FLOW INJECTION ANALYSIS

(FIA)

By Assoc. Prof. Dr. Jaroon Jakmunee

20 September 2012
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Wet Chemical Analysis

(wet
chemical analysis)

.......

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ANALYSIS

COST OF ANALYSIS

Accuracy
Precision
Sensitivity
Selectivity

Chemical(s)
Chemical reaction
Timing
Instrument
system cost
operating cost
maintenance cost
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  !  
"


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(turbulence
flow) =>

(carry over)

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steady state
(equilibrium)

......

steady state
...

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Reaction time resident time

reaction time

(
steady state)

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OVERVIEW
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1.1. Basics, 1.2. Principles, 1.3. Methods & Applications. 1.4. Instruments & Components

Flow Injection (FI), the first generation of FIA techniques, is the one
most widely used. In its simplest form , the sample zone (red) is
injected into a flowing carrier stream of reagent (blue). As the injected
zone moves downstream, the sample solution disperses into reagent,
while a product begins to form at interfaces between the sample zone
and the reagent. A detector placed downstream records a change of
color or of another parameter as it changes due to the passage of the
derivatized sample material through the flow cell ( Ruzicka & Hansen 1975).
J. Ruzicka & E.H.Hansen, Anal. Chim. Acta , 78, 145 (1975)
J. Ruzicka & E.H.Hansen, Flow Injection Analysis 2nd ed. J. Willey, N.Y. 1988

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1.1.1

SINGLE STREAM MANIFOLD

SIMPLEST , MANUALLY OPERATED SYSTEM
IS COMPRIZED OF:

peristaltic pump
manually operated two position injection valve
manifold of connectors tubing and reactors
flow through detector
Basic FI instrument furnished with a tungsten light source and
spectrophotometer, is well suited as a training tool in an
undergraduate laboratory or for assay of small sample series.
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1.1.2.

TRAVEL TIME

Peak height is the most frequently used response for construction of a calibration
curve. Depending on the flow rate and reaction rate this readout is often available
within less than 30 seconds after sample injection. With a sampling frequency of up
to 120 s/hour, thousands of samples are analyzed within a week in routine
Laboratories, where FI system is usually coupled with an autosampler. Peak width
(W) is readout for FI titration, while peak area (A) is used infrequently.
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1.1.3.

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1.1.4.

Since many assays use several reagents that must be added in a given sequence,
FI systems use multichannel pumps that propel carrier stream along with reagent
streams. This allows reagents to be added continuously to injected sample at a
desired concentration, so that reactions can be carried out in sequence as the
sample zone passes through the first and second reactor. A majority of FI systems
use peristaltic pumps that allow flow rates of carrier and reagents to be controlled
by choosing pump revolution rate, and by selecting pump tubes of a desired
diameter. A typical flow rate is 0.5 -1,5mL/min of individual streams, while an
injected volume is selected in a range of 25 to 100L.
Multistream FI system are routinely combined into multichannel systems, where
each channel is dedicated to a different chemical assay. Typically a three channel
system allows , phosphate, nitrate and nitrite to be analyzed simultaneously in
water and soil sample extracts.
Yet another advantage of multistream FI systems is their versatility, that allows
automation of solvent extraction, dialysis, and gas diffusion based assays.
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1.1.5.

Fully automated FI analyzer

furnished with an autosampler
comprises a four channel peristaltic
pump, injection valve and an
integrated manifold with a z-type
flow through cell. For spectrophotometic
measurements, the flow cell is
connected by fiber optics to
a tungsten lamp and
a scanning spectrophotometer.
A@540nm 8
The attached readout shows a
calibration record of 0,2,5 and
8ppm nitrate, followed by a
routine run of nitrate assay
in soil samples using cadmium
reduction column and sulfanilamide
reagent.

2
0
seconds
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1.1.6

OPTICAL
FIBER

WASTE
MIXING
COIL #2

SAMPLE

MIXING
COIL #1
SAMPLE
LOOP
CARRIER

REAGENTS

FLOW CELL INJECTION VALVE

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PRINCIPLE
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1.2.1.

This section is designed to provide an understanding of processes

that yield FI response curve, and to offer tools for optimizing sensitivity,
detection limit and sampling frequency of flow injection based assays.
We begin with definition of three cornerstones on which all
flow injection techniques are based:
sample injection
controlled dispersion
reproducible timing
and will continue with examples how these parameters are controlled
and manipulated through change of injected volumes, flow rates and
manifold configurations. While single reagent assays can be performed
using the simplest, single stream manifold, it will be shown why a majority
of FI techniques use multistream manifolds, where several reagents are
sequentially merged with a carrier stream that moves the injected sample
zone through the manifold and a flow cell. Note that discussion in the following
sections deals with a single stream system. For multistream systems
D-values have to be corrected by dilution caused by additional streams.
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1.2.2

Blue dye injected into a single stream manifold forms a concentration gradient, as it
flows through a coiled reactor. Peak profile (B) recorded by a colorimeteric
measurement @620 nm shows a gradient profile, on which reproducibility of flow
injection assays is based. Profile of a concentration gradient is shaped by injected
volume, dispersion processes in the flow channel and by the resident time of the zone
traveling between injector and detector.

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1.2.3.

SAMPLE INJECTION

CONTROLLED DISPERSION

REPRODUCIBLE TIMING

Sample injection provides the initial

Co
square input serving as a staring point
for initial concentration ( Co) and startup
o
T
time.
SAMPLE
Controlled dispersion takes place as the sample zone moves downstream through the manifold.
This process forms a well defined concentration gradient that can be viewed as continuum of elements
of fluid with different concentrations, where the highest one ( Cmax) corresponds to peak maximum.
Since it is convenient to locate peak maximum, most FI methods use this element of fluid as a readout
In order to optimize a given assay it useful to know how much the sample has been diluted in the FI
system and how much time was available for chemical reactions to proceed. Therefore the dispersion
coefficient has been defined a s D= Co/ Cmax allowing the degree of sample dilution
to be estimated . Similarly T max is the time elapsed from the moment of injection
To to the moment of peak maximum T max. Reproducible timing of sample travel
from injection to detection yields repeatable value of Tmax.
Cmax

Tmax
DISPERSED SAMPLE ZONE
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1.2.4.

Four stages of dispersion process shown above depict the underlying physical principle of FI
at continuous forward flow. For success of a reagent based assay, it is essential to design the
flow system in such a way that the degree of the axial dispersion is controlled to suit the
purpose of a planned assay, while the radial dispersion is designed to provide an efficient
mixing of sample zone with reagent supplied by carrier stream.

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1.2.5.

Flow Injection response is a result of two processes, both kinetic in nature:

the physical process of dispersion of the sample zone and the chemical process
of formation of a detectable species. These two processes occur simultaneously
and they yield , together with the dynamic characteristics of the detector the FI
response curve. Note that the reaction product (yellow), is gradually formed at the
interface between the sample zone (red) and carrier stream of reagent (blue).
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1.2.6.

Reaction product (yellow) is formed at interface

between sample (red) and reagent (blue).

Injection of increasing sample volumes causes

formation of a double peak..

It is important to select the degree of axial dispersion in such a way that a sufficient
amount of reagent is available through entire zone length to form a reaction product. In
a s single stream manifold (A), a double peak) will be recorded if the injected sample
volume (and its length) will cause a lack of reagent in the center of the zone (B), and
formation of a double peak (C).
To avoid this problem, dispersion coefficient of a system should be determined
and adjusted accordingly. This can be done by either injecting a smaller sample
volume, or, by using a two stream system furnished with a confluence point

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1.2.7.

As the injected zone (A) moves

downstream, it disperses
forming a concentration
gradient that can be viewed as
composed of a continuum of
concentration segments of
individual concentrations C.
Of these segments, the one
situated at peak apex ( Cmax) is
the one on which peak height
measurement, and calibration,
will be based.
Dispersion coefficient ( D )has been defined as a ratio of C0 / C max and the
flow injection systems are designed to yield
dispersion of the injected sample
Note that for D=2 a sample segment situated atop the peak, has been diluted to half of
its original concentration, by carrier solution .
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1.2.8.

When the distance between injector

and detector is minimized in a single
stream FI system, injection of a
sufficiently large sample volume
will produce a square peak that
will have a horizontal steady state
section with C max concentration
situated at its top.
Systems with limited dispersion are
designed for automation of all
reagent based assays.
or time
conductivity measurement
for direct sample injection in high sensitivity ICP and AA based assays
for bioligand interaction studies by surface plasmon resonance (BIA)
for functional receptor binding assays on live cell for drug discovery.
pH measurement
Note that in a multistream system, D value is always > 2 as the flow
rates of at confluence points have to be taken into account.
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1.2.9.

By adjusting the volume of injected

sample, of the volume of reactors
between injector and flow cell and
of flow rates in single or multistream
manifolds, dispersion can be adjusted
to a medium value. Resulting
concentration gradient will have
a form of a smooth peak that will be
only slightly skewed.
Systems with medium dispersion are
designed for automation of all reagent based assays. Note that in order
to reach high sensitivity of a colorimetric assays:
sample volume should be maximized
reagents should be added by reagent streams via confluence points
long path flow cell should be used
dispersion coefficient should be adjusted to between 2 and 5
NOTE: Sensitivity and detection limit of reagent based assays can
be further enhanced by Bead Injection Technique .
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1.2.10.

By decreasing volume of injected

sample, and by increasing the volume
of conduits between injector and flow
cell, dispersion can be increased
to a large value. Resulting
concentration gradient will have
a form of a smooth long peak that will
have an exponentially decreasing tailing
edge. In order to obtain very large D
values a mixing chamber should be
integrated into flow manifold.
If the volume of a mixing chamber dominates the volume of the flow channel
the resulting concentration gradient will have a exponentially decreasing
trailing edge if the system is operated t continuous constant flow rate.
Systems with large dispersion are used for process control monitoring
when extensive sample dilution is required and for automated titrations.
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1.2.11.

Increasing the injected sample volume increases

peak height, until a steady state plateau is reached.
Up to D =2 value (in a single stream system), peak
height increases linearly with the injected sample
volume. The sample volume needed to reach 50% of
the steady state depends on the volume, geometry
and flow rates in the channel between injector and
flow cell. For conventional FI systems this value is
around 50L, for micro SI systems as low as 5 L .
If the radial mass transfer is incomplete, the resulting
peak shape is composed of two exponential curves,
as shown here. With increasing efficiency of radial
mixing, gradients are reshaped and follow erf function, ultimately approaching
Gaussian shape ( see Section 0.2.2. ).Changing injected sample volume is
versatile, and convenient tool for adjusting dispersion coefficient and for
optimizing the sensitivity of flow injection based assays.
Recording shows traces obtaining by injection bromothymol blue solution into 0.5mmI.D tubing, 20 cm
long, at a flow rate of 1,4mL/min in a single stream system. Spectropotometry at 620nm.
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1.2.12.

Selection of injected sample volumes

is a powerful tool for optimization of all FIA
techniques. It allows :
. Selection of sensitivity and detection limit

2.0

1.5
2.0

40

Identification of the linear range of a detector

1.0
1.5
Automated dilution of sample material Absorbance

in Sequential Injection sample volume is

determined by the volume of stroke reversal of
a syringe pump. This allows automated selection
of injected volumes by means of software control.

L BTB

30

(as shown here on spectrophotometry of a dye)

Volume of a sample solution injected into

conventional FI system is accomplished by
manually changing volume of the sample loop.

50

516nm
567nm

20

0.5
0.5
10

0.0
0

100 200 300 400

TIME, sec
Automated injection of increasing sample volumes.
Bromothymol Blue dye monitored at two
wavelengths. (Sample 0.002%
002% BTB, carrier 0.005M
005M
sodium teraborate.

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1.2.13.

Increasing length of a tubular channel decreases

peak height while peak shape undergoes a change
from asymmetrical to symmetrical shape. At the
same time the resident time of the peak maximum
increases with the distance traveled and the peak
base broadens. This is the principal limitation of
FI based on constant forward flow, as the constant
flow rate limits the incubation time for chemical
reaction to about 20 seconds, with a total conduit
length of about 250cm, and combined flow rates
around 3 mL/min. (The recording here was obtained at a flow rate of
1,4mL/min, sample volume of 60L in a way described in the previous
slide).
The use of programmable, instead of continuous flow, allows incubation
time to be prolonged by stopping the flow, and speeding up the system
wash by accelerating the flow. Programming the flow makes Sequential
Injection technique more versatile than FI.
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1.2.14.

Since all chemical reactions are time dependent, reproducible timing

of sample handling operations is critical to success of flow based chemical
assays, as in this format reaction equilibrium is not necessarily achieved.
INJECTOR

INJECTOR

DETECTOR

DETECTOR

STOP

At continuous flow the time interval

available for chemical reaction to
take place is defined by linear flow
velocity and is limited by the length
of conduit between point of
injection and detector. Although
longer tubes allow longer reaction
time the yield is offset by dilution
due to increase in sample zone
dispersion.

Stop flow allows longer reaction time

without penalty of dilution, thus yielding
higher sensitivity. It saves reagents and
generates less waste than continuous
pumping. It allows miniaturization by
minimizing the length between injector
and detector. It provides information on
reaction kinetics through reaction rate
measurement.
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TECHNIQUES
IDEA
APPLICATIONS
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1.2.15.

DETECTOR

The stop flow mode is

based on arresting a selected
portion of the sample zone in the
detector. Provided that the reaction
did not reach equilibrium while the
zone was on the way to detector,
reaction rate curve will be recorded
while the reaction product (yellow)
is being formed in the detector.
Next, flow is resumed and
reacted sample zone is flushed out
of the detector, while the baseline
is restored.
While stop flow technique has been
used in FI format, it is difficult to
carry out reproducibly when
peristaltic pumps are used propel
carrier and reagent streams.
Syringe driven systems either FI or
SI are reliable and their use in
stopped flow mode is highly
recommended.
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1.2.16.

FLOW
BLANK

DELAY TIME

Since the analyte (red) disperses as the

sample zone travel downstream, the thus
formed concentration provides numerous
sections from which an analytical signal
can be recorded. This can be in following
ways
Zone sampling relies on diverting a
desired diluted section from the
mainstream by a valve into a secondary
manifold for further processing
Electronic dilution is based readout
obtained at the tail section of the peak,
rather than on peak maximum. This is
useful, when high analyte concentration
causes readout to be out of detector
range .
Stop flow is the most useful and effective
approach for reaction rate based assays.

S REPRESENTS SAMPLE PROFILE

I IS INJECTION POINT. DELAY TIME
DEFINES SELECTION OF GRADIENT PORTION.
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1.2.17.

ANALYTE

BLANK

DELAY TIME

FLOW

Delay time between sample injection

and commencement of the stop flow
period determines which section ( )
of the sample zone will be arrested in
the observation field of a detector ( )
for reaction rate measurement. Since
the analyte (red) disperses within the
reagent stream (blue) on the way to
the detector while the product (yellow)
is being formed, it is essential that
the delay time is perfectly reproduced
for each assay. In the example shown,
longer delay times will yield lower
slopes since tail sections of the
sample zone are more diluted, while
shorter delay times ( up to peak
maximum) will yield steeper slopes. In
the absence of analyte horizontal
(blank) line will be observed.
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1.3.1.

GLUCOSE OXIDASE

Glucose + 2H2O + O2

Gluconic Acid + H2O2

PEROXIDASE

2 H2O2 + 4-Aminoantipyrine + p-Hydroxybenzene

Sulfonate Quinoneimine Dye + 4H2O

Enzymatic assay of glucose, monitored at 505 nm

is carried out by reaction rate measurement
during a stopped flow period lasting 20 seconds
while data are collected for construction of a calibration curve.
Using a single stream flow scheme, FIAlab 2500 and
associate software series of standards containing 500,
1000, 1500, 2000 and 2500 ppm glucose were injected
yielding response curves shown on the right.

Note that the travel time between injector

and detector has been minimized in order
to carry our the reaction within the flow
cell, while the pump has been stopped.

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1.3.2.

A majority of FI assays are carried at continuous flow, when carried and reagents
are pumped simultaneously at a constant flow rate. Sample is injected into carrier
stream of water (or appropriate buffer) while reagent streams are added at confluence
points. Advantages of this approach are:
even addition of reagents to entire sample zone length
steady baseline
minimized carryover
simplicity of operation and transparency to user.
The main drawback of FI is continuous reagent consumption and waste generation
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1.3.3.

Merging of reagent and carrier stream

Two reagents, three stream FI system

Almost all FI instruments employ multichannel peristaltic pumps to move carrier and reagent
( R1,R2) streams that merge at confluence points ( ) where reagent merges with sample
zone. Sample is injected by means of a two position injection valve with a fixed injection
loop. The valve is furnished with a bypass (not shown) that allows carrier solution to pass
through the valve, while the sample is being filled into the loop. The pump moves solutions
continuously in forward direction, thus providing a repeatable time frame for samples and
standards as they are serially injected. In this way all samples and standards are processed in
exactly the same way and the standards yield a readout used for construction of a calibration
curve.
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1.3.4.

Three stream FI manifold where

at the first confluence point ( )
molybdate is added to stream of water,
that carries injected sample of phosphate
to be analyzed. At the second confluence
point ( ), ascorbic acid is added to form
phosphomolybdenum blue. Since
molybdate/ascorbic acid mixture
A@720nm
decomposes rapidly, forming a blue
product, these reagents must be stored
separately and added sequentially to the
carrier stream. Use of water as carrier
stabilizes baseline and improves detection
limit for assay of phosphate in water
based samples.
This method, yields sample
frequency of 80 s/h and is one of the most
frequently performed FI assays. It is even
more effective when miniaturized into SILOV format, as waste generation and
reagent consumption is reduced 50 times.

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1.3.5.
Agricultural and environmental analysis of water, soil and fertilizers for assay of
ammonia, nitrate, nitrite, phosphate, chloride, iron , chromium ( IV and VI) and
cyanide are routinely carried in test laboratories on a very large number of
samples by FI. These reagent based assays are all based on spectrophotometric
detection in VIS region and their limit of detection is adjusted by selecting
reaction conditions and the length of flow cell path. A comprehensive review
( Puchades 1991)of sea water FI analysis of anionic and organic species lists
detection limits for nitrate 0.1M, sulfide 1,2mM and phosphate 0.05M.
Trace analysis of metals by atomic spectroscopies (AA, HGAAS, ICP and ICP-MS)
uses FI as a front end sample processing system in two ways. The most
significant method is based on hydride generation, that converts target analytes
into volatile metal hydrides, leaving matrix interferences in a sample solution.
Three monographs and a large number of papers deal in detail with FI based
hydride generation ( see FI based separations). Yet another, simple use of FI is in
assaying sea water, saline, fertilizers and serum, i.e. samples that cannot be
continuously pumped into nebulizers of AA or ICP instruments, as high content of
water soluble salts will crystallize and block the nozzle. By injecting small, well
defined sample volumes into carrier stream of water this problem is eliminated.
Pharmaceutical and enzymatic assays with UV-VIS or fluorescence detection is
yet another area, where FI is routinely used in a large scale for quality and
process control. These application are reviewed and summarized in monographs
of Catalyud and Trojanowicz.
J. Atienza, M.A.Herrero, A.Maquieira and R. Puchades, Critical Rew. Nal. Chem 22(5) 331-344 (1991)
Calatayud J.M.: (Ed.), Flow Injection Analysis of Pharmaceuticals,, Taylor & Francis, London, 1997.
Trojanowicz M.: Flow Injection Analysis, Instrumentation and Applications, World Scientific Ltd., Singapore,
2000.
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1.3.6.

Multistream FI systems are ideally suited for automation of all separations

based on partition between two phases. This section deals briefly only with
gas/ liquid separations and
solvent extraction
while ion exchange, sorbent extraction and other microcolumn based
separation and conversions (enzymatic, redox etc) are too numerous
to be reviewed here.
An excellent , detailed review of FI based separations is found in the
following monographs:
Fang Z-L.:, Flow-Injection Separation and Preconcentration, VCH
Verlagsgesellschaft mbh, Weinheim, 1993.
Trojanowicz M.: Flow Injection Analysis, Instrumentation and Applications,
World Scientific Ltd., Singapore, 2000.
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1.3.7.

Hydride generation based Atomic Spectroscopies are routinely used for trace analysis of
As, Bi, Ge, Hg, Pb, Se, Sn and Te, while assay of volatile compounds of Ag, Co, Cu, Ni, and
Zn has been reported in research publications. Advantages of hydride generation:
separation of the trace metals from complex matrices, analyte enrichment, fast reaction
speed, and ease of automation were first demonstrated by Astrom in his pioneering work
on FI based hydride AA assay of bismuth. By combining an acidified sample stream with
a strong reducing agent (sodium borohydride), hydrogen and metal hydride is rapidly
released and the gaseous phase is separated with aid of purging gas ( air or argon) and
swept into the detector. Atomic absorption spectroscopy , cold
vapor atomic absorption spectroscopy , inductively coupled
plasma spectroscopy , as
well as inductively coupled
plasma mass spectrometry
have been used s detectors
The key component of the
design is the gas-liquid
separator.

Fang Z-L.: Flow Injection Atomic Spectrometry, Wiley, Chichester, 1995.

Sanz-Medel A.: (Ed.), Flow Analysis with Atomic Spectrometric Detectors, Elsevier, Amsterdam, 1999
Burguera J.L: (Ed.) Flow Injection Atomic Spectroscopy, Marcel Dekker, New York, 1989
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1.3.8.
There are two types of separators: gas expansion separator and membrane separator.
Gas expansion separators are most frequently used, as they are robust and easy to
construct and maintain. The entire separator, or at least its vertical tubular body is
made of glass, and often partially filled with large glass beads, as hydrophilic surface
of glass assists in gas liquid separation. Carrier/hydrogen/hydride stream is confluenced
with purging gas ( air, nitrogen, or argon) that sweeps the liquid within the separator and
carries the released volatiles into the detector. The level of liquid in the separator is
maintained by external pump. Gas expansion separators are operated at high flow rates;
combined flow of sample, reagent and carrier is up to 15mL/min and purging gas flow rate
of 30mL/min is not unusual. Membrane separators rely on gas diffusion through a
hydrophobic membrane and offer higher sensitivity at lower flow rates, since their
internal gas volume is much smaller. When integrated with a flow cell for cold Hg
assay, they offer an excellent sensitivity and detection limit ( Fang 1988 ).

Membrane separator.
Gas
expansion
separator
Fang, Z.-l.; et.al., Anal. Chim. Acta
1988, 214, 41-55.

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1.3.9.

In a two stream FI system, sample containing carbonate (or dissolved carbon

dioxide) is acidified, releasing carbon dioxide, that diffuses across a silicone
rubber made membrane from a donor ( blue) to an acceptor (green) stream
changing color of an acidobasic indicator, monitored at 430nm (Baadenhuijsen
1979). Membranes made of Teflon are hydrophobic, with up to 50%porosity,
forming an air gap between carrier and donor stream through which gases like
ammonia, sulphur dioxide, chlorine, ozone or volatile compounds rapidly
permeate into an acceptor stream where they are detected by means of a
suitable reagent. Flat plate diffusers, ( as the one shown above) are easy to
assemble. The drawback hydrophobic membranes is that they can be fouled by
surfactants that destroy the air gap barrier. When miniaturized and integrated with
a fiber optic detector, placed into acceptor channel, asandwich cell construction
allows increase of sensitivity of an assay.
Another, innovative approach to gas separation is gas pervaporation, that offers a
robust alternative to gas diffusion in parallel plate diffuser (Castro 1998)
H. Baadenhijsen & H.E.H. Seuren-Jacobs, Clin. Chem. 25, 443, (1979)
M. D. L. de Castro & I. Papaefstathiou, TRAC, 17, 41, (1998)
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1.3.10.

This flow cell design uses a bifurcated optical cable to

illuminate a white surface and to collect reflected light
as it passed twice through the monitored aqueous layer.
This flow cell can be used to monitor either a single ,
liquid stream, or if furnished with a gas permeable membrane,
(M) mounted between two spacers (A,B) it is useful to monitor
volatile species emanating from a donor stream.
Note that Teflon membrane may be furnished with an opening ( )
situated downstream from the fiber, to alleviate pressure differences
between acceptor and carrier streams. Note that stopping the flow of
acceptor (indicator) stream allows accumulation of analyte and increase
of sensitivity of measurement. (Pavon et. al. 1992)

A
B

M
J.L.P.Pavon et.al. Anal. Chem. 64, 923 (1992)
C. G. Pinto, M. E. F. Laespada, J. L. P. Pavon and B. M. Cordero Analytical applications of separation techniques
through membranes Lab. Autom. Inf. Managem., 34(2) 115-130 (1999)

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1.3.11.

Two stream manifold for

automated solvent extraction. Sample
(S) is injected into a moving carrier
stream of water (AQ), which is merged
(a) with an organic phase (ORG) and
pumped through a Teflon made
extraction coil (b). In separator (c) the
aqueous phase is discarded into
waste, while organic phase is led into a
flow cell. Detail showing circulation of
extracted dye within segment of
organic phase (Nord & Karlberg 1984),
as it moves through a Teflon tubing, provides
clue to mechanism of hydrodynamics of solvent extraction.
This method, applicable to assay of hormones, pharmaceuticals and
numerous hydrophobic compounds, (Karlberg & Thelander 1978), revolutionized
solvent extraction technique, that up to that time was mostly carried manually.
Miniaturization and automation of solvent extraction minimizes exposure to
harmful solvents and reduces consumption of reagents and generation of
hazardous waste.
B. Karlberg & S.Thelander, Anal. Chim. Acta 98, 1 (1978)

L. Nord & B. Karlberg, Anal. Chim. Acta, 164, 233 (1984)

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1.3.12.

ORGANIC PHASE
HEAVIER THAN
WATER

b
ORGANIC PHASE
LIGHTER THAN
WATER

Choice of materials for manifold components and their orientation is critical because
aqueous phase (aq) adheres to glass, while organic phase adheres to Teflon.
In segmentor organic phase enters through a glass fitting and adheres to Teflon tubing (1).
In separator a thin Teflon strip (3) serves to guide organic phase through a glass made T piece. In the
membrane separator Teflon made membrane allows only the organic phase to penetrate through
hydrophobic pores, while aqueous phase is discarded.
Karlberg B. Pacey C.E.: Flow Injection Analysis, A Practical Guide, Elsevier, Amsterdam, 1989.
Fang Z-L.:, Flow-Injection Separation and Preconcentration, VCH Verlagsgesellschaft Weinheim, 1993.
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INSTRUMENT
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1.4.1.

In the early days from 1974 up to mid 80 a vast majority of Flow Injection instruments was home
made from components found in the lab or purchased piecemeal. This was because most researchers
found challenge and joy in innovative design of their own systems and also because the commercially
available systems were quite expensive. With advent of computers, however, a significant change took
place, since software became a key component of a successful design.
Initially, in research laboratories, and especially in Academia , a whole generation of graduate students
became victim of necessity to create home made software, while their supervisors became in turn
victims of their former graduate students, who left behind software bundles, that no one could unravel
It is not a trivial task to design and to write software package that does control instrument functions,
that does provide flexible timing of events, and controls peripherals such as spectrophotometers,
external pumps and valves while collecting and evaluating data in a real time.
Today versatile software is commercially available that accommodates peripherals added to core
Instrument. Such open architecture allows FI instrument to be assembled for virtually any research task
or a specilaized assay. For advanced detectors ( AA, ICP), patches are available that allow to bridge the
gap between FIAlab or LABview software and detector with proprietary software drive. Therefore it is no
longer necessary to waste time by composing home made programs. Indeed, to do so is irrational as
would be writing of a personal version of a word processing or slide presenting program.
For routine, serial assays such as soil water or environmental analyses, a several commercial
instrument packages from FIAlab or Lachat Instruments is available.
All commercially available FI instruments were recently reviewed (Smith 2002), including
prices, special features and available peripherals.
J.P.Smith & V. Hinson-Smith, Anal. Chem. 74, 385A ( 2002)
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1.4.2.

Single channel, three stream FI system, with two reaction coils and fixed volume loop injection valve
is the configuration, most frequently used for automation of reagent based chemical assays. While
continuously pumping FI systems were in the past operated manually, and their response was recorded
on a chart , modern systems use automated two position injection valves, and computer controlled
peristaltic pumps as well as computerized data collection.
Since UV-VIS spectrophotometry is the most frequently used detection technique, fiber optic flow cells
with a 10mm optical path coupled to software controlled solid state spectrophotometer are now
common, replacing earlier designs with filter photometers. For teaching and single purpose
assays, where a single wavelength is sufficient light emitting diodes offering yet another practical
alternative.
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1.4.3.

Integration of manifold components allows miniaturization

and optimization of flow channel dimensions in order to
minimize sample and reagent consumption. Integration of
valve with the sample processing channel and a flow cell
was originally suggested as a tool for miniaturization of
Sequential Injection technique ( See Section 2). In FI format
such lab-on-valve platform is used to streamline manifold
components ( valve, tube fittings, confluence points and
flow cell), while reactor coils #1 and#2 are mounted externally.
The advantage of this construction is that it makes function
of the manifold transparent to the user, and for routine
assays provides a format that is easy to reproduce, so that
when a standard serial assays ( such as phosphate, nitrate etc.)
is optimized on one instrument, it can be transferred to other
instruments in another location with ease.
The FI-LOV configuration shown here is designed for two
reagent assay using 50 cm and 100cm long reaction coils,
fiber optic flow cell with 10mm light path and 50 L sample
Injection loop. Eight roller four channel peristaltic pump
is used to fill sample loop, to propel the carrier stream
and two reagent streams.

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1.4.4.

Replacing peristaltic pump with four channel syringe pump is a logical extension of FI-LOV
instrument development. Advantages of using syringe pumps for FI applications have been
recognized by Japanese researchers long time ago (Yoza 1977) and the use of Multisyringe
Flow Injection Systems (MSFIA) has been proposed in numerous publications (Cerda 1999).
However, use of peristaltic pumps for FI applications is deeply entrenched, and it is likely to
prevail in routine laboratories, because of cost, convenience of operation, and ease of
replacement of peristaltic tubing. Yet, an instrument build around individually driven syringe
pumps combined with solvent resistant LOV
module has following advantages:
resistance to corrosive chemicals
precise control of liquid delivery and manipulation
capability of programmable flow, including
stop flow FI for reaction rate measurement.
The main drawback of using multiple syringes
is mechanical complexity, as compared to the
conventional FI system. Also microSI instrument,
is far less complex as it operates with a only a single
pump and a single valve. Indeed, unless all four
pumps will be run in a fully synchronized and
automatically cycled mode, the flow programming
of this novel instrument configuration will be
a challenging task.
Yoza N., Ishibashi K., Ohashi S. J. Chromatography134, 497 (1977)
Cerda V. et. al. Talanta 50, 695 (1999)

Miro M., Estela J.M., Cerda V., TRAC 21, 199 (2002)
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1.4.5.

For teaching applications one may wish to construct a simple robust inexpensive
system, with replaceable components. Such instrument does not have to be
computer controlled, if it uses peristaltic pump and two position manually operated
injection valve . An interesting alternative to peristaltic pumping and valve injection
is the use of solenoid driven pumps (1.4.6) that, however, need a simple software
and computer control for flow rate selection and sample injection.
For research applications there are almost infinite combinations possible of
available components. To begin with, the most important is the choice of software,
as it has to be compatible not only with the instrument, detector and other
peripherals, but also with the user itself. Buying valves, pumps etc and connecting
them with a tubing is the easiest step. To make these components work in concert
is quite another matter. The key to success is in designing simplest possible system
with smallest number of components and then simplify it further. Remember that:
Once you exhausted all possibilities, there is a simple solution highly visible to
everybody else, but you ( Murphys Law).
The most practical way to approach construction of a research instrument is to
purchase a core unit, driven by software with and open architecture and to add
desired peripherals as the project gradually develops. Make sure that the peripherals
you intend to use are compatible with the software before purchasing the core unit.
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1.4.6.
Peristaltic pumps are still the most frequently used drives for FI systems, since they generate
continuous flow in any desired number of parallel channels. while the flow rates can be easily adjusted
by rotation rate and I.D of peristaltic tubing. It is important to use a pump furnished with at least eight
rollers, in order to generate a flow with small regular pulses as otherwise resulting irregular flow rate
will affect dispersion and repeatability of assay. Contributing factor to popularity of peristaltic pumping
is its apparently low cost, although cost of peristaltic tubing exceeds many times the price of a pump
over its lifetime. The largest drawback of peristaltic pumping is due to elasticity of peristaltic tubing as
the flow rates gradually change as the tubing is stretched out, requiring frequent recalibration of the
analyzer.
Stepper motor driven syringe pumps generate highly reproducible flow that can be computer controlled
in a programmable way. They cover a very wide range of flow rates as the piston speed and syringe size
can be varied. They are durable and chemically resistant, their only drawbacks being cost and inability
to generate continuous flow beyond the capacity of the syringe that has to be refilled.
Solenoid activated micro pumps generate flow by delivering well defined pulses the frequency and
volume of which controls the flow rate. A typical FI pulsed flow system (Rangel 2005) used 8L pulses
in three stream, three pump system generating flow between 0.48 to 1.92mL/min., depending on pulsing
frequency (60 to 240 pulses/min). The weakness of this truly innovative approach is durability of these
pumps that must generate about 300.000 pulses/day while exposed to aggressive chemicals.

Solenoid Pump.
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Santos J.L.M, Clausse, A.,Lima J.L.F.C., Saraiava M.L.M.F.S.,Rangel A.O.S., Analyt. Sci. 21, 461 (2005)

Peristaltic pump

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1.4.7.

While I.D. of 0.5mm to 0.8mm is typical for majority of FI and SI systems, there is a wide
variety of tubing materials available for constructing reactor coils and connection lines.
Teflon and Peek are the most frequently used polymers. Stainless steel is yet another
material that has advantage of heat conductivity gas impermeability and surface
properties that minimize protein adsorption. A majority of polymer made tubing is
transparent and often available
color coded, so that tubing I.D. can be identified at glance.
Connectors made of colored coded polymers are fitted with ferrules that are designed
to grip tubing while the connector nut is being tightened. Since all FIA systems operate
at a low pressure, there is not necessary to use connectors designed for HPLC. It is,
however very important to use nuts, ferrules and fitting from a single manufacturer as
products from different sources are often incompatible, resulting in a leak.

Tubing connectors, ferrule

and T-connector

Teflon made reactor coil .

Heated reactor coil with

temperature controller.

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1.4.8.

LOAD

LOOP

Two position, six port injection valve with a fixed loop is the most frequently used tool for injection of well defined
sample volumes. Volume of the external loop (shown above) can is selected between 20 and 100L by changing the
length and I.D of the loop tubing. The valve can be switched from load to inject mode manually or automatically and the
loop can be filled either manually by syringe, or automatically from an autosampler by means of a pump (above). It is
important to keep the length of the conduit between sample container and port #4 as short as possible in order to
save sample material, and to avoid sample to sample cross contamination. Introducing air bubble and wash between
samples is useful, but requires exact timing so that the injected volume is air free and contains undiluted sample.

Six port multiposition valve combined with a stepper motor driven syringe pump is the key component of all
Sequential Injection systems ( See Section 2). It allows injected volumes to be chosen at will, and at a selected
flow rate. This injection mode is an ideal tool for automated optimization of FI and SI based assays.
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1.4.9.

Sample volume and reaction time are the most

important parameters of flow injection experimental
protocol. By changing injected volumes and reaction
times sensitivity and detection limit of reagent based
assays can be adjusted to desired level.
Since conventional FIA employs a two position valve
furnished with fixed sample loop volume, injected
volumes cannot be automatically selected by
a computer.
Variable volume injection removes this limitation
allowing automated optimizationof assay
parameters. The key difference is in that
the injection system based on a multiposition valve
and the volume of injected sample is
controlled by a syringe pump.
Injected volumes are controlled by high precision syringe pump that aspirates selected volume of
sample solution from sample cups, while the central port is connected to port #4. (The auxiliary
pump serves to transport sample solution from sample cup just past port #4, whenever next
sample change is to be injected).
The volume of sample solution to be injected is determined by:
the volume of the reversal stroke of the syringe pump, and
the volume of the forward stroke of the syringe pump, when central port is connected to port #2 .
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1.4.10.
FLOW
INJECTING AN ENTIRE SAMPLE VOLUME
If selected volumes of reversal and forward stroke are
identical, not all sample material will be injected into
the sample processing manifold, because the sample
forms a concentration gradient (A) in the sample
holding coil. Since the central stream moves at a
double of average flow velocity, sample zone occupies
in the holding coil twice the length of aspirated volume (B)
the upstream end of sample zone being diluted by
carrier solution. Thus, if entire sample is to be
injected into the sample processing channel, the
forward stroke should be at least twice of the reversal
stroke volume. (C).

A
B
C
D

INJECTING ONLY PART OF SAMPLE

DISCARD
ANALYZE

Smaller volumes of the forward stroke can be, injected into sample
processing manifold, but then the tail section of the sample zone
must be flushed to waste (through port # 1) in order to avoid carryover of sample
material remaining in the holding coil into the next sample processing cycle.

DILUTING SAMPLE (D).

If sample is to be diluted prior to injection into the sample processing manifold, a desired portion of
sample solution that has been aspirated by flow reversal, adjacent to the valve is directed via port #1
into waste, and than a selected section of the remaining diluted sample zone is injected into the sample
processing manifold.
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1.4.11.

Fiber optics and solid state spectrophotometers revolutionized the

way in which all FIA techniques are carried out, since this technology
allowed optimization by bringing light and collecting data from any
position of the sample flow path. While this change impacted mostly
Sequential Injection, also more traditional FI systems benefit from versatility
and robustness of fiber optic technology. A typical system comprises a
z-type flow cell connected with quartz fibers to a spectrophotometer and
a tungsten or deuterium lamp. For a single purpose systems, a light emitting
diode is mounted directly onto the flow cell.

Ocean Optics
Spectrophotometer
and a Tungtsen light source.

Z-cell with 10mm

light path

Z-cell with 10 cm light path.

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There is a vast experimental material accumulated in FI literature, yet it would be mistake to conclude
all has been done already and therefore a further original research cannot be done in this area. The
recent work of Brazilian ( Lapa 2002) and Portugese ( Rangel 2005) teams on pulsed flow FI is an
outstanding example of an innovative research, that opens a novel, practical way to miniaturization of
FI systems. Their work has a special significance, since downscaling of FI to submicroliter
level , although much tried within last ten years, has not gained acceptance, as it failed to become
applicable to real life assays. Indeed it is puzzling , why almost all microfluidic systems described
in TAS literature so far, have been designed to function on continuous flow basis, while their
proponents rediscover well known limitations. The central problem, mixing of sample with reagents at
conditions of stabilized laminar flow remains unsolved. Attempts to use osmotic or electrophoretic
pumping fail, due to different electrolytic properties of sample and reagent materials, or because the
conduit walls become fouled by real life samples.
Microreactor technology, that aims at exploring novel ways how to synthesize small amounts of rare
chemicals, or to study flow through reactor design in microscale is a research field closely related to FI
technology. (Haswell & Skelton,2000 ). In appropriately scaled version, and carried out within robust
conduits made of steel, glass or Teflon, using syringe pump drive it will benefit from
technology transfer of solvent extraction, ion exchange and gas pervaporation (Castro 1998),
techniques originally designed for FI. A joint meeting of Flow Analysts with Microreactor
Synthetists would surely not only be only inspiring, but will also advance progress of both fields.
Lapa R.A.S, Lima J.F.L.C, Reis B.F., Santos J.L.M. Zagatto, E.A.G. Anal. Chim. Acta. 466, 125, (2002)
Santos J.L.M, Clausse, A.,Lima J.L.F.C., Saraiava M.L.M.F.S.,Rangel A.O.S., Analyt. Sci. 21, 461 (2005)
S.J.Haswell & V. Skelton, TRAC, 19, 389 (2000)
M.D. L de Castro & I. Papaefstathiou, TRAC, 17, 41, (1998

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Conventional flow injection is a mature technique, that has a wide range of applications,
described in over 13.000 publications. It provides an unprecedented versatile sample
handling, along with strict control of reaction conditions. It has been applied as a front end
to practically all spectroscopic and electrochemical detectors, of which UV-VIS
spectroscopy, Atomic Absorption and Inductively Coupled Plasma Spectroscopy are most
prominent examples.
The chief advantage of FI is the transparency of its experimental setup, where sample
injection and movement through reagent addition and product detection follow a simple
route, traveled by means of continuous flow. That allows automated assay to be carried out
even without computer control, since it is the flow generated by a pump, along with
sample injection, that provide strict time framework for reaction conditions. Such control
of mixing and timing allows reagent based assays to be carried reproducibly, even if
chemical reactions involved do not reach completion.
While manually operated experimental setup would be, understandably, frowned upon by
well heeled technician armed with PC and autosampler, it should be remembered that
manually operated FI has been a workhorse of serial assays in developing countries, and it
is in any setting the best tool for teaching of principles of flow analysis, as it allows
students to perceive the interplay of kinetics of physical dispersion and chemical
reactions without unnecessary distraction provided by software or PC.
Advances in computerization has enhanced FI mainly through automation of data
collection and of calibration routines, while majority of commercial analyzers still uses
continuous flow platform, where computer control has nothing to offer. Yet, continuous
flow operation is the main drawback of conventional FI as is consumes reagents, and
creates chemical waste continuously, from the moment of instrument startup, even when
no samples are being injected and analyzed.

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The unique feature of all flow injection methods is the well defined concentration gradient
formed when analyte is injected into a carrier (or reagent ) stream. Surprisingly, this feature
still remains relatively unexplored, although it offers opportunity to automate:
analyte dilution
optimization of analyte / reagent ratio
titration
Stopped flow FI format exploits concentration gradients through exact timing of microfluidic
operations, controlled by computer and programmed through dedicated software. Stoppedflow gradients are ideal for enzymatic assays since they allow automated selection of a proper
reagent/analyte ratio for reaction rate measurement of either substrate concentration or
enzymatic activity. Stopped flow FI should be carried out using syringe pump, or solenoid
activated micro pump driven systems, as elasticity of peristaltic pump tubing makes selection
of reagent/analyte ratio difficult to maintain as the flow rate changes during the day.
In the future choice between FI mode or microSI mode will be mainly a matter of a personal
preference. Since FIA technology is already fully computerized, and since advantages of
computer control of microfluidic manipulations, are now recognized, the deciding factor might
be a more transparent mode of operation of FI, compared to microSI , since SI is logistically
more complex. This apparent drawback is , however, much offset by unprecedented savings of
time, of reagent consumption and of waste generation and versatility of programmable flow.
For a researchers microSI is the way to proceed, as it offers unexplored avenues for novel
discovery. Bead injection (BI) and SI Chromatography are a stellar examples of avenues that
opened new approaches to enhancement of immunoassays, trace analysis, pharmaceutical
assays, drug discovery and cell biology.

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