J. Flow Injection Anal., Vol. 19, No.1 (2002)

PSI: Optimization of Reagent ased Chloride Assayin Lab-on-Valve System. .

Syouhei ish hi ha ma,^^^'* Louis S ~ a r n ~ a v ianda , ~Jaromir ~uzicka'~ e ~ a r t r n eof
n t~ h e m i s t r ~ , ' ~35'1700,
ox
University of washington, Seattle, WA 98195-1700, USA
Department of Chemical Processes and Environments, Faculty of Environmental Engineering,
The University of Kitakyushu, Hibikino 1-1,Kitakyushu 808-0135, Japan
Abstract
A spectrophotometric chloride assay has been investigated, employing an integrated lab-on-valve manifold in a micro fluidic
sequential injection format (pSI). By optimizing the chemical and physical parameters a limit of detection of 45.02 ppb was
obtained with r2 = 0.9917 (0-1000 ppb). The assay for higher concentrations (0-100 ppm) was also achieved by microfluidic
manipulation of the sample zone within the holding coil. Thus, this method is useful for chloride analysis over a 3-decadeconcentration range. The method described serves as a template for downscaling other colorimetric assays where addition of theanalyte results in a change of absorbance but not the color.

Keywords PSI, lab-on-valve, chloride assay
1. Introduction
The chloride assay is one of the most important indexes ofwater quality, and many spectrophotometric techniques havebeen developed [I]. The Utsumi's method [2,3], using
mercury(I1) thiocyanate and iron(II1) ion, is now widely applied
to the chloride assay. This method is based on the following
reactions.
Hg(SCN)2 + 2C1- -* HgCI, + 2SCN"~e~~ + SCN- -+ F ~ S C N ~ 'This. method was automated for clinical and industrial
laboratories [4], and adapted to flow injection (FI) by utilizing
the stream sample splitting technique [5,6]. This colorimetricmethod, however, has disadvantages in that a toxic reagent isneeded and that the dark yellowish color of the reagent interfereswith the detection of the red colored product.
Miniaturization of flow-based assays is at present anactively pursued topic in analytical research. Based on flow
injection, sequential injection (SI) has been developed for
reducing reagent and sample consumption [7,8~. The SI system
consists of the central processing unit (lab-on-valve manifold),the syringe pump, holding and mixing coil, and the detector. In
a conventional FI system, samples are injected into a continuous
flow that is always generating waste. The sample and reagent
solutions in a SI system, are sequentially aspirated only as
needed, into the lab-on-valve manifold, and then are dispensed tothe detector using a minimal of carrier solution. Thus, the SI
method can substantially reduce waste production and reagent
consumption. Another advantage to the SI system is thatdifferent reagent based assays can be performed using the same
hardware structure, since variations in the experimental
protocols (sample size, flow rates, mixing periods) areprogrammable. The greatest advantage of the SI method is its
ability to handle wider dynamic concentration ranges throughprogrammable dispersion. There are several reports [8,9] andreviews {10,11] for analysis using SI system. The extension ofthe's1 system to the chloride assay with the Utsumi's method
may be expected to reduce the amount of the toxic reagent,achieve lower detection levels and handle a wider concentration*Corresponding author.
E-mail: nishihama@env.kitakyu-u.ac,jp

range. The analytical protocol described in this work will also
be applicable to other SI analyses where the reagent has highbackground color.

2. Experimental
2.1 Instrument

The sequential injection system (FlAlab-3000, FIAlab
Instruments, http://www.flowinjection.com) consists of a stepper
motor driven syringe pump (1000 pL), a six-port selector valve,
and a single speed unidirectional peristaltic minipump, as shownin Figure 1. A tungsten-halogen lamp (LS-1, Ocean Optics Inc.,

Kg
coil

Car

ReagenspacerFigure 1. Schematic layout of the @I instrument with a
precision syringe pump (1000 pL) connected to the holding
coil and the mixing coil. These coils are where the stacked
zone of reagentlsample is made. The lab-on-valve device
was mounted atop a six-way, multi-port valve, w h i a allows
for the fluidic operations of sample (port #5) and reagent(port #3) selections as well as the selection of the flow cell
(port #2). An auxiliary peristaltic pump serves as theflow-through port (port #5) for rapid sample exchange.
The flow cell is shown in absorbance configuration using
two optical fibers facing each other. The internal diameter(I.D.) of all the t~bing was 0.5mm (O.D.: 1.6mm).

http://www.oceanoptics.com) was used as the VIS light source
for the UV-VIS spectrophotometer (USB2000, Ocean Optics
Fiber optic cables,
Inc., http://www.oceanoptics.com).
furnished with a stainless steel clad tip (400 micron, tip 0.0625"
OD) were used to connect the flow cell to the light source andthe spectrometer. Data was collected by FIAlab for Windows
98 (version 5.9.30) installed on a PC (Pentium I1 300 MHz, 64MBRAM) with Windows
98(Microsoft
Inc.,
http://www.microsoft.com) as the operating system.
The lab-on-valve manifold was fabricated in-house andinterfaced to match a six-port selector valve as described earlier[8]. The central inlet was connected to a holding coil. The
flow cell was designated to connect port #2 of the six-port valve
to the integrated flow through detector. The sampling port was
set to port #5 while the waste was designated to #1, the reagent
was designed to #3, and spacer was designated to #4. The flow
cell was configured for absorbance measurements at 460 run, as
shown in Figure 1, and the pass length was set with 10 mm forlow concentration (0-1000 ppb) assay or 5 mm for highconcentration (0-100 ppm) assay.

the slope of the calibration curve in low sample concentration
region (0-1000 ppb) was investigated in order to optimize theassay for the highest sensitivity. In this case, the light pass was
set as 10 mm, the aspiration rate for reagent, sample, and spacer
was set as 50 uJL/sec, and the integration time of thespectrophotometer was set as 500 msec. Figure 3 shows theeffect of the concentration of iron(II1) nitrate and mercury(I1)thiocyanate on the slope of the calibration curve. For bothiron(1II) nitrate and mercury(I1) thiocyanale, the sensitivity isgreater with increased reagent concentration. The improvementof the sensitivity is no longer obtained around 1.2 M of iron(II1)

Holding
cO1l<Aspirating

Reagent

SampleSpacerFlow
Despensing

2.2 Reagents and standards

A 100 ppm chloride stock solution was made by dissolving
sodium chloride (J.T. Baker Inc., http://www.jtbaker.com) in DI
water, and then the standard solutions were made by dilution
from this stock solution. The reagent solution was made bydissolving appropriate amount of iron(II1) nitrate nonahydrate(Fisher Scientific USA, http://www.fishersci.com), mercury(I1)nitricthiocyanate (Sigma USA, http://www.sigma-aldrich.com),acid (Fisher Scientific) and methanol (Fisher Scientific) into the
DI water to a final volume of 100 mL. DI water was used forboth carrier and spacer. All assays were carried out triplicate
and the average value was used for the calibration.

3. Results and Discussion
3.1 @I protocol
The pS.1 technique allows sample and reagent solutions tobe selected, mixed, and diluted automatically. This is achievedby sequentially aspirating, from the multi-port valve, sample andreagent into stacked zones into a holding coil (Figure 1). It has
been shown that it is preferable to inject the sample zone first,
followed by the reagent zone in the sequence required by thechemistry [7]. To promote good samplelreagent zone overlap
and mixing, an additional spacer zone of carrier solution is
aspirated into the holding coil. In this study, the $1 method
starts by stacking the zones into the holding coil in the following
order: carrier, reagent, sample, and spacer. Following the delay
time of 10 sec for the reaction, the composite zone was pushed
into the detector cell. These series of executions are controlled
simply by the software, as shown in Table 1. Figure 2(a) shows
the typical peak shapes obtained with the protocol shown in
Table l(a). There is another possible protocol which uses thereagent as the spacer instead of DI water. Figure 2(b) shows
the typical peak shapes obtained when the reagent was used as
the spacer. In this case, the product peak is hidden due to thelarge absorbance of the reagent, and the concentration cannot becalibrated. As shown, the background of the reagent during theprotocol should always be considered carefully, when thereagent has similar color to the product.
3.2 Optimization of the reagent and the protocol
The reagent concentration was changed and its effect onTime ( sec )Time ( sec )Figure 2. The peak shape obtained for the blank control
and the 100 ppm sample. The stacked zones of sample,
reagent, and spacer are depicted for the chloride assay.
Spacer is (a) water or (b) reagent. Light pass andintegration time are (a) 5 mm and 500 msec, and (b) 1 mm
and 200 msec.

Figure 3. The effect of the concentration of iron(II1) nitrate
and mercury(I1) thiocyanate on the slope of the calibration
curve. The volumes of the injected zones are 50 pL(sample), 10 pL (reagent), and 50 pL (spacer). Inset:
Typical calibration curve of chloride in low concentration
region. The volumes of each injected zone are 100 pL(sample), 20 biL (reagent), and 50 jtL (spacer). Light passis 10 mm.

Table 1. @I protocola in F I A L ~ ~ ~ ,ProtocolNote

Syringe Pump Command (?) KOR

Turn off backlash
compensation ofsyringe pump

Peristaltic Pump On
Delay (sec) 10
Peristaltic Pump Off

Cleaning and reagent
preparation

Loop Start (#) 3

Repeat the same assay
three times

Aspiration of carrier
Syringe Pump Valve In
Syringe Pump Flowrate (pL/sec) 100 solution
Syringe Pump Aspirate (pL) 400
Syringe Pump Delay Until Done
Syringe Pump Valve Out
Multiposition Valve Reagent
Syringe Pump Flowrate ( G s e c ) 10
Syringe Pump Aspirate (pL) 20
Syringe Pump Delay Until Done

Aspiration of reagent
solution

Multiposition Valve Sample
Syringe Pump Flowate ( ~ U s e c 10)
Syringe Pump Aspirate (pL) 10
Syringe Pump Delay Until Done

Aspiration of sample

Multiposition Valve spacer
Syringe Pump Flowrate (pL1sec) 10
Syringe Pump Aspirate (pL) 100
Syringe Pump Delay Until Done

Aspiration of spacer

Note
Delay 1 sec for thereaction~ u l t i ~ o s i t i ovalvenFlow cell
Syringe Pump Flowrate (pL/sec) 10
Syringe Pump Dispense (pL) 70
Syringe Pump Delay Until Done

Dispense the solutions
to tlowcell

Spectrometer Reference Scan
Spectrometer Absorbance Scanning

Start the ref.
and abs. scan

Syringe Pump Flowrate (pL/sec) 3
Syringe Pump Dispense
80
Syringe Pump Delay Until Done
Syringe Pump Flowrate ( ~ U s e c 100)
Syringe Pump Empty
Syringe Pump Delay Until Done
Spectrometer Stop Scanning(a)Continued to (c)(4Syringe Pump Valve In
Syringe Pump Flowrate (pusec) 200
Syringe Pump Aspirate (pL) 500
Syringe Pump Delay Until Done
Syringe Pump Valve Out
Multiposition Valve Flow Cell
Syringe Pump Empty
Syringe Pump Delay Until Done

Restoring the initialcondition of theflowcell using highflow rate and flushingwith additional carrier
solution

Loop End

Continued to (a) or (b)(a)
Delay (sec) 10

ProtocolDelay 10 sec for thereactionSpectrometer Reference Scan
Spectrometer Absorbance Scanning

Start the ref.
and abs. scan

Multiposition Valve Flow Cell
Syringe Pump Flowrate (pL/sec) 3
Syringe Pump Empty
Syringe Pump Delay Until Done
Spectrometer Stop Scanning

Dispense the solutions
to flowcell

Continued to (c)Actual experimental protocol

nitrate and 0.01 M of mercuryfll) thiocyanate. In the case ofiron(II1) nitrate, concentrations above 0.8 M can cause air
bubbles to form during the aspiration of the reagent solution,
since the reagent solution becomes viscous. The effect of
methanol and nitric acid in the reagent solution was also
investigated. In these cases, the amount of methanol waschanged from 40 to 70 rnL, and that of the nitric acid waschanged from 2 to 7 mL in 100 mL of the reagent solution. For
both cases, the smaller amount gave good sensitivity. However,
mercury(1I) thiocyanate is difficult to dissolve in methanol
volumes less than 40 mL. Based on these results, the reagent
solution containing 0.8 M of iron(II1) nitrate, 0.01 M of

mercury(I1) thiocyanate, 2 mL of nitric acid (in 100 mL of thereagent), and 50 mL of methanol (in 100 mL of the reagent) wasused hereafter.
The effect of the volume of each injected reagent zone,
sample and spacer, was also investigated. In the case of both
reagent and sample, the slope of the calibration curve increased
with increase in each injected volume, where 20 pL of reagent
and 100 pL of sample gave good sensitivity. In the case of thespacer, the sensitivity increases with injected volume to -50 pL,
where it then decreases for volumes greater than 50 pL. This isdue to excess sample and reagent dispersion since the spacer
creates a longer path-length to the detector. Therefore, theOne of the difficulties of this system is that the absorbance
protocol with 20 pL of the reagent, 100 (xLof sample, and 50 pLof spacer was used hereafter. The effect of physical parameters of the background is high, since the color of the reagent itself is
was finally investigated, for improvement the limit of the similar to the product. For decreasing background absorbance,i t is effective to set the reference scan (100% Transmittance)
detection (LOD) value. The LOD value was defined as 30&,
where ohis the standard deviation of the background signal and after 70 pL of the mixed solution is dispensed. The calibration
m is the slope of the calibration curve. The LOD value with curve obtained with this protocol is shown in Figure 5(a). The
above condition is 371.2 ppb. The LOD value improves to linear relationship between the concentration and absorbance is104.0 ppb by decreasing the aspiration speed of each fragment also obtained, at a cost to reproducibility. The sampling ratefrom 50 m s e c to 10 pL/sec, due to the greater aspiration for each assay is also one of the important parameters. Thereprecision possible. The LOD value also improves to 45.03 ppb are three possible ways to improve the sampling rate; (1)with increasing integration time of the spectrometer to 2000 increase the flow rate immediately after peak identification, (2)msec, although the data collection speed was compromised. decrease the delay time for the reaction, and (3) increase flow
The calibration curve obtained here is shown in the inset of rate prior to reference scan. The protocol used is shown in
Figure 3. A good linear relationship of Y = 6.150 X I O - ~ X Table l(b). Figure 5(b) shows the calibration curve obtainedwith this protocol. A linear relationship is also obtained.
+ 4 . 0 6 8 ~10" where r2 = 0.9917 was obtained.
Compared to Figure 5(a), higher background absorbance is
3.3 Calibration of chloride
Based on the above studies, the chloride assay was carriedout with the detection zone setup in the "high concentration"
mode. In this case, the light path was set to 5 mm, and theintegration time of the spectrometer was set to 500 msec. The
aspiration rate of each zone was set as 10 pL/sec. Figure 4
shows the calibration curves obtained by changing the volumesof sample and spacer, while the injected reagent volume
remained fixed at 20 pL. In the case of (a) 100 pL of sample
and 50 pL of spacer, the calibration curve deviates from thelinear relationship at around 25 ppm chloride. This deviation
appears to be due to the limitation of Beer's law. The range
where the linear relationship can be obtained thus increases to 50
pprn in the case of (b) 20 (xL of sample and 100 pL of spacer,
and to 100 ppm in the case of (c) 10 pL of sample and 100 pL ofspacer, by the improved dispersion of the sample with the spacer.
This is one of the advantages to the SI system, sample dilution
can be achieved easily by changing the ratio of sample to spacer.

ref. scan at 70 uLw

Holding*Aspirating

Reagent

SampleSpacerFlow
Despenslng

Figure 5 . The calibration curves based on the reference
scan with the spacer. The stacked zones of 10 pL of
sample, 20 [iL of reagent, and 100 pL of spacer (Dl water)
are depicted. The dispense rate until reference scan is (a) 3
pL/sec and (b) 10 pL, and the delay time for the reaction is(a) 10 sec and (b) 1 sec.
(a) Y = 2.990xl0-~x + 6.562~10:~,r2 = 0.9995.
(b) Y =3.803xlo-~X+2.145x10-' , 2 = 0.9988.

Figure4. The calibration curve of chloride in the highconcentration region. The stacked zones used were (a) 100{J~L(sample), (b) 20 yL (sample), and (c) 10 uL (sample), 20
pL (reagent), and (a) 50 pL (spacer) and (b) & (c) 100 pL(spacer). Calibration curves obtained were:(a) Y = - ~ . O ~ O X I O+ "2 ~. 3X9 ~2 ~ 1 0 - ~ ~ + 2 . 8 7 1 x l 0 - ~r2 = 0.9995 (0-50 ppm).
(b) Y = - 2 . 3 5 1 ~ 1 0 -+ ~8 ~. 2~ 8 6 ~ 1 0 t-2~. 7~4 1 ~ 1 0 - 'r L 0.9997 (0-100 ppm).
(c) Y = 3 , 6 8 2 x l 0 -x
~ + 2.793~10-I
2 = 0.9992 (0-100 ppm).

Time ( sec )Figure 6 . The actual peak shape for the Figure 5(b).

observed because of limited reagent dispersion due to the short Literature Cited
delay time. In this case, the reproducibilities are also decreased,[I] 2. Marcenko: "Separation and Spectrophotometricand the relative standard deviations increase about 1.5-3 times
Determination of Elements", Chapter 16 (1986), (John Wiley &compared to those of Figure 4(c). Figure 6 shows the actual
Sons, New York).
peaks obtained by the same protocol for Figure 5(b). The
sampling rate is improved and a throughput frequency of 57 [2] S. Utsumi: Nippon Kagaku Kaishi (in Japanese), 73, 835(1952).
samples/h can be obtained, while it was 17 samplesh with the[3] I. Iwasaki, S. Utsumi and T. Ozawa: Bull. Chem. Soc.
protocol shown in Table l(a).
Japan, 25,226 (1952)[4] L.T. Skeggs and H. Hochstrasser: d i n . Chem., 10, 918
4. Conclusion( 1 964).
The chloride assay with the sequential injection system has 1.51 J. Ruzicka, J.W.B. Stewart and E.A. Zagatto: Anal. Chim.
been investigated, with the following results.
The Acta, 81, 387 (1976).
determination of chloride both in the low and high concentration [6] M. Cheregi and A.F. Danet: Anal. Lett., 30, 2847 (1997).
region can be carried out. Indeed, this method is useful for [7] J. Ruzicka and G.D. Marshall, Anal. Chim.Acta, 237, 329
chloride analysis over a 3-decade concentration range, while (1990).
most colorimetric assays can only be performed over 1-2 [8] J. Ruzicka, Analyst, 125, 1053 (2000).
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detection value of 45 ppb was obtained with the appropriate run Analyst, 126, 291 (2001).
conditions. I n the case of a high concentration region, chloride [lo] V. Cerda, A. Cerda, A. Cladera, M.T. Oms, F. Mas, E.
can be determined by increasing sample dispersion, which is Gomez, F. Bauza, M. Miro, R. Forteza and J.M. Estela, Trends
easily achieved by changing the volumetric ratio of sample to Anal. Chem., 20,407 (2001).
spacer. This (iSl approach reduces the use of the toxic reagent [ l l ] T. Sakai and N. Teshima, Bunseki (in Japanese), (6), 289
to just 20 pLfsampIe, which is more than a 200-fold reduction to (2001)
conventional FIA techniques [5] and more than a 50-fold
reduction to the original Utsumi's method [2].
(Received January 9,2002)

Acknowledgement
S.N. is grateful to the Research ell ow ships of the Japan
Society for Promotion of Science for Young Scientists and to the
Ogasawara Foundation for the Promotion of Science andEngineering.

(Accepted February 28,2002)