Chapter III
AUTOMATIC ANALYTICAL METHODS FOR
ENVIRONMENTAL MONITORING AND
CONTROL
287
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.
289
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:
290
-
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.
1.
2.
Types of continuous flow methods
The continuous flow methods can be classified into three groups:
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.
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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P
S
RC
C
D
W
A
B
(a)
P
KL
RC
D
W
C
S
A
(b)
RC
D
S
A
P2
W
Analytical signal
P1
ttr
ttr
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) continuousflow 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.
P
Buffer (pH=6.3)
Chloraminne T
Sample
RC1=25 cm
PyridineBarbituric acid
Reagent
35° C
D
W
RC2=525 cm
Figure III.2.2. Diagram of CFA system used for cyanide spectrophotometric
determination. P – peristaltic pump; RC – reaction coil; D – detector; W –
waste.
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.
Pump
Air
Reaction
coil
Flow-cell
Detector
Air
Sampler
Separation
unit
Debubber
Thermostated
bath
Diluent
Reagent
Waste
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 flowrate 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 highperformance computer.
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Liquid drawn
through pump
Light
source
Segmented
stream
Air or same liquids
to waste
Photocell
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:
dC
 k (C E  C t )
dt
where C is the equilibrium concentration ant Ct that corresponding to a given time, t.
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Analytical signal
tL
tw1/2
tr
tr
tin
timp (s)
tout
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 halfwashing 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)
-10
0
10
20
30
40
50
tL
(Lag phase)
-0.5
log Ct
log 2
-1.0
log 2
-1.5
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 steadystate 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

tb  t L
t w1 / 2
t t 
L
 . Clearly, the smaller the
gives the so-called degree of interaction,     b
t
w1 / 2 

value of tL and tw1/2 the higher is the performance of the analyzer.
Flow
Time
Air
Air
S2
Air
S1
Air
Air
S2
Air
S1
Air
(a)
S3
S2
S1
S2
S1
time (s)
(i)
S3
Analytical signal
Analytical signal
Analytical signal
S3
S2
S1
time (s)
time (s)
(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.
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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).
Air
2
1
6
5
4
3
WS
Air
S2
Air
WS
Air
2
S1
1 Sequence
Air
Flow
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:
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.
Pump
Waste
(air)
Washing
solution
Air
EDTA
Sample
630 nm
Sampler
Phenolate
Hypochlorite
Nitroprusside
Waste
(a)
Waste
(air)
Pump
Washing
solution
Waste
Air
Dialyzer
Water
Sample
480 nm
Sampler
Air
Water
Colour
reagent
Waste
(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 selfadaptation, 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.
P
S
RC
C
D
W
Vi
R
Analytical signal
(a)
T
H
S
tb
time (s)
(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.
305
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 L 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.
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/CmaxR
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.
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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
Injected sample volume (L)
Carrier flow rate (mL/min)
Reaction coil length (cm)
Tubing inner diameter (mm)
Flow-cell volume (L)
Sample throughput (h-1)
Time for one determination (sec)
Value
10 – 200
0.3 – 2.5
10 – 200
0.3 – 0.8
8 – 40
60 – 360
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
4
3
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.
309
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 injection
In 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, connectors
Pump 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
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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.
LOADING STEP
1
L
INJECTION STEP
FT
6
to RC
S
2
1
S
6
2
5
3
W
L
to RC
5
3
4
FT
4
W
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.
Inner view
Bolt holes
Top
block
Flow channel
Bottom
block
Waste
PTFE
thread
Bottom
block
Top
block
Organic
phase
Aqueous phase
+ analyte
Acceptor
outlet
Acceptor
inlet
(i)
(ii)
Membrane
Donor
inlet
Organic phase
+ analyte
Donor
outlet
Side view
(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 L
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.
312
h
(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 predilution 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
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.
Analytical Response
Analytical Response
10 s
t1 t2 t3 t4
D1
100%
D2
75 %
D3
S
50 %




t3



S
D4
t1
t2




25
25 %
time (s)



t4
100
%


50
75
(a)

10 s


S

0.25 0.50 0.75
1
%
t4 t3 t2 t1
Analytical response
Analytical response
3 min
b
c
25 s
S
d
a
t0
time (s)
Scan
(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.
314
Multiple-determination and multiple-detection FIA techniques
Another trend in FIA development has been the design and optimization of
systems 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 sample is injected and two or more signals are
recorded in different ways:
- using a single detector, the signals being delayed;
- using a dynamic detector, a physical parameter is measured continuously within a
certain range (e.g. absorbance vs. wavelength, current vs. potential, etc., along the
dispersed sample zone;
- using more detectors of the same type displaced in series or in parallel.
The multiple-detection (Figure III.2.18) may be successively performed,
obtaining more data of a certain measured parameter, or simultaneously, using a single
detector that performs simultaneous measurements at different working parameters of
the instrument.
Multiple-determination – of one or more analytes from a sample may be
sequentially 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 of these two FIA techniques, it results that the
multiple-determination may be realized by the multiple-detection, but the multipledetection does not means multiple-determination, strictly.
Two examples of simultaneous determinations are presented below:
(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)
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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
S
RC1
RC2
C
Analytical signal
Vi
W
D
RC3
P1
P2
P3
S
T1
T2
time (s)
(a)
P
S
C
D1
D2
D3
D4
D5
W
Vi
(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.
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P
S
S
RedC
C
D
Vi1
W
Vi2
R
(a)
P
S
C
D
W
RedC
R1
Vi
R2
(b)
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
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).
III.2.1.4. Sequential Injection Analysis
Sequential injection analysis (SIA) was first reported in 1990 by J. Ruzicka
and G.D. Marshall at the University of Washington, as an evolution of the flow
analysis 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
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
physical reconfigurations of the manifold, allowing to perform determinations of
different analytes.
Principles
An operational manifold design for SIA is illustrated in Figure III.2.20. The
heart of the system is a multi-port selection valve, used in place of a conventional
injection 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 holding coil by the pump moving in a forward motion. Each
measuring cycle begins by switching the multi-port valve to the sample line and
aspirating a precise measured volume (few L) 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.
HC
W
D
P
St
R
Vp
R
S
HC
W
D
P
St
R
Vp
R
S
HC
W
D
P
St
R
Vp
R
S
Figure III.2.20. Schematic diagram of a SIA system. P – single-line
reversible (high-precision bi-directional) pump; HC – holding coil; Vp – multiport 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
reconfiguration of the flow path. The injected sample volume, reaction time, sample
dilution, reagent/analyte ratio or system calibration are controlled from a computer
319
keyboard. 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 (L/analysis).
Operational parameters in SIA
“How does SIA differ form FIA and what are its drawbacks?”
The characteristic feature of FIA patterns is that the injected sample zone
flows to a confluence point where two streams merge in a continuous fashion and in
this manner; an equal volume of reagent is added to each element of the passing
carrier stream. 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 multiport 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
R
S
S
H
H
I
I
time (s)
(a)
time (s)
(b)
Figure III.2.21. (a) Formation of sample zone concentration gradient, S –
sample line concentration, in FIA mode via a confluence point supplying a
steady reagent, R – reagent line concentration. (b) Mutually penetration of
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
(or overlay) of the adjacent zones. This is dependent on the relative volumes, in
addition to the usual parameter of tubing size and length, reaction coil geometry and
320
the flow 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 reagent) the overlapping decreases, but
the dispersion 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. Straight reactors allow a greater zone penetration through axial dispersion
than the coiled reactors preferred in FIA.
Instrumentation
In previous SIA works a low-pressure syringe was used as liquid driver that
provided a sinusoidal flow as a result of non-uniform motion of the piston in the camdriven piston pump, illustrating that the flow is not constant but reproducible.
Peristaltic 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 flow-cell detector, tubing/reaction coils and connectors (as those used in FIA)
and a personal 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.
321
III.2.1.5. Hyphenated Systems
The combination of the different continuous flow techniques described above
with powerful instrumentation is of great interest area. It is well-known the success of
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.
III.2.2. AUTOMATED FLOW ANALYZERS
The acceptance of the existence of a correlation between environmental
preservation and standard of living has led to the need of vigilance and continuous
control 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 offline 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, 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 and online process 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 underused 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 handling of solutions, and provides strict
control 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 techniques. New online UV-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 be different in technology, costs and sample types or number.
323
Table. III.2.2.
characteristics.
Commercial
continuous
Product
QuikChem 8000
QuikChem® FIA+
QuikChem®FIA +
IC QuikChem®IC
+ LabTOC2100
FIAstar 5000
FIAstar5010
Company
LACHAT®
INSTRUMENTS,
INC.
6645 W. Mill
Road,
Milwaukee, WI
53218 U.S.A.
TECATOR AB,
Box 70, S-263 21
Hoganas
SWEDEN
Website address www.lachatinstrume
nts.com
Inorganics and
Applications
total organic
carbon in
environmental,
industrial,
agronomy, food
and beverage
analysis.
6 - 90 samples/h
Sample
throughput
Injected volume
2 L – 2 mL
Detector
Photometric, ISE,
flame
photometric,
amperometric,
pH, fluorimetric
www.foss.dk
Wet chemical
analysis of
nutrients and
other parameters
in water, soil,
meat, food
analysis
flow
analyzers
CNSolution 3000
Flow Solution IV
Flow Solution
3000
and
their
SIA2000-S
FIAflo2000
SFA 2000
DUOflo2000
WATERLAB 2000
ALERT 2000 – S
(industrial process
monitor)
BURKARD
SCIENTIFIC Ltd.
PO Box 55,
Uxbridge, Middx,
UB8 2RT, U.K.
ALPKEM -OI
ANALYTICAL
HEADQUARTERS
151 Graham Road,
PO Box 9010
College Station,
Texas 77842-9010,
U.S.A
www.oico.com
www.burkardcientific.
co.uk
Environmental,
Water, soil, food
oceanographic,
and beverage,
agricultural and
industrial process,
industrial
agricultural and
analysis.
biochemical
analysis
60 – 180
samples/h
20 – 400 L
30 – 75 samples/h
30 – 120 samples/h
40 – 200 L
Digital dualwavelength
photometer that
reduce baseline
disturbances for
higher accuracy
and lower
detection limits
Amperometric,
ISE, pH,
photometric,
tandem detector
design, expended
range detection
technology
Variable loop,
minimum 10 L
Photometers,
fluorescence, flame
photometer,
chemiluminescence
detector
324
(Table III.2.2. continued)
Product
FIMS 100
FIMS 400
FIAS Flow
Injection Systems
FI-MH
Company
The PERKIN
ELMER
Corporation
761 Main
Avenue,
Norwalk, CT
06859-0012
U.S.A
EVOLUTION II
SINGLE: “oneshut” Analyzer
The INTEGRAL
Integral FUTURA
CHEMLINE
ALLIANCE
INTRUMENTS
Zone d’activites
les bosquets 4,
BP 31, 95540
Mery-sur-Oise,
FRANCE
ASIA Flow
Injection analyzer
(modular FIA)
FIAlab 2500
FIAlab 3000
ISMATEC SA
FIAlab®
Feldeggstrasse 6, INSTRUMENTS
CH-8152
Inc.
Glattbrugg/Zurich 1440 Bel-Red
GERMANY
Riad, Suite 208
Bellevue WA
98007-3926
U.S.A.
Website address www.perkin-elmer.comwww.inforoute.cgs.fr/a www.ismatec.com
lliance
Applications
Environmental
control, medicine,
food, agriculture,
geology, industry,
research and
teaching
Water, wine,
tobacco,
agriculture, food,
chemical
industries
analyses
Environmental
monitoring, quality
control of food and
beverage, bio- and
chemical-process
monitoring and
control, sensor
testing
Sample
throughput
120 – 180
samples/h
6 – 120 samples/h
30 – 120
samples/h
Injected volume
Detector
< 500 L
Atomic
absorption, ICPOES, ICP-MS
10 L
Photometer with
optical fibers,
20 – 1000 L
Photometer, IES,
biosensors
www.flowinjection.co
m
Environmental
studies,
laboratory
research in
chemistry,
biotechnology,
drug screening,
industrial process
control.
Photometric,
fluorescencemicroscopy,
luminescence,
FTIR, MS,
electrochemical
325
(Table III.2.2. continued)
Product
Company
Skalar San+
Analyzes:
San++
FormacsLT
FormacsTN
PrimacsSC
Primacs SN
Robotic Analyzer
SP 100
BODcompact
Fluorecence
Analyzer
FluoImager
Toxicity Analyzer
ToxTracer
PISCESTM
9000 (portable
SIA system)
FloPro 4P
FloPro 9P
ASI/Eppendorf
Variable
Analyzers
SKALAR’s Head
Office
PO Box 3237, 3800
DE Breda,
NEATHERLANDS
CONSTELLA
TION
TECHNOLOG
Y, 7887 Bryan
Dairy Road,
Suite 100,
Largo, Florida
33777, U.S.A.
Global FIA, Inc.
PO BOX 480, Sixth
St, Fox Island, WA
98333, U.S.A.
www.constech.
com
Environmental
waters,
industrial
process
analyses
www.globalfia.co
m
Inorganic and
organic analytes,
on-line processes;
industrial analysis;
lab and field
analysis
AMKO
Systems, Inc.,
250 W. Beaver
Creek Road,
Unit 6,
Richmond Hill,
Ontario,
CANADA L4B
1C7
www.amkosyste
ms.com
Chemical,
environmental,
biotechnology,
pharmaceutical,
food and
beverage.
Website address www.skalar.com
Application
Waters, detergents,
fertilizers,
pharmaceuticals,
soil, plants, beer,
wine, food, tobacco
Sample
throughput
Injected volume
20 – 140 samples/h
Detector
Photometer, flame
photometer,
fluorimeter, IES,
pH-meter,
conductivity-meter;
UV- VIS; IR;
chemiluminescence
detector
Up to 0.3 mL/min
1 – 3 min/sample
<2.5 L
Variable sample
number and
volume
Integrated UV- UV-VIS spectro
VIS
photometer;
spectrophotome chemiluminescence,
ter for
amperometric
absorbance and
fluorescence
detection.
20 samples/h
with 2 – 100 L
samples
Colorimeters,
ISEs;
conductivity
detector;
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III.2.2.1. Continuous and Discontinuous Systems
There are many variations of flow analysis and most of them can be classified
in: segmented flow analysis (SFA), completely continuous flow analysis (CCFA); and
flow injection 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 uneconomical in terms of reagent consumption and waste
generation, 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 m),
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. Commercial Automated Flow Analyzers
The analysis system may be classified as continuous or discrete (batch)
instruments. The continuous system constantly measures some physical or chemical
properties 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: airsegmented, unsegmented continuous flow; flow injection analysis and sequential
327
injection 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 analytes. Automated instruments perform the following
operations:
1.
Sampling (from a small cup on auto-sampler, or assembly line);
2.
Sample dispensing;
3.
Dilution and reagents adding;
4.
Chemical reaction development;
5.
Directing the reaction product in the detection system;
6.
Reading the recorded data;
7.
Processing the data.
The distinguishing feature of the commercial flow analyzers is their
automated sampling handling capability and many analysts and instrument
manufacturers 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 new alternative in the use of individual computercontrolled 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 automated flow analyzer:
a.
Sample throughput – the number of samples processed per unit of time should
be estimated in association with specified carry-over level. It is also
recommended to specify the consumption of sample and/or reagents when
sample throughput is calculated.
b.
Analytical characteristics – accuracy, sensitivity, detection limit, selectivity,
dynamic range of concentration and precision should be calculated from the
data according to the IUPAC regulations. The analyzer should be validated
using a standard method with confidence limits and tests of significance.
c.
Robustness – is discussed from two points of view: the dependence of the
analytical signal on the variation of system parameters (temperature, reagent
concentrations, 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.
d.
Portability – to another operational environment and consequent suitability for
in situ, in vivo or on –site analyses, as process analyzer for on-line monitoring.
After the automated flow analyzer has been characterized, it is recommended
to furnish some additional information: zone sample sampling; merging zones and
how to get them; sample electrostatic dissolution, in-line gas sampling; multicommutation; adherence to well-established variants of flow analysis, etc.
Examples of commercial analyzers and their features
1. BURKARD Scientific is an English company. BURKARD Series 2000
automatic chemical analyzers are a range of high specification systems meeting the
needs of 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 control are catered for
in single 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 has been designed for economy and ease of use to
the modern 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 following 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, rivers, sea-water, sewage) and effluents.
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, threeterm 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 online 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
completion 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 even a single sample and to analyze 60-180 samples/h
(Figure III.2.22). The standard digital dual-wavelength (DDW) detector used allows
measurements in a wide dynamic range, from -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, subppb 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 set-up into a fully automatic system, able to evaluate, print out and store the
routine testing results and to control the entire FIAstar system from the computer
332
keyboard. Calibration and recalibration can be done quickly and easily, and displayed
on the screen.
Figure III.2.23. SoFIA software for FIAstar 5000 analyzer.
3. ALLIANCE Instruments is a French company providing automatic
analyzers to the industrial market and for routine and research laboratories.
The CHEMLINE is the ALLIANCE Instruments’ first on-line monitor and it
gives a correct answer to the problems of reliability, accuracy and low maintenance.
CHEMLINE is unique with its innovative-patented reactor based on a new
principle of analysis called STEP-CHEM ALLIANCE Instruments. The principle is
the following: samples and reagents are mixed in the reactor equipped with 10
motorized valves and piston managed by a stepper motor that delivers very precise
volumes (up to 10 L 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. 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.
d. 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.
g. After closing this valve, there are three possibilities:
A new cycle is started;
A complete washing operation is performed;
Calibration is performed
h. If it is important to accelerate the development of the reaction product by heating,
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.
All the operations: step number of the motor, opening and closing of the
valves are 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 orings 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.
334
Figure III.2.24. Schematic new principle of analysis: STEP-CHEM.
335
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
336
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 multianalyte 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.
337
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www.flowinjection.com
www.fia.unf.edu/fad/fad/html
www.foss.dk
www.perkinelmer.de
www.zelana.com/lachat
www.fia.unf.edu
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,
semiconductorsimpedance, 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.
339
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, multisyringe, 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.
340
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
341
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 photoreduction 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 photodegradation) 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.
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Sample
Prefilter
0.45 m
Syringe
filter
Cu/Cd
reactor
Carrier
P
Iv
Sulphanilamide
NED
D
reactor
W
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
L; 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.
Power connector
“O” ring
SV
Standard
inlet W
Sample
inlet
C
NED
Sulphanilamide
PVC cover
plate
P2 :
Standard/sample
FC/SSD
Iv
Reducing
column
Reactor
“T” Conector
Distribution
manifold
P1 : reagents
Sample
loop
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
343
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 g L-1 N up to 100 - 2000 g L-1
N; the detection limit is 2.8 g L-1 N with a light-path 2 mm long and 250 L 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
successfully applied in either laboratory and field situations.
Figure III.2.27. FIA system for determination of the dissolved oxygen
1, solution of MnSO4 to inject 50 l; 2, water sample (as carrier); 3, mixture of KI and
NaOH; 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
P, peristaltic pump.
344
III.2.3.3. Monitoring and Control in Rain Water
The hydrogen peroxide and due to its oxidative characteristics has relevant
effects on atmospheric chemistry; like the quick conversion of dissolved SO2 to
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 provided with aquarium pumps for producing the flow.
The manifold 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 channels; one contains the solid-phase reactor to eliminate the
hydrogen 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 Amberlite (IRA – 743);
then 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, at pH 4.8 and at –1.00 V
during 15 min.
The life span was of 15 and 7 days for the reactor and Au-Pt electrodes,
respectively. Sample throughput was 90 h-1 and reproducibility under 1%. The
assembly 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
Iv
E
E
AP
D
TR
W
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: waste.
345
A former article was also dealing with the amperometric determination of
hydrogen 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 is Ag/AgCl and placed at
the tip 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 second, all the SMS is converted in S(IV) by
increasing 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 auxiliary enzymatic
process 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.
ME
AE
P
D
Water
Iv
Figure III.2.29. FIA system for determination of H2O2, HSO3- and hydroxyl
methane sulfonate
ME: 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 HSO3- and HMS respectively; AE: main electrolyte flow,
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 L sample loop); D: electrochemical
cell and polarograph.
346
III.2.3.4. Water Quality, Wastewater
As a summary of automatic flow-procedures is depicted a SIA assembly
suitable of monitoring different quality parameters using the usual analytical
chemistry.
A multi-parametric SIA assembly designed for wastewater quality in-situ and
real-time monitoring is depicted in Figure III.2.30. It is designed to determine total
organic 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. Atmospheric Monitoring and Control
The dramatic increase in the environmental pollution is a challenge for the
analytical monitoring and control; a challenge in which the analyst is “aided” by the
new 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 retain the
pollutant 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
quality in-situ and real-time monitoring.
A primary caution in sampling, sample transport and storage should be kept in
mind. Sampling 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 pollution require different sampling procedures according to the
type 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 other two parts
(sampler and trap) to configure a complete in-line monitoring system.
Monitoring ethanol in ambient air
There are many anthropogenic actions which consequence is a change in
atmospheric 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
representative example is the extensive use of ethanol as vehicle fuel (very important
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
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. During air sampling the ethanol reacts with
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):
(a) Air is aspirated through a KI filter to eliminate the ozone influence
(b) A scrubber coil (about 100 cm long) made of borosilicate glass is the sampling
device. This is treated with acetone and 40% HF; then is washed with pure water to
obtain 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 at pH 9 with a phosphate buffer.
(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 nm).
(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 excess of solution is
eliminated through channel C.
(f) The flow assembly (detector and pump excluded) fitted into a thermostated box for
field experiences.
Flowmeter, pump
A
Ozone filter
scrubber-coil
air
C
B
A
P
V
D
C
Figure III.2.31. A: absorbing solution: ADH, NAD+, phosphate buffer; P:
peristaltic pump; D: fluorescence detector; V: injection valve. See explanation
in the text.
349
The system was calibrated with the references:
QA= 0.23 mL min-1; QB= 0.15 mL min-1; gas flow rate=1 L min-1 and 70 % relative
humidity. Calibration over the range 0 -112 mg mL-1. The dynamic range in not linear
as usual in enzymatic reactions. The calculated deviation was 6.5 % (c = 44 mg mL -1,
n = 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 level of analytes should be very low and
the sample cannot be analyzed as is it in the environment, which obliges to include an
on-line sample pre-treatment to obtain a continuous and automated procedure. The socalled 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 is
proposed 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 stopped. A second pump is used to
force the sample air (20 L) into the cell at 7 mL min-1. The NO2 is transferred from
the air to the absorbing reagent and transformed into nitrite ions.
2. 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 spectrophotometer.
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
is of the LED type (light emitting diode) with a power source (12 V battery) (for
laboratory determinations the FIA manifold was formed with PTFE tubing 0.5 mm
internal diameter). The detection limit is 0.9 g L-1 and the complete cycle is about 5
min.
350
RS
SL
P1
AS
AS
D
V1
DG
V2
P2
CMC
air
P3
Figure III.2.32. Flow system with the chromato-membrane. RS, reagent
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 FIA 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.
Ozone flow-analyzer
The authors do not clearly recommend a reagent; several were tested. They
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 detection zone:
(a) The hanging droplet is formed by a PTFE capillary 3 cm long and 200 nm internal
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.
(b) the film-reagent is a smooth glass surface, 80 x 6 mm, provided with a triangular
distributing device to avoid irregularities of the liquid film over the whole surface.
Analytical applications are only studied in the film-reactor mode.
(c) The reactor consists of a 35 x 200 mm chamber build in black PVC. The ozone
flowing at 2 L min-1 reacts with the suitable reagent (according to the operator
choice).
(d) The chemiluminescent reagent is pumped to the detector device placed at about 20
mm of the entrance window of the Photon Counting Module.
The flow system was as shown Figure III.2.33. With 1 mmol L-1
phenosafranine in ethanol the following analytical figures were obtained: application
range, 5.2-330 g m-3; quantification limit, 2.1 g m-3 and time per sample 5 s.
Comparing results with the UV method this detector present lesser sample
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 SO2
It is one of the relevant polluting compounds to be controlled to test the air
quality and 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 provided with on-line sampling
and pre-concentration and able to be field applied is reported.
A micro-pore hydrophobic membrane made of poly(vinyldene) difluoride,
sandwiched 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 reagent is inserted by the injection
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).
V
P
Reagent
waste air
RC
D
air
waste
solution
Figure III.2.33. Flow assembly for ozone determination. RC: Reaction
chamber; D: photo counting module; V: valve; P: peristaltic pump
352
Sample gas
W
DTNB
P
DTNB
D
V
W
Gas inlet
110 mm
Gas outlet
20 mm
4 mm
40 mm
60 mm
25 mm
10 mm
Liquid inlet
Liquid
outlet
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 g 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 g m3
. 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 photochemiluminescence.
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
Atrazine
Metalachlor
Alachlor
Methyl bromide
Cyanazine
Dichloropropene
2,4-D
Metam Sodium
Trifluralin
Glyphosate
Tones
31,500-33,750
27,000-29,250
20,250-22,500
13,500-15,750
13,500-15,750
13,500-15,750
11,250-13,500
11,250-13,500
9,000-11,250
6,750-9,000
Pesticide
Chlorpyrifos
Chlorothalonil
Propanil
Dicamba
Terbufos
Bentazone
Mancozeb
Parathion
Simazine
Butylate
Tones
4,500-6,750
4,500-6,750
3,150-5,400
2,700-4,500
22,250-3,600
1,800-3,150
1,800-3,150
1,800-3,150
1,350-2,700
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 physicchemical 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
354
potential water pollutant.
Monitoring photo-degradation processes has recently proved an effective
method for the in situ control of environmental pollutants. The potential of sunlightbased photocatalytic decontamination has lately aroused much interest.
The flow manifold depicted in Figure III.2.35 comprised three solenoid valves
each of 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.
Figure III.2.35. Flow assembly optimized for pesticide determination.
Q2: photodegradation medium (glycine buffer at pH 8.3); Q1: Aqueous
solution 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)63- 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.
The optimum insertion profile for each of the two tested oxidant systems is
depicted in Figure III.2.36. With the potassium permanganate, an overall of 20
alternate micro-insertions of pesticide and photo-degradation medium were
355
performed. During each micro-insertion, valve V1 was kept ON for 0.3 s to aspirate
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
OFF


SV1 ON
SV1= 0, 20*(0.3,0.1), 0.1
SV3 ON
SV2= 97.5, 17

SV2
OFF
ON
OFF
SV3= 0, 8, 90, 17*(0.7,0.2), 0.5
Cycle: 123
20 segments; 8 s
90 seconds
123 seconds
17 segments;
15.3 s
+s35 s
pesticide (0.7 s segments) and the oxidant (0.2 s segments) in 17 ON/OFF cycles.
Figure III.2.36. Optimized insertion profile for obtaining a typical transient
analytical signal for MnO4-/H2SO4 systems.
Two oxidant systems were studied: potassium permanganate and ferrycianide.
With the selected MnO4–/H2SO4 system, the response was linear up to an asulam
concentration 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 with the Fe(CN)63-/NaOH system and the photo-degradation
of nitrate 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 g 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 as in the text)
1. Field-portable flow-injection analysers for monitoring of air and water pollution.
P. W. Alexander, L. T. Di Benedetto, T. Dimitrakopoulos, D. B. Hibbert, J. C.
Ngila, M. Sequeira and D. Shiels, Talanta, 1996, 43(6), 915 – 925.
2. B. Karlberg, B. Pacey, Flow Injection Analysis, a practical guide, Elsevier, New
356
York, 1989.
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
and P. J. Worsfold, Talanta, 2002, 58(6), 1015 – 1027.
4. A reverse-flow injection analysis method for the determination of dissolved oxygen
in fresh and marine waters. S. Muangkaew, I. D. MaKelvie, M. R. Grace, M.
Rayanakorn, K. Grudpan, J. Jakmunce and D. Nacapricha, Talanta, 2002, 58(6),
1285 – 1291.
5. Flow-injection system with enzyme reactor for differential amperometric
determination of hydrogen peroxide in rain water, R. Camargo Matos, J.J. Pedrotti
and L. Angnes, Anal. Chim. Acta, 2001, 441(1), 73-79.
6. Amperometric flow-injection technique for determination of hydrogen peroxide and
sulphur(IV) in atmospheric liquid water, I.G.R. Gutz and D. Klockow, Fresenius'
Z. Anal. Chem. 1989, 335(8), 919-923.
7. Application of flowing stream techniques and related compounds to water analysis.
Part I. Ionic species: dissolved inorganic carbon, nutrients, M. Miró, J. M. Estela
and V. Cerdá, Talanta, 2003, 60(5), 867 – 886
8. An enzymic-fluorimetric method for monitoring of ethanol in ambient air. M.
Schilling, G. Voigt, T. Tavares and D. Klockow, Fresenius' J. Anal. Chem., 1999,
364(1-2), 100-105.
9. Absorption, concentration and determination of trace amounts of air pollutants by
flow injection method coupled with a chromatomembrane cell system: application
to nitrogen dioxide determination. Y. L. Wei, M. Oshima, J. Simon, L.N. Moskvin
and S. Motomizu, Talanta, 2002, 58(6), 1343-1355.
10. Determination of ozone in ambient air with a chemiluminescence reagent film
detector. C. Eipel, P. Jeroschewski and I. Steinke, Anal. Chim. Acta, 2003, 491(2),
145-153.
11. Flow injection determination of gaseous sulfur dioxide with gas permeation
denuder-based online sampling and preconcentration. Z.X. Guo, Y.Z. Li, X.X.
Zhang, W.B. Chang and Y.X. Ci, Anal. Bioanal. Chem., 2002, 374(6), 1141-1146.
12. 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.
357
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 photocounts, 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
359
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 m. 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 m), 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.
361
Table III.3.1. Typical parameters of the DIAL LIDAR system
Species
Laser wavelength
Nitric oxide, NO
Nitrogen dioxide, NO2
Sulfur dioxide, SO2
Ozone, O3
Mercury vapor, Hg
Benzene, C6H6
Toluene, C7H9
Methane, CH4
Ethane, C2H6
Ethylene, C2H4
Acetylene (C2H2)
Hydrogen chloride (HCl)
Nitrous oxide, N2O
Methanol, CH3OH
226 nm
450 nm
300 nm
289 nm
254 nm
253 nm
267 nm
3.42 m
3.36 m
3.35 m
3.02 m
3.42 m
2.90 m
3.52 m
Measurement
sensitivity (ppb)
5
10
10
5
0.5
10
10
50
20
10
40
20
100
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, 31453164.
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, 115.
7. Milton, M. J. T., P. T. Woods, B. W. Jolliffe et al., Appl. Phys. B, 1992, 55, 41-45.
362
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
363
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)
Ammonia, NH3
Nitric oxide, NO
Nitrogen dioxide, NO2
Nitrous oxide, N2O
Sulfur dioxide, SO2
Formaldehyde, CH2O
Benzene, C6H6
Toluene, C7H8
Phenol, C6H6O
Ethylbenzene, C8H10
Benzaldehyde, C7H6O
Xylene, C8H10
Cresol, C7H8O
Dimethylphenol, C8H10O
Trimethyphenol, C9H13O
Trimethylbenzene, C9H12
Methybenzaldehyde, C8H8O
200 - 230
200 - 230
400 - 500
325 - 390
280 - 320
280 - 350
236 - 263
250 - 270
250 - 280
238 - 270
257 - 290
243 - 275
253 - 285
255 - 287
260 - 290
240 - 290
266 - 306
Detection limit
(ppb)
0.8
1.8
1.0
0.9
0.2
1.2
0.9
1.5
0.1
2.4
0.4
1.2
0.5
0.6
1.8
2.4
1.8
365
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.
366
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
367
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.
368
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 timeconsuming, 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
369
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 semiautomated 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
370
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) 321352.
[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.
371
[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 µTechniques 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) 124132.
[26] E. Mallat, C. Barzen, A. Klotz, A. Brecht, G. Gauglitz and D. Barceló, Environ.
Sci. Technol., 33 (1998) 965.
372
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.
373
III.4.1. NUTRIENTS:
MEASUREMENT
ENVIRONMENTAL
SIGNIFICANCE
AND
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
Nitrite/nitrate
ammonia
Phosphate
Calcium
Automated methodology
Chemical method
Lab-on-a valve
Griess modified
Salycilate
Colorimetric analyzer
Molybdate
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 FIA and r-FIA
Cr(VI)/1,5(VI)
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.
374
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
L) and minor flow rate; or a method to be applied to higher concentrations by using a
smaller sample (25L) 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).
375
SO2NHR
2
SO2NHR
+ NO2- + 2 H+
N=N+
NH2
Sulfanilamide
SO2NHR
2
Diazonium ion
NHCH2CH2NH2
+
N=N
+ 2 H2
RHN-O2S
N=N
NHCH2CH2NH2 + H+
+
NED
Azo Dye
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
376
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 g L-1 and nitrite, 0.7 g 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.
Nitrite / Nitrate Assay
1.000
Absorbance
Blank
2 ppm NO2NO22ppm
lmax = 542 nm
0.800
0.600
0.400
0.200
0.000
300
350
400
450
500
550
600
650
700
750
800
850
Wavelength (nm)
Figure III.4.2. Absorption spectrum of the nitrite assay. Reproduced from J.
Ruzicka, flow Injection 2nd Edition (CD) FIAlab Instruments, with
permission.
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.
377
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 bluegreen 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
ammonia
Cl
OCl
N Cl
N
+ OH
-
chloramine
-
O
+
N
Cl
hypochloride
chloramine
O
-
O
N Cl
phenol
+
-
O
O
N
-
O
378
The modification of the phenate method means the reagent phenol is
substitute by the salicylate:
COOH
OH
Salicylic 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 NH 3 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.).
FIA2000 Series
b
Light Source
f
V alve
WW
as ates te
c
d
3
4
2
5
1
M ix ing Co il 1
6
S am ple
Line 1
a
e
g
h
M ix in g Co il 2
W a ste
Tee
Z F lo w C ell
L in e 2
Spectrophotometer
Pe rista ltic P um p
Figure III.4.3. Flow Injection Hardware Setup for ammonia determination for
use in a unidirectional and/or stopped flow procedure.
Note: the schematic above shows the sample loop in the load mode.
379
Reproduced from J. Ruzicka, Flow Injection 2nd Edition (CD) FIAlab
Instruments, with permission.
5-Aminosalicylate Complex
1.500
NH3: 50 ppm
NH3: 0 ppm
Absorbance
1.200
675nm max
0.900
0.600
0.300
0.000
400
450
500
550
600
650
700
750
800
Wavelength (nm)
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
Phosphorus is biologically important; it is an essential nutrient for the
phytoplankton growth. Excessive inputs can lead to eutrophication of coastal marine
waters, which is accompanied of abnormal growth of algae.
There are also condensed forms of phosphate as pyrophosphate and
polyphosphates and organic phosphates. The environmental parameter known as “total
phosphorous” 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); being the chemical distribution of phosphorus between
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 analyte (phosphate)
380
with molybdate in acidic medium (Figure III.4.5.). In a second step the heteropolyacid
is then reduced to an intensely colored blue compound with a maximum absorbance
band at 840 nm (McKelvie, Peat, and Worsfold, 1995).
PO43- + 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
tin(II) chloride and being important potential interferences silicate and arsenate.
The phosphorus determined is defined as “molybdate reactive” or soluble
reactive 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.
The following automatic apparatus is a commercially designed colorimetric
analyzer 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 parameters (small design changes) are copper, chromate, high range
silica, 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
are very 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 spectrophotometric examples on metal determination are presented
below.
Determination of Calcium in Drinking Water Samples
The method is based on the formation of the complex calcium with ocresolphthalein [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 L 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.
Figure III.4.6. Flow assembly for determination of calcium in drinking water
samples. 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)
Cr (VI) reacts with 1,5-diphenylcarbazide (DCP) yielding a colored complex
with maximum absorbance at 540 nm. Cr (III) do not interfere with this reaction and
its determination 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 required.
A flow assembly similar to the depicted has also been proposed for the
speciation of As(III) and As(V). The oxidant was the potassium iodate and the
colorimetric 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 substituted by a splitting point and
a double flow-cell.
Figure III.4.7. Flow assembly for speciation of Cr (III) and Cr (VI). 1, DCP
solution; 2, oxidant; 3, sample; 4, sulfuric acid as carrier. a, the way for
oxidant; and b, way for DCP. P, peristaltic pump; Iv, injection valve; S,
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
applied for the continuous monitoring of water (rivers, irrigation channels, estuaries,
383
etc.). The Figure III.4.8 depicts an FIA assembly for continuous monitoring of Cr (VI)
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
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 volume to be inserted 169 μL.
Figure III.4.9. Type of outputs from the r-FIA for continuous monitoring of
Cr (VI) (continuous output, a) and periodic measurements of Cr (III) (b peaks)
(transient signals) equivalent to the total chromium concentration.
384
To obtain a sequentially monitoring of Cr(III) and Cr(VI) the alternative
manifold is depicted in Figure III.4.10, in which the carrier (water sample) splits in
two different streams for a two separated manifold branches one for each 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
presented the outputs form the Cr measurements.
Figure III.4.10. A r-FIA assembly for “continuously sequential” monitoring
of Cr (III) and Cr (VI). Upper Pump: 1, DCP solution; 3, acidic medium at 0.3
mL 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 flowing at
1.9 mL min-1. Reactor lengths: Upper branch, 20 and 50 cm for R1 and R2,
respectively. Lower branch; 600 and 40 cm, for R1 and R2, respectively.
385
Figure III.4.11. Outputs from the sequential continuous monitoring of
Cr(VI), outputs a; and, Cr (III): total chromium outputs b.
386
Determination of Lead in Water by a Sequential Injection Analysis (SIA)
Flow Assembly
The method is based on the catalytic effect of the Pb(II) 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 phasereactor (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 L 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.
387
III.4.1.5. 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.
MeO
+
H3N
OMe
+
NH3
Dianisidine in acidic medium
MeO
- 2 e+ 2 H+
+
H2N
OMe
+
NH2
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. The operating cycle for
obtaining a typical analytical transient signal can be easily programmed by means of
friendly software running in the Windows environment.
The manifold comprises a set of three solenoid valves acting as an
independent switch. See Figure III.4.13. The sample (channel Q3) merges with a 0.5
mol L-1 HCl solution (Q4); the global chlorine is converted into chlorine and
transported through the gas diffusion membrane. Valves 2 and 3 control the time of
stopped-flow and then the time of pre-concentration (volume of sample and diffusion
of chlorine to a basic solution (Q2) acting as acceptor solution). The resulting basic
solution causes the quantitative decomposition of chlorine into hypochlorite, which
facilitates the gradient solution and transport process through the membrane. After this
pre-concentration step the volume comprised between valves 2 and 3 is forced to the
flow system and merges with the solution of dianisidine (Q1). A previous solenoid
valve is used for recycling the non-inserted reagent solution.
The high sensitivity and selectivity of the method is due to the manifold is
provided with a gas-diffusion unit which permits the removal of interfering species as
well as the pre-concentration of chlorine. The separation unit was made with two
pieces of methacrylate being screwed together, the groove carved in the pieces formed
a channel that is split by the fluoropore membrane filter of 0.5 m pore size, which is
held firmly between the two blocks (Figure III.4.14).
388
Q1
W
Q2
V3
W
ON
ON
V2
ON
P
OFF
V1
OFF
D
W
OFF
Q3
W
Q4
Figure III..4.13. Tandem-flow assembly for the determination of chlorine. Q1=
Q2= 5.2 mL min-1, Q3= 0.6 mL min-1, Q4= 5.0 mL min-1. Q1: 10-3 mol L-1 odianisidine in 1 mol L-1 acetic acid; Q2: 0.005 mol L-1 NaOH, Q3: sample; Q4:
0.5 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 g mL-1 of chlorine; the limit of detection is 0.05 g
mL-1; the reproducibility (as the rsd of 42 peaks of a 0.72 g mL-1 chlorine solution) is
1.5 % and the sample throughput is 38 h-1.
III.4.1.6. Cyanide
Measurements by a segmented-flow assembly
Cyanide is highly toxic to mostly living organisms by preventing the normal
activity of metal-containing molecules due to its property of strongly complexing
389
metal cations. However, in low concentrations it is biodegradable by some bacteria. It
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 by
flushing 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
sampling air may lower the pH and facilitate the releasing of hydrogen cyanide gas.
Other problems appear from the oxidizing agents in solution, which may transform
cyanide during storage and handling. It is recommended for the storage of cyanide
samples to collect the samples at pH 12-12.5 in tightly closed dark bottles and store
them as soon as possible in a cool, dark place. It is also recommended that the samples
be analyzed immediately upon collection.
Particulate cyanides are known to decompose in moist air with the liberation
of 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;
the hydrogen cyanide ion is retained by the diluted alkaline flow stream.
The resulting alkaline stream merges with the chloramine T (N-chloro-ptoluene sulfonamide sodium salt) stream at pH less than 8 and the cyanide is converted
into cyanogen 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 “closed” between two consecutive air bubbles
are homogeneous (as contrary in FIA with the gradient concentration) and the
transient signal is not a peak; it is theoretically speaking a rectangle, in practice a
curved rectangle.
390
1
2
3
4
5
6
7
L
W
m
R1
P
R2
D
C
W
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
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1. H. Muller, B. Frey and B. Schweizer, Techniques for flow analysis in UV-vis
Spectroscopy, Perkin Elmer, Publication B2304, 30E; Part Number B050-7757.
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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, 1984.
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6. G. D. Christian, Analytical Chemistry, (sixth edit.) J. Wiley, New York, 2004.
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