FLOW INJECTION ANALYSISFLOW INJECTION ANALYSIS

(FIA)

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By Assoc. Prof. Dr. Jaroon Jakmunee20 September 2012

Wet Chemical Analysis

• พัฒนาการของการวิเคราะห์ทางเคมี ที่วิเคราะห์ทางเคมี ที่อาศัยการเกิดปฏิกิริยาในสารละลาย (wet chemical analysis)

• ทําไมจึงต้องมีการพัฒนาวิธีการวิเคราะห์

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พัฒนาวิธีการวิเคราะห์.......

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• Accuracy • Chemical(s)

ANALYSIS COST OF ANALYSIS

• Accuracy

• Precision

• Sensitivity

• Selectivity

• Chemical(s)

• Chemical reaction

• Timing

• Instrument

• system cost

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• system cost

• operating cost

• maintenance cost

• วัตถุประสงค์ของการพัฒนาระบบวิเคราะห์อัตโนมัติ

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������������ ������������������� �������������� � ��!�����������"������������

สารที่ไหลในท่อจะถูกคั่นด้วยฟองอากาศ ทําให้เกิดการไหลแบบหมุนวน (turbulence flow) => ช่วยให้สารflow) => ช่วยให้สารผสมกันได้ดี

แต่ข้อเสียคือเสียเวลาเพราะต้องรอให้

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เพราะต้องรอให้ปฏิกิริยาเกิดสมบูรณ์ก่อนทําการตรวจวัดและมีการปนเปื้อนระหว่างตัวอย่าง (carry over) สูง

•ปฏิกิริยาส่วนใหญ่ต้องการเวลาในการเกิดปฏิกิริยาจนเข้าสู่ steady state หรือเข้าสู่steady state หรือเข้าสู่สมดุล (equilibrium)

•การวิเคราะห์หาปริมาณสารให้ได้ค่าที่ถูกต้อง จะต้องทําการตรวจวัดที่

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เวลาเท่าไร......

•จะตรวจวัดก่อนเข้าสู่ steady state ได้หรือไม่...

•Reaction time หรือ resident time คือเวลาตั้งแต่ผสมสารเข้าด้วยกันจนกระทั่งนําสารผสมที่ได้ไปตรวจวัดด้วยเครื่องมือวัด•การตรวจวัดที่เวลา reaction time คงทีแ่น่นอน จะสามารถใช้ในการหาปริมาณสารได้อย่างถูกต้อง (โดยไม่จําเป็นต้องรอให้เกิดปฏิกิริยาจนเข้าสู่ steady state)เกิดปฏิกิริยาจนเข้าสู่ 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

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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

1.1.1

•peristaltic pump

SINGLE STREAM MANIFOLD

SIMPLEST , MANUALLY OPERATED SYSTEMIS COMPRIZED OF:

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•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 anundergraduate laboratory or for assay of small sample series.

1.1.2.

TRAVEL TIME

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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.

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

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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 100µµµµL.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.

Fully automated FI analyzerfurnished with an autosamplercomprises a four channel peristaltic

1.1.5.

comprises a four channel peristaltic

pump, injection valve and an

integrated manifold with a z-typeflow through cell. For spectrophotometicmeasurements, the flow cell is connected by fiber optics toa tungsten lamp anda scanning spectrophotometer.

A@540nm

58

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The attached readout shows acalibration record of 0,2,5 and

8ppm nitrate, followed by a

routine run of nitrate assayin soil samples using cadmiumreduction column and sulfanilamidereagent.

seconds

0

2

5

OPTICALFIBER

1.1.6

SAMPLE

MIXING

MIXING COIL #2

FIBER

WASTE

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CARRIER REAGENTS

MIXING COIL #1

FLOW CELL INJECTION VALVE

SAMPLELOOP

PRINCIPLE

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1.2.1.

This section is designed to provide an understanding of processesthat 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 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

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and manipulated through change of injected volumes, flow rates andmanifold configurations. While single reagent assays can be performed

using the simplest, single stream manifold, it will be shown why a majorityof FI techniques use multistream manifolds, where several reagents are sequentially merged with a carrier stream that moves the injected samplezone 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.

1.2.2

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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.

SAMPLE INJECTION CONTROLLED DISPERSION REPRODUCIBLE TIMING

Sample injection provides the initial Co

1.2.3.

Sample injection provides the initialsquare input serving as a staring point for initial concentration ( Co) and startup time.

C

To

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 elementsof 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 readoutIn 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 dilutionto be estimated . Similarly T max is the time elapsed from the moment of injection

SAMPLE

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Cmax

Tmax

to be estimated . Similarly T max is the time elapsed from the moment of injectionTo to the moment of peak maximum T max. Reproducible timing of sample travel from injection to detection yields repeatable value of Tmax.

DISPERSED SAMPLE ZONE

1.2.4.

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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.

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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 processof formation of a detectable species. These two processes occur simultaneouslyand they yield , together with the dynamic characteristics of the detector the FIresponse curve. Note that the reaction product (yellow), is gradually formed at the interface between the sample zone (red) and carrier stream of reagent (blue).

A C

1.2.6.

Injection of increasing sample volumes causes Injection of increasing sample volumes causes Injection of increasing sample volumes causes Injection of increasing sample volumes causes formation of a double peak..formation of a double peak..formation of a double peak..formation of a double peak..

Reaction product (yellow) is formed at interface Reaction product (yellow) is formed at interface Reaction product (yellow) is formed at interface Reaction product (yellow) is formed at interface between sample (red) and reagent (blue).between sample (red) and reagent (blue).between sample (red) and reagent (blue).between sample (red) and reagent (blue).

B

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

formation of a double peak..formation of a double peak..formation of a double peak..formation of a double peak..

As the injected zone (A) movesdownstream, it disperses forming a concentration

gradient that can be viewed as composed of a continuum of

1.2.7.

composed of a continuum of concentration segments ofindividual 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

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Dispersion coefficient ( D )has been defined as a ratio of C0 / C max and theflow injection systems are designed to yield

Note that for D=2 a sample segment situated atop the peak, has been diluted to half ofits original concentration, by carrier solution .

dispersion of the injected sample

When the distance between injector and detector is minimized in a single stream FI system, injection of a

1.2.8.

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 aredesigned for automation of all

reagent based assays.or time

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•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

or time

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.

By adjusting the volume of injectedsample, of the volume of reactorsbetween injector and flow cell and

of flow rates in single or multistreammanifolds, dispersion can be adjusted

1.2.9.

manifolds, dispersion can be adjusted to a medium value. Resulting concentration gradient will have

a form of a smooth peak that will beonly 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:

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•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 .

By decreasing volume of injectedsample, and by increasing the volume of conduits between injector and flow

1.2.10.

of conduits between injector and flow

cell, dispersion can be increased to a large value. Resulting concentration gradient will havea 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.

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If the volume of a mixing chamber dominates the volume of the flow channelthe resulting concentration gradient will have a exponentially decreasingtrailing edge if the system is operated t continuous constant flow rate.

Systems with large dispersion are used for process control monitoringwhen extensive sample dilution is required and for automated titrations.

1.2.11.

Increasing the injected sample volume increasespeak 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 sampleheight increases linearly with the injected samplevolume. The sample volume needed to reach 50% ofthe steady state depends on the volume, geometryand flow rates in the channel between injector and

flow cell. For conventional FI systems this value isaround 50µµµµL, for micro SI systems as low as 5 µµµµL .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

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as shown here. With increasing efficiency of radialmixing, 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.

1.5

2.0 A 516nm

567nm

2.040

50

µµµµL BTB

Selection of injected sample volumesis a powerful tool for optimization of all FIA techniques. It allows :

1.2.12.

Absorbance

0.0

0.5

1.0

1.5

0.5

1.5

0

2.0

10

20

30

40

•. Selection of sensitivity and detection limit (as shown here on spectrophotometry of a dye)

• Identification of the linear range of a detector• Automated dilution of sample material

Volume of a sample solution injected into conventional FI system is accomplished by manually changing volume of the sample loop.

in Sequential Injection sample volume is

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0.0100 200 300 400

0TIME, secAutomated injection of increasing sample volumes. Automated injection of increasing sample volumes. Automated injection of increasing sample volumes. Automated injection of increasing sample volumes. Bromothymol Blue dye monitored at two Bromothymol Blue dye monitored at two Bromothymol Blue dye monitored at two Bromothymol Blue dye monitored at two wavelengths. (Sample wavelengths. (Sample wavelengths. (Sample wavelengths. (Sample 0000....002002002002% BTB, carrier % BTB, carrier % BTB, carrier % BTB, carrier 0000....005005005005M M M M sodium teraborate.sodium teraborate.sodium teraborate.sodium teraborate.

in Sequential Injection sample volume is determined by the volume of stroke reversal ofa syringe pump. This allows automated selectionof injected volumes by means of software control.

Increasing length of a tubular channel decreasespeak height while peak shape undergoes a changefrom asymmetrical to symmetrical shape. At the

1.2.13.

from asymmetrical to symmetrical shape. At the

same time the resident time of the peak maximumincreases with the distance traveled and the peakbase broadens. This is the principal limitation of FI based on constant forward flow, as the constant

flow rate limits the incubation time for chemicalreaction 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

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around 3 mL/min. (The recording here was obtained at a flow rate of 1,4mL/min, sample volume of 60µµµµL 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 systemwash by accelerating the flow. Programming the flow makes Sequential Injection technique more versatile than FI.

INJECTOR INJECTORDETECTOR DETECTOR

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.

1.2.14.

At continuous flow the time interval available for chemical reaction to take place is defined by linear flow

Stop flow allows longer reaction time without penalty of dilution, thus yielding higher sensitivity. It saves reagents and

STOP

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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.

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.

TECHNIQUESTECHNIQUESIDEA

APPLICATIONS

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APPLICATIONS

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

1.2.15.

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

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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.

Since the analyte (red) disperses as the sample zone travel downstream, the thus formed concentration provides numerous sections from which an analytical signal

1.2.16.

BLANK

FLOWFLOWFLOWFLOW

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

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DELAY TIME

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 PROFILEI IS INJECTION POINT. DELAY TIMEDEFINES SELECTION OF GRADIENT PORTION.

ANALYTE

Delay time between sample injection and commencement of the stop flow period determines which section ( ) of the sample zone will be arrested in

1.2.17.

BLANK

DELAY TIME

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

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FLOWFLOWFLOWFLOW

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.

Glucose + 2H2O + O2 Gluconic Acid + H2O2

2 H2O2 + 4-Aminoantipyrine + p-Hydroxybenzene Sulfonate Quinoneimine Dye + 4H2O

GLUCOSE OXIDASE

PEROXIDASE

Enzymatic assay of glucose, monitored at 505 nm

1.3.1.

Enzymatic assay of glucose, monitored at 505 nmis carried out by reaction rate measurementduring a stopped flow period lasting 20 secondswhile 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.

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Note that the travel time between injectorand detector has been minimized in orderto carry our the reaction within the flow cell, while the pump has been stopped.

1.3.2.

A majority of FI assays are carried at continuous flow, when carried and reagentsare pumped simultaneously at a constant flow rate. Sample is injected into carrierstream of water (or appropriate buffer) while reagent streams are added at confluencepoints. Advantages of this approach are:

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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

1.3.3.

Almost all FI instruments employ multichannel peristaltic pumps to move carrier and reagent

Merging of reagent and carrier streamMerging of reagent and carrier streamMerging of reagent and carrier streamMerging of reagent and carrier stream Two reagents, three stream FI systemTwo reagents, three stream FI systemTwo reagents, three stream FI systemTwo reagents, three stream FI system

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( 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.

Three stream FI manifold where

at the first confluence point ( ) molybdate is added to stream of water, that carries injected sample of phosphate

1.3.4.

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 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

A@720nm

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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 SI-LOV format, as waste generation and reagent consumption is reduced 50 times.

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 ( Puchades 1991)of sea water FI analysis of anionic and organic species lists

detection limits for nitrate 0.1µµµµM, sulfide 1,2mM and phosphate 0.05µµµµM.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

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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.

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.

J. Atienza, M.A.Herrero, A.Maquieira and R. Puchades, Critical Rew. Nal. Chem 22(5) 331-344 (1991)

1.3.6.

Multistream FI systems are ideally suited for automation of all separationsbased 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:

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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.

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 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 , aswell as inductively coupledplasma mass spectrometryhave been used s detectors

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Fang Z-L.: Flow Injection Atomic Spectrometry, Wiley, Chichester, 1995.Sanz-Medel A.: (Ed.), Flow Analysis with Atomic Spectrometric Detectors, Elsevier, Amsterdam, 1999Burguera J.L: (Ed.) Flow Injection Atomic Spectroscopy, Marcel Dekker, New York, 1989

The key component of thedesign is the gas-liquidseparator.

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 toconstruct and maintain. The entire separator, or at least its vertical tubular body ismade of glass, and often partially filled with large glass beads, as hydrophilic surfaceof 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 iscarries the released volatiles into the detector. The level of liquid in the separator ismaintained 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 rateof 30mL/min is not unusual. Membrane separators rely on gas diffusion through a hydrophobic membrane and offer higher sensitivity at lower flow rates, since theirinternal gas volume is much smaller. When integrated with a flow cell for cold Hgassay, they offer an excellent sensitivity and detection limit ( Fang 1988 ).

Membrane separator.

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Gasexpansionseparator

Fang, Z.-l.; et.al., Anal. Chim. Acta1988, 214, 41-55.

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

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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, a“sandwich 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)

This flow cell design uses a bifurcated optical cable to illuminate a white surface and to collect reflected lightas it passed twice through the monitored aqueous layer.This flow cell can be used to monitor either a single ,

1.3.10.

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 monitorvolatile species emanating from a donor stream.Note that Teflon membrane may be furnished with an opening ( )situated downstream from the fiber, to alleviate pressure differencesbetween acceptor and carrier streams. Note that stopping the flow ofacceptor (indicator) stream allows accumulation of analyte and increaseof sensitivity of measurement. (Pavon et. al. 1992)

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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 techniquesthrough membranes Lab. Autom. Inf. Managem., 34(2) 115-130 (1999)

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

1.3.11.

(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 ofextracted 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.

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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)

ORGANIC PHASE

a b

1.3.12.

Choice of materials for manifold components and their orientation is critical because

ORGANIC PHASE

HEAVIER THAN

WATER

ORGANIC PHASE

LIGHTER THAN

WATER

b

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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.

INSTRUMENT

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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 researchersfound 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.

1.4.1.

place, since software became a key component of a successful design. Initially, in research laboratories, and especially in Academia , a whole generation of graduate studentsbecame 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 unravelIt 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 nolonger necessary to waste time by composing home made programs. Indeed, to do so is irrational as

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longer necessary to waste time by composing home made programs. Indeed, to do so is irrational aswould 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 commercialinstrument packages from FIAlab or Lachat Instruments is available. All commercially available FI instruments were recently reviewed (Smith 2002), includingprices, special features and available peripherals.

J.P.Smith & V. Hinson-Smith, Anal. Chem. 74, 385A ( 2002)

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

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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 cellswith 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.

1.4.3.

Integration of manifold components allows miniaturizationand optimization of flow channel dimensions in order tominimize sample and reagent consumption. Integration ofvalve with the sample processing channel and a flow cell valve with the sample processing channel and a flow cell was originally suggested as a tool for miniaturization ofSequential Injection technique ( See Section 2). In FI formatsuch “lab-on-valve” platform is used to streamline manifoldcomponents ( valve, tube fittings, confluence points andflow 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 routineassays 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 otherinstruments in another location with ease.

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instruments in another location with ease.The FI-LOV configuration shown here is designed for tworeagent assay using 50 cm and 100cm long reaction coils,fiber optic flow cell with 10mm light path and 50 µµµµL sampleInjection loop. Eight roller four channel peristaltic pumpis used to fill sample loop, to propel the carrier streamand two reagent streams.

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 MultisyringeFlow 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 toprevail in routine laboratories, because of cost, convenience of operation, and ease of

1.4.4.

prevail in routine laboratories, because of cost, convenience of operation, and ease of replacement of peristaltic tubing. Yet, an instrument build around individually driven syringepumps combined with solvent resistant LOVmodule 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 theconventional FI system. Also microSI instrument,

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conventional FI system. Also microSI instrument,is far less complex as it operates with a only a singlepump and a single valve. Indeed, unless all fourpumps will be run in a fully synchronized andautomatically cycled mode, the flow programmingof this novel instrument configuration will bea 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)

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 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 softwareand 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 concertis quite another matter. The key to success is in designing simplest possible systemwith smallest number of components and then simplify it further. Remember that:

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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 “( Murphy’s 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.

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 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 8µµµµL pulsesin 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.

Table of ContentsSantos 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)

Solenoid Pump.

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 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.

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Tubing connectors, ferrule

and T-connector

Teflon made reactor coil . Heated reactor coil withtemperature controller.

LOAD

1.4.8.

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 100µµµµL by changing thelength 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 isimportant 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.

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

Sample volume and reaction time are the most important parameters of flow injection experimental protocol. By changing injected volumes and reactiontimes sensitivity and detection limit of reagent based assays can be adjusted to desired level.

1.4.9.

assays can be adjusted to desired level.Since conventional FIA employs a two position valvefurnished with fixed sample loop volume, injectedvolumes cannot be automatically selected by a computer.Variable volume injection removes this limitation allowing automated optimizationof assayparameters. The key difference is in that the injection system based on a multiposition valveand the volume of injected sample is controlled by a syringe pump.

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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 .

FLOW

A

B

C

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 sampleforms a concentration gradient (A) in the sample

holding coil. Since the central stream moves at a

1.4.10.

C

D

holding coil. Since the central stream moves at a

double of average flow velocity, sample zone occupiesin 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 beinjected into the sample processing channel, the

forward stroke should be at least twice of the reversal stroke volume. (C).

INJECTING ONLY PART OF SAMPLE

Smaller volumes of the forward stroke can be, injected into sample’processing manifold, but then the tail section of the sample zone

DISCARDDISCARDDISCARDDISCARD

ANALYZEANALYZEANALYZEANALYZE

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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 samplematerial 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 ofsample solution that has been aspirated by flow reversal, adjacent to the valve is directed via port #1into waste, and than a selected section of the remaining diluted sample zone is injected into the sample

processing manifold.

1.4.11.

Fiber optics and solid state spectrophotometers revolutionized the way in which all FIA techniques are carried out, since this technologyallowed optimization by bringing light and collecting data from any

position of the sample flow path. While this change impacted mostly position of the sample flow path. While this change impacted mostly Sequential Injection, also more traditional FI systems benefit from versatilityand 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 emittingdiode is mounted directly onto the flow cell.

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Ocean Optics Spectrophotometerand a Tungtsen light source.

Z-cell with 10mmlight path

Z-cell with 10 cm light path.

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 µµµµTAS 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

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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.

S.J.Haswell & V. Skelton, TRAC, 19, 389 (2000)

M.D. L de Castro & I. Papaefstathiou, TRAC, 17, 41, (1998

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)

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 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

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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.

The unique feature of all flow injection methods is the well defined concentration gradientformed 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•titrationStopped flow FI format exploits concentration gradients through exact timing of microfluidicoperations, controlled by computer and programmed through dedicated software. Stopped-flow 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

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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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