Aspects of sample dispersion for optimizing flow-injection analysis systems

Analytrca Chumca Acta, 2610992)

539-548

539

Elsevler Science Publishers B V . Amsterdam

Aspects of sample dispersion for optimizing flow-injection analysis systems Takashl Korenaga Center for Environmental Scrence and Technology, Faculty of Engmeenng, Okayama Unrverstty, Tsushuna, Okayama 700 (Japan)

(Received 1st August 1991, revised manuscript received 26th November 1991)

Abstract Solute dispersion of an injected sample plug was studied by using an experimental apparatus with ideal lammar flow m order to develop a hydrodynamic model for the design of sensitwe and precise flow-injection analysis systems The dispersion behaviour of the sample slug under different manifold conchtions was first studied m detail to evaluate the effects of various operating conditions such as tube radius, tube length, flow-rate and molecular diffusion coefficient of sample solute The capillary flow propertIes were also exammed for some commercial micropumps to select the most suitable pumping method Mwng profiles and baseline stability m short, straight tubes were mvestlgated A double-plunger micropump having a hnear cam mechamsm and a fast, short reciprocation time was proposed to obtain smoother mlxlng and more stable pumping with good reproduck& Complete nuxmg and low flow-rate pumping are strongly desired for reliable flow-injection methods for Industrial process use, blosensmg devices for protem and enzyme bioassays require lower consumption of valuable reagents Keywords Flow system, Dispersion

Because blosensmg devices have become smaller, and reduction of the consumption of valuable blochemlcal reagents IS desirable, there 1s a need for stable pumping at very low flow-rates when usmg flow-mjectlon systems for the determination of macromolecular bloproducts with low molecular dlffuslvlty Therefore, solute dlsperslon properties of the inJected sample plug have to be fundamentally examined by using an ideal flow system to develop a hydrodynamic model and designing guldelmes for high senatlvlty, precision and rehablhty m chemical and blochemlcal process uses of practical flow-injection apparatus Some workers have discussed sample dlsperslon by pointing out the differences between dlsperslon and mung In evaluating lmpulse-response functions of mwng tees, Van NugterenOsmga et al [l] found that mwng tee geometry had a negligible effect on dispersion, whereas another study m which the murmg efflclencles of

similar tee geometries were evaluated by Clark et al [2] indicated maJor differences m mwng time among the tees The difference between mwng and dispersion has also been illustrated by the appearance of double peaks when sample volumes become large enough to isolate a section of the injected sample from the reagent [3] A theoretical evaluation by Hungerfold and Christian [4] predicted these peaks by consldermg the mfluence of sample volume on mwng Given the essentially “mcomplete” and contmuous nature of mwng in flow-injection analysis (FIA) it is not appropriate simply assume adequate mwng, and a theoretical dlscusslon of sample dispersion behavlour should also be presented Previous studies have included the development of a finite element method (FJZM) [51 and an improved numerical solution [6] by means of advanced computation techniques for the fundamental Taylor convection-diffusion equation [7]

0003-2670/92/%05 00 0 1992 - Elsewer Science Pubhshers B V All rights reserved

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An apparatus has also been constructed 181 for the analysis of capillary flow properties and to evaluate the solute dispersion behavrour of the injected sample plug m order to be able to design appropriate mmtaturrzed flow-mJectton systems When a dye solutron plug was introduced mto an aqueous carrier stream of the apparatus, the breakthrough signal showed drrectly the mean concentratrons of the resultmg solutron over a cross-section at each moment [9,10] After alternately feeding a coloured dye solutron and colourless water, mwng profiles m the tube were mvestrgated spectrophotometrrcally In order to evaluate capillary flow properties, whtch were shown to be effective for high-sensrtmrty flow-mJectron techniques with small-bore and shortlength tubes, the degree of mwng was also measured by usmg the same apparatus [ll] The results were useful for the development of an advanced mrcropump with double plungers, havmg a lrnear cam and fast recrprocatron mechamsm With this pump, complete mwng and stable pumpmg over a wide regton of flow-rates could be accomplished The system described by Korenaga and Stewart [12] combmes a small-bore (1 d < 0 5 mm) and short (length < 100 mm) reactron tube wrth a small flow cell (volume < 8 ~1) and low flow-rate (< 50 pi/mm) to attam hrgher sensmvrty and precrslon m flow determmattons of both macromolecules and morgamc ions 112-161 Although various mrcropumps based on several pumping mechanisms are avallable for use m tradrtronal flow-inJection analysis (FIA), it is not easy to realize such micro flow properties In this work, the prevtous apparatus 181 was improved m order to control the reaction temperature precrsely even when using short reaction tubes Mass transfer of sample molecules m a lammar capillary flow was then mvestrgated m detail to evaluate the solute dispersion behavlour of the Injected sample plug The results were used for the development of a flow sensmg device and capillary stream sensor to be used m flow-mJectron systems Baseline stabrlmes and mwng profiles examined downstream are of practical use for process momtormg and control purposes m brotechnology Followmg these theoretical and

Chm Acta 261 (1992) 539-548

experimental studres, there have recently been great demands to develop flow-mJectron and other non-segmented contmuous-flow systems based on theoretical connderatrons, such as (capillary) column liquid chromatography [lo,171 and capillary electrophorests [lo] for the purpose of m-hne momtormg m brotechnologrcal processes

EXPERIMENTAL

Apparatus A schematic diagram of the improved apparatus 1s shown m Fig 1 An essential part is the laboratory-made flow-through mrcrophotometrtc detector which allows measurements to be carried out without any disturbance of the lammar flow A patr of hght sources conastmg of a mmrature fluorescent lamp and light acceptor with a photodrode (S-1033-01, 15 mm width, 6 mm length) (Hamamatsu Photon4 are trghtly mounted parallel on the outside wall of a transparent poly(tetrafluoroethylene1 (PTFE) tube (e g , 0 806 mm 1 d and 15 mm o d 1 The solute mean concentration over a cross-sectron is measured by the photometrtc system vra a Model 757N logarrthmrc amplifier (Analog Devrces) The photodtode 1s so large that all the hght through a cross-section can be received to ensure that the measured concentratton 1s really the mean concentratron over the cross-section To reduce the _---

1

L Fig 1 Block diagram of the Improved theoretlcal expenmental apparatus A, Syrmge mlcropump, B, lomt, C, PTFE tube, D, hght source, E, photodlode, F, logarlthmlc ampbfier, G, recorder, H, air-bath, I, water-bath, J, temperature-controlled room

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Chrm Acta 261 (1992) 539-548

influence of any possible natural convection resulting from the dens@ difference between the sample solute and water carrier, a vertical and downflow system was adopted m place of the hoIlzonta1 system [9] The flow-rate was calculated by measurmg the weight of effluent fluid at the tube exit Before the expenment was carried out, a mean concentration calibration graph was obtained by contmuously passing a Basic Blue 3 (BB3) dye solution of known concentration through the tube The callbratlon graph found was almost lmear The slug length used must be much shorter than the travellmg distance between the mjectlon and detection pomts, a ratlo of travellmg distance to slug length of more than 150 has no effect on the hydrodynamic treatment, as the sample slug 1s rapldly diluted to a very low mean concentration at the detection point because of the lammar (axml) dispersion behavlour Further, the unproved apparatus includes a syringe micropump (JP-V-W, Furue Saence), a straight PTFE capillary tube of various inner diameter, a precisely controlled air-bath (CRB6AS, Shlmadzu) and a multi-range chart recorder (U-228, Nippon Denshl Kagaku) The internal temperature of the air-bath 1s precisely controlled by means of temperature-controlled water obtamed from a precise water-bath (LCH-19, Advantec Toy01 and flowing through copper heatexchanger tubes installed m the internal wall of the an-bath The expenmental set-up 1s located m a temperature-controlled room eqmpped with a laboratory air condltloner Ma tenals

A dilute solution of BB3 dye (Aldrich) and dlstllled water were mainly used as coloured sample slug and carrier fluid, respectively The molecular dlffusron coefficient (D) of BB3 m pure water at 20°C 1s 3 78 X 10e4 mm* s-l [18] Other coloured morgamc chemicals such as KMnO, (D = 12 X 10m3 mm* s-l), K2Cr20, (D = 124 x 10e3 mm* s-l> and K,Fe(CN), (D = 9 6 x 10e4 mm2 s-l) [19,201, and commercial pigment mk containing a coloured macromolecule [pigment cobalt (DIG-578P type), D = 5 x 10e5 mm* s-l, estimated value by the authors [19],

541

obtained from Sakura Colour Products] were used as received All reagents except BB3 and the pigment mk were of analytlcal-reagent grade from Wako Procedure

A small portion (2-8 ~1) of sample dye solution is Injected into the stopped-flow system at Jomt B m Fig 1 Before injection, the upper tube 1s removed and a small amount of water IS removed from the lower tube Thus a free space 1s formed that can be filled with the sample The sample solution is injected mto the free space with a glass mlcrosyrmge By such a space mJectlon method, an almost Ideal input can be realized The syrmge mlcropump 1s then started nnmediately In order to ensure a fully developed flow, the two tubes are reJoined after several seconds, and at the same time the chart recorder starts to monitor the breakthrough profile curve The sample slug flows according to lammar flow condltlons [lo], which was checked by mjectmg a sample mto a flowing or a stopped system The response curves found from the two mJectlon manners do not vary The real mean concentration dlstrlbutlon can be obtained usmg a cahbratlon graph

RESULTS AND DISCUSSION

Improvement of the expenmental apparatus The apparatus described m the previous study

[81 was Improved by locatmg the laboratory-made flow-through mlcrophotometrlc detector and straight, transparent PTFE tubing m the precisely temperature-controlled air-bath (preaslon, *O l”C), which was specially made for this work The temperature of the air-bath could be controlled by mtroducmg temperature-controlled water which was obtamed from a precise water-bath (precision of water temperature, f0 05°C) and the water was pumped mto copper tubes installed m the internal wall of this air-bath Moreover, the experimental space containing all the expenmental equipment was controlled with the laboratory air condltloner (preaslon of room temperature, ca *l”C)

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The Improved apparatus was found to be useful for the theoretical treatment of breakthrough peak profiles and the study of the optlmlzatlon The experrmental results for solute dispersion behavlour of the inJected sample solution obtamed m these experiments were satisfactory and effective for developing guidelines for the design of optlmlzed FIA systems Breakthrough profile of dye solute plug m capdlary tube

To study peak broadening and controlling properties m lammar capillary tube flow, breakthrough response profiles of injected sample plugs were examined with the improved experimental apparatus (Fig 1) under various operating condltlons m order to design theoretically based FIA manifolds In this work the operating condltlons were restrlcted to the followmg parameters and operating ranges travellmg distance (L) from the inJection port to mlcrophotometrlc detector 1502000 mm, tube radius CR) 0 23-O 90 mm, volume flow-rate 3 4-60 ~1 s-l and molecular dlffuslon coefficient of the sample solute CD) 5 x 10e5-1 24 x 10e3 mm* s-l Directly after mtroducmg the dye solution plug mto the stopped water carrier, it was propelled as a slug in a continuous stream of water carrier The syringe mlcropump formed parabolic profiles between the sample slug and water carrier when small amounts of sample (less than 8 ~1) were

inJected into water at a controlled temperature (e g ,2o”C) Representative breakthrough curves obtained are shown m Fig 2 for given operating condltlons The expernnental profiles agreed satlsfactardy with the theoretical breakthrough curves obtained by the authors’ improved numerical solution [6] Therefore, it was found that the expenmental breakthrough curves were useful for treatmg theoretically solute dispersion behavlour for the purpose of the design of advanced FIA apparatus Effect of sample volume

In order to ensure the optimum slug length of the mmal input, the volume of coloured sample solution qected into the stopped-flow system of the apparatus was varied from 2 to 20 ~1 The nutlal slug length was readily calculated from uqectlon volume and tube diameter From Fig 3, it can be seen that a volume change of the sample seldom affected either the baseline-to-baseline time dispersion value (At, total time between uutlal appearance of a peak and its final disappearance at the detector) or breakthrough profile, as the slug length CL,> was much smaller than the travellmg distance (L) Followmg the above procedure for defined flow conditions, the slug length of the mltlal input did not affect the experimentally found breakthrough curve profiles For nearly constant baselme-to-

z x

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200

400 Time

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800

(s)

Rg 2 TypIcal breakthrough peak profiles of expenmental (sohd hne) and theoretlcal (broken hne) results for drfferent operatmg condltlons (L, = 9 7 mm, R = 0 403 mm, D = 3 78 x 10m4 mm* s-l) (A) L = 400 mm, U = 20 4 mm s-l, (B) L = 800 mm, u = 3 4 mm s -‘,(C)L=1250mm,U=34mms-’

T Korenaga/Anal Chrm Acta 261 (1992) 539-548

0

smaller bore sues and shorter lengths than those described above Larger slopes were obtamed at tube radu R > 0 33 mm The slope also corresponded with the authors’ recent slmulatlon result [6] obtamed by usmg the volume flow-rate value as the parameter (see Eqn 3) The present results indicate that mmlmlzmg the tube radius was much more effective for radn larger than 0 33 mm and less effective for smaller radu Therefore, the tube radius was one of the possible controllmg factors for optlmlzmg sample dispersion m theoretical FIA manifolds using straight reaction tubes

L...l...“.“J 10

20

Sample iniectlon

volume

543

(mm31

value for 4 5, 8, 7 8, Q, 14 7, @, 22 5, 0, 424

Effect of travelhg dutance The travelhng distance CL) was defined as the

baseline time dlsperslon (At,) values, the optlmum flow velocity (U) must be greater than 14 7 mm s-l, the sample mjectlon volume (V) must be 2-8 ~1 and the L,/L values should be smaller than 0 2 In this experiment, however, none of the flow condltlons gave measurable errors for the measurement of the At, value m the breakthrough curve

straight tube length from the mJectlon port to the point of cross-sectron view of the micro detector The effect of travellmg distance was investigated by changing parameters such as velocity flow-rate (U) and tube radius (R) m order to evaluate different operating condltlons Typlcal examples are given m Fig 5 A logarithmic linear relationship was observed between travellmg distance (the same as the tube length L m common cases) and baseline-to-baseline time dispersion (At,) values for various flow-rates at a tube radius R = 0 42 mm For the sx tube radu

Rg

3 Effect of sample mjection volume on hi,

various flow-rates mm s-l

0,

Effect of tube radrus

Figure 4 shows the relationship between baseline-to-baselme time dispersion (At,) and tube radius (R) (0 23-O 90 mm) for various tube lengths These experimental results show that At, 1s proportional to R2 m the range R =033-O 90 mm, but 1s proportional to Ro72 m the range R = 0 23-O 33 mm In Fig 4, critical turning points were clearly observed for a tube radius of 0 33 mm for all the tube lengths examined The slopes obtained for At, vs R were almost constant, being about 0 72 for tube radu between 0 23 and 0 33 mm The value of 0 72 did agree with that obtained m the authors’ recent slmulatlon [6] with Taylor’s convection-diffusion equation [7] This was also suggested from previous FEM apphcatlon results [5] with the same equation, although the slmulatlons were made for

I

01

02

“,’

0'

05 IO R (nn)

20

Fig 4 Effect of tube radws CR) on At b value for various tube lengths o, 2oo0, O, 1200, @, 600,q 300, 8, 150 mm

544

T Korenaga /Anal

200 -

5-

100

500

200

L Fig

5

Effect

1000 2000 (mm)

of travelhng distance (L) on At, 0, 6 3, 0, 11 0, 0, 20 8, 0, 32,

various flow-rates

value for A,

45, A,

60 mm s-l

examined (0 23, 0 28, 033, 0 53, 0 76 and 0 90 mm), almost the same relatlonshlps were obtamed for At, vs L For each tube radms, the slopes of the seven graphs were about 0 64, but the extrapolation pomts at L = 100 mm were different From these experunental results it can be concluded that At, value was proportional to Lo64 m the examined range Effect of flow velocity

Figure 6 shows a typical relationship between baseline-to-baseline time dispersion (At,) and

WJ51 Effect of molecular d&%suxty

Molecular dlffuslvlty of the solute 1s one of the most important parameters controlling basehneto-baseline time dispersion m FIA It 1s also an effective parameter for high-resolution separation and for the design of capillary chromatographic systems [17] A correlation study of molecular dlffuslvlty with baseline-to-baseline time dispersion phenomena also formed the basis for a rapid measurement of D values [19,20] For R = 0 42 mm, both theoretical and expenmental results show a linear logarlthmlc relatlonship between At b and D values, as shown m Fig

I III11.I 2

5

10

20 D

50

100

Fig 6 Effect of flow-rate (U) on At, value for various lengths o, 2000, 0, 1200, 0, 600, 0,300, 8, 150 mm

5

I IO

Dx104

(mm/s)

tube

Acta 261 (1992) 539-548

mean flow velocity G-0 values for various L values at constant radius (R = 0 42 mm) The L values examined were from 150 to 2000 mm There 1s a linear loganthmlc relationship between At, and U values with a slope of - 0 64 Hence flow velocity 1s an attractrve factor for the optlmlzatlon of peak broadening and controlling phenomena m constructing optnnum flow-mjection manifolds This experunent shows that At,, 1s proportional to U-064 m the range exammed between U = 4 5 and 60 mm s-l For other constant tube radu examined (R = 0 23, 0 28, 0 33, 0 53, 0 76 and 0 90 mm), the same relationship between At, and U was obtained Even at lower flow velocltles the At, values of the breakthrough curves showed a similar relationship m such Ideal experiments

10 3

Chm

Fig 7 Effect of molecular various flow-rates 0, 6 3, 60 mm s-l

20

,I 50

(mm21s) dtislvity (D) on AI, 110, 0, 20 8, 0, 32,

c) ,

value for 45, A,

A,

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7 All the slopes of the log At, vs log D plots for different U values were -0 36 m the D range exammed All the results show that At, IS proportlonal to D-o36 m the range U = 6 3-60 mm s-l For different tube radu (R = 0 23,O 28,O 53,O 76 and 0 90 mm), the same proportlonahty between At,, and D was observed Effect of mean restdence time Mean residence time (t,) was experlmentally

defined for the sample slug as the time between the inJection and the detectlon of the peak maximum of the breakthrough curve, and theoretically as the L/U value of the dispersed sample solute slug The latter gives a theoretlcal residence time of the mltlal sample solute mput without any molecular diffusion Figure 8 shows the logarlthmlc relatlonshlp between At, and t, (= L/U) values This relationship IS nearly linear over a wide range of L/U values and all the results obtamed for dlfferent R values show almost the same dependence However, the At, values seems to deviate from the theoretical relatlonshlp (broken line) at small R values, as the radial molecular dlffuslvlty of the solute shows a larger effect m suppressmg axial dlsperslon of the slug These experimental results suggest that At, values are nearly proportional to (L/U)o61 for the examined range of R values

24

0 2

4JD

a

0

10

10

100 (s)

1000

Fig 8 Effect of mean resldencc time (t,,,) on AI, value for various tube radn A, 0 90, l, 0 76, 0, 0 53, 0, 0 42, 0, 0 23

1

05>

O

0 005

III./ 2UR2

Fig 9 RelatIonship between dlmenslonless travelhng distance (DL/ZUR*) and dlmenslonless baselme-to-basehne time dlsperslon (At,D/R*) for theoretical (sohd hne) and expenmental (symbols) results for various flow-rates 0, KMnO,, 0, BB3 dye, 0, K,Cr,O,, A, K,Fe(CN),

Evaluation of peak broademng and controllmg propertw m FL4 The At, value 1s the most important factor for

attammg high sensltlvlty The At, values are inversely proportional to peak height because the peak-area values measured with the present setup are nearly constant for each breakthrough curve Hence the At, value could be optumzed as functions of R, L, U and D values with satisfactory results Accordmg to the authors’ slmulatlon results, the At, value can be described as functions of R, L, U and D values as indicated by the lme m Fig 9 Figure 9 shows that dlmenslonless At, D/R* values are satlsfactorlly proportional to dlmenslonless (DL/2UR2)061 values for all the expenments m the range examined The experiments shown were carried out for R = 0 42 mm and L = 300 mm The same relatlonshlps were obtamed for other tube radu and lengths The basic equation for At, IS At,

t, as L/U

mm

"a 202

=

1 24RO

72Lo

MU-0

64D-0

36

(1)

Equation 1 could be easdy modified to Eqns 2 and 3 by usmg a time parameter as L/U and a flow velocity as Q, respectively At, = 1 93R072(L/U)owD-036

(2)

At,=2

(3)

58R*LO"Q-o"D-"""

546

Equation 1 was also supported by the previous differential analysis method of Vandershce et al [21] and the FEM slmulatlon [5], although total optlmlzatlon of At, values could not be carried out m this work because it was not easy to select the most successful controlling factor from the above four functions and other imaging parameters because these functions were interrelated m all the given flow systems This first part led to the followmg conclusions R should be as small as possible because the At, value 1s effectlvelly mmlmlzed m proportion to Ro7* (or R*) However, for tube radn less than R = 0 33 mm, it 1s problematic to ensure contmuous operatablhty of practical flow-mnjectlon systems When L decreases, At, becomes smaller because it 1s proportional to Lo@ if adequate mwng and mmlmum reaction time have to be guaranteed However, a mmlmum reaction time should be maintained m common chemical and blochemlcal reactions Mmnmzatlon of U is very effective for obtaining small At, because it 1s proportional to U- 064 No problems a re to be expected except for bubble formation at high reaction temperatures Mmlmlzatlon of the D value 1s also effective for controlling the At,, value with good processablhty of the FIA apparatus, At, being proportional to D-o36 Hence a low vlscoslty of the carrier fluid and high temperature of the FIA reaction system are to be preferred with a view to design guldelmes and mstrumentatlon hydrodynamics for a theoretical FIA manifold construction Performance of a theoretical FL4 manlfold system

All the factors examined were evaluated to develop design gmdelmes and to fmd suitable hydrodynamic condltlons for setting up advanced FIA systems Consldermg peak broadenmg and controllmg propertles, D values of the given flow system have to be mmlmlzed by use of water-hydrophlhc organic solvent murtures as the carrier solution 1171 and/or adoptlon of hlgh-temperature reaction media Further, the improvement of pumping properties for the purpose of mmlmlzatlon of At,, 1s proposed Provided that the volume flow-rate

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was mmlmlzed to ca 1 ~1 s-l with stable pumpmg properties, molecular dlffuslvlty of the solute molecule seemed to contribute successfully to overcommg axial dispersion caused by the lamlnar flow velocity profile in flow-injection systems Hence the development of a new mlcropump with an advanced pumping mechanism 1s one of the most important aspects for developing a FIA apparatus with high sensltmty, precision and rehability Importance of pumpmg technology with regard to destgn guuielmes and hydrodynamic condrtlons m FL4 It was concluded that a long-period, steady-

flow mlcropump must be developed for a flow-mJectlon apparatus that can be used m a variety of mdustrlal processes The properties of the mlcropump necessary for mmumzmg At, are as follows improved mwng between reagent and carrier streams has to be reahzed without any increase m dead volume (current mncmg chambers and/or mwng Joints have dead volumes that cause wide At, values), a completely stable baseline, which is obtamed Just after mwng between the reagent and carrier streams without any drift, will give the posslblhty of attammg high senntmlty by electrical amphflcatlon, higher reproduclblhty of flow-rates of reagent and carrier streams 1s desirable to attam more reliable determinations with higher precision and lower relative standard deviations, and long-life operation of the mlcropump with mmlmum operator care and mamtamance of the instrument 1s necessary Based on these connderatlons, attempts were made to use smaller flow-rates for optlmlzmg At,, values m FIA systems for the determination of macromolecular compounds with high sensitivity [12,15] However, Eqn 2 also suggested a broadening of At, m proportion to (L/U)‘@ when the mean residence time (as L/U) becomes larger Therefore, the importance of micro-flow pumping technology for the purpose of developmg an advanced FIA system oriented to practical use such as m mdustrlal process momtormg 1s emphasized again A double-plunger mlcropump with an advanced linear cam mechanism and short/fast reclprocatlon instead of the traditional

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eccentric cam mechanism for LC use has been successfully developed, the former 1s avallable for the ordinary total flow-rate range of 3 3-33 ~1 s-l (1 e , 0 2-2 0 ml mm-‘) [221 In such a FIA apparatus for practical use, the basic specifications of the micropump are listed m Table 1 Mung properties of a hear-cam double-plunger mlcropump The mlcropump, which IS considered appropn-

ate for FIA, can be applied to many conventional FIA methods (total flow-rate 0 2-2 0 ml mm-‘) Recently, a micro-flow double-plunger pump for flow rates smaller than ~1 s-l was developed 1111 This mlcropump was found useful for highly sensitive, precise and reliable bloassays for macromolecular bloproducts such as enzymes and protems [23] Mlxmg profiles III the straight tube were mvestlgated spectrophotometrlcally from the baselme stablhty, which was shown as the result of reclprocal mwng of coloured dye solution and colourless dlstllled water The breakthrough signals were recorded after dye solution and water were mtroduced by each double micropump and then mtxed Every mlcropump forms alternate parabolic profiles between the sample plug and water carrier when small amounts of sample (less than 8 ~1) are InJected into water

547

Total interface areas of the flowmg sample slug were estimated for each method of pumping, total Interface areas ImplIed a complete contact surface area between the dye sample and water carrier when the sample and water were reaprotally pumped A lmear-cam double-plunger mlcropump possessed the broadest total interface areas between sample and water streams Just after mwng, because of its lmear cam pumpmg mechamsm with very short but fast plunger strokes No auxlhary damping tube is needed to disperse its solute This micropump therefore gave a well mixed baselme The pumpmg IS so stable for each flow-rate that noise IS hardly observed m any flow-mjectlon mode The reproduclblhty of the mxropump was also tested by usmg the apparatus under condltlons where double peaks can be obtamed The micropump gave reproducible breakthrough peak profiles that reflected stable feeding, other mlcropumps showed Irregular feedmg at relatively small flow-rates The lmear-cam double-plunger micropumps should be useful over a wide flow-rate range for developmg future theoretlcally based FIA systems such as a mmlatured flow sensing device and capillary stream sensor, bemg apphcable for m-lme momtormg and prolonged operation because of its smoother pumping and lower reagent consumption

TABLE I Basic cornposItIons and speclficatlons of an ordmaly double-plunger mlcropump developed wrth hnear cam mechanism Pumpmg type Flow-rate range Cam system Motor Plunger materlal Plunger diameter Stroke length Stroke volume Pumpmg mechamsm Flow-rate change mechamsm Pressure resistance Llquld contactmg material Lme degasser

Double plunger pump 02-2Omlmul(3 3-33 PI s-l) Lmear cam with hnear curve rotation D c brushless motor with d c sewo-mechamsm feedback system Sapphire 25mm 1Omm 4 9 ~1 per stroke Dual-head reclprocatlon with phase shift of 180” Variable rotation 600 kgf cm-* PTFE cyhnder and ruby balls Glass air trap with total volume of ca 1 ml for ehmmatlon of air bubbles m stream hnes

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REFERENCES

Rg 10 Sample elutlon behavlour and its breakthrough peak profiles using the Ideal-flow experlmental apparatus at various flow velocltles Sample 2 ~1 of BB3 dye aqueous solution Carrier dlstdled water Flow-rate (a) 0 33, (b) 0 83, (c) 17, (d) 3 3, (e) 8 3, (f) 16 7, (g) 30 ~1 s-l

In conclusion, often there 1s a gap between theoretlcal treatments and actual FIA practice, the result being a theoretical treatment that does not reflect the actual state of the art from a practical pomt of view For instance, the condltlons required to match experiments to theory really may not be optunum for analyses for hlghmolecular-weight compounds In fact, secondary flow induced by deformed channels (especially meandering or knitted tubes rather than simple colled tubes) ~111probably lead to improved mvrmg as radial mwng by dlffuslon alone 1s mefficlent for large, sluggish blomolecules In order to indicate the degree of orlgmahty on the basis of this work alone, the most representatlve example of how to apply this theory to an analytlcal mstrumentatlon problem is presented m Fig 10 The author thanks to Professors T Takahashi of Okayama University and K K Stewart of Vu-gmla Polytechnic Institute and State Umverslty for helpful dlscusslons He also thanks Dr F Shen, Mrs M Izawa, Mr X Zhou, T Fupwara, H Yoshlda and Y Yokota and Miss R Zuraldah for slulful experlmental assistance, and the Mmlstry of Education, Science and Culture of Japan for partial fmanclal support over several years

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