By Assoc. Prof. Dr. Jaroon Jakmunee
20 September 2012
Table of Contents

Wet Chemical Analysis
• พัฒนาการของการ
วิเคราะห์ทางเคมี ที่
ในสารละลาย (wet
chemical analysis)
• ทําไมจึงต้องมีการ

Table of Contents

Table of Contents

ANALYSIS COST OF ANALYSIS • Accuracy • Precision • Sensitivity • Selectivity • Chemical(s) • Chemical reaction • Timing • Instrument • system cost • operating cost • maintenance cost Table of Contents .

• วัตถุประสงค์ของการพัฒนาระบบวิเคราะห์อัตโนมัติ Table of Contents .




 Table of Contents .

สารที่ไหลในท่อจะถูก คั่นด้วยฟองอากาศ ทํา ให้เกิดการไหลแบบ หมุนวน (turbulence flow) => ช่วยให้สาร ผสมกันได้ดี แต่ข้อเสียคือเสียเวลา เพราะต้องรอให้ ปฏิกิริยาเกิดสมบูรณ์ ก่อนทําการตรวจวัด และมีการปนเปื้อน ระหว่างตัวอย่าง (carry over) สูง Table of Contents .

... Table of Contents .. •จะตรวจวัดก่อนเข้าสู่ steady state ได้หรือไม่ .•ปฏิกิริยาส่วนใหญ่ ต้องการเวลาในการ เกิดปฏิกิริยาจนเข้าสู่ steady state หรือเข้าสู่ สมดุล (equilibrium) •การวิเคราะห์หาปริมาณ สารให้ได้ค่าที่ถูกต้อง จะต้องทําการตรวจวัดที่ เวลาเท่าไร....

•Reaction time หรือ resident time คือเวลาตั้งแต่ผสมสารเข้า ด้วยกันจนกระทั่งนําสารผสมที่ได้ไปตรวจวัดด้วยเครื่องมือวัด •การตรวจวัดที่เวลา reaction time คงทีแ ่ น่นอน จะสามารถใช้ ในการหาปริมาณสารได้อย่างถูกต้อง (โดยไม่จําเป็นต้องรอให้ เกิดปฏิกิริยาจนเข้าสู่ steady state) Table of Contents .

OVERVIEW Table of Contents .

J. Ruzicka & E. 145 (1975) J.4. As the injected zone moves downstream.Hansen. Willey. while a product begins to form at interfaces between the sample zone and the reagent. “Flow Injection Analysis” 2nd ed.2. Ruzicka & E. the sample zone (red) is injected into a flowing carrier stream of reagent (blue). 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). 1988 Table of Contents . Instruments & Components Flow Injection (FI).H. J.Hansen. N. 78.1. 1. Anal. In its simplest form . 1. Principles.Y.1. the first generation of FIA techniques. the sample solution disperses into reagent. Basics. Methods & Applications. Acta .3. Chim. is the one most widely used. 1.H.

1. MANUALLY OPERATED SYSTEM IS COMPRIZED OF: •peristaltic pump •manually operated two position injection valve •manifold of connectors tubing and reactors • flow through detector Basic FI instrument furnished with a tungsten light source and spectrophotometer. is well suited as a training tool in an undergraduate laboratory or for assay of small sample series. Table of Contents .1 SINGLE STREAM MANIFOLD SIMPLEST .1.

TRAVEL TIME Peak height is the most frequently used response for construction of a calibration curve. With a sampling frequency of up to 120 s/hour.2.1. where FI system is usually coupled with an autosampler. Table of Contents . thousands of samples are analyzed within a week in routine Laboratories.1. while peak area (A) is used infrequently. Depending on the flow rate and reaction rate this readout is often available within less than 30 seconds after sample injection. Peak width (W) is readout for FI titration.

3. Table of Contents .1.1.

dialysis. and gas diffusion based assays. and by selecting pump tubes of a desired diameter. phosphate. nitrate and nitrite to be analyzed simultaneously in water and soil sample extracts. Since many assays use several reagents that must be added in a given sequence. Multistream FI system are routinely combined into multichannel systems. Yet another advantage of multistream FI systems is their versatility.1.4.5 -1.5mL/min of individual streams. Typically a three channel system allows . that allows automation of solvent extraction. while an injected volume is selected in a range of 25 to 100µL. This allows reagents to be added continuously to injected sample at a desired concentration.1. Table of Contents . A majority of FI systems use peristaltic pumps that allow flow rates of carrier and reagents to be controlled by choosing pump revolution rate. where each channel is dedicated to a different chemical assay. so that reactions can be carried out in sequence as the sample zone passes through the first and second reactor. A typical flow rate is 0. FI systems use multichannel pumps that propel carrier stream along with reagent streams.

injection valve and an integrated manifold with a z-type flow through cell. 5 2 0 seconds Table of Contents . For spectrophotometic measurements.1. Fully automated FI analyzer furnished with an autosampler comprises a four channel peristaltic pump.5. followed by a routine run of nitrate assay in soil samples using cadmium reduction column and sulfanilamide reagent. the flow cell is connected by fiber optics to a tungsten lamp and a scanning spectrophotometer.1. A@540nm 8 The attached readout shows a calibration record of 0.5 and 8ppm nitrate.2.


PRINCIPLE Table of Contents .

and to offer tools for optimizing sensitivity.1. flow rates and manifold configurations.1. This section is designed to provide an understanding of processes that yield FI response curve. Table of Contents . it will be shown why a majority of FI techniques use multistream manifolds. Note that discussion in the following sections deals with a single stream system. While single reagent assays can be performed using the simplest. 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. single stream manifold. For multistream systems D-values have to be corrected by dilution caused by additional streams.2. detection limit and sampling frequency of flow injection based assays. where several reagents are sequentially merged with a carrier stream that moves the injected sample zone through the manifold and a flow cell.

2 Blue dye injected into a single stream manifold forms a concentration gradient. Profile of a concentration gradient is shaped by injected volume. Table of Contents . Peak profile (B) recorded by a colorimeteric measurement @620 nm shows a gradient profile. on which reproducibility of flow injection assays is based.1. as it flows through a coiled reactor.2. dispersion processes in the flow channel and by the resident time of the zone traveling between injector and detector.

Reproducible timing of sample travel from injection to detection yields repeatable value of Tmax. where the highest one ( Cmax) corresponds to peak maximum.1. This process forms a well defined concentration gradient that can be viewed as continuum of elements of fluid with different concentrations. most FI methods use this element of fluid as a readout In order to optimize a given assay it useful to know how much the sample has been diluted in the FI system and how much time was available for chemical reactions to proceed. Since it is convenient to locate peak maximum.2. Similarly T max is the time elapsed from the moment of injection To to the moment of peak maximum T max. Therefore the dispersion coefficient has been defined a s D= Co/ Cmax allowing the degree of sample dilution to be estimated .3. SAMPLE Controlled dispersion takes place as the sample zone moves downstream through the manifold. Cmax Tmax DISPERSED SAMPLE ZONE Table of Contents . SAMPLE INJECTION CONTROLLED DISPERSION REPRODUCIBLE TIMING Sample injection provides the initial Co square input serving as a staring point for initial concentration ( Co) and startup o T time.

For success of a reagent based assay. Four stages of dispersion process shown above depict the underlying physical principle of FI at continuous forward flow. 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.1.4.2. Table of Contents . while the radial dispersion is designed to provide an efficient mixing of sample zone with reagent supplied by carrier stream.

Table of Contents .

Table of Contents .

These two processes occur simultaneously and they yield .2.1.5. both kinetic in nature: the physical process of dispersion of the sample zone and the chemical process of formation of a detectable species. is gradually formed at the interface between the sample zone (red) and carrier stream of reagent (blue). together with the dynamic characteristics of the detector the FI response curve. Table of Contents . Note that the reaction product (yellow). Flow Injection response is a result of two processes.

1. Injection of increasing sample volumes causes formation of a double peak. In a s single stream manifold (A). by using a two stream system furnished with a confluence point Table of Contents . This can be done by either injecting a smaller sample volume.6. and formation of a double peak (C). or. To avoid this problem. 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. dispersion coefficient of a system should be determined and adjusted accordingly.2. 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). A C B Reaction product (yellow) is formed at interface between sample (red) and reagent (blue)..

2. the one situated at peak apex ( Cmax) is the one on which peak height measurement. Table of Contents . by carrier solution . Dispersion coefficient ( D )has been defined as a ratio of C0 / C max and the flow injection systems are designed to yield dispersion of the injected sample Note that for D=2 a sample segment situated atop the peak.7. it disperses forming a concentration gradient that can be viewed as composed of a continuum of concentration segments of individual concentrations C.1. and calibration. Of these segments. As the injected zone (A) moves downstream. has been diluted to half of its original concentration. will be based.

When the distance between injector and detector is minimized in a single stream FI system.2. Table of Contents . 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. •pH measurement Note that in a multistream system. Systems with limited dispersion are designed for automation of all reagent based assays. or time •conductivity measurement •for direct sample injection in high sensitivity ICP and AA based assays •for bioligand interaction studies by surface plasmon resonance (BIA) •for functional receptor binding assays on live cell for drug discovery. D value is always > 2 as the flow rates of at confluence points have to be taken into account.1.8.

dispersion can be adjusted to a medium value. Systems with medium dispersion are designed for automation of all reagent based assays. Resulting concentration gradient will have a form of a smooth peak that will be only slightly skewed. Note that in order to reach high sensitivity of a colorimetric assays: •sample volume should be maximized •reagents should be added by reagent streams via confluence points •long path flow cell should be used •dispersion coefficient should be adjusted to between 2 and 5 NOTE: Sensitivity and detection limit of reagent based assays can be further enhanced by Bead Injection Technique . Table of Contents .2.9. By adjusting the volume of injected sample. of the volume of reactors between injector and flow cell and of flow rates in single or multistream manifolds.1.

1. In order to obtain very large D values a mixing chamber should be integrated into flow manifold. By decreasing volume of injected sample. If the volume of a mixing chamber dominates the volume of the flow channel the resulting concentration gradient will have a exponentially decreasing trailing edge if the system is operated t continuous constant flow rate. Systems with large dispersion are used for process control monitoring when extensive sample dilution is required and for automated titrations. Table of Contents . and by increasing the volume of conduits between injector and flow cell.10. Resulting concentration gradient will have a form of a smooth long peak that will have an exponentially decreasing tailing edge.2. dispersion can be increased to a large value.

Up to D =2 value (in a single stream system).2. Increasing the injected sample volume increases peak height.D tubing.1. Spectropotometry at 620nm. peak height increases linearly with the injected sample volume. 20 cm long. ). the resulting peak shape is composed of two exponential curves. at a flow rate of 1. gradients are reshaped and follow erf function. Table of Contents . until a steady state plateau is reached.2. and convenient tool for adjusting dispersion coefficient and for optimizing the sensitivity of flow injection based assays.Changing injected sample volume is versatile.2. With increasing efficiency of radial mixing.4mL/min in a single stream system. as shown here. For conventional FI systems this value is around 50µL. ultimately approaching Gaussian shape ( see Section 0. geometry and flow rates in the channel between injector and flow cell. for micro SI systems as low as 5 µL . If the radial mass transfer is incomplete.11. Recording shows traces obtaining by injection bromothymol blue solution into 0. The sample volume needed to reach 50% of the steady state depends on the volume.5mmI.

Table of Contents .5 10 0. Selection of sensitivity and detection limit 2.5 • Automated dilution of sample material Absorbance in Sequential Injection sample volume is determined by the volume of stroke reversal of a syringe pump.2.0 1.12. 50 516nm 567nm 20 0. sec Automated injection of increasing sample volumes.0 0 100 200 300 400 TIME. This allows automated selection of injected volumes by means of software control. µL BTB 30 (as shown here on spectrophotometry of a dye) Volume of a sample solution injected into conventional FI system is accomplished by manually changing volume of the sample loop. It allows : •.5 2.5 0.005M 005M sodium teraborate.0 40 • Identification of the linear range of a detector 1. (Sample 0. Bromothymol Blue dye monitored at two wavelengths. carrier 0.1.002% 002% BTB.0 A 1. Selection of injected sample volumes is a powerful tool for optimization of all FIA techniques.

Increasing length of a tubular channel decreases peak height while peak shape undergoes a change from asymmetrical to symmetrical shape.2. as the constant flow rate limits the incubation time for chemical reaction to about 20 seconds. with a total conduit length of about 250cm. This is the principal limitation of FI based on constant forward flow.13. The use of programmable. Programming the flow makes Sequential Injection technique more versatile than FI. and combined flow rates around 3 mL/min. allows incubation time to be prolonged by stopping the flow.1. instead of continuous flow. (The recording here was obtained at a flow rate of 1.4mL/min. sample volume of 60µL in a way described in the previous slide). and speeding up the system wash by accelerating the flow. Table of Contents . At the same time the resident time of the peak maximum increases with the distance traveled and the peak base broadens.

2. Table of Contents . It saves reagents and generates less waste than continuous pumping. It provides information on reaction kinetics through reaction rate measurement. It allows miniaturization by minimizing the length between injector and detector. Since all chemical reactions are time dependent. Although longer tubes allow longer reaction time the yield is offset by dilution due to increase in sample zone dispersion. as in this format reaction equilibrium is not necessarily achieved. INJECTOR INJECTOR DETECTOR DETECTOR STOP At continuous flow the time interval available for chemical reaction to take place is defined by linear flow velocity and is limited by the length of conduit between point of injection and detector.1. Stop flow allows longer reaction time without penalty of dilution.14. thus yielding higher sensitivity. reproducible timing of sample handling operations is critical to success of flow based chemical assays.


it is difficult to carry out reproducibly when peristaltic pumps are used propel carrier and reagent streams.1. reaction rate curve will be recorded while the reaction product (yellow) is being formed in the detector. Table of Contents . Next. Syringe driven systems either FI or SI are reliable and their use in stopped flow mode is highly recommended.15. DETECTOR The stop flow mode is based on arresting a selected portion of the sample zone in the detector. Provided that the reaction did not reach equilibrium while the zone was on the way to detector. while the baseline is restored.2. While stop flow technique has been used in FI format. flow is resumed and reacted sample zone is flushed out of the detector.

S REPRESENTS SAMPLE PROFILE I IS INJECTION POINT.16. •Stop flow is the most useful and effective approach for reaction rate based assays. 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. when high analyte concentration causes readout to be out of detector range . DELAY TIME DEFINES SELECTION OF GRADIENT PORTION.2. This is useful.1. FLOW BLANK DELAY TIME Since the analyte (red) disperses as the sample zone travel downstream. rather than on peak maximum. Table of Contents . the thus formed concentration provides numerous sections from which an analytical signal can be recorded.

1. longer delay times will yield lower slopes since tail sections of the sample zone are more diluted. ANALYTE BLANK DELAY TIME FLOW Delay time between sample injection and commencement of the stop flow period determines which section ( ) of the sample zone will be arrested in the observation field of a detector ( ) for reaction rate measurement. it is essential that the delay time is perfectly reproduced for each assay. while shorter delay times ( up to peak maximum) will yield steeper slopes. Table of Contents . Since the analyte (red) disperses within the reagent stream (blue) on the way to the detector while the product (yellow) is being formed. In the absence of analyte horizontal (blank) line will be observed. In the example shown.17.2.

Note that the travel time between injector and detector has been minimized in order to carry our the reaction within the flow cell. 1500. while the pump has been stopped. FIAlab 2500 and associate software series of standards containing 500. 1000. Table of Contents . monitored at 505 nm is carried out by reaction rate measurement during a stopped flow period lasting 20 seconds while data are collected for construction of a calibration curve.1. 2000 and 2500 ppm glucose were injected yielding response curves shown on the right.1.3. GLUCOSE OXIDASE Glucose + 2H2O + O2 Gluconic Acid + H2O2 PEROXIDASE 2 H2O2 + 4-Aminoantipyrine + p-Hydroxybenzene Sulfonate Quinoneimine Dye + 4H2O Enzymatic assay of glucose. Using a single stream flow scheme.

A majority of FI assays are carried at continuous flow.1. when carried and reagents are pumped simultaneously at a constant flow rate. Sample is injected into carrier stream of water (or appropriate buffer) while reagent streams are added at confluence points. The main drawback of FI is continuous reagent consumption and waste generation Table of Contents .2.3. 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.

Table of Contents . The valve is furnished with a bypass (not shown) that allows carrier solution to pass through the valve.3. Sample is injected by means of a two position injection valve with a fixed injection loop. Merging of reagent and carrier stream Two reagents.1. three stream FI system Almost all FI instruments employ multichannel peristaltic pumps to move carrier and reagent ( R1. 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. while the sample is being filled into the loop. The pump moves solutions continuously in forward direction.3.R2) streams that merge at confluence points ( ) where reagent merges with sample zone.

At the second confluence point ( ). ascorbic acid is added to form phosphomolybdenum blue. Since molybdate/ascorbic acid mixture A@720nm decomposes rapidly.3. these reagents must be stored separately and added sequentially to the carrier stream. as waste generation and reagent consumption is reduced 50 times. Three stream FI manifold where at the first confluence point ( ) molybdate is added to stream of water.4. forming a blue product. Table of Contents . Use of water as carrier stabilizes baseline and improves detection limit for assay of phosphate in water based samples. that carries injected sample of phosphate to be analyzed. This method. It is even more effective when miniaturized into SILOV format.1. yields sample frequency of 80 s/h and is one of the most frequently performed FI assays.

Singapore. simple use of FI is in assaying sea water.2mM and phosphate 0. Trace analysis of metals by atomic spectroscopies (AA.1µM. Critical Rew. iron . fertilizers and serum. World Scientific Ltd. Trojanowicz M. These application are reviewed and summarized in monographs of Catalyud and Trojanowicz. that converts target analytes into volatile metal hydrides. well defined sample volumes into carrier stream of water this problem is eliminated.A. sulfide 1. saline. The most significant method is based on hydride generation. 2000. Three monographs and a large number of papers deal in detail with FI based hydride generation ( see FI based separations). phosphate. Atienza.1. Nal.Maquieira and R.: (Ed. HGAAS. nitrate. Instrumentation and Applications.Herrero.M. leaving matrix interferences in a sample solution. A comprehensive review ( Puchades 1991)of sea water FI analysis of anionic and organic species lists detection limits for nitrate 0. 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. 1997. Puchades. Taylor & Francis. Flow Injection Analysis of Pharmaceuticals. M. By injecting small. Yet another. A. Pharmaceutical and enzymatic assays with UV-VIS or fluorescence detection is yet another area.. London.: Flow Injection Analysis. chloride. Agricultural and environmental analysis of water. Table of Contents .05µM. nitrite.. i. ICP and ICP-MS) uses FI as a “front end” sample processing system in two ways.3. as high content of water soluble salts will crystallize and block the nozzle. samples that cannot be continuously pumped into nebulizers of AA or ICP instruments.e. soil and fertilizers for assay of ammonia. Chem 22(5) 331-344 (1991) Calatayud J.). J.5. chromium ( IV and VI) and cyanide are routinely carried in test laboratories on a very large number of samples by FI. where FI is routinely used in a large scale for quality and process control.

Table of Contents . An excellent . Trojanowicz M. sorbent extraction and other microcolumn based separation and conversions (enzymatic..: Flow Injection Analysis.1. 2000. detailed review of FI based separations is found in the following monographs: Fang Z-L. redox etc) are too numerous to be reviewed here.3. 1993. VCH Verlagsgesellschaft mbh. Flow-Injection Separation and Preconcentration.:. Multistream FI systems are ideally suited for automation of all separations based on partition between two phases.6. Weinheim. World Scientific Ltd. Instrumentation and Applications. This section deals briefly only with •gas/ liquid separations and •solvent extraction while ion exchange. Singapore.

Hg. Marcel Dekker. Advantages of hydride generation: separation of the trace metals from complex matrices. Amsterdam.).3. Ni.L: (Ed. and ease of automation were first demonstrated by Astrom in his pioneering work on FI based –hydride AA assay of bismuth. 1999 Burguera J. 1995. Atomic absorption spectroscopy .: (Ed. 1989 Table of Contents . Hydride generation based Atomic Spectroscopies are routinely used for trace analysis of As. Flow Analysis with Atomic Spectrometric Detectors. New York. Wiley. fast reaction speed.1. Ge. cold vapor atomic absorption spectroscopy . 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.: Flow Injection Atomic Spectrometry. Bi. Sanz-Medel A.7. while assay of volatile compounds of Ag. Sn and Te. Elsevier. and Zn has been reported in research publications. inductively coupled plasma spectroscopy . Fang Z-L. By combining an acidified sample stream with a strong reducing agent (sodium borohydride). Cu. Pb.) Flow Injection Atomic Spectroscopy. analyte enrichment. as well as inductively coupled plasma mass spectrometry have been used s detectors The key component of the design is the gas-liquid separator. Co. Se. Chichester.

Acta 1988. as hydrophilic surface of glass assists in gas liquid separation.al. 214. combined flow of sample. Gas expansion separator Fang. Membrane separators rely on gas diffusion through a hydrophobic membrane and offer higher sensitivity at lower flow rates. or argon) that sweeps the liquid within the separator and carries the released volatiles into the detector..1. Chim. or at least its vertical tubular body is made of glass. There are two types of separators: gas expansion separator and membrane separator. Gas expansion separators are most frequently used. Anal. The level of liquid in the separator is maintained by external pump. as they are robust and easy to construct and maintain. reagent and carrier is up to 15mL/min and purging gas flow rate of 30mL/min is not unusual. 41-55.-l. When integrated with a flow cell for cold Hg assay. Membrane separator. they offer an excellent sensitivity and detection limit ( Fang 1988 ). since their internal gas volume is much smaller. et. Gas expansion separators are operated at high flow rates. The entire separator. Carrier/hydrogen/hydride stream is confluenced with purging gas ( air.3. nitrogen. Z.8. Table of Contents . and often partially filled with large glass beads..

monitored at 430nm (Baadenhuijsen 1979). Baadenhijsen & H. chlorine. Another. Membranes made of Teflon are hydrophobic. D. The drawback hydrophobic membranes is that they can be fouled by surfactants that destroy the air gap barrier. Seuren-Jacobs. that diffuses across a silicone rubber made membrane from a donor ( blue) to an acceptor (green) stream changing color of an acidobasic indicator. that offers a robust alternative to gas diffusion in parallel plate diffuser (Castro 1998) H. 41. L. releasing carbon dioxide. 443. (1979) •M.E.9. (1998) Table of Contents . TRAC. with up to 50%porosity. sample containing carbonate (or dissolved carbon dioxide) is acidified. ( as the one shown above) are easy to assemble. When miniaturized and integrated with a fiber optic detector. Chem. ozone or volatile compounds rapidly permeate into an acceptor stream where they are detected by means of a suitable reagent. Clin. 25. 17.3. innovative approach to gas separation is gas pervaporation. forming an air gap between carrier and donor stream through which gases like ammonia. In a two stream FI system. Flat plate diffusers. a“sandwich cell” construction allows increase of sensitivity of an assay. Papaefstathiou.1. sulphur dioxide.H. de Castro & I. placed into acceptor channel.

al.B) it is useful to monitor volatile species emanating from a donor stream. 1992) A B M J. Inf. 64.10. to alleviate pressure differences between acceptor and carrier streams. or if furnished with a gas permeable membrane. J. Autom. L. Cordero Analytical applications of separation techniques through membranes Lab.L. (Pavon et. 34(2) 115-130 (1999) Table of Contents . (M) mounted between two spacers (A. Chem. This flow cell can be used to monitor either a single . 923 (1992) C. Pinto. P. M. Managem. Pavon and B. Note that stopping the flow of acceptor (indicator) stream allows accumulation of analyte and increase of sensitivity of measurement.P. E. al. This flow cell design uses a bifurcated optical cable to illuminate a white surface and to collect reflected light as it passed twice through the monitored aqueous layer. Laespada. Anal. F. M. G.3.. Note that Teflon membrane may be furnished with an opening ( ) situated downstream from the fiber. liquid stream.1.Pavon et.

1 (1978) L. Miniaturization and automation of solvent extraction minimizes exposure to harmful solvents and reduces consumption of reagents and generation of hazardous waste. applicable to assay of hormones. provides clue to mechanism of hydrodynamics of solvent extraction. revolutionized solvent extraction technique.Thelander. Anal. (Karlberg & Thelander 1978).11. Anal. Sample (S) is injected into a moving carrier stream of water (AQ). which is merged (a) with an organic phase (ORG) and pumped through a Teflon made extraction coil (b). as it moves through a Teflon tubing. Chim. Detail showing circulation of extracted dye within segment of organic phase (Nord & Karlberg 1984). 164. Karlberg. This method. Acta. Nord & B. Acta 98. while organic phase is led into a flow cell. pharmaceuticals and numerous hydrophobic compounds. Karlberg & S. 233 (1984) Table of Contents . B. Chim. Two stream manifold for automated solvent extraction. In separator (c) the aqueous phase is discarded into waste.1. that up to that time was mostly carried manually.3.

In segmentor organic phase enters through a glass fitting and adheres to Teflon tubing (1).:. In the membrane separator Teflon made membrane allows only the organic phase to penetrate through hydrophobic pores. 1993.3. A Practical Guide. Pacey C. Elsevier. while organic phase adheres to Teflon. 1989. Flow-Injection Separation and Preconcentration. Amsterdam.: Flow Injection Analysis. b a ORGANIC PHASE HEAVIER THAN WATER b ORGANIC PHASE LIGHTER THAN WATER Choice of materials for manifold components and their orientation is critical because aqueous phase (aq) adheres to glass.1. while aqueous phase is discarded.E. Karlberg B. In separator a thin Teflon strip (3) serves to guide organic phase through a glass made T piece. Table of Contents . Fang Z-L.12. VCH Verlagsgesellschaft Weinheim.

INSTRUMENT Table of Contents .

serial assays such as soil water or environmental analyses.P. “patches” are available that allow to bridge the gap between FIAlab or LABview software and detector with proprietary software drive. J. All commercially available FI instruments were recently reviewed (Smith 2002). who left behind software bundles. Hinson-Smith. Initially. Today versatile software is commercially available that accommodates peripherals added to core Instrument. ICP). Chem. and especially in Academia . including prices. 74. With advent of computers. that does provide flexible timing of events.1. In the early days from 1974 up to mid 80’ a vast majority of Flow Injection instruments was “home made” from components found in the lab or purchased piecemeal. special features and available peripherals. in research laboratories. and controls peripherals such as spectrophotometers. 385A ( 2002) Table of Contents . since software became a key component of a successful design. For routine. For advanced detectors ( AA. a whole generation of graduate students became victim of necessity to create “home made” software. however. Indeed. while their supervisors became in turn victims of their former graduate students. external pumps and valves while collecting and evaluating data in a real time. that no one could unravel It is not a trivial task to design and to write software package that does control instrument functions. to do so is irrational as would be writing of a personal version of a word processing or slide presenting program.4. Such open architecture allows FI instrument to be assembled for virtually any research task or a specilaized assay.Smith & V. a several commercial instrument packages from FIAlab or Lachat Instruments is available. This was because most researchers found challenge and joy in innovative design of their own systems and also because the commercially available systems were quite expensive. a significant change took place.1. Therefore it is no longer necessary to waste time by composing home made programs. Anal.

Table of Contents . three stream FI system. Since UV-VIS spectrophotometry is the most frequently used detection technique.2. replacing earlier designs with filter photometers. where a single wavelength is sufficient light emitting diodes offering yet another practical alternative. While continuously pumping FI systems were in the past operated manually.4. Single channel.1. and their response was recorded on a chart . and computer controlled peristaltic pumps as well as computerized data collection. For teaching and single purpose assays. with two reaction coils and fixed volume loop injection valve is the configuration. modern systems use automated two position injection valves. fiber optic flow cells with a 10mm optical path coupled to software controlled solid state spectrophotometer are now common. most frequently used for automation of reagent based chemical assays.

nitrate etc. while reactor coils #1 and#2 are mounted externally. The advantage of this construction is that it makes function of the manifold transparent to the user. and for routine assays provides a format that is easy to reproduce. Integration of valve with the sample processing channel and a flow cell was originally suggested as a tool for miniaturization of Sequential Injection technique ( See Section 2). confluence points and flow cell). Eight roller four channel peristaltic pump is used to fill sample loop. Integration of manifold components allows miniaturization and optimization of flow channel dimensions in order to minimize sample and reagent consumption. tube fittings.) is optimized on one instrument. The FI-LOV configuration shown here is designed for two reagent assay using 50 cm and 100cm long reaction coils. so that when a standard serial assays ( such as phosphate. Table of Contents . to propel the carrier stream and two reagent streams.3. In FI format such “lab-on-valve” platform is used to streamline manifold components ( valve.1. it can be transferred to other instruments in another location with ease. fiber optic flow cell with 10mm light path and 50 µL sample Injection loop.4.

Yoza N. 199 (2002) Table of Contents . because of cost.1. and it is likely to prevail in routine laboratories. as compared to the conventional FI system. unless all four pumps will be run in a fully synchronized and automatically cycled mode. the flow programming of this novel instrument configuration will be a challenging task.. al. Replacing peristaltic pump with four channel syringe pump is a logical extension of FI-LOV instrument development. Advantages of using syringe pumps for FI applications have been recognized by Japanese researchers long time ago (Yoza 1977) and the use of Multisyringe Flow Injection Systems (MSFIA) has been proposed in numerous publications (Cerda 1999). Estela J.. 695 (1999) Miro M. Chromatography134. The main drawback of using multiple syringes is mechanical complexity. Ishibashi K. 497 (1977) Cerda V. convenience of operation. is far less complex as it operates with a only a single pump and a single valve. including stop flow FI for reaction rate measurement. Also microSI instrument. Ohashi S. et.4... However. an instrument build around individually driven syringe pumps combined with solvent resistant LOV module has following advantages: •resistance to corrosive chemicals •precise control of liquid delivery and manipulation •capability of programmable flow. use of peristaltic pumps for FI applications is deeply entrenched. Yet. Indeed. and ease of replacement of peristaltic tubing. J. Cerda V.M.. Talanta 50.4. TRAC 21.

Table of Contents .6) that. For teaching applications one may wish to construct a simple robust inexpensive system. Buying valves. there is a simple solution highly visible to everybody else.4. as it has to be compatible not only with the instrument.5. detector and other peripherals. Make sure that the peripherals you intend to use are compatible with the software before purchasing the core unit.4. To begin with. need a simple software and computer control for flow rate selection and sample injection. with replaceable components. but also with the user itself. if it uses peristaltic pump and two position manually operated injection valve . The most practical way to approach construction of a research instrument is to purchase a core unit. Such instrument does not have to be computer controlled. however. To make these components work in concert is quite another matter. The key to success is in designing simplest possible system with smallest number of components and then simplify it further. An interesting alternative to peristaltic pumping and valve injection is the use of solenoid driven pumps (1. the most important is the choice of software. pumps etc and connecting them with a tubing is the easiest step. Remember that: “Once you exhausted all possibilities. but you “( Murphy’s Law). For research applications there are almost infinite combinations possible of available components. driven by software with and open architecture and to add desired peripherals as the project gradually develops.1.

L. Saraiava M.4..L. They cover a very wide range of flow rates as the piston speed and syringe size can be varied.Rangel A.Lima J. A. Clausse.S.M. Solenoid activated micro pumps generate flow by delivering well defined pulses the frequency and volume of which controls the flow rate. although cost of peristaltic tubing exceeds many times the price of a pump over its lifetime. Analyt. Stepper motor driven syringe pumps generate highly reproducible flow that can be computer controlled in a programmable way.F. Peristaltic pumps are still the most frequently used drives for FI systems. 21. Table of Contents Santos J. 461 (2005) .000 pulses/day while exposed to aggressive chemicals.48 to 1.6. requiring frequent recalibration of the analyzer. since they generate continuous flow in any desired number of parallel channels.. Contributing factor to popularity of peristaltic pumping is its apparently low cost.O.L. It is important to use a pump furnished with at least eight rollers. The weakness of this truly innovative approach is durability of these pumps that must generate about 300..S.D of peristaltic tubing.1.M.F. depending on pulsing frequency (60 to 240 pulses/min).. their only drawbacks being cost and inability to generate continuous flow beyond the capacity of the syringe – that has to be refilled. A typical FI pulsed flow system (Rangel 2005) used 8µL pulses in three stream.. Sci. Solenoid Pump.92mL/min.C. They are durable and chemically resistant. 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. three pump system generating flow between 0. in order to generate a flow with small regular pulses – as otherwise resulting irregular flow rate will affect dispersion and repeatability of assay. while the flow rates can be easily adjusted by rotation rate and I.

Peristaltic pump Table of Contents .

however very important to use nuts. resulting in a leak.4. of 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.1. Tubing connectors. Connectors made of colored coded polymers are fitted with ferrules that are designed to grip tubing while the connector nut is being tightened. Stainless steel is yet another material that has advantage of heat conductivity gas impermeability and surface properties that minimize protein adsorption.7. Since all FIA systems operate at a low pressure.D. ferrules and fitting from a single manufacturer as products from different sources are often incompatible.D. While I. Heated reactor coil with temperature controller. ferrule and T-connector Teflon made reactor coil .5mm to 0. A majority of polymer made tubing is transparent and often available color coded. It is. can be identified at glance. Table of Contents . so that tubing I. there is not necessary to use connectors designed for HPLC. Teflon and Peek are the most frequently used polymers.

The valve can be switched from load to inject mode manually or automatically and the loop can be filled either manually by syringe. six port injection valve with a fixed loop is the most frequently used tool for injection of well defined sample volumes. Introducing air bubble and wash between samples is useful. It allows injected volumes to be chosen at will.1. or automatically from an autosampler by means of a pump (above).8. and to avoid sample to sample cross contamination. Volume of the external loop (shown above) can is selected between 20 and 100µL by changing the length and I. LOAD LOOP Two position.D of the loop tubing. It is important to keep the length of the conduit between sample container and port #4 as short as possible in order to save sample material.4. and at a selected flow rate. Table of Contents . but requires exact timing so that the injected volume is air free and contains undiluted sample. Six port multiposition valve combined with a stepper motor driven syringe pump is the key component of all Sequential Injection systems ( See Section 2). This injection mode is an ideal tool for automated optimization of FI and SI based assays.

Table of Contents .4. and •the volume of the forward stroke of the syringe pump. The key difference is in that the injection system based on a multiposition valve and the volume of injected sample is controlled by a syringe pump. Injected volumes are controlled by high precision syringe pump that aspirates selected volume of sample solution from sample cups.9. By changing injected volumes and reaction times sensitivity and detection limit of reagent based assays can be adjusted to desired level. Variable volume injection removes this limitation allowing automated optimizationof assay parameters. injected volumes cannot be automatically selected by a computer. Sample volume and reaction time are the most important parameters of flow injection experimental protocol. when central port is connected to port #2 .1. 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. (The auxiliary pump serves to transport sample solution from sample cup just past port #4. while the central port is connected to port #4. Since conventional FIA employs a two position valve furnished with fixed sample loop volume.

and than a selected section of the remaining diluted sample zone is injected into the sample processing manifold. injected into sample’ processing manifold. if entire sample is to be injected into the sample processing channel. FLOW INJECTING AN ENTIRE SAMPLE VOLUME If selected volumes of reversal and forward stroke are identical. A B C D INJECTING ONLY PART OF SAMPLE DISCARD ANALYZE Smaller volumes of the forward stroke can be.10. sample zone occupies in the holding coil twice the length of aspirated volume (B) the upstream end of sample zone being diluted by carrier solution. (C). the forward stroke should be at least twice of the reversal stroke volume. not all sample material will be injected into the sample processing manifold. adjacent to the valve is directed via port #1 into waste.1. Thus. but then the tail section of the sample zone must be flushed to waste (through port # 1) in order to avoid carryover of sample material remaining in the holding coil into the next sample processing cycle.4. Since the central stream moves at a double of average flow velocity. If sample is to be diluted prior to injection into the sample processing manifold. DILUTING SAMPLE (D). a desired portion of sample solution that has been aspirated by flow reversal. Table of Contents . because the sample forms a concentration gradient (A) in the sample holding coil.

Table of Contents . a light emitting diode is mounted directly onto the flow cell. A typical system comprises a “z-type” flow cell connected with quartz fibers to a spectrophotometer and a tungsten or deuterium lamp. also more traditional FI systems benefit from versatility and robustness of fiber optic technology. For a single purpose systems. Ocean Optics Spectrophotometer and a Tungtsen light source. Fiber optics and solid state spectrophotometers revolutionized the way in which all FIA techniques are carried out.4.11. While this change impacted mostly Sequential Injection.1. since this technology allowed optimization by bringing light and collecting data from any position of the sample flow path. Z-cell with 10mm light path Z-cell with 10 cm light path.

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

traveled by means of continuous flow. even if chemical reactions involved do not reach completion. and it is in any setting the best tool for teaching of principles of flow analysis. While manually operated experimental setup would be. Atomic Absorption and Inductively Coupled Plasma Spectroscopy are most prominent examples. from the moment of instrument startup. where sample injection and movement through reagent addition and product detection follow a simple route. Table of Contents . that provide strict time framework for reaction conditions. while majority of commercial analyzers still uses continuous flow platform. The chief advantage of FI is the transparency of its experimental setup. since it is the flow generated by a pump. even when no samples are being injected – and analyzed. Yet. it should be remembered that manually operated FI has been a workhorse of serial assays in developing countries. continuous flow operation is the main drawback of conventional FI as is consumes reagents. of which UV-VIS spectroscopy. It has been applied as a front end to practically all spectroscopic and electrochemical detectors. along with sample injection. understandably. Such control of mixing and timing allows reagent based assays to be carried reproducibly. It provides an unprecedented versatile sample handling. that has a wide range of applications. and creates chemical waste continuously. That allows automated assay to be carried out even without computer control. frowned upon by well heeled technician armed with PC and autosampler.Conventional flow injection is a mature technique. where computer control has nothing to offer. along with strict control of reaction conditions.000 publications. as it allows students to perceive the interplay of kinetics of physical dispersion and chemical reactions without unnecessary distraction provided by software or PC. described in over 13. Advances in computerization has enhanced FI mainly through automation of data collection and of calibration routines.

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

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