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TUTORIAL
LESSON 7
Sequential Injection Analysis
- Lesson 1: Introduction
- Lesson 2: Fundamentals of Flow
Injection Analysis
- Lesson 3: Membrane Sampling Devices
- Lesson 4: Dispersion
- Lesson 5: Enrichment
- Lesson 6: Chemistry
- Lesson 7: Sequential Injection
Analysis
- Bibliography
WHAT IS SIA?
SIA is an automated approach to sample handling that will allow
you to automate manual wet chemistry procedures in a rapid, precise,
and efficient manner. Small solution zones are manipulated under
controlled dispersion conditions in narrow bore tubing.
Sharing many characteristics with FIA, some would
argue that it is simply an extension of FIA. Nevertheless, more
than 100 journal articles have been published on SIA since the
first paper in 1990. While, like FIA, it is fundamentally dependent
on the dispersion of zones in a flowing stream, conceptually,
the practice of SIA is different from FIA.
Lets look at a simple FIA experiment and compare
it to its SIA equivalent. Consider a single line FIA experiment
where we inject a sample into a carrier stream containing a reagent.
The FIA manifold is depicted in the above diagram. A sample is
pumped into the sample loop of a two-position injection valve
and the carrier is flowing constantly through the detector. The
length of the sample loop determines the volume of sample injected.
When the sample loop is loaded, the valve is switched and the
sample is introduced into a flowing carrier stream. The carrier
carries the sample through the reactor (usually a reaction coil)
to the detector. En route, the sample reacts with the reagent
to form a detectable species. The detectable species gives rise
to a peak when it passes through the flow cell of the detector.
A calibration curve is then used with the peak height, area, or
width to determine the concentration of the analyte in the sample.
SIA on the other hand does not make use of an injection
valve. Rather, a multi-position selection valve replaces the injection
valve. Usually, the frequently used FIA peristaltic pump is replaced
with a syringe pump and an additional coil called the holding
coil is added. Compare the schematic of an SIA manifold to that
of the FIA manifold. To achieve the same measurement as described
above, the syringe is filled with carrier solution containing
the reagent. Then the selection valve is advanced to a port that
is connected to the sample line. A small volume of sample is drawn
up into the holding coil. The flow program determines the volume
of sample; viz. the volume of sample that is drawn up by the pump
into the holding coil. The selection valve is then advanced to
a port that is connected to the detector, and the carrier transports
the sample through the reactor to the flow cell of the detector.
Again, a detectable species is formed and is registered as a peak
by the detector. The concentration of the analyte in the sample
is determined in a similar manner as for FIA.
It is worth noting that at the moment of injection
in an FIA experiment, an undispersed plug of sample is introduced
into the carrier stream. In SIA, already during aspiration of
the sample into the holding coil, dispersion begins to take place
and the flow reversal that takes place when the sample is sent
off to the detector plays a dramatic role in mixing the sample
with the carrier. The next figure depicts graphically what happens
at the point of sample injection (1) in an FIA manifold (top)
and during the flow reversal inherent to SIA manifold (bottom)
and a few seconds after the carrier has started to move (2) the
sample towards the detector. This phenomenon may cause an SIA
peak to look slightly different to an FIA peak. As long as there
has been good mixing between sample and reagent, this will not
affect quantification because samples and standards are treated
alike. In fact, it has been shown that the flow reversal contributes
significantly to the mixing of zones.
To further ensure good mixing of reagent and sample,
we recommend the use of a knotted reactor such as our Super
Serpentine Reactor.
More often than not, instead of including the reagent
in the carrier, an SIA experiment is expanded so that the reagent
is loaded as a separate zone. In this case the syringe is first
filled with a simple carrier or buffer. After the sample zone
has been drawn up into the holding coil, the selection valve is
advanced to a port connected to a reagent reservoir and a small
reagent zone is drawn up into the holding coil. In this way, it
is possible to construct a stack of well defined zones which can
be mixed together to give rise to a detectable species. You will
appreciate that unlike FIA, which requires re-plumbing when a
more complex chemical addition scheme is required, in SIA, all
that is required is a change to the flow program. The manifold
remains the same. The additional advantages of lowering reagent
consumption and minimizing the production of potentially hazardous
wastes are both important advantages of SIA.
The other ports of the selection valve can be used
for calibration standards, additional reagents and as locations
where more sophisticated operations such as dilution, trace enrichment,
and incubation of reactants can take place. More about this later
in the lesson.
A variation of SIA is sequential injection titration
(SIT). In SIT, the reactor and detector in the above diagram are
replaced with a stirred titration cell. Appropriate sensors, electrochemical
or colorimetric, are placed in the titration cell which acts as
flow cell as well as titration chamber. Sequential injection titrations
are the subject of another lesson.
HOW DID SIA COME ABOUT?
SIA was developed by Ruzicka and Marshall at the University of
Washington in response to an industry-initiated requirement for
a more robust automated wet chemistry technique than FIA. At the
Center for Process Analytical Science (CPAC), industry representatives
challenged faculty and researchers to take FIA to the next level.
Develop it to the point where its strengths were maintained and
enhanced, but its limitations in the process environment were
remedied. At other institutions, the problem was tackled by investigating
the use of sensors. Other researchers explored miniaturization
of FIA. Another group at CPAC looked at the development of a hybrid
flow sensor called the Flow Probe.
In Ruzicka and Christian's group, the goal of simplifying
the manifold, reducing reagent consumption, and relying more on
micro processor controlled flow programming gave rise to SIA.
Early workers in the field had to broaden their understanding
of what makes for a good flow manifold. It soon became apparent
that a key factor was going to be an understanding of factors
that impact on zone penetration. Unlike FIA where the reagent
zone is often merged with the carrier and therefore with the sample
at a confluence point, in SIA interaction between the sample and
the reagents takes place through zone penetration. In the following
diagram, the zone of overlap represents the zone where chemical
reactions will take place and the detectable species will form.
The larger this zone of overlap, the more sensitive the measurement
will be.
Since its description in 1990, researchers have
exploited SIA for the analysis of compounds as diverse as radionuclides
and several different bio-compounds. In many cases, its advantages
when applied in the process environment have been exploited.
WHAT ARE THE ADVANTAGES AND
DISADVANTAGES OF SIA?
While SIA makes use of a simpler flow manifold (this is particularly
so for multi component chemistries), development of the SIA method
is not as straight forward. Careful attention needs to be given
to the design of the measurement sequence to ensure that adequate
zone penetration has taken place.
Accurate measurement of sample and reagent zones
necessitates microprocessor control. Of course, once the method
has been developed, the microprocessor ensures slavish repetition
of the optimized sequence.
Previous limitations associated with the use of syringe pumps (notably
the need for a syringe fill cycle, and poor precision for sample
volumes smaller than 10 l), no longer apply. Global FIA has developed
a new pump called the milliGAT™ that
has all the advantages of a syringe pump but eliminates many of
its limitations. In particular, the fill cycle and need to compromise
syringe diameter in order to allow sufficient carrier volume for
an experiment are conveniently overcome in the milliGAT™.
SIA has several advantages over FIA.
- Reagent use is drastically reduced. Typical FIA experiments
make use of at least 1mL of reagent per measurement. SIA typically
makes use of 50l. This means that in a 24-hour period assuming
one measurement per minute, the FIA analyzer would consume 1440ml
of reagent. The SIA analyzer would consume 72ml. It has been
noted that the most frequent reason for process analyzer failure
is running out of reagents.
- Flow manifolds are simple and robust typically comprising
a pump, selection valve, and detector connected by tubing. The
same manifold can be used for widely different chemistries simply
by changing the flow program rather than the plumbing. Analyzer
maintenance is therefore simplified.
- The selection valve replaces the injection valve and provides
a means for selecting different sample streams and calibrants.
This enables convenient automated calibration.
- Components used in a SIA manifolds are amenable to laboratory,
field, and plant operation. In addition to these, SIA enjoys
all of the benefits of FIA.
WHAT EQUIPMENT IS NEEDED FOR
SIA?
A typical SIA manifold has been described above and comprises
the following main components:
- Pump
- Selection valve
- Reactor
- Detector
- Software
The following picture shows a four panel FloPro
analyzer configured to do SIA.
Pump
Syringe pumps have been most widely used to aspirate zones and propel
the stack of zones through the detector. Some researchers have used
peristaltic pumps. The requirements for the pump are that it is
precise, reproducible, bi-directional, and able to measure small
volumes. Computer control is imperative. Cavro makes a reliable
syringe pump and the Global FIA milliGAT™
pump is expected to replace syringe pumps in future SIA instruments.
Selection Valve
The selection valve must allow random access of the ports. Small
dead volume and zero cross contamination between ports are essential
features of a good selection valve. Valco
valves have proved to be most suitable for SIA and these are
available with between 6 and 28 ports. The 10 port multi-position
valve is by far the most widely used.
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Reactor and Holding coil
Although the idea of knotting reactors in FIA has long been
advocated, many users of these flow-based techniques make use
of reaction coils. We have shown that if excellent mixing without
increasing dispersion is required, then a serpentine flow path
provides the optimum conditions. We have developed a Super
Serpentine Reactor and have shown that it is superior to
all present mixing reactors. |
Detectors
The wide range of detectors that are employed for FIA are suitable
for SIA. The only requirement is that they be equipped with a
flow cell. As for FIA, low dead-volume and immunity to bubbles
are key requirements. We have a range of colorimetric and electrochemical
detectors which have been developed for FIA and SIA.
Software
The crux of SIA is the flow program. This sequence of events results
in the assembly of the stack of zones in the holding coil and subsequent
transport to the detector flow-cell. Microprocessor control is imperative.
Several packages have been written to achieve this. We have a DOS-based
package that has the functionality to provide for the requirements
of a process analyzer. It is called Flow
TEK™. A Windows package called FloStar(tm) is under development.
How would I develop an SIA method?
The development of an SIA method follows a similar procedure to
the development of an FIA method. It would be worth reviewing
the Chemistry tutorial at this point to refresh your memory on
how method development is approached for FIA.
In general, the key steps in the development of
a SIA method are as follows:
Identify a suitable chemistry of measurement
- A good starting point is to establish whether there are manual
methods already in use for your samples. In addition to providing
useful insights to a chemistry that has been proven, if there
is an existing method, method validation is more easily achieved
if the chemistry applied is common to both the manual and automated
SIA method.
- Review the literature for methods to see whether there is any
published literature on the analyte of interest. In this regard,
the Flow Analysis Database
(FAD) is a valuable resource. Most of the papers on SIA thus far
have been devoted to the description of methods of analysis. Search
for both FIA and SIA methods. If a paper is found, it will provide
a useful starting point but you will probably have to optimize
conditions for your particular sample.
- Refer to a good general reference on wet chemistry measurement
chemistries. At the end of the Chemistry chapter in the Tutorial,
there is a list of recommended references. Identify a suitable
chemistry of measurement.
- If none of these resources provide a method, then the task
becomes quite challenging and requires the development of the
chemistry of measurement from first principles. That goes beyond
the scope of this Tutorial.
Determine appropriate reagent concentrations
and stochiometry
- A careful study of the method will soon reveal the reagents
that are to be used and their relative quantities.
- The sequence of additions can be conveniently duplicated in
an SIA experiment by aspirating appropriate zones of sample
and reagent and building a stack of zones in the holding coil
that will be mixed together as they are transported to the detector.
Of course, in some instances, it may be necessary to pre-mix
two reagents before exposing the mixture to the sample. It will
now be apparent that this is easily achieved using SIA. Indeed
this is one of the strengths and characteristics of SIA. Sample
manipulation is controlled by flow programming or stated in
another way, by manipulating the pump and selection valve under
microprocessor control.
- Typical volumes used in SIA are on the l scale. It is not
uncommon for the sample and reagent zones to be 20l
- A useful means of establishing what the mixing patterns are
is to sequentially introduce a suitable dye as each of the zones.
You can simply use water or any non-detectable solution for
the other zones. By overlaying the resultant dye profiles as
recorded at the detector, you can see how each zone overlaps
the others. Of course, this experiment does not take into account
the affect of chemical reactions. It simply describes the physical
zone penetration.
- Once the flow program has been established, you will have
to test whether you have sufficient reagent to ensure that the
method is not reagent starved. You do this by increasing the
reagent concentration until further increase does not result
in an increase of the signal. If it is reagent starved, you
will either have to increase the reagent concentration, or the
zone volume, or you could sandwich the sample between two zones
of reagent. Of course, increasing the zone volume is only affective
if there is adequate mixing.
Optimize all experimental parameters
- All parameters typically optimized in the development of the
chemistry of a wet chemical procedure also should be optimized
in the SIA methodology. These include the strength and pH of buffers,
addition of masking agents, reaction and incubation times, and
temperature. (Global FIA has a Reactor
Heater that conveniently accepts metal-potted Super Serpentine
reactors.)
- Physical parameters such as reagent volume, pump speed, and
reactor length also affect the performance and robustness of
the method.
Test the method using samples of known concentration
- Once the method has been developed, its analytical figures
of merit can be determined. These include
- Precision of the method. Usually 10 replicates of a sample
with concentration at mid range are measured and the %RSD
(std deviation / mean expressed as a percentage)
- Long term and inter-laboratory precision. A good measure
of the day to day a repeatability of the method and inter-laboratory
repeatability provides a measure of the robustness of the
method.
- Linear range. Standards spanning the useful range of the
method are measured and a linear regression analysis is
carried out. Goodness of fit as determined by the correlation
coefficient r2 is frequently calculated and found to be
better than 0.98.
- Detection limit. A common measure of detection limit is
the concentration that corresponds to the signal equal to
3 times the standard deviation of the detector baseline.
- Determination limit. The determination limit is usually
set at 10 times the standard deviation of the detector baseline.
- Measurement frequency. The number of measurements that
can be carried out in an hour is quoted. When quoting this
figure, any sample preparation prior to injection into the
SIA manifold should be reported separately. This figure
is derived from the time it takes the SIA instrument to
do a single measurement.
- The accuracy of the method is tested by comparing the results
obtained by an independent technique or by the analysis of certified
reference materials.
- Explore whether commonly occurring matrix elements interfere
with the measurement
What can I do with SIA?
A review of the present literature on SIA will give you
a good feel for what is presently achievable. Some of the techniques
that have been employed in sequential injection analyzers include:
- Dilution
- Trace enrichments
- Chemical masking
- pH adjustment
- Medium exchange miniature columns
- Phase exchange using membrane sampling devices
- Use of solid reagents
- Titration
A wide range of detectors can be employed with SIA. These include
- UV, vis, and ir photometers
- Fluorescence
- Chemiluminescence
- pH
- Ion selective electrodes
- Conductivity
- Amperometry
- Potentiometric stripping analysis
- Other electrochemical techniques
This completes this session of our Web Tutorial.
© Global FIA, Inc, 2003
253-549-2223
800-581-6298 (TOLLFREE)
253-549-2283
info@GlobalFIA.com
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