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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 .

Hansen.H. 78. 1. Anal.Hansen. Principles. “Flow Injection Analysis” 2nd ed. J.Y. N. Instruments & Components Flow Injection (FI). Acta .4.3. J. is the one most widely used. the first generation of FIA techniques. 1988 Table of Contents . In its simplest form . As the injected zone moves downstream. Willey.1.2. Basics.H.1. Methods & Applications. while a product begins to form at interfaces between the sample zone and the reagent. 1. 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). the sample solution disperses into reagent. Chim. Ruzicka & E. 1. Ruzicka & E. 145 (1975) J.

1.1 SINGLE STREAM MANIFOLD SIMPLEST .1. is well suited as a training tool in an undergraduate laboratory or for assay of small sample series. Table of Contents . 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.

With a sampling frequency of up to 120 s/hour.1. thousands of samples are analyzed within a week in routine Laboratories. Peak width (W) is readout for FI titration. TRAVEL TIME Peak height is the most frequently used response for construction of a calibration curve.1. Depending on the flow rate and reaction rate this readout is often available within less than 30 seconds after sample injection. Table of Contents .2. where FI system is usually coupled with an autosampler. while peak area (A) is used infrequently.

1.1.3. Table of Contents .

4. phosphate. where each channel is dedicated to a different chemical assay. Typically a three channel system allows . nitrate and nitrite to be analyzed simultaneously in water and soil sample extracts. This allows reagents to be added continuously to injected sample at a desired concentration. Since many assays use several reagents that must be added in a given sequence. A typical flow rate is 0. and gas diffusion based assays.1. Multistream FI system are routinely combined into multichannel systems. so that reactions can be carried out in sequence as the sample zone passes through the first and second reactor.5 -1. that allows automation of solvent extraction.1. Yet another advantage of multistream FI systems is their versatility. FI systems use multichannel pumps that propel carrier stream along with reagent streams. while an injected volume is selected in a range of 25 to 100µL. Table of Contents . dialysis. and by selecting pump tubes of a desired diameter.5mL/min of individual streams. A majority of FI systems use peristaltic pumps that allow flow rates of carrier and reagents to be controlled by choosing pump revolution rate.

5. For spectrophotometic measurements.2. Fully automated FI analyzer furnished with an autosampler comprises a four channel peristaltic pump.5 and 8ppm nitrate. injection valve and an integrated manifold with a z-type flow through cell. A@540nm 8 The attached readout shows a calibration record of 0.1. the flow cell is connected by fiber optics to a tungsten lamp and a scanning spectrophotometer.1. followed by a routine run of nitrate assay in soil samples using cadmium reduction column and sulfanilamide reagent. 5 2 0 seconds Table of Contents .


PRINCIPLE Table of Contents .

For multistream systems D-values have to be corrected by dilution caused by additional streams. While single reagent assays can be performed using the simplest. and to offer tools for optimizing sensitivity. 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. flow rates and manifold configurations.2. 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.1. This section is designed to provide an understanding of processes that yield FI response curve. Table of Contents . single stream manifold.1. Note that discussion in the following sections deals with a single stream system. it will be shown why a majority of FI techniques use multistream manifolds.

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

most FI methods use this element of fluid as a readout In order to optimize a given assay it useful to know how much the sample has been diluted in the FI system and how much time was available for chemical reactions to proceed. Therefore the dispersion coefficient has been defined a s D= Co/ Cmax allowing the degree of sample dilution to be estimated .1.3. SAMPLE INJECTION CONTROLLED DISPERSION REPRODUCIBLE TIMING Sample injection provides the initial Co square input serving as a staring point for initial concentration ( Co) and startup o T time. Since it is convenient to locate peak maximum. SAMPLE Controlled dispersion takes place as the sample zone moves downstream through the manifold.2. where the highest one ( Cmax) corresponds to peak maximum. This process forms a well defined concentration gradient that can be viewed as continuum of elements of fluid with different concentrations. Reproducible timing of sample travel from injection to detection yields repeatable value of Tmax. Similarly T max is the time elapsed from the moment of injection To to the moment of peak maximum T max. Cmax Tmax DISPERSED SAMPLE ZONE Table of Contents .

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. For success of a reagent based assay. Table of Contents . while the radial dispersion is designed to provide an efficient mixing of sample zone with reagent supplied by carrier stream.2.

Table of Contents .

Table of Contents .

2. Flow Injection response is a result of two processes.1. together with the dynamic characteristics of the detector the FI response curve. Table of Contents .5. is gradually formed at the interface between the sample zone (red) and carrier stream of reagent (blue). Note that the reaction product (yellow). These two processes occur simultaneously and they yield . both kinetic in nature: the physical process of dispersion of the sample zone and the chemical process of formation of a detectable species.

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

will be based. the one situated at peak apex ( Cmax) is the one on which peak height measurement. 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. and calibration.7. Of these segments. by carrier solution . Table of Contents .1. it disperses forming a concentration gradient that can be viewed as composed of a continuum of concentration segments of individual concentrations C.2. As the injected zone (A) moves downstream. has been diluted to half of its original concentration.

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.2. Table of Contents . •pH measurement Note that in a multistream system. When the distance between injector and detector is minimized in a single stream FI system. 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.8. D value is always > 2 as the flow rates of at confluence points have to be taken into account. Systems with limited dispersion are designed for automation of all reagent based assays.1.

1. 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 . 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.9. Table of Contents . dispersion can be adjusted to a medium value.2. of the volume of reactors between injector and flow cell and of flow rates in single or multistream manifolds. By adjusting the volume of injected sample.

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

gradients are reshaped and follow erf function.2. Table of Contents . For conventional FI systems this value is around 50µL.4mL/min in a single stream system. If the radial mass transfer is incomplete. The sample volume needed to reach 50% of the steady state depends on the volume. the resulting peak shape is composed of two exponential curves. 20 cm long. and convenient tool for adjusting dispersion coefficient and for optimizing the sensitivity of flow injection based assays. ultimately approaching Gaussian shape ( see Section 0.1. Increasing the injected sample volume increases peak height.5mmI. peak height increases linearly with the injected sample volume. at a flow rate of 1.Changing injected sample volume is versatile. Spectropotometry at 620nm. ). for micro SI systems as low as 5 µL .2. Recording shows traces obtaining by injection bromothymol blue solution into 0. geometry and flow rates in the channel between injector and flow cell. as shown here.2. Up to D =2 value (in a single stream system). until a steady state plateau is reached.11.D tubing. With increasing efficiency of radial mixing.

0 0 100 200 300 400 TIME. Selection of sensitivity and detection limit 2.002% 002% BTB.0 1. carrier 0. Table of Contents .5 2.1.5 10 0.5 0. 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 : •. This allows automated selection of injected volumes by means of software control.0 40 • Identification of the linear range of a detector 1.5 • Automated dilution of sample material Absorbance in Sequential Injection sample volume is determined by the volume of stroke reversal of a syringe pump. sec Automated injection of increasing sample volumes.005M 005M sodium teraborate. 50 516nm 567nm 20 0. Bromothymol Blue dye monitored at two wavelengths.0 A 1. Selection of injected sample volumes is a powerful tool for optimization of all FIA techniques. (Sample 0.2.12.

Programming the flow makes Sequential Injection technique more versatile than FI. Increasing length of a tubular channel decreases peak height while peak shape undergoes a change from asymmetrical to symmetrical shape. sample volume of 60µL in a way described in the previous slide). The use of programmable. allows incubation time to be prolonged by stopping the flow. At the same time the resident time of the peak maximum increases with the distance traveled and the peak base broadens. Table of Contents .4mL/min.2.1. This is the principal limitation of FI based on constant forward flow. (The recording here was obtained at a flow rate of 1.13. and speeding up the system wash by accelerating the flow. as the constant flow rate limits the incubation time for chemical reaction to about 20 seconds. instead of continuous flow. and combined flow rates around 3 mL/min. with a total conduit length of about 250cm.

It saves reagents and generates less waste than continuous pumping. thus yielding higher sensitivity.2. 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. It allows miniaturization by minimizing the length between injector and detector. Table of Contents . It provides information on reaction kinetics through reaction rate measurement. reproducible timing of sample handling operations is critical to success of flow based chemical assays.14. 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. Since all chemical reactions are time dependent. Stop flow allows longer reaction time without penalty of dilution.


Provided that the reaction did not reach equilibrium while the zone was on the way to detector. Syringe driven systems either FI or SI are reliable and their use in stopped flow mode is highly recommended.15. flow is resumed and reacted sample zone is flushed out of the detector. Next. Table of Contents . while the baseline is restored. While stop flow technique has been used in FI format. it is difficult to carry out reproducibly when peristaltic pumps are used propel carrier and reagent streams.2.1. reaction rate curve will be recorded while the reaction product (yellow) is being formed in the detector. DETECTOR The stop flow mode is based on arresting a selected portion of the sample zone in the detector.

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

Since the analyte (red) disperses within the reagent stream (blue) on the way to the detector while the product (yellow) is being formed. 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.2. In the absence of analyte horizontal (blank) line will be observed. it is essential that the delay time is perfectly reproduced for each assay.17.1. In the example shown. Table of Contents . while shorter delay times ( up to peak maximum) will yield steeper slopes. longer delay times will yield lower slopes since tail sections of the sample zone are more diluted.

FIAlab 2500 and associate software series of standards containing 500. 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. 1000. 2000 and 2500 ppm glucose were injected yielding response curves shown on the right.1.1. while the pump has been stopped. Using a single stream flow scheme.3. 1500. Table of Contents . Note that the travel time between injector and detector has been minimized in order to carry our the reaction within the flow cell. GLUCOSE OXIDASE Glucose + 2H2O + O2 Gluconic Acid + H2O2 PEROXIDASE 2 H2O2 + 4-Aminoantipyrine + p-Hydroxybenzene Sulfonate Quinoneimine Dye + 4H2O Enzymatic assay of glucose.

The main drawback of FI is continuous reagent consumption and waste generation Table of Contents .2. A majority of FI assays are carried at continuous flow.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. 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.1.

1. 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. Sample is injected by means of a two position injection valve with a fixed injection loop.R2) streams that merge at confluence points ( ) where reagent merges with sample zone. Merging of reagent and carrier stream Two reagents.3. thus providing a repeatable time frame for samples and standards as they are serially injected. three stream FI system Almost all FI instruments employ multichannel peristaltic pumps to move carrier and reagent ( R1.3. Table of Contents . 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.

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

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

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

Bi. fast reaction speed.: (Ed. Elsevier. 1999 Burguera J.1.3. Hg. Ni. and ease of automation were first demonstrated by Astrom in his pioneering work on FI based –hydride AA assay of bismuth. Cu. Fang Z-L.L: (Ed. Chichester. Ge. 1995. Flow Analysis with Atomic Spectrometric Detectors. Pb. while assay of volatile compounds of Ag.: Flow Injection Atomic Spectrometry. analyte enrichment.) Flow Injection Atomic Spectroscopy. as well as inductively coupled plasma mass spectrometry have been used s detectors The key component of the design is the gas-liquid separator. Sanz-Medel A.7. Wiley. 1989 Table of Contents . Hydride generation based Atomic Spectroscopies are routinely used for trace analysis of As. Amsterdam. and Zn has been reported in research publications. Co.). Atomic absorption spectroscopy . By combining an acidified sample stream with a strong reducing agent (sodium borohydride). cold vapor atomic absorption spectroscopy . Advantages of hydride generation: separation of the trace metals from complex matrices. New York. Se. 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. Marcel Dekker. inductively coupled plasma spectroscopy . Sn and Te.

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

E. Chem. In a two stream FI system. Baadenhijsen & H. D. Another. forming an air gap between carrier and donor stream through which gases like ammonia. L. sample containing carbonate (or dissolved carbon dioxide) is acidified. ozone or volatile compounds rapidly permeate into an acceptor stream where they are detected by means of a suitable reagent. (1998) Table of Contents . de Castro & I. The drawback hydrophobic membranes is that they can be fouled by surfactants that destroy the air gap barrier. with up to 50%porosity. (1979) •M. that offers a robust alternative to gas diffusion in parallel plate diffuser (Castro 1998) H.1. innovative approach to gas separation is gas pervaporation. TRAC. Clin. ( as the one shown above) are easy to assemble. 443. a“sandwich cell” construction allows increase of sensitivity of an assay. placed into acceptor channel. Seuren-Jacobs. monitored at 430nm (Baadenhuijsen 1979). chlorine. that diffuses across a silicone rubber made membrane from a donor ( blue) to an acceptor (green) stream changing color of an acidobasic indicator. Papaefstathiou.H. 25. releasing carbon dioxide. 17. sulphur dioxide.9.3. When miniaturized and integrated with a fiber optic detector. Membranes made of Teflon are hydrophobic. 41. Flat plate diffusers.

This flow cell can be used to monitor either a single . F. Pinto. Chem. Anal. E.B) it is useful to monitor volatile species emanating from a donor stream. 1992) A B M J. (M) mounted between two spacers (A. 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.Pavon et. Autom. M. Note that Teflon membrane may be furnished with an opening ( ) situated downstream from the fiber. al.P. P. 64.3. 923 (1992) C.. (Pavon et. G. M. Laespada.L. Cordero Analytical applications of separation techniques through membranes Lab.10. Note that stopping the flow of acceptor (indicator) stream allows accumulation of analyte and increase of sensitivity of measurement.1. Managem. liquid stream. J. Pavon and B. L. to alleviate pressure differences between acceptor and carrier streams. 34(2) 115-130 (1999) Table of Contents . or if furnished with a gas permeable membrane.

1 (1978) L. Anal.1.3. Miniaturization and automation of solvent extraction minimizes exposure to harmful solvents and reduces consumption of reagents and generation of hazardous waste.11. This method. Chim. applicable to assay of hormones. Detail showing circulation of extracted dye within segment of organic phase (Nord & Karlberg 1984). Sample (S) is injected into a moving carrier stream of water (AQ). Nord & B. which is merged (a) with an organic phase (ORG) and pumped through a Teflon made extraction coil (b). In separator (c) the aqueous phase is discarded into waste. while organic phase is led into a flow cell. 164. as it moves through a Teflon tubing. Anal. Chim. pharmaceuticals and numerous hydrophobic compounds. Acta. (Karlberg & Thelander 1978). Karlberg & S. Two stream manifold for automated solvent extraction. Acta 98. that up to that time was mostly carried manually. provides clue to mechanism of hydrodynamics of solvent extraction. B. Karlberg. revolutionized solvent extraction technique.Thelander. 233 (1984) Table of Contents .

In the membrane separator Teflon made membrane allows only the organic phase to penetrate through hydrophobic pores. Pacey C.12.E. A Practical Guide. 1989. In segmentor organic phase enters through a glass fitting and adheres to Teflon tubing (1).: Flow Injection Analysis. Flow-Injection Separation and Preconcentration.1. Elsevier.3. In separator a thin Teflon strip (3) serves to guide organic phase through a glass made T piece. Karlberg B. while aqueous phase is discarded. Table of Contents . while organic phase adheres to Teflon. 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. 1993. Amsterdam. Fang Z-L.:. VCH Verlagsgesellschaft Weinheim.

INSTRUMENT Table of Contents .

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

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

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

Indeed. et. J. Yoza N.4. 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). Yet.. TRAC 21. Estela J.M. 497 (1977) Cerda V. al. 695 (1999) Miro M. unless all four pumps will be run in a fully synchronized and automatically cycled mode. However. and it is likely to prevail in routine laboratories. Chromatography134. 199 (2002) Table of Contents . and ease of replacement of peristaltic tubing. is far less complex as it operates with a only a single pump and a single valve. Talanta 50. Cerda V.1.. Also microSI instrument. The main drawback of using multiple syringes is mechanical complexity. convenience of operation. because of cost. as compared to the conventional FI system. use of peristaltic pumps for FI applications is deeply entrenched.... Ohashi S. the flow programming of this novel instrument configuration will be a challenging task. Ishibashi K.4. including stop flow FI for reaction rate measurement. Replacing peristaltic pump with four channel syringe pump is a logical extension of FI-LOV instrument development. 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.

as it has to be compatible not only with the instrument. The most practical way to approach construction of a research instrument is to purchase a core unit. there is a simple solution highly visible to everybody else. however. pumps etc and connecting them with a tubing is the easiest step. To begin with. with replaceable components. detector and other peripherals. For teaching applications one may wish to construct a simple robust inexpensive system. Such instrument does not have to be computer controlled.4. the most important is the choice of software.1. but also with the user itself. need a simple software and computer control for flow rate selection and sample injection. The key to success is in designing simplest possible system with smallest number of components and then simplify it further. Make sure that the peripherals you intend to use are compatible with the software before purchasing the core unit. Buying valves. driven by software with and open architecture and to add desired peripherals as the project gradually develops. An interesting alternative to peristaltic pumping and valve injection is the use of solenoid driven pumps (1. but you “( Murphy’s Law).5.4. For research applications there are almost infinite combinations possible of available components. Table of Contents .6) that. if it uses peristaltic pump and two position manually operated injection valve . To make these components work in concert is quite another matter. Remember that: “Once you exhausted all possibilities.

The weakness of this truly innovative approach is durability of these pumps that must generate about 300. They cover a very wide range of flow rates as the piston speed and syringe size can be varied.S.Lima J.M. Clausse. although cost of peristaltic tubing exceeds many times the price of a pump over its lifetime.F. Contributing factor to popularity of peristaltic pumping is its apparently low cost.C. A. It is important to use a pump furnished with at least eight rollers.L.1. 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. 21. depending on pulsing frequency (60 to 240 pulses/min). requiring frequent recalibration of the analyzer. since they generate continuous flow in any desired number of parallel channels..6. A typical FI pulsed flow system (Rangel 2005) used 8µL pulses in three stream. They are durable and chemically resistant.F.D of peristaltic tubing. 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.48 to 1.L.. Analyt. Solenoid Pump. Stepper motor driven syringe pumps generate highly reproducible flow that can be computer controlled in a programmable way.M.S. Saraiava M.L.92mL/min. Solenoid activated micro pumps generate flow by delivering well defined pulses the frequency and volume of which controls the flow rate. Sci.. their only drawbacks being cost and inability to generate continuous flow beyond the capacity of the syringe – that has to be refilled. Table of Contents Santos J. Peristaltic pumps are still the most frequently used drives for FI systems. while the flow rates can be easily adjusted by rotation rate and I.4.O.000 pulses/day while exposed to aggressive chemicals.Rangel A... 461 (2005) .

Peristaltic pump Table of Contents .

there is a wide variety of tubing materials available for constructing reactor coils and connection lines.D. Heated reactor coil with temperature controller.5mm to 0. so that tubing I. Teflon and Peek are the most frequently used polymers. Tubing connectors. It is.8mm is typical for majority of FI and SI systems.D. ferrule and T-connector Teflon made reactor coil . can be identified at glance. While I. Since all FIA systems operate at a low pressure. resulting in a leak. ferrules and fitting from a single manufacturer as products from different sources are often incompatible. Connectors made of colored coded polymers are fitted with ferrules that are designed to grip tubing while the connector nut is being tightened. however very important to use nuts.4. Table of Contents . there is not necessary to use connectors designed for HPLC.1. Stainless steel is yet another material that has advantage of heat conductivity gas impermeability and surface properties that minimize protein adsorption. of 0.7. A majority of polymer made tubing is transparent and often available color coded.

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

and •the volume of the forward stroke of the syringe pump.1. whenever next sample change is to be injected). Variable volume injection removes this limitation allowing automated optimizationof assay parameters. Injected volumes are controlled by high precision syringe pump that aspirates selected volume of sample solution from sample cups.4.9. By changing injected volumes and reaction times sensitivity and detection limit of reagent based assays can be adjusted to desired level. Since conventional FIA employs a two position valve furnished with fixed sample loop volume. injected volumes cannot be automatically selected by a computer. Sample volume and reaction time are the most important parameters of flow injection experimental protocol. (The auxiliary pump serves to transport sample solution from sample cup just past port #4. Table of Contents . while the central port is connected to port #4. when central port is connected to port #2 . 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. The volume of sample solution to be injected is determined by: •the volume of the reversal stroke of the syringe pump.

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

1. For a single purpose systems. a light emitting diode is mounted directly onto the flow cell. also more traditional FI systems benefit from versatility and robustness of fiber optic technology. since this technology allowed optimization by bringing light and collecting data from any position of the sample flow path. A typical system comprises a “z-type” flow cell connected with quartz fibers to a spectrophotometer and a tungsten or deuterium lamp. While this change impacted mostly Sequential Injection.11. Table of Contents .4. Z-cell with 10mm light path Z-cell with 10 cm light path. 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.

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

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

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

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