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Automated methods of

AnalysisThe automation of some processes or technologies presents

great advantages, which will not be mentioned here, allowing

the achievement of great performance and low costs for the

products obtained, which would otherwise be

inaccessible.Automation found a wide application domain even

in the eld of analyticalchemistry, presently being harder and

harder, and in some cases even impossible, to resolve theproblems arising for a chemist without using automated analysis

Generally, the automation of the analytical processes can be

made in severalways. Thus, automated analyzers for discrete

samples were created, together with owways. analyzers and

robotic analyzers. From these, of etreme importance are the

analyzers based on the principle of ow analysis.

!n the last decades special attention was paid to the ow analysismethods being applied for the solving of routine or research

analytical problems. The commoncharacteristic of these methods

is the fact that the measurements are made in ananalytical

channel through the sample to be analyzed and the reagents

circulate.The use of computers to command, control and

diagnose the e"uipment used,and for the processing of the

obtained eperimental data, has increased even furtherthe

performances of the ow analyzers, many of them being able tooperate in a fully automated regime.

FLOW ANALYSIS TECHNIQUES


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#ontinuous Flow Analysis

#ontinuous ow analysis $#FA% refers to any process in which theconcentration of the analyte is measured uninterruptedly in a

stream of li"uid or gas. The basic principle of continuous ow

analysis is to eliminate chemical analysis by hand&miing of

reagents in individual items of glassware and to substitute a

continuously owing stream of li"uid reagents circulating through

a closed system of tubing. Therefore, in #FA the sample is

converted into a owing stream by a pumping system and the

necessary reagent additions are made by continuous pumpingand merging of the sample and reagent streams. The miing and

the chemical reactions ta'e 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 di(culty to

overcome is to prevent intermiing of successive samples during

their passage through the analyzer, this intermiing causing

overlap and loss of discrimination at the recording stage. !n

general, to minimize this so&called carry&over, the design of thetiming se"uence 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.

a pump $)% that is used to propel the carrier stream through a

thin tube* & a coil of tubing also named reaction coil $+#% in whichthe sample zone disperses and reacts with the components of the

carrier, forming species that are detected by a ow&through

detector.


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Types of continuous ow

and either sending to the waste $open systems% or r.

#ontinuous miing methods $Figure !!!.-..a% that involve the

sample insertion into the system, miing it with the carrier or

reagent, measuring the reaction plug as it passes through a

suitable detector ecirculating 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.

-. /topped&ow continuous miing methods $Figure !!!.-..b%

the ow is stopped at various stages during the process in orderto prevent air from entering the system between sample

aspiration and reagent aspiration or washing. A 'inematically

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 reagent0washing solution reservoir and the pump is

restarted. !n 'inetic methods, the ow is stopped into the

detector ow&cell to monitor the evolution of the reaction.

Flow in1ection analysis

Flow in1ection analysis $F!A% is a form of ow analysis that does

not rely on air segmentation to prevent the interactions between

successive samples. !nstead, it employs a non&segmented ow

under conditions such that sample spreading is minimized and

successive samples can be introduced at short time intervals/ince F!A was born, it found many applications both in the

laboratory and in process control and became 'nown simply as

F! as it was realized that F! is not only a tool for analysis, but also

a generally applicable solution handling techni"ue. !ts ability to

control and monitor 'inetic aspects of automated assays has


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been recognized, and identied as 2'inetic advantage3. 4y now

its scope is broadening into environmental research and into a

tool of biotechnology and for the study of the chemistry of life.

F!5s versatility, or self& adaptation, and perfect computer

compatibility ma'es it an ideal interface between a computerand a $bio%chemical system.

!6/T+7896TAT!:6

)rinciplesFlow&in1ection analysis is based on the insertion0in1ection of a

li"uid sample into a moving non&segmented carrier stream of asuitable li"uid. The in1ected 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, p;, etc.% that changes

continuously as a function of time as the sample passes through

the ow 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 adetectable 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 F!A response curve is a result

of two processes, both of 'inetic nature, the physical process of

dispersion of the sample zone within the carrier stream, and the

chemical process of the formation of chemical species.

)umpThe 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 ow, but periodic

rell is necessary. The most used and suitable device for

propelling the li"uid ows is the peristaltic pump, because it may


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accommodate several channels whereby, according to individual

tube diameters, e"ual or di a% stop and start

instantaneously, allowing the precise control of all moving

streams for stopped&ow or intermittent pumping functions.

/ample !n1ection

/ample in1ection !n order to save reagent, to increase the

residence time with a minimum sample dispersion, and to

accomplish zone sampling ingenious ow manifolds have been

designed with di


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rotary in1ection valve and its operating mode. The loading and

in1ection 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

in1ection because they will modify the pattern of the ow in F!Asystem, a


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compatible with a owing stream is suitable for ow in1ection.

/pectrophotometry, nephelometry, uorescence,

chemiluminescence, atomic absorption, ame photometry,

potentiometry with ion&selective0modied electrodes or eld

e


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detector used. The gradient calibration techni"ue is an etension

of the above described one. !ts 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 uid originating from a sample zone of themost concentrated standard sample solution $Figure !!!.-.H.b%.

The F!A stopped&ow approach is based on a combination of

stopped&ow measurements and of gradient dilution. !t is based

on the increase of residence time, 'eeping the reactor short and

decreasing the ow&rate of the carrier stream. #hoosing

appropriate stop&go time intervals, the reaction time will

increase and the reaction rate.

A))E!#AT!:6/

!n fow in1ection literature the terms limited dispersion medium

dispersion and large dispersion are fre"uently encountered

where they refered to dispersions of to = , = to and greater

then respectivily.

.limited dispersion analysislimited dispersion ow techni"ues have found considerable

application for high speed feeding of such detectors system as

ame atomic absorption and emmission as well as inductively

coupled plasma.

-.stopped ow methods

it is used for 'inetic measurement

=.ow in1ection titration

titration can also be performed continuously in a ow in1ection

apparatus.


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/egmented Flow Analysis /FA

introduction

The segmented ow analysis $/FA% was the earliest contribution

in the eld of automated methods development. !t originated

from the scientic paper of Cr. E /'eggs $7niversity of #alifornia,

7/A% published in IBH and has found widespread use in almost

every facet of analytical chemistry. /'eggs5s studies were

materialized in the design of the rst continuous dynamic

measuring system with se"uential introduction of samples and

the use of a ow&cell. /ample carry&over was prevented bysegmentation with air bubbles introduced between successively

aspirated samples.

!nstrumentation

/FA is characterized by the use of one or several li"uid streams

where the samples are introduced by se"uential aspiration and

separated by means of air bubbles aimed at avoiding the carry&

over. Therefore, the nal li"uid stream is segmented into small

discrete li"uid slugs by bubbles of air or other gas that entirely

lls the stream tubing bore. The sample and reagents are mied

by passing through glass coils and through a temperature

controlled heating coil if heat is re"uired to speed the

development of the reaction product before detection. !n the

initial wor', 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 techni"ue has become

one of the most reliable and widely used methods for automatic

chemical analysis in routine and research analytical laboratories.

This techni"ue has the advantage of for measuring large


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batches of samples for up to analytes simultaneously at

speeds of up to - samples.

/ampling device which consists of a sample turntable

and moving articulated aspiration probe. )ropelling device aimed to provide the sample and reagent and air streams. These

are generally multi&channel peristaltic pumps $Figure !!!.-.=% but

may also be used piston pumps and the pressure eerted by a

gas or gravitational force. The ow& rate of the streams can be

ad1usted and maintained as constant as possible by using

eible plastic tubes that withstand the mechanical pressure to

which they are sub1ected.

+eaction&miing coils sample and reagents merge inthe appropriate stages through 2T3&connectors and then pumped

through the glass, )TF9 or polyethylene tubing where the miing

of reactants and the analytical reaction ta'es place. The tubing

is coiled and horizontally mounted. !t provides the reactants

miing by repetitive inversions of the li"uid phase and its length

governs the time over which the reacting miture Jresides5 in it

and hence the sampling fre"uency.

;eating device, continuous separation

device are introduced in /FA system, if re"uired, and theyare 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, li"uid&li"uid

etractors, sorption or ion&echange micro&columns, lters areused. These devices are placed before the reaction coils to

remove potentially interfering species.

Cebubbler its aim is to remove the introduced air bubbles1ust before the li"uid stream reaches the ow&cell of the


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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 undesirablesignals and those actually corresponding to the reaction miture.

#ontinuous detection system any optical orelectrochemical detector that can be e"uipped with a ow&cell is

used as detection unit in /FA. The air bubbles are e(ciently

removed from the li"uid stream if the waste tubing of the ow&

cell is coupled to a channel of the peristaltic pump.

The ow rate of the li"uid entering the ow&cell must be higher

than the ow rate of the li"uid drawn through the pump.

Cata recording and treatment unit whichshould be prepared to operate in continuous mode and be as

simple as a typical Kt recorder or a sophisticated as an

advanced microprocessor0computer carrying out both operations

or delivering directly the re"uired results. The most modern /FA

systems are fully operated by a high& performance computer.

)rinciples and 'inetics of /FA

An operational feature of the /FA system is that, in addition tosample and reagents, air is drawn into the system through one

of the pump tubes and it produces segmentation of the li"uid

stream once it has been merged with it. This segmentation is

maintained through the succeeding stages of the analysis up tothe 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

li"uid slugs and this presents several advantages. First, the air

segments are responsible for maintaining a sharp concentration


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prole at the leading and following edges of each individual

sample. /econd, the presence of air bubbles promotes the

miing of the phases. 9ach stream slug can invert e(ciently as

it rises and falls through each turn of the miing coil. For

maimum miing e(ciency the length of each li"uid slug mustbe less than half the diameter of the coil. !n addition, the wiping

action of the air along the tube wall prevents the build&up at the

surface of residues from the preceding li"uid slug. The

measurements carried out in /FA systems are made under

physical $homogenization of the sample&reagent slug between

two consecutive bubbles% and chemical $analytical reaction

reaches the e"uilibrium state before the reacting slug reaches

the detector% e"uilibrium. Therefore, in these systems thesteady&state signals are recorded and hence their design and the

operation should achieve these e"uilibria. The main advantage

of a determination realized in a /FA analyzer, namely precision

and rapidity are drastically inuenced by operational

parameters.

/ample carry&over

The sample carry&over occurs mainly in unsegmented streams

and in terms of the standard /FA system design this implies>

a. the aspiration device, the probe is contaminated internally

and eternally by the previous sample unless a washing

mechanism is employed*

b. ow system, a static, thin li"uid lm prevents direct contact

of air with the tubing walls, thereby generating a heading

sample residue. 7pon arrival at the same point, part of the rstsample is mied with and incorporated into the segment of the

second $Figure !!!.-.L.a%.

c. connection between the debubbler and the detector input. !t

favors miing through the absence of any physical separation


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between successive samples. The connection must be as narrow

and short as possible in order to avoid aial di


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b. sample variables> viscosity* surface tension* molecularor ionic di a. the compressibility of the air bubbles

gives rise to a turbulent ow regime which fosters miing b. the

helically coiled tubing favors the radial di viscosity* density*

reactants di


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followed by !/9 potentiometry $ &@M% and much less

nephelometry, uorimetry. Figure !!!.-..a depicts an assembly

used for the determination of ammonia in see and tap water. The

sample is aspirated $eventually after ltration% in the system and

mied with 9CTA $metal ion mas'er% and then with phenolateand hypochlorite streams to form the dye indophenol blue,

whose color is intensied by a nitroprusside stream. After de&

aeration, the absorbance is measured at = nm. !n this manner,

nitrogen can be determined over the range of concentration .-

& - mg0mE at a sample throughput of samples0h.

/e"uential in1ection analysis $/!A%

introduction

this techni"ue or automatic sample analysis, is based on the

same principles as F!A, namely controlled partial dispersion and

reproducible sample handling, and it o


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/!A presents two ma1or disadvantages> the sample throughput is

lower then that of the usual ow systems and ma1or di(culties

in the miture of sample and reagents. /!A is a single&line

system, completely microcomputer controlled, that can be

congured to perform most operations of conventional F!A.

)rinciples

this is the primary di


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environmental preservation and standard of living has led to

need of vigilance and continuous c 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 havebeen performed by o 2This is a techni"ue that allows chemists to easily


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automate and optimize well&developed wet chemical methods

for routine laboratory use. Kou can even program an analyzer to

switch from one analyte to another during the analysis of a

batch of sampleR 4ut F!A was never been a popular product of

larger instrument companies, so smaller rms produce most ofthese analyzers3. !n spite of all, the techni"ue is surprisingly

under& used by laboratory chemists and this perhaps automation

was too epensive or too' too long when F!A was introduced in

IHB. F!A o it is computer&compatible, allows automated

of reaction conditions. Also, because of its versatility in sampling

handling, F!A serves as front end to practically all

spectrophotometric and electrochemical detectors and tovarious environmental, clinical and industrial assay. :ther

applications include 2real&time3 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.

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