FLOW INJECTION ANALYSIS

Flow injection analysis (FIA) is an automated method in which a sample (analyte) is injected into a
continuous flow of a carrier solution, which mixes with other flowing solutions (reagents) before
reaching a detector. Flow injection analysis has been used to analyze a wide variety of samples,
including environmental, clinical, agricultural, industrial, and pharmaceutical samples. The majority of
analyses involve environmental and clinical samples, which is the focus of this section.

Flow analysis is the generic name for all analytical techniques that are based on the introduction,
processing, and detection of liquid samples in flowing media.

Principles

A sample (analyte) is injected into a flowing carrier solution stream that is forced by a peristaltic pump.
The injection of the sample is done under controlled dispersion in known volumes. The carrier solution
and sample then meet at mixing points with reagents and react. The reaction time is controlled by a
pump and reaction coil. The reaction product then flows through a detector. Most often, the detector is
a spectrophotometer as the reactions usually produce a colored product. One can then determine the
amount of an unknown material in the sample as it is proportional to the absorption spectrum given by
the spectrophotometer. After moving through the detector, the sample then flows to waste.

Detail of sample dispersion

When a sample is injected into the carrier stream it has the rectangular flow. As the sample is carried
through the mixing and reaction zone, the width of the flow profile increases as the sample disperses
into the carrier stream. Dispersion results from two processes: convection due to the flow of the carrier
stream and diffusion due to a concentration gradient between the sample and the carrier stream.
Convection of the sample occurs by laminar flow, in which the linear velocity of the sample at the tube's
walls is zero, while the sample at the center of the tube moves with a linear velocity twice that of the
carrier stream. The result is the parabolic flow profile, before the sample passes through a detector to a
waste container.

Detectors

A flow-through detector is located downstream from the sample injector and records a chemical
physical parameter. Many types of detector can be used such as:

1. spectrophotometer

2. fluorimeter

3. ion-selective electrode

4. biosensors

5. mass spectrometer
Instrumentation

The basic components of a flow injection analyzer are shown in Figure 13.16 and include a unit for
propelling the carrier stream, a means for injecting the sample into the carrier stream, and a detector
for monitoring the composition of the carrier stream. These units are connected by a transport system
that provides a means for bringing together separate channels and that enables an appropriate mixing
of the sample with the carrier stream. Separations modules also may be incorporated in the flow
injection analyzer. Each of these components is considered in greater detail in this section.

Propelling Unit

The propelling unit is used to move the carrier stream in the main channel, as well as any additional
reagent streams in secondary channels, through the flow injection analyzer. Although several different
propelling units have been used, the most common is a peristaltic pump. A peristaltic pump consists of a
set of rollers attached to the outside of a rotating drum (Figure 13.20). Tubing from the reagent
reservoirs is placed in between the rollers and a fixed plate. As the drum ro- tates the rollers squeeze
the tubing, forcing the contents of the tubing to move in the direction of the rotation. Peristaltic pumps
are capable of providing a constant flow rate, which is controlled by the drum’s speed of rotation and
the inner diame- ter of the tubing. Flow rates from 0.0005–40 mL/min are possible, which is more than
adequate to meet the needs of FIA, for which flow rates of 0.5–2.5 mL/min are common. One limitation
to peristaltic pumps is that they produce a pulsed flow, particularly at higher flow rates, which may lead
to oscillations in the signal.

Injector

The sample, typically 5–200 μL, is placed in the carrier stream by injec- tion. Although syringe injections
through a rubber septum are used, a more com- mon means of injection is the rotary, or loop, injector
used in HPLC. This type of injector provides reproducible injection volumes and is easily adaptable to
automation, a feature that is particularly impor- tant when high sampling rates are desired.

Detector

Detection in FIA may be accomplished using many of the electrochemi- cal and optical detectors used in
HPLC. These detectors were discussed and are not considered further in this section. In addition, FIA
detectors also have been designed around the use of ion-selective electrodes and atomic ab- sorption
spectroscopy.

Transport System

The heart of a flow injection analyzer is the transport system used to bring together the carrier stream,
the sample, and any reagents that must react with the sample to generate the desired signal. Each
reagent reservoir con- nected to the flow injection analyzer is considered a separate channel. All
channels must merge before the carrier stream reaches the detector, with the merging points
determined by the chemistry involved in the method. The completed assembly of channels is called a
manifold.

The simplest manifold includes only a single channel, the basic outline of which is shown in Figure 13.21.
This type of manifold is commonly used for direct analyses that do not require a chemical reaction. In
this case the carrier stream only serves as a means for transporting the sample to the detector rapidly
and repro- ducibly. For example, this manifold design has been used as a means of sample in- troduction
in atomic absorption spectroscopy, achieving sampling rates as high asDW700 samples/h. This manifold
also is used for determining a sample’s pH or the concentration of metal ions using ion-selective
electrodes.

A single-channel manifold also can be used for systems in which a chemical re- action generates the
species responsible for the analytical signal. In this case the car- rier stream both transports the sample
to the detector and reacts with the sample.

Because the sample must mix with the carrier stream, flow rates are lower than when no chemical
reaction is involved. One example is the determination of chlo- ride in water, which is based on the
following sequence of reactions.

The carrier stream consists of an acidic solution of Hg(SCN)2 and Fe3+. When a sample containing
chloride is injected into the carrier stream, the chloride displaces the thiocyanate from Hg(SCN)2. The
displaced thiocyanate then reacts with Fe3+ to form the reddish colored Fe(SCN)2+ complex, the
absorbance of which is moni- tored at a wavelength of 480 nm. Sampling rates of approximately 120
samples/h have been achieved with this system.

Most flow injection analyses requiring a chemical reaction use a manifold containing more than one
channel. This provides more control over the mixing of reagents and the interaction of the reagents with
the sample. Two configura- tions are possible for dual-channel systems. The dual-channel manifold
shown in Figure 13.22a is used when a mixture of the reagents is unstable. For example, in acidic
solutions phosphate reacts with molybdate to form the heteropoly acid H3P(Mo12O40). In the presence
of ascorbic acid the molybdenum in the het- eropoly acid is reduced from Mo(VI) to Mo(V), forming a
blue-colored complex that is monitored spectrophotometrically at 660 nm. Solutions of molybdate and
ascorbic acid are maintained in separate reservoirs because they are not sta- ble when mixed together.
The two reagent channels are merged and mixed just before the point where the sample is injected.
A dual-channel manifold can also be used to mix a reagent in a secondary chan- nel with a sample that
has been injected into the primary channel (Figure 13.22b). This style of manifold has been used for the
quantitative analysis of many analytes, including the determination of chemical oxygen demand (COD) in
wastewater.24 The COD is a measure of the amount of oxygen required to completely oxidize the
organic matter in the water sample and is an indicator of the level of organic pollu- tion. In the
conventional method of analysis, COD is determined by refluxing the sample for 2 h in the presence of
acid and a strong oxidizing agent, such as K2Cr2O7 or KMnO4. When refluxing is complete, the amount
of oxidant consumed in the re- action is determined by a redox titration. In the flow injection version of
this analy- sis, the sample is injected into a carrier stream of aqueous H2SO4, which merges with a
solution of the oxidant from a secondary channel. The oxidation reaction is kineti- cally slow. As a result,
the mixing and reaction coils are very long, typically 40 m, and are submerged in a thermostated bath.
The sampling rate is lower than that for most flow injection analyses, but at 10–30 samples/h it is
substantially greater than that for the conventional method.

More complex manifolds involving three or more channels are common, but the possible combination of
designs is too numerous to discuss in this text. One ex- ample of a four-channel manifold is shown in
Figure 13.23.

Separation Modules

Incorporating a separation module in the flow injection manifold allows separations, such as dialysis,
gaseous diffusion, and liquid–liquid extraction, to be included in a flow injection analysis. Such
separations are never complete, but are reproducible if the operating conditions are carefully
controlled.

Dialysis and gaseous diffusion are accomplished by placing a semipermeable membrane between the
carrier stream containing the sample and an acceptor stream (Figure 13.24). As the sample stream
passes through the separation mod- ule, a portion of those species capable of crossing the
semipermeable membrane do so, entering the acceptor stream. This type of separation module is
common in the analysis of clinical samples, such as serum and urine, for which dialysis separates the
analyte from its complex matrix. Semipermeable gaseous diffusion membranes have been used for the
determination of ammonia and carbon diox- ide in blood. For example, ammonia is determined by
injecting the sample into a carrier stream of aqueous NaOH. Ammonia diffuses across the
semipermeable membrane into an acceptor stream containing an acid–base indicator. The re- sulting
acid–base reaction between ammonia and the indicator is monitored spectrophotometrically.

Liquid–liquid extractions are accomplished by merging together two immis- cible fluids, each carried in a
separate channel. The result is a segmented flow through the separation module, consisting of
alternating portions of the two phases. At the outlet of the separation module, the two fluids are
separated by taking advantage of the difference in their densities. Figure 13.25 shows a typical
configuration for a separation module in which the sample is injected into an aqueous phase and
extracted into a less dense organic phase that passes through the detector.

Purpose of flow analysis?

Electrochemical flow analysis systems are widely used to automate and improve the throughput of
clinical, food, or pharmaceutical analysis. The design of the flow cell is a key point for analyte delivering
to the electrodes surface and enhancing the detection sensitivity.

The pumps used in flow injection systems

Peristaltic pumps are the most commonly used devices in FIA. These pumps are based on the
compression of a flexible tube by rotating rollers. Rollers are attached to a rotor in motion and compress
the tube moving the liquid inside.

Application of FIA

Flow injection analysis has been used to analyze a wide variety of samples, including environmental,
clinical, agricultural, industrial, and pharmaceutical samples. The majority of analyses involve
environmental and clinical samples, which is the focus of this section.

Typical applications of flow injection analysis include the following fields

1. Pharmaceutical application

2. Environmental analysis

E.g Sea water, Waste water, Sediments

3. Food analysis

E.g Fruit juice, soft drinks, Wine, Milk and dairy products

4. Biological material

E.g Plants, animal

5. Mineral material

E.g Soil, Fertilizers, Alloys.

6. Clinical assay

E.g Serum, Plasma, Whole blood d. Urine.

7. Bio analytical chemistry

E.g Proteins, Amino acids, Ammonia, Glucose.

Application Areas of Flow Injection Analysis

Flow injection analysis has been applied to several analysis techniques and main applications areas are
mentioned in this article.

Atomic Absorption Spectroscopy

FIAS has been adapted to AAS mainly for analysis of volatile hydride forming elements and mercury. A
stream of argon gas drives the volatile hydrides to the quartz cell which is in the beam path of AAS.
Detection limits of some elements like arsenic and antimony are improved around 100 times.

UV-Visible Spectroscopy

Analysis is carried out either by direct absorption by the absorbing species or by the product of reaction
with a reagent. Commercial UV- VIS systems with flow through detector or HPLC detector are commonly
used. Direct and indirect determininations by complexation with metal chelation agents are generally
adopted.

FT-IR Spectroscopy

Majority of applications are in the mid-and far- IR regions. Flow cells have been used with KBr windows
or Zn Se windows for aqueous samples. Gaseous compounds have been analysed using gas cells.

Turbidimetry

Turbidimetric detection is based on principle of light scattering by solutions containing solid

suspensions. Majority of applications have been developed around measurement of light scattering by
insoluble precipitates such as barium or lead sulphates.

Luminescence

Luminescence methods show a wider response and better selectivity than absorption methods.
Fluorescence agents or dyes have also been used to enhance the fluorescence on complexation.
Biotechnology

Flow injection systems have been developed along with biosensors for online monitoring of glucose and
enzyme induced processes online.

Flow injection analysis is finding its way into numerous application areas. It makes possible analysis of
samples containing high levels of dissolved solids. Sample pre-concentration or matrix removal is
achieved by adding a pre-columnl in the solvent flow path. This extends scope to speciation studies for
elements like Cr (III) and Cr (VI).

Advantages of flow injection analysis

1. Saving of time which is a major consideration for process quality control and commercial testing
laboratories

2. Improve precision over manual operations involved in sample preparation and detection

3. Reduction in analysis costs due to reduced sample and reagent consumption

4. Lowering limits of detection thereby offering ready solutions for ultra trace analysis

5. Reduced burden on disposal of waste

Disadvantages of flow injection analysis

Flow injection analysis has the drawback of being less sensitive than manual analytical methods for the
following reasons. Short reaction time; reaction may not reach equilibrium. 2. Dilution of the sample in
the carrier results in a decrease in signal intensity.
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