it does not require a specific laser wavelength, but has two main disadvantages: Raman backscatter is very weak and the rotational-vibrational bands of O2 and N2 can mask Raman lines from minor constituents. A typical application of the Raman technique has been to search for leaks from gas pipelines – concentrations of ~ 1 % methane may readily be detected 2 km away.

Recent work in this field, however has almost exclusively exploited the DIAL technique. As new and better tuneable laser transmitters are developed – especially in

360

the infrared – new applications for DIAL are opened up. The two wavelength regions most commonly exploited are the ultraviolet between 230 and 300 nm, and the infrared between 3 and 5 μm. In the former, gases such as nitric acid (226 nm), sulfur dioxide (287 nm), toluene (267 nm) and benzene (253 nm) have sharp absorption lines making them suitable for DIAL detection. This spectral region has also the advantage that is solar-blind and permits the operation for daytime.

Another pollutant that can be detected readily by UV LIDAR is mercury as vapor. Mercury is released from its ore, cinnabar, by roasting it, and in regions where this extraction is practiced (particularly the huge cinnabar deposits at Almadén in Spain). Since mercury has a very strong electronic transition at 253.6 nm, it is readily detected (in high enough concentration) by DIAL LIDAR.

The development of high-quality infrared non-linear optical materials has led to a new class of bright, stable infrared laser beams. These infrared sources are tuneable and have very narrow line widths, opening up the possibility of DIAL measurements in the infrared. Many molecules of atmospheric and environmental interest have vibrational – rotational bands in the near infrared (2 - 5 μm), and provided that interference with water vapor and CO2 bands can be avoided, can be measured with infrared DIALs. Examples are methane (CH4), acetylene (C2H2), ethylene (C2H4) and ethane (C2H6). The most important application for such LIDARs is to measure emissions from petrochemical plants and storage facilities, although they are also useful for tracking plumes for several kilometers downwind of industrial sources.

An example of a mobile LIDAR designed for pollution measurements is that operated by the UK National Physical Laboratory, London. This uses two lasers, one for UV measurements and one for IR measurements. In Table III.3.1 are presented the gases measurable with this instrument (together with their typical detection limits for a 100 m diameter plume centered at 200 m range). The system is calibrated by reference to standard cells with known concentrations of the measured gases. It has been used to measure volatile organic compound emissions from more than twenty different petrochemical facilities, including oil refineries, retail petrol stations and ethylene processing plants.

In conclusion, we can appreciate that LIDARs are valuable instruments for atmospheric research and environmental monitoring because they provide an active remote sensing technique that can probe atmospheric regions inaccessible to other instruments, and at high spatial and temporal resolutions. LIDARs operating from ground and space provide complementary information: where satellite-borne LIDARs provide global coverage but at lower horizontal resolution, ground-based instruments reveal the fine detail required for atmospheric process research.

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Table III.3.1. Typical parameters of the DIAL LIDAR system

Species Laser wavelength Measurement sensitivity (ppb)

Nitric oxide, NO 226 nm 5 Nitrogen dioxide, NO2 450 nm 10

Sulfur dioxide, SO2 300 nm 10 Ozone, O3 289 nm 5

Mercury vapor, Hg 254 nm 0.5 Benzene, C6H6 253 nm 10 Toluene, C7H9 267 nm 10 Methane, CH4 3.42 μm 50 Ethane, C2H6 3.36 μm 20

Ethylene, C2H4 3.35 μm 10 Acetylene (C2H2) 3.02 μm 40

Hydrogen chloride (HCl) 3.42 μm 20 Nitrous oxide, N2O 2.90 μm 100 Methanol, CH3OH 3.52 μm 200

REFERENCES 1. Encyclopedia of Atmospheric Sciences, Ed. J.R. Holton, J. Pyle, J.A. Currie,

Academic Press, 2002. 2. Optical and Laser Remote Sensing, D. K. Killinger, A. Mooradian, eds., Springer

Verlag, New York, 1982. 3. Sunesson, J. A., A. Apituley, D. P. J. Swart, Appl. Opt., 1994, 33, 7045-7058. 4. Kempfer, U., W. Carnuth, R. Lotz, T. Trickl, Rev. Sci.Instrum., 1994, 65, 3145-

3164. 5. Ferrara, R., B. E. Maserti, H. Edner et al., Atmos. Environ., 1992, 26A, 1253-1258. 6. Edner, H., P. Ragnarson, S. Svanberg et al., Science Total Environ., 1993, 133, 1-

15. 7. Milton, M. J. T., P. T. Woods, B. W. Jolliffe et al., Appl. Phys. B, 1992, 55, 41-45.

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III.3.2. DOAS FOR ENVIRONMENTAL CONTROL

Mihaela BADEA, Mihaela-Carmen CHEREGI, Andrei Florin DĂNEŢ

DOAS stands for Differential Optical Absorption Spectroscopy, a name first applied in Europe in the 1980’s, but an analytical technique that has been used in laboratories for at least 50 years. Its primary benefits are the ability to quantitatively and simultaneously measure many different analytes within a sample with low detection limits.

In environmental control DOAS is used as a method of measurement of gases. The idea of measuring gases with light may sound peculiar to laymen, but based on this idea in 1985 two Ph.D. students from the University of Lund, Sweden founded the company OPSIS (www.opsis.se) which became the leading supplier in the area. For this, many times the DOAS concept is identified with OPSIS. III.3.2.1. Principle of DOAS Operation

In classical DOAS system (Figure III.3.2), the light emitted by a light source (i.e. a Xenon short arc lamp) is passed by the transmitter along the open path; (distance from 200 to 1000 meters) to a (fused silica) retro-reflector and is reflected back to the receiver (telescope). Then light passes through the fiber optic guide to the monochromator and spectrum is detected by the diode array. The spectrum is analyzed to determine the average concentration of gas pollutants along the open path.

Figure III.3.2. The scheme of a DOAS instrument

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In an OPSIS system, the light from an emitter is projected through ambient air to a receiver, which may be up to 2 km away; the light is then transferred to the OPSIS analyzed via a fiber optic cable where it is analyzed and the concentration of a wide range of compounds determined.

The Beer-Lambert law (See Chapter II.4.3) can not directly be applied to atmospheric measurements because of several reasons:

a) Besides the absorption of the trace gases light extinction occurs also due to scattering on molecules and aerosols. Especially for aerosols this extinction can not be corrected for with the desired accuracy.

b) In the atmosphere the absorptions of several species always add up to the total absorption. Thus in most of the cases it is not possible to measure one specific species.

c) In the case of satellite measurements the detected intensity can strongly depend on the ground.

These restrictions can be avoided by applying the method of differential optical absorption spectroscopy (DOAS). The DOAS technique relies on the measurement of absorption spectra instead of the intensity of monochromatic light. Thus it is possible to separate the absorption structures of several atmospheric species from each other as well as from the extinction due to scattering on molecules and aerosols.

This technology is used in instruments that can measure a number of different pollutants along a single light beam that may be up to 2000 meters long.

If two analytes both absorb at the same wavelength, for example, the resulting absorption will be the sum of the two individual absorptions. Therefore it is possible to mathematically treat the signal produced, to eliminate interferences, and to produce the spectrum for each analyte being sought. The ability to differentiate between adjacent absorption features can be improved with increased system resolution.

DOAS procedure can only be applied to species the spectrum of which contains reasonably narrow absorption features, thus limiting the number of molecules detectable by this technique. A possible continuous absorption of the trace gas will be neglected by this procedure. Nevertheless, slow varying absorption attenuates the total available light intensity that has an influence on the detection limit.

III.3.2.2. Spectral Regions Usable for DOAS Measurements

The most DOAS instruments operate in ultraviolet (UV) and nearest visible part of spectrum from 200 to 460 nm. At shorter wavelengths the usable spectral range is limited by rapidly increasing Rayleigh scattering and O2 absorption. Although only a limited number of gases have absorption spectrum in this spectral range, UV spectroscopy has some important practical advantages in comparison with infrared measurements, in particular, the existence of powerful light sources with continuous spectrum and sensitive photo-receivers. Moreover, the interpretation of absorption

364

spectra is not so complicated and requirements to spectral resolution of spectrometers is not so high as in infrared region, since only a few of the main atmospheric gas constituents have structured absorption in the 200-460 nm spectral range.

At producing measurements a spectral range of about 60 nm is chosen from the overall region 200-460 nm and the spectrum is registered in this range. Generally, at any spectral range a number of gases simultaneously have absorption bands. The only exception is the NO2 spectrum in 400-500 nm region. The least square method provides for simultaneous determination of concentration of all gases having absorption in the selected spectral range. Nevertheless, inevitable small errors in knowledge of gases cross-section gives rise to specific interference errors in determination of gas concentration. For minimization of this type and other measurement errors the selected spectral range have to satisfy to following conditions: 1. maximum possible light source spectral intensity 2. absence of sharp peaks or another fine structures in light source spectrum 3. maximum absorption cross section for gas component to be detected 4. minimum absorption cross section for other gases.

The spectral ranges in which the pollutant gases have absorption spectrum are presented in Table III.3.2 along with the detection limits (of level S= 3s).

Table III.3.2. The list of gases defined with the help of the DOAS instrumentation.

Gas Wavelength range (nm) Detection limit

(ppb) Ammonia, NH3 200 - 230 0.8 Nitric oxide, NO 200 - 230 1.8 Nitrogen dioxide, NO2 400 - 500 1.0 Nitrous oxide, N2O 325 - 390 0.9 Sulfur dioxide, SO2 280 - 320 0.2 Formaldehyde, CH2O 280 - 350 1.2 Benzene, C6H6 236 - 263 0.9 Toluene, C7H8 250 - 270 1.5 Phenol, C6H6O 250 - 280 0.1 Ethylbenzene, C8H10 238 - 270 2.4 Benzaldehyde, C7H6O 257 - 290 0.4 Xylene, C8H10 243 - 275 1.2 Cresol, C7H8O 253 - 285 0.5 Dimethylphenol, C8H10O 255 - 287 0.6 Trimethyphenol, C9H13O 260 - 290 1.8 Trimethylbenzene, C9H12 240 - 290 2.4 Methybenzaldehyde, C8H8O 266 - 306 1.8

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III.3.2.3. How does a DOAS Based Instrument work?

Standards Preparation - The first step is to record (or otherwise obtain) spectra for the specific analytes being sought, as well as the other analytes that might be present, at the same set of system operating parameters [i.e. resolution, etc.] over a range of concentrations above and below the levels anticipated or sought in the samples. These spectra are stored in memory.

Sample Analysis - Modern analyzers allow the capture of a spectrum generally in less than 0.1 sec. Multiple spectra may be collected and added to improve Signal/Noise, and/or individual spectra may be analyzed to record changes as a function of time. Specific pre-selected analytes may be quantified by analyzing their specific absorption features, but also other “unknowns” can be analyzed by searching through a library of absorption spectra. In addition, the spectra can be stored for subsequent analysis for even a broader list of potential sample components.

Spectral Analysis - Since the absorption spectrum is a fundamental physical property, it is possible to compute the concentration of the absorbing gas directly from the measured spectra, without “calibrating” the analyzer each time with known concentrations of reference gases. This significantly reduces the time and cost of the analysis.

III.3.2.4. DOAS Application in Pollution Monitoring

DOAS is successfully applied in continuous emissions monitoring (CEM), ambient quality monitoring (AQM), dust and mercury monitoring. The monitoring systems developed by OPSIS, AIM and other companies are approved by international institutes and authorities and they are found in a range of applications worldwide.

The OPSIS system has been designated an Equivalent Method by the U.S. EPA for the monitoring of O3, NO2 and SO2 in ambient air. In addition to these gases a wide variety of inorganic and organic gases can also be measured.

The DOAS based instrumentation is characterized by: - Total monitoring solution - Cost-effective, open-path technology - Multi-gas and multi-path system - High-performance monitoring of criteria pollutants - No sample required - Real time measurements - Easily calibrated - Operates with a minimum of maintenance - Low detection limits - Fully meeting the EU requirements - Withstands aggressive environment with high levels of particulates and gases - Remote service functions and servicing by highly skilled service network.

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REFERENCES

1. Donald L. Fox, Air Pollution, Anal. Chem., 1991, 63, 291R-301R. 2. Pasquale Avino, Domenico Brocco, Luca Lepore, Mario V. Russo, Ida Ventrone,

Annali di Chimica, 2004, 94, 704 – 714. 3. Axelsson,H., A.Eilard, A.Emanuelsson, B.Galle, H.Edner, P.Ragnarson, H. Kloo,

Appl.Spectr., 1995, 49, 1254. 4. Edner,H., P. Ragnarson, S.Spännare, S.Svanberg, Appl.Opt., 1993, 32, 327,. 5. Evangelisti, F., A.Baroncelli, P.Bonasoni, G.Giovanelli, and F.Ravegnani,

Appl.Opt., 1995, 34, 2737. 6. www.opsis.se 7. www.epa.gov/ttn/emc/tmethods.html 8. http://www.epa.gov/compliance/civil/programs/caa/caaenfpriority.html#Fence

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III.3.3. AUTOMATION IN IMMUNOASSAY

Jenny EMNEUS The concept of immunoassay was first described in 1945 when Landsteiner suggested that antibodies could bind selectively to small molecules (haptens) when they were conjugated to a larger carrier molecule [1]. This hapten-specific concept was explored by Yalow and Berson in the late 1950s, and resulted in an immunoassay that was applied to insulin monitoring in humans [2, 3]. The first application of based immunological technology in the environmental field was reported in 1970, when Centeno and Johnson developed antibodies that selectively bound malathion [4]. A few years later, radioimmunoassays were developed for aldrin and dieldrin [5] and for parathion [6]. In 1972, Engvall and Perlman introduced the use of enzymes as labels for immunoassay and launched the term enzyme-linked immunosorbent assay (ELISA) [7]. In 1980, Hammock and Mumma [8] described the ELISA potential for agrochemical and environmental pollutants. Since then, the use of immunoassay for pesticide analysis has increased dramatically. Immunoassay technology has become a primary analytical method for the detection of products containing genetically modified organisms (GMOs) [9]. In the 1990s, the immunoassay laboratories were faced with many challenges, which included reengineering of instrumentation, space limitation in laboratory, limited available resources for analysis, cost compression and also increased regulation of testing laboratory. Despite these challenges, the users expected the immunoassay laboratory to provide better services. In these conditions, the immunoassay laboratory needed to be come more efficient by incorporating creative solutions and adapting to changes. One solution was automation and system reintegration. Since most immunoassay procedures are labor-intensive, automation reduces the dependency of the labor equipment. Furthermore, the analysis can be performed outside of the laboratory near to "sample source", when the automatic system is portable [10]. During the last 20 years, major advances have been achieved in the automating routine, for environmental chemistry procedures. Discrete and random access analyzers provided a wide spectrum of immunochemistry tests around the clock to meet the demands of rapid testing. The first attempt was to automate radioimmunoassay (RIA) and regarding that several automatic systems were introduced in the late 1970s. Those included the Centria (Union Carbide), Concept 4 (Micromedic), ARIA II (Becton-Dickinon), and Gammaflow (Suibb), with limited throughput and testing menu, were not as reliable and cost-effective as the users wanted [10]. Automation in immunoassay became successful when non-isotopic systems were introduced.

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Generally, an automated immunoassay system has the instrument (as only one part or multiple parts), the reagents and the computer. These three components are interdependent, since the format of the reagent will determine the instrument design, while the limits of the instrument design may require modification of the reagents and immunoassay procedure. The computer program could optimize the reaction conditions, the sequence of the reagent addition and the order of the sample testing. It will expedite data processing and management as well as result reporting. A system will not be successful unless all three components are functioning well as a unit, which can be named integrated system [10]. The traditional idea of automation in immunoassay is to adapt reagent to an automated instrument for immunoanalysis. Such instrumentation mechanized all the necessary steps in immunoassay procedure (e. g. pipetting, incubation, washing and detecting the signal). The automated system performs large variety of tests, mixes at the same time with fairly high throughput. The instrument can be a general chemistry analyzer when the procedure is a homogeneous immunoassay (i. e. an assay requiring no physical separation of bound and unbound antigens) or a dedicated analyzer for heterogeneous immunoassay (i. e. an assay requiring physical separation between bound and unbound antigens). The automated homogeneous immunoassay systems use small sample size and low reagent volume, and provide fast turnaround time. The calibration curve is stable from several days to weeks, which allows performing analysis at all hours without having a recalibration of the system. The efficiency is enhanced by saving technical time, quality control and reagent expenses. In this area, an example is the TDx analyzer (Abbott Laboratories, USA) based on the PFIA (Polarization Fluorescence Immunoassay) principle. The discrimination between bound and free analyte is read indirectly involving a fluorescent tracer, which emitted differently the polarized light in free and bound form. Therefore, TDx analyzer is useful for environmental analysis (e. g. organophosphoric pesticides [11], DDT insecticide [12], propanil herbicide [13]). The heterogeneous immunoassay is more versatile than homogeneous immunoassay in terms of automation, since there is not limitation of analyte size (small and large molecule can be analyzed). The analyte fractions (bound and unbound) are separated by a simple washing step, which can eliminate also the interfering substances present in the sample before quantification, and in this way the method sensitivity wins several orders compared with the sensitivity of homogeneous immunoassays. But, heterogeneous immunoassays are more labor-intensive and time-consuming, which indeed requires a dedicated immunoassay analyzer, semi-automated or fully automated. The semi-automated immunoassay system is an automated instrument built on multiple blocks. These building blocks may be either linked by computer program or mechanically attached. In most semi-automated systems, these blocks function separately (e. g. the pipetting / injecting of reagents, the incubation of the reaction

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mixture, the bound/free separation by washing the solid phase). ELISA is one of the batch immunoassay methods, which fit well with the semi-automated systems, but the instrument is used frequently in clinical analysis (e. g. Amerlite, Kodak [14], Photon QA, Hybritech [15], Commander, Abbott [16]). For environmental analysis, the semiautomatic systems are set up usually in flow injection systems, based on heterogeneous competitive immunoassay. J. Yakovleva et al [17] reported in 2002 a semi-automated immunoassay method for atrazine analysis, which involves offline preparation of sample (mixture of analyte and enzyme tracer) followed by automated analysis. The sample is passed over the silicon chip surface with anti-atrazine antibody immobilized and then an enzyme substrate is injected to quantify the tracer concentration bound on the immuno active surface, which gives an indication on the analyte concentration from the sample. Atrazine was also analyzed using the semi-automated system consisting from a protein G modified disc composite from monolithic metacrylate and polyethylene [18]. Alkylphenol ethoxylate surfactants [19], alkylbenzene sulfonates [20], 4-nitrophenol [21] and 2,4,6-trichlorophenol [22] are other examples of automated immunoassay systems involved in environmental analysis. Fully automated system for heterogeneous immunoassay link all the separate components of the semi-automated systems and allow the testing to be completed from sample addition to result reporting. Depending upon the ability of the system to conduct the analysis, the fully automated system can be further subdivided into batch and "in flow". An example of a batch-automated system is the portable immunochemical sensor for field screening used in TNT, atrazine and diuron determination based on ELISA principle [23]. The instrument consists in a µ-fluidic part and ground plate with automated control and one-way chip, which hosts all immunoreagents (antibody, enzyme tracer). The user has only to add the sample and the instrument will show directly the analyte concentration detected in the sample content. Another example of fully automated immunoassay system is BIACORE (Biacore, Uppsala, Sweden), which can be named "in flow" system. The instrument function is based on the SPR (Solid Plasmon Resonance) principle. It is equipped with a continuous flow system in which four channels are coupled in series. It has an automatic sample needle to deliver buffer and sample to the sensor chip surface. The continuous flow ensures that no changes in analyte concentration occur during the measurement. It can be used for kinetic measurement as well as for environmental analysis. The "River Analyzer" (RIANA) is another example from this subclass of fully automated systems, which was applied for environmental pollutant and their metabolites analysis (e.g. atrazine, 2,4-dichlorophenoxyacetic acid, isoproturon, pentachlorophenol, alachlor) [24-26]. RIANA is based on specific immunoassays for analyte recognition and Total Internal Reflection Fluorescence (TIRF) as transducer principle. The device consists of an optical detection unit, a flow cell and an integrated fluid handling based on flow injection (FIA). The sample and anti-analyte antibody labeled with fluorescence marker are mixed together and after the incubation time, the

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mixture is injected into the system and the unbound antibody is retained on the transducer surface covered with immobilized antigens. The bound antibody fraction on the transducer surface will indicate the analyte concentration from the sample. The impact of automation on immunoassay operation was especially on the mechanization of the testing procedure and the consolidation of the workstations. The random access feature of the automation will facilitate the workflow and improve the turnaround time. Automation will change the function of a technologist from technician to data manager and quality control officer. Through workstation consolidation, it will reduce the labor equipment, as well as the skill level and number of workers. The ability to perform simple as well as multiple analysis with better sensitivity, accuracy and precision are other important advantages of the automation process in immunoassay, which can prove the benefits of an automated immunoassay system. REFERENCES [1] L. Landsteiner, The specificity of Serological Reactions, (1945) . [2] R.S. Yalow and S.A. Berson, Nature (London), 184 (1959) . [3] R.S. Yalow and S.A. Berson, J. Clin. Invest., 39 (1960) . [4] E.R. Centeno and W.J. Johnson, Int. Arch. Allergy Appl. Immunol., 37 (1970) 1. [5] J.J. Langone and H.v. Vunakis, Res. Common. Chem. Pathol. Pharmacol., 10 (1975) 163. [6] C.D. Ercegovich, R.P. Vallejo, R.R. Gettig, L. Woods, E.R. Bogus and R.O. Mumma, J. Agric. Food Chem., 29 (1981) 559. [7] E. Engvall and P. Perlmann, J. Immunol., 109 (1972) 129. [8] B.D. Hammock and R.O. Mumma, Pesticide Analytical Methodology, (1980) 321-352. [9] G. Shan, C. Lipton, S.J. Gee and B.D. Hammock, Immunoassay, biosensors and other chromatographic methods, Handbook of Residue Analytical Methods for Agrochemicals, (2002) 623-679. [10] D.W. Chan, Automation of Immunoassay, Immunoassay, (1996) 483-505. [11] A.Y. Kolosova, J.-H. Park, S.A. Eremin, S.-J. Kang and D.-H. Chung, Journal of Agricultural and Food Chemistry, 51 (2003) 1107-1114. [12] S.A. Eremin, A.E. Bochkareva, V.A. Popova, A. Abad, J.J. Manclus, J.V. Mercader and A. Montoya, Analytical Letters, 35 (2002) 1835-1850. [13] A.I. Krasnova, S.A. Eremin, M. Natangelo, S. Tavazzi and E. Benfenati, Analytical Letters, 34 (2001) 2285-2301. [14] J.D. Faix, Amerlite immunoassay system, Immunoassay automation: A practical guide, (1992) 117-127. [15] R.F. Frye, The photon-ERA immunoassay analyzer, Immunoassay automation: A practical guide, (1992) 269-292.

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[16] P.P. Chou, IMx system, Immunoassay automation: A practical guide, (1992) 203-219. [17] J. Yakovleva, R. Davidsson, A. Lobanova, M. Bengtsson, S. Eremin, T. Laurell and J. Emnéus, Anal. Chem., 74 (2002) 2994-3004. [18] S.R. Jain, E. Borowska, R. Davidsson, M. Tudorache, E. Pontén and J. Emnéus, Biosensors & Bioelectronics, 19 (2004) 795-803. [19] M. Badea, C. Nistor, G. Yasuhiro, F. Shigeru, D. Shin, D. Andrei, D. Barceló, V. Francesc and J. Emnéus, Analyst (Cambridge, United Kingdom), 128 (2003) 849-856. [20] M. Franek, J. Zeravík, S.A. Eremin, J. Yakovleva, M. Badea, A. Danet, C. Nistor, N. Ocio and J. Emnéus, Fresenius J. Anal. Chem., 371 (2001) 456-466. [21] C. Nistor, A. Oubina, D. Barceló, M.-P. Marco and J. Emnéus, Anal. Chim. Acta, 426 (2001) 185-195. [22] C. Nistor and J. Emnéus, Analytical and Bioanalytical Chemistry, 375 (2003) 125-132. [23] P.M. Krämer, I.M. Ciumasu, C.M. Weber, G. Kolb, D. Tiemann, I. Frese, H. Löwe and A.A. Kettrup, A new, automated, portable immunochemical sensor system for field screening, IAEAC: The 6th Workshop on Biosensors and BioAnalytical µ-Techniques in Environmental and Clinical Analysis, ENEA - University of Rome "La Sapienza", October 8-12, 2004 - Rome, Italy (2004) . [24] E. Mallat, C. Barzen, R. Abuknesha, G. Gauglitz and D. Barceló, Anal. Chim. Acta, 426 (2001) 209-216. [25] E. Mallat, D. Barceló, C.G. Barzen, G. and R. Abuknesha, TrAC, 20 (2001) 124-132. [26] E. Mallat, C. Barzen, A. Klotz, A. Brecht, G. Gauglitz and D. Barceló, Environ. Sci. Technol., 33 (1998) 965.

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III.4. AUTOMATIC SPECTROPHOTOMETRY José Martínez CALATAYUD

The most common method of detection in automated analysis is colorimetry and its close relative UV-spectrophotometry; this is a natural fact because the UV-VIS absorption measurements are by far, the most usual detector in analytical instrumentation. The well-known parts of a colorimeter or an spectrophotometer are: (a) the light source, which can be as frequent as a tungsten-filament light bulb; (b) the required optics for focusing the light into a colored filter (colorimeter) or a monochromator to disperse the electromagnetic radiation according to the different wavelengths into a “rainbow” of “colors” (UV light is included in the definition) by using a prism or a diffraction grating. By rotating the prism or grating, the wavelength of light can be selected to match the wavelength with that absorbed by the sample; (d) a sample compartment to hold a transparent (glass or silica) cell, (e) a light-sensitive detector, the photomultiplier tube (PMT) to convert the light intensity into an electric current, and (f) electronics for measuring and displaying the output from the PMT. The analytical method involves the reaction of the analyte to match with two main objectives: (a) to obtain a new chemicals presenting higher molecular absorptivity to increase the sensitivity; and, (b) to avoid interferences.

The automatic methods of analysis can be classified in three main groups: batch (discrete), flow and robotics, with an “explosive” growth during the last decades of the methods based on some kind of flow: flow injection analysis (FIA); stopped-flow; segmented flow analysis; sequential injection analysis (SIA); and the emerging new methodology known as multi-commutation. Most spectrophotometric procedures can be found within any of the related methods. Most of them are the result of “translating” a classic batch procedure into an automated method. Due to that, the chemical processes are always very similar as they start from the same sample and analyte to process and have the same goal, the spectrophotometric measurement of the same final chemical. Differences among them are mostly due to the adaptation of the chemical process to the automation type. Due to the large number of the existing automatic analytical methodologies and the also a large number of environmental interest analyses; this chapter tries to give a panoramic vision by describing the determination of different analytes in water samples with the aid of different type of automatic procedures.

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III.4.1. NUTRIENTS: ENVIRONMENTAL SIGNIFICANCE AND MEASUREMENT

Nutrients are mainly compounds of nitrogen or phosphorus, although other elements (iron, magnesium, and potassium, among others) are also necessary for bacterial and plant growth. The nutrient concentrations are very relevant for life in natural waters because, in excess, they cause nuisance growth of algae or aquatic weeds. A higher concentration of one of them involves the abnormal growth of the population of definite algae. In some industrial wastewaters treatment plants, ammonia or phosphoric acid must be added as a supplement bearing in mind that a deficiency of nutrients limits the effectiveness of biological treatment processes.

Table III.4.1. Examples of automated spectrometric methods for pollutants determination

Analyte Automated methodology Chemical method

Nitrite/nitrate ammonia

Lab-on-a valve Griess modified Salycilate

Phosphate Colorimetric analyzer Molybdate Calcium Flow Injection Analysis, FIA Ca(II) / o-cresolphthalein

at pH 10.0 Cyanide Segmented flow pyridine + barbituric acid Pb (II) Sequential Injection Analysis, SIA Sulfide-resazaurine,

Pb (II) as catalyst Chlorine Multi-commutation Oxidation of dianisidine Cr (III) and Cr (VI)

FIA and r-FIA Cr(VI)/1,5-diphenylcarbazide

Nitrogen occurs primarily in the oxidized forms like nitrate or nitrite or the

reduced form of ammonia or "organic nitrogen". The latter means, the nitrogen is part of an organic compound such as an amino acid, a nucleic acid, a protein, etc. and can be used as nutrient after the organic nitrogen decomposes to a simpler form.

The classical batch spectrophotometric methods for nitrogen compounds, which have been translated into automatic methods, are the following:

Ammonia is determined by the Nessler, phenate or salycilate methods, after distillation from an alkaline solution to separate it from interferences. In recent automatic methods, the distillation is substituted by volatilization methods and separation from the matrix by a membrane separator.

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Organically-bound nitrogen can be determined by the same ways after a digestion (the Kjeldahl procedure) which converts the nitrogen into ammonia.

Nitrite is determined colorimetrically by the Griess method, which suffered many changes from different authors who proposed more suitable reagents.

The most popular method for nitrate is reducing nitrate to nitrite chemically using copperized cadmium (other reducing metals or amalgams have been also proposed) then analyzing the nitrite. Nitrate can be also converted into nitrite by photoirradiation (UV region) with a low pressure Hg lamp; or, by the homogeneous way with dissolved reducing chemicals.

Trends in Analytical Chemistry are in the way to put together automation with miniaturization. A “natural son” from miniaturization of FIA and SIA is the so called Lab-on-a-valve; an automatic and miniaturized compact set-up designed by J. Ruzicka and based on the SIA principles with the aid of a syringe pump instead of the usual peristaltic pump.

The general characteristics of this novel emergent technology are previously reported in the section Application of flow techniques in environmental monitoring and control. The miniaturization reaches to the detector; a diode spectrophotometer (LED) provided with optical fibers from light source to the sample (rest of the set) and from sample to computer. The instrumental set-up (detector excepted) is always the same for any procedure or detection way; versatility is obtained through the software.

Two spectrophotometric methods for water samples have been selected to illustrate the use of this new methodology: namely, nitrite/nitrate and ammonia. III.4.1.1. Nitrite / Nitrate The method is based on the reaction of nitrites with sulfanilamide to form and azo dye that will then couple with N-(1-naphthyl) ethylenediamine dihydrochloride to form magenta colored solution which can be quantified in the spectrophotometer at 540 nm. The linear working range and sensitivity of this procedure are variable, depending on minor changes like sample size and flow rates: namely, greater sensitivity is easily obtained by using larger sample volumes (over the range 80-200 μL) and minor flow rate; or a method to be applied to higher concentrations by using a smaller sample (25-80 μL) and fast flow rate. The determination of nitrates is based on the same chemical reaction with prior reduction of nitrate to nitrite. In this method is used a solid-phase reactor integrated into the flow assembly; the reactor is filled with copperized cadmium. The use of the reducing column will result in a transient signal proportional to the total of nitrate and nitrite concentrations present in the sample (Figure III.4.1).

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NED

SO2NHR

NH2

+ NO2- + 2 H+

SO2NHR

N=N+

+ 2 H2

2

Sulfanilamide Diazonium ion

Azo Dye

SO2NHR

N=N+

+

NHCH2CH2NH2

RHN-O2S N=N NHCH2CH2NH2 + H+

2

Figure III.4.1. Set-up for the spectrophotometric automated determination of nitrite and nitrate. Reproduced from J. Ruzicka, Flow Injection 2nd Edition (CD) FIAlab Instruments, with permission.

In other flow-methods have been substituted the nitrate reduction to nitrite by a photo-reactor which means clear advantages for the environmental safety; namely: (a) to avoid large amounts of toxic metals to the public sewage. (b) Suspended matter or turbid samples can clog the Cu-Cd reactor. Previous

filtering is advisable. (c) The Cu-Cd column will be degraded by samples containing soluble mercury or

thiosulfate. Samples containing soluble oils can inactivate the catalytic surface. A sample suspected of containing oil or grease requires a pre-treatment; oil or

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grease should be extracted by a liquid-liquid process with an organic solvent layer. Cadmium should be re-copperized.

(d) The lamp performs with a remarkable stability leading to improve the reproducibility.

Homogeneous reduction with solved chemicals is also widely accepted. The analytical figures of merit of the procedure are as follows: Linear dynamic range: 0.04 - 10.0 mg (N) L-1, nitrate; 0.015 - 2.0 mg (N) L-1, nitrite. Detection limits: nitrate, 1.4 μg L-1 and nitrite, 0.7 μg L-1. Deviation: 2% and Sample throughput: Maximum of 98 h-1 (36.6 s/assay). Sample volume, 200 μL. This chemistry is relatively free from interferences supposing that the sample does not present strong absorbance at 540 nm (Figure III.4.2). Na2-EDTA has been included to the buffer (ammonium chloride) to minimize potential interference from metallic ions; large amounts of iron, nickel, and copper reduce the transient outputs. The problem due to the atmospheric oxidation of nitrites can be prevented for sample stored more than 24 hours; samples should be protected form daylight and added HgCl2 as preserver.

Figure III.4.2. Absorption spectrum of the nitrite assay. Reproduced from J. Ruzicka, flow Injection 2nd Edition (CD) FIAlab Instruments, with permission.

Nitrite / Nitrate Assay

0.000

0.200

0.400

0.600

0.800

1.000

300 350 400 450 500 550 600 650 700 750 800 850

Wavelength (nm)

Abso

rban

ce

Blank2ppm NO2-

λmax = 542 nm2 ppm NO2

-

The photo-reduction of nitrate into nitrite with a low-pressure mercury lamp

has occasionally been used for different analytical purposes; like for “in-situ” preparation of nitrite as reagent for spectrophotometric determination of pharmaceuticals. In this context and recently, this kind of conversion has been exploited for the simultaneous determination of nitrate and nitrite in water samples.

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Nitrate can be photo-reduced to nitrite in the 200 - 300 nm regions where nitrate exhibits two UV bands; a low-pressure 8 W lamp as UV source coiled with 697 cm of PTFE coil 0.8 mm internal diameters served for the goal. The pH affects the photochemical process; it is especially favorable the alkaline region. The temperature had shown a negative influence the room temperature being the finally selected one. Is important the irradiation time to fix the optimum found time period, 3 min 11 sec, is necessary to keep a flow-rate (for the selected reactor length) of 1.1 mL min-1. The presence of sensitizers, scavengers etc was tested only best results were obtained with the presence of 0.001 mol L-1 Na2-EDTA as an activator for the reaction.

III.4.1.2.Ammonia

Ammonia is a principal excretion product of fishes, resulting from the metabolism of nitrogenous (nitrogen containing) compounds in their food. Ammonia is also formed from the bacterial degradation of nitrogen containing organic materials. Ammonia can be determined using the salicylate method, a variation of the former phenate method but does not require the use and disposal of toxic mercury salts and phenol. The method is based on the following reaction sequence: (a) The first reaction is the addition of chlorine to convert ammonia to

monochloroamine. (b) The second step follows with the reaction of monochloroamine with salicylate to

form 5-aminosalicylate. (c) Finally the 5-aminosalicylate is oxidized by sodium nitroprussiate to form a blue-

green colored compound that can be monitored over the wavelength range from 650 nm to 710 nm. In the following scheme are presented the reactions involved in the determination

of ammonia by phenate method.

N + OCl - +NCl-OH

NCl + O- O N Cl

O N Cl + O- O N -O

ammonia hypochloride chloramine

chloramine phenol

N + OCl - +NCl-OH

NCl + O- O N Cl

O N Cl + O- O N -O

ammonia hypochloride chloramine

chloramine phenol

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The modification of the phenate method means the reagent phenol is substitute by the salicylate: COOH

OH

Salicylic acidSalicylic acid Avoid working with extreme acidity or alkalinity; the sample should be close

to neutral pH value Several components interfere with the determination of ammonia. First is a usual interferent, the turbidity of the sample. Sulfide, hydrazine and glycine will intensify the final blue-green color; they should be previously eliminated. Other interfering are the metallic ions iron, calcium and magnesium; and the inorganic anions sulfate, phosphate, nitrate and nitrite. The usual method of preparing doped standard solutions for the calibration or using the standard addition procedure can minimize or eliminate the influence of the interferents.

Analytical figures of merit are: linear dynamic range 10 to 1000 mg NH3 L-1 [0.59 m mol L-1 – 58.8 m mol L-1 NH3; deviation: 10 to 1000 mg range: 3%. Sample throughput: 112 samples per hour. The following figures depicted the schematic flow assembly in which ammonia analysis is performed in the Z flow cell above using a stop flow procedure (Figure III.4.3); and, the visible spectrum of the resulting complex (Figure III.4.4.).

Figure III.4.3. Flow Injection Hardware Setup for ammonia determination for

load mode. ) FIAlab

Instruments, with permission.

V alveW aste

Line 1L ine 2

T ee

W aste

Z Flow C ell

M ixing Coil 1

Perista lt ic Pum p

M ixing Coil 2

Sam ple

4

1

23

65

FIA2000 Series

a

b

c

d

ef g h

Spectrophotometer

Light Source

Waste

use in a unidirectional and/or stopped flow procedure. Note: the schematic above shows the sample loop in the Reproduced from J. Ruzicka, Flow Injection 2nd Edition (CD

379

5-Aminosalicylate Complex

0.000

0.300

0.600

0.900

1.200

1.500

400 450 500 550 600 650 700 750 800Wavelength (nm)

Abso

rban

ce

NH3: 50 ppmNH3: 0 ppm

675nm max

Figure III.4.4. Absorption spectra of the 5-aminosalicylate complex (heavy line) generated by using 50 mg L-1 NH3 and analyzed after a 5-minute reaction time. The lighter line is the reference. Absorption spectrum of the nitrite assay. Reproduced from J. Ruzicka, flow Injection 2nd Edition (CD) FIAlab Instruments, with permission.

III.4.1.3. Phosphate

important; it is an essential nutrient for the hytoplankton growth. Excessive inputs can lead to eutrophication of coastal marine

arameter known as “total

being the chemical distribution of phosphorus between

analyte (phosphate) ith mo

Phosphorus is biologicallypwaters, which is accompanied of abnormal growth of algae. There are also condensed forms of phosphate as pyrophosphate and polyphosphates and organic phosphates. The environmental pphosphorous” TP, means the sum of all these forms. Other operational parameters are total reactive phosphorus (TRP), filterable reactive phosphorus (FRP), and total filterable phosphorus (TFP). The natural phosphorus cycle does not depend on a significant atmospheric component (unlike nitrogen); water and particulate components via adsorption and precipitation processes. Other bulk phosphorous sources are marine sediments, soils and rocks. Most methods of phosphorus determination are based on the formation of phosphomolybdate heteropolyacid through the reaction of the w lybdate in acidic medium (Figure III.4.5.). In a second step the heteropolyacid

380

is then reduced to an intensely colored blue compound with a maximum absorbance band at 840 nm (McKelvie, Peat, and Worsfold, 1995).

PO4

3- + 12 MoO42- + 27 H+ → H3PO4 (MoO3)12 + 12 H2O

H3PO4 (MoO3)12 → Phosphomolybdenum blue Mo(V)

As reducing reagents for the second step have been proposed ascorbic acid or

luble

e following automatic apparatus is a commercially designed colorimetric alyze

(small design changes) are copper, chromate, high range

tin(II) chloride and being important potential interferences silicate and arsenate. The phosphorus determined is defined as “molybdate reactive” or soreactive phosphorus (SRP). When suspected the presence of other phosphorus containing organic compounds and condensed phosphates the process initiates with chemical, photochemical, thermal or microwave digestion prior to the molybdate reaction. Than r for different parameters in water samples being phosphorus one of them (Model 31 500). The sample is introduced into the analyzer by a piston pump and mixed with measured amount of reagent that is introduced by another piston pump. The mixture develops the corresponding color, which will be monitored when introduced into the cell. Other parameterssilica, permanganate, free chlorine, hardness, phenolphthalein alkalinity, ozone, hydrazine, chlorine dioxide, etc.

Figure III.4.5. Schematic view of a commercially available colorimetric analyzer for phosphate determination. L-s, lamp source; W, waste; pmt, photomultiplier tube; r, reactor; c-c, colorimetric cell; r-p, reagent pump; s-p, sample pump.

381

III.4.1.4. Metals

There are numerous colorimetric methods for metals. Most of these methods e very

etric examples on metal determination are presented below.

Determination of Calcium in Drinking Water Samples um with o-

igure III.4.6. Flow assembly for determination of calcium in drinking water

ar useful to analyze samples like drinking waters; however, for other type of samples, like wastewater, the presence in high contents of interfering substances brings to select other analytical measurements. The most popular method in use today involves one form or another of atomic spectroscopy especially flame atomic spectrophotometry. The X-ray emission spectroscopy is useful primarily for solid samples. Electrochemical methods, like polarography and anodic stripping voltammetry, which are quite sensitive, are mostly confined to research purposes rather than routine analyses.

Some spectrophotom

The method is based on the formation of the complex calcicresolphthalein [Ca-C32H32N2O12] at pH 10.0. The borax buffer and the reagent (through 2 and 3, respectively at 0.8 mL min-1) merge in a 50 cm length reactor R1 (Figure II.4.6). 20 μL of the sample are inserted through the insertion valve into the resulting mixture and, after flowing through R2 (50 cm length), are leaded to the flow cell were absorption is recorded at 575 nm.

Fsamples. R, reactors; P, peristaltic pump; I v, injection valve; D, detector; and, W, waste. 2, buffer; 3, reagent; both streams 1 and 2, flowing at 0.8 mL min-1. All PTFE tubing is 0.5 mm internal diameter.

382

Determination of Chromium, Speciation of Cr (III) and Cr (VI) r (VI) reacts with 1,5-diphenylcarbazide (DCP) yielding a colored complex with ma eaction and

s dete

d.

iodate and the

substituted by a splitting point and

for

d b, way for DCP. P, peristaltic pump; Iv, injection valve; S,

applied continuous monitoring of water (rivers, irrigation channels, estuaries, tc.). The Figure III.4.8 depicts an FIA assembly for continuous monitoring of Cr (VI)

Cximum absorbance at 540 nm. Cr (III) do not interfere with this r

it rmination is based on the same reaction after being oxidized to Cr(VI) by Ce (IV). A flow assembly provides the simultaneous or sequential determination of both chromium valences. Figure IV.4.7 depicts the flow-assembly for the sequential speciation. A selecting key allows the passage of the reagent or the oxidant. The first step consists in inserting the sample into a sulfuric acid stream and then merging with the reagent for the determination of Cr(VI). The second step is a new sample insertion to be oxidized by Ce(IV) before merging with the reagent and results in a transient signal proportional to the total chromium amount. Flow–rates (in mL min-1) were: 0.37, 0.30 and 1.22 for DPC, oxidant and sulfuric acid, respectively. A water-bath for the oxidation is require A flow assembly similar to the depicted has also been proposed for the speciation of As(III) and As(V). The oxidant was the potassiumcolorimetric reaction was with molybdenum blue. An alternative to the sequential determination is a simultaneous speciation in a flow manifold in which the selecting key has been a double flow-cell.

oxidant; an

Figure III.4.7. Flow assembly for speciation of Cr (III) and Cr (VI). 1, DCPsolution; 2, oxidant; 3, sample; 4, sulfuric acid as carrier. a, the way

selecting key; R, reactors; D, detector; W-b, water-bath at 42 ºC; and W, waste.

The reported chemical fundamentals for chromium speciation have been also for the

e

383

and periodic measurements of Cr(III); the assembly is of the reverse-FIA type in which aliquots of reagent solutions are inserted through the injection valve instead of the sample; the sample stream is the carrier. The basic manifold presented in the above mentioned figure illustrates the continuous passing of the sample (the carrier) which merges with the DCP and allowing the continuous monitoring of Cr(VI) by giving a continuous output; when the injection valve is actuated the insertion of Ce(IV) oxidizes the Cr (III) and resulting in transient signals proportional to the total chromium presence (Figure III.4.9).

Figure III.4.8. Continuous monitoring of Cr(VI) and periodic measurement of Cr(III) by a reverse FIA assembly. 1, oxidant Ce(IV); 2, water sample as carrier; and 3, DCP solution. Flow-rates

olume to be inserted 169 μL.

g of peaks)

ransient signals) equivalent to the total chromium concentration.

were 1.2 and 1.4 mL min-1 for channel 2 and 3, respectively. Reactor length: 600 cm R1 and 50 cm R2. Oxidant v

Figure III.4.9. Type of outputs from the r-FIA for continuous monitorinCr (VI) (continuous output, a) and periodic measurements of Cr (III) (b (t

384

manifoltwo dif chromium oxidation status. A selecting key allows or prevents the passage of the resulting reaction. Absorption measurements were done at 540 nm. In Figure III.4.11 are

onitoring

at 0.3

flowing at anch, 20 and 50 cm for R1 and R2,

spectively. Lower branch; 600 and 40 cm, for R1 and R2, respectively.

Figure III.4.11. us monitoring of

To obtain a sequentially monitoring of Cr(III) and Cr(VI) the alternative d is depicted in Figure III.4.10, in which the carrier (water sample) splits in ferent streams for a two separated manifold branches one for each

presented the outputs form the Cr measurements.

1.9 mL min

Figure III.4.10. A r-FIA assembly for “continuously sequential” mof Cr (III) and Cr (VI). Upper Pump: 1, DCP solution; 3, acidic mediummL min-1. Lower pump: 1; DCP solution; 3, oxidant Ce(IV) in acidic medium at 0.3 mL min-1. For both pumps, 2, water sample as carrier and

-1. Reactor lengths: Upper brre

Outputs from the sequential continuoCr(VI), outputs a; and, Cr (III): total chromium outputs b.

385

Determination of Lead in Water by a Sequential Injection Analysis (SIA)

ions on the redox reaction resazurine – sulfide in alkaline media. The catalytic reactions are very sensitive though lack selectivity. Several metals interfere with this redox reaction: namely, Cu(II), Co(II), Ni(II) and Fe(III) even at very low concentrations. The analytical former applications of this reaction implied the use of a certain number of tedious steps including toxic reagents to avoid interferences. To improve the selectivity and bearing in mind lead forms iodo-complexes in an acid medium unlike the other cited ions, the reported method uses a previous sample pre-treatment with potassium iodide and then the formed iodo-complexes are retained in a solid phase-reactor (placed in the external loop of an auxiliary injection valve) filled with the anionic resin exchanger AG1 X8 (Figure III.4.12). The retained lead iodo-complexes were eluted by 90 μL of 0.2 mol L-1 NaOH solution. The method was applied to drinking water samples and results were compared with the obtained with the electrothermal atomization Atomic Absorption Spectrometry.

Figure III.4.12. SIA assembly for spectrophotometric Pb (II) determination. P, peristaltic pump; h-c, holding coil; D, detector; s-v, 8-port selecting valve; W, waste; Iv, injection valve provided with the solid-phase reactor filled with the anionic resin; m-c, mixing chamber; and D, detector. The manifold is implemented with a 0.8 internal diameter PTFE tubing.

Flow Assembly The method is based on the catalytic effect of the Pb(II)

386

III.4.1.5

The operating cycle for btaining a typical analytical transient signal can be easily programmed by means of

friendl

a 0.5 mo ne and

e of

r this

d

s

ell as the pre-concentration of chlorine. The separation unit was made with two pieces oa channheld firm

. Chlorine

Tandem-flow multi-commutation assembly An automated method for determination of free chlorine in water samples

can be performed based on the oxidation of dianisidine as colorimetric reagent to release a colored product that can be spectrophotometrically monitored at 445 nm.

Dianisidine in acidic medium Oxidized dianisidine by chlorine The automation of the method is based on the “tandem flow” approach, which uses a set of solenoid valves acting as independent switches.

MeO

H3N

OMe

NH3+ + - 2 e-

+ 2 H+H2N NH2

OMeMeO

+ +

oy software running in the Windows environment.

The manifold comprises a set of three solenoid valves acting as anindependent switch. See Figure III.4.13. The sample (channel Q3) merges with

l L-1 HCl solution (Q4); the global chlorine is converted into chloritransported through the gas diffusion membrane. Valves 2 and 3 control the timstopped-flow and then the time of pre-concentration (volume of sample and diffusionof chlorine to a basic solution (Q2) acting as acceptor solution). The resulting basicsolution causes the quantitative decomposition of chlorine into hypochlorite, which facilitates the gradient solution and transport process through the membrane. Aftepre-concentration step the volume comprised between valves 2 and 3 is forced to theflow system and merges with the solution of dianisidine (Q1). A previous solenoivalve is used for recycling the non-inserted reagent solution.

The high sensitivity and selectivity of the method is due to the manifold isprovided with a gas-diffusion unit which permits the removal of interfering species aw

f methacrylate being screwed together, the groove carved in the pieces formed el that is split by the fluoropore membrane filter of 0.5 μm pore size, which is

ly between the two blocks (Figure III.4.14).

387

Figure III..4.13. Tandem-flow assembly for the determination of chlorine. Q1=

mL min-1, Q4dianisidine in 1 mol L acetic acid; Q : 0.005 mol L NaOH, Q : sample; Q : 0

I.4.1.

Measurements by a segmented-flow assembly Cyanide is highly toxic to mostly living organisms by preventing the normal

ctivity of metal-containing molecules due to its property of strongly complexing

Q2= 5.2 mL min-1, Q3= 0.6 = 5.0 mL min . Q-11: 10 mol L o-

-1 -1

-3 -1

2 3 4.5 mol L-1 HCl ; P, peristaltic pump; V1, V2, and V3, solenoid valves; D,

detector; and, W, waste.

Fig III.4.14. Lateral view of one block from the gas-diffusion unit .

The method for a concentration step of 30 seconds, resulted in a dynamic

linear range from 0.05 to 1.30 μg mL-1 of chlorine; the limit of detection is 0.05 μg mL-1; the reproducibility (as the rsd of 42 peaks of a 0.72 μg mL-1 chlorine solution) is

.5 % and the sample throughput is 38 h1 -1. II 6. Cyanide

a

DP V3

1

V2

WW

Q1

Q4

V

W

WQ3

OFF

OFF

Q2 OFF

ONONON

DP V3

1

V2

WW

Q1

Q4

V

W

WQ3

OFF

OFF

Q2 OFF

ONONON

388

metal cations. However, in low concentrations it is biodegradable by some bacteria. It

sampling air may lower the pH and facilitate the releasing of hydrogen cyanide gas. Other cyanidsamplthem abe ana ollection.

Particulate cyanides are known to decompose in moist air with the liberation f hydrogen cyanide. Filters are usually used to trap particulate cyanides, which can

be quantified separately after acid distillation. Inorganic cyanides in water sample can be present both as complexed and free cyanide and the determination of cyanide in water is usually classified in free cyanide, cyanide amenable to chlorination, and total cyanide. Cyanides are usually measured by a sensitive colorimetric/ spectrophotometric procedure that can detect levels down to about 5 parts per billion in water. Since much of the cyanide in an industrial plant effluent is likely to be bound to metal ions, the sample is acidified and irradiated with UV light to convert metallo-cyanides into simple cyanides. The hydrogen cyanide formed is easily separated from the rest of the matrix when the sample flows through the dialyser provided with a porous membrane;

e hydrogen cyanide ion is retained by the diluted alkaline flow stream. -chloro-p-

oluene converted to cy

ed” between two consecutive air bubbles ntrary in FIA with the gradient concentration) and the

ot a peak; it is theoretically speaking a rectangle, in practice a urved rectangle.

can be found in industrial wastewater effluents, it is used in mining and different industries. Hydrogen cyanide and cyanide salts are important environmental problems and there are numerous methods for determination of cyanide in air, water, workplace, etc. Hydrogen cyanide in environmental or workplace air is usually collected byflushing the sample (filtered to distinguish from the particulate cyanide) in sodium hydroxide solution, and then measured by the spectrophotometric procedure. It should be pointed out the problems derived from the instability of the samples (hydrogen cyanide is highly volatile). However, the presence of carbon dioxide from the

problems appear from the oxidizing agents in solution, which may transform e during storage and handling. It is recommended for the storage of cyanide

es to collect the samples at pH 12-12.5 in tightly closed dark bottles and store s soon as possible in a cool, dark place. It is also recommended that the samples lyzed immediately upon c

o

th The resulting alkaline stream merges with the chloramine T (N

sulfonamide sodium salt) stream at pH less than 8 and the cyanide is tin anogen chloride (CNCl). Finally the resulting cyanogen chloride gives a reddish-violet color compound by reaction with pyridine and barbituric acid. The Figure III.4.15 depicts the segmented-flow method for the determination of the cyanide. In this methodology the liquid streams are segmented by air bubbles (periodically inserted). The solutions “closare homogeneous (as cotransient signal is nc

389

Figure III.4.15. Cyanide determination by stopped-flow W, waste; m, porous membrane; D, detector; C, computer; P, peristaltic pump; L, UV lamp; and, R, reactors. Solutions: 1 and 4, air; 2, sample or reference; 3, acidic medium; 5, alkaline medium; 6, chloramines T; 7, pyridine and barbituric acid solution. Wavelength measurement at 570 nm

30E; Part Number B050-7757. May

84. K. A

orsfold. Envi

0. Hansen, E.H., Ruzicka J. and Ghoe A. K., Anal Chim Acta 1978, 100, 151, Ruzicka)

REFERENCES 1. H. Muller, B. Frey and B. Schweizer, Techniques for flow analysis in UV-vis

Spectroscopy, Perkin Elmer, Publication B2304, 92

2. M. Valcarcel and M. D. Luque de Castro, Automatic Methods of Analysis, Elsevier, Amsterdam, 1988.

3. J. Martínez Calatayud, Flow Injection Analysis of Pharmaceuticals. Automation in the Laboratory. Taylor and Francis, Oxford, 1996.

4. P. J. Elving, E. Grushka and I. M. Kolthoff (editors) Treatise on Analytical Chemistry, (2nd Edition). Part I, Volume 4, Interscience, New York, 19

5. . Robinson and J. F. Rubinson, Contemporary Instrumental Analysis, Prentice Hall, 2000.

6. G. D. Christian, Analytical Chemistry, (sixth edit.) J. Wiley, New York, 2004. 7. Grady Hanrahan, Paulo C.F.C. Gardolinski, Martha Gledhill and Paul J. W

ronmental Monitoring of Nutrients. University of Plymouth, Plymouth (U.K.) 1994.

8. J. Ruzicka, Flow Injection 2nd Edition (CD) from FIAlab Instruments. Method adapted from Anderson (Analytica Chimica Acta 110 (1979) p129–137

9. Gil Torró, J.V. García Mateo and J. Martínez Calatayud, Analytica Chimica Acta, 1-9 (1998)

1

P

4567

mR1 R2 D C

123

LW

WP

4567

mR1 R2 D C

123

LW

W

390

391

,

11. J. Ruz, A. Rios M. D. Luque de Castro and M. Valcarcel, Anal. Chim. Acta, 186,1986, 139.

12. N. C. Aracama, A. N. Araujo and R. Perez-Olmos. Analytical Sciences, 2004, 20,679

13. M. Catalá Icardo, J.V. García Mateo, J. Martínez Calatayud, Anal. Chim. Acta2001, 443, 153-163.