Anal. Chem. 1992, 6 4 , 923-929 (6) Marshall, A. 0. Mspersbn vs. Absorptlon (DISPA): A Magic Ckcle for SpectroscopiC Line Shape Analysls. Chemom. Intell. Lab. Syst. 1066, 3, 261-275. (7) Bracewell, R. N. The Hertby Tmnsfffm; Oxford University Press: New York, 1966. (6) Wllllams, C. P.; Marshall, A. Q. Anal. Chem. 1060, 87. 428-431. (9) Kramers, H. A. Esdgtto &@ AtU dd Congress0 Intmazkmehs de FisIcl Coma; Nicob Zonlchelll: Bologna, 1927. (10) Krkrlg, R. Ned. TBschr. New& 1042. 9 . 402. (1 I)Bode, H. W. M W Ana&& end Feedback AmpMw Design; D. Van Nostrand Company: New York, 1940. (12) Sommetfeld, A. Ann. phys. 1014, 4 4 , 177-202. (13) Brlllouln, L. Ann. php. 1014, 44, 203-240. (14) Toll, J. S. ~ J SRev. . 1056, 104, 1760-1770. (15) Emst, R. R. J . M g n . Reson. 1060, 7 , 7-26. (16) Ohta, K.; Ishlda, H. A&. SpeclroS~.1966, 42, 952-957.
929
(17) Wang, T.C. L.; Marshall, A. G. Anal. Chem. 1063, 55, 2346-2353. (16) Herring, F. G.; Marshall, A. 0.; PhHllps, P. S.; Roe, D. C. J . &p?. Reson. 1060, 37, 293-303. (19) Press, W. H.: Flannery, B. P.; Teukdsky, S. A.; Vetterlkrg, W. T. Numer/a/Reclpes; Cambrklge University Press: New York, W66. (20) Dunbar, R. C. Appl. Speclrosc. 1000, 4 4 , 1547-1551. (21) Roth, K.; Klmber, B. J . &gn. Reson. Chem. 1005, 23, 632-833. (22) Verdun, F. R.; Mullen, S. L.; Ricca. T. L.: Marshall. A. Q. A&. &ss Spectrom. 1066. 17A, 870-671. (23) Bertie. J. E.;E y d , H. H. A w l . Speclros~.lo=, 39, 392-401. (24) Ohta, K.; Ishlda, H. Appl. SpecbosC. 1066. 42. 952-957.
RECEIVED for review August 21,1991. Accepted January 2, 1992.
Universal Sandwich Membrane Cell and Detector for Optical Flow Injection Analysis Jose Luis Perez Pavbn,+Encarnacidn Rodriguez Gonzalo,t Gary D. Christian, and Jaromir Ruzicka* Department of Chemistry, University of Washington, BG-10,Seattle, Washington 98195
Spectrophotometric detectlon, chemllumlnescence,gas dlffurlon, membrane separation, and thelr comblnatlons have h n wccerrfully performed In a flow cell, whlch comblnes H(HI ogtlcr, flow channels, spacers, reflecting sutfacos, and separation membranes Into a robust mndwlch type senslng unlt. Appllcatlons whlch Include a oeparatlon step were carded out urlng a rultable membrane, whlch acted as an opaque rw(ac@,reflectlng the tranmffled llght Into a blfurcated optkal cable.
INTRODUCTION The original concept of flow injection analysis (FIA) was based on the injection of a liquid sample into a moving unsegmented continuous stream of a suitable liquid. The injected sample forms a zone which is then transported toward a detector that continuously records a physical parameter that changes as a result of the passage of sample material through the flow cell.' Although the idea of detection "after" reaction or physical process has never been specified, the different steps involved (reaction, separation, detection) take place in different parts of the manifold and at different times in most of the FIA applications. Recent trends are toward integrating these processes in a single module in order to best suit the requirements of the intended assay, and as a way to obtain more complete information, to facilitate miniaturization,and to save time and reagent consumption. Some studies in which either preconcentration or separation has been integrated with detection in FIA have been reported,2b and recently, continuous analytical systems that integrate reaction and spectroscopic detection4or separation and detection5have been reviewed. One of the latest attempts to simplify reaction4etection schemes is the work of Wilson and co-workerss in which a flow injection approach is used for solid-phase chemiluminescent immunoassay with a memPermanent address:. Departamento de Quimica Analhica, Nutricidn y Bromatologia, Facultad de Quimica, Universidad de Salamanca, 37008 Salamanca, Spain. 0003-2700/92/0364-0923$03.00/0
brane-based reactor. Other reported membrane-based flow cells for chemiluminescent and/or gas diffusion measurements include those of Smart' and Pilosof and Nieman.8 Another approach, capturing the analyte within the observation field of a detector by stopping the flow, while transforming the analyte by a chemical reaction into a detectable species, has numerous indisputable advantages. It allows multipoint reaction rate measurements, which is more selective and informative than a singlepoint readout obtained at continuous flow. Higher sensitivity is obtained, since the yield of chemical reaction is increased during the stopped-flow period in the course of which the reactants are not being diluted by the dispersion process, which otherwise would take place during the continuous flow. It also allows analyte preconcentration via membrane transport into a stationary acceptor stream or on a stationary phase, where the analyte can be optosensed in situ. Finally, it allows, in combination with fiber optics, an overall miniaturization of the flow system, thereby leading to improved economy of sample and reagent consumption. Ever since these advantages of stopped-flowg and flow injection optosensing1° were conceptualized several years ago, various experimental designs have been proposed to accommodate reaction rate measurements," pH measurement by means of immobilized indicat~rs,'~J~ enzymatic assays,l' gas-diffusion separation and detection of ammonia,1° gas diffusion-enzymatic assays of urea,15 solid-phase analyte preconcentration by sorbent extractionls and by ion exchange,17and even anion determination by catalyzed reduction on a solid phase.2 These papers, along with recent review a r t i c l e ~ , ~underline ~J~ the importance of flow injection optosensing, which can be viewed as a crossbreed between classical reagent-based chemistry and flow injection-assisted chemical sensor technology. Yet an ideal flow cell capable of accommodating all of the above techniques has not been designed. In fact, even simple photometric flow through cell designs, such as %cells and U-cells, are known to entrap microbubblea and disturb the flow pattern by secondary mixing, while tending to be expensive and difficult to disassemble for cleaning or reconfiguration of the flow path. 0 1992 American Chemical Society
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The sandwich reflectance cell presented here (Figure 1) is
a logical outcome of several less practical designs described by us in the past.10J2J5J8The present construction is unique in that it allows flow injection to be performed in conjunction with spectrophotometry, gas diffusion, and membrane separation, as well as chemiluminescence aided by membrane diffusion, within the same basic cell configuration, using modifications easily implemented with minimum use of time and tools. In addition, the same cell construction can undoubtedly be used for UV spectrophotometry, fluorescence, and solid-phase separation/detection as supported by the experimental evidence given below.
EXPERIMENTAL SECTION Reagents. For the spectrophotometric measurements, a 0.1 M borate buffer solution (pH = 9.2) was prepared and used as carrier. A stock solution of bromothymol blue (BTB) was prepared by dissolving 0.1 g of the solid product (Aldrich) in 100 mL of the buffer solution; the samples were prepared by dilution of the stock solution with the same buffer. For the pH optosensing measurements, MacIlvaine buffer solutions were prepared according to the tables in the monograph of Perrin and Dempseymand used as samples. The carrier stream was M HC1. For the chemiluminescencemeasurements, a luminol solution was prepared by dissolving 0.080 g of luminol (Sigma) in 100 mL of 0.10 M carbonate buffer to control the pH; the amount of KOH was varied to achieve the desired pH. A stock cobalt(I1) solution (240 ppm) was prepared by dissolving the appropriate amount of reagent-grade cobalt sulfate in 0.01 M hydrochloric acid; working solutions were prepared fresh daily by appropriate dilution of the stock solution with water. A stock hydrogen peroxide solution (30%,Baker Analyzed) was used to prepare the working solutions. Hypochlorite standards were prepared just before use from a stock sodium hypochlorite solution (5% w/v available chlorine, Aldrich). Luminol and all other reagents were used without further purification. All solutions were prepared in Nanopure Water. M For the determination of ammonia by gas diffusion, a NaOH solution was prepared from the solid product (Baker Analyzed) and used as reagent for the donor stream; the acceptor stream was prepared by dissolving the appropriate amount of bromothymol blue (Aldrich) in deionized water, and the pH was adjusted to 6.5 with HCl; a stock solution of ammonium chloride was prepared by dissolving the solid product (Baker Analyzed) in water, and the samples were prepared by dilution of the stock solution. For the determination of phenol, a stock solution of phenol was prepared by dissolving the appropriateamount of the solid product (Fluka) in deionized water. Standards were prepared by diluting the stock solution with water. A stock buffer solution (pH = 10.4) was prepared by dissolving 23 g of NaHC03, 27 g of and 35 g of KOH in 1000 mL of deionized water. The 4-aminoantipyrine (4-AAP) reagent solution was prepared by dissolving 0.40 g of the solid product (Pierce Chemical Co.) in 16 mL of the stock buffer solution, and the volume was made up to 100 mL. The oxidant solution was prepared by dissolving 2.5 g of K2S208 (Baker Analyzed) in 100 mL of deionized water; the pH was adjusted to about 11 with KOH (Mallinckrodt). Apparatus. Figure 1 shows the sandwich flow cell as assembled for reflectance measurements and membrane separation. It comprises two blocks made of PVC (1-cm thick, 1.5-cm wide, and 3-cm long) with holes drilled for the inlet and outlet of the donor and acceptor streams. An additional hole made in the block at the acceptor side, into which the common end of the bifurcated optical fiber was pressfitted. The spacers placed between the two blocks have slots which determine the width of flow path, while the effective light path length is determined by the thickness of the spacers. The membrane is placed between two such spacers, and the entire sandwich is assembled together by means of two screws. By using more than one spacer at a time, the effective volume of the cell and the light path length can be modified. The detector used in all experiments was a single-beam Milton Roy Co. Minispectronic-20 connected to a Radiometer Rec. 80 Servograph (Copenhagen)recorder. Note that any single-beam
Donor stream
Flgure 1. Fiber optics flow-through cell. LS, light source: D, detector; S, spacers; M, membrane.
spectrophotometer can be modified for the present purpose by removing the light source and replacing it by the outlet end of the bifurcated optical cable. Except for the chemiluminescence applications, a bifurcated optical fiber (Twardy Technology Incorporated, 61-cm length, 2.4-mm bundle diameter) was used, the light source being a homemade housing that contains a commercial halogen lamp, the outlet of which was regulated by changing the voltage of a stabilized power source. The output signal was directed to the recorder through a homemade logarithmic converter. For the chemiluminescencemeasurements, a straight optical cable (Twardy Technology Incorporated, 61-cm length, 5.1-mm bundle diameter) was used, the end of which was directly coupled to the detector of the Minispectronic-80,the output of which was connected to the recorder. The light emission was observed directly without any wavelength discrimination. Alitea C8/2-XV Model (Alitea USA) peristaltic pumps and a Rheodyne 5020 six-port valve were used for all experiments. The pump tubing was Tygon (1.2-mm i.d.), and 0.5-mm-i.d. Teflon was used for the manifolds. The individual manifolds and cell configuration are described in detail for every application separately. Membrane separations were accomplished with a Celgard 2500, 0.025 mm thick, 45% porosity, 0.04-pm effective pore size membrane. Procedures. Spectrophotometric Measurements. Samples, prepared by serial dilution of stock BTB solution in borax buffer (0.01 M, pH = 9.2), were injected in the carrier stream (borax buffer) while absorbance at 620 nm was continuously recorded at a flow rate of 0.75 mL/min. The injection volume was 100 pL using the manifold shown in Figure 2. p H Optosensing. A 600-pL portion of the sample, a series of MacIlvaine buffer solutions with the pH value obtained as listed by Perrin and Dempsey,20was injected into the carrier stream while the reflectance signal (A = 600 nm) was recorded. Chemiluminescent Assay of CobaZt(1l). Measurements were carried out with the flow system described in Figure 3; the configuration of the flow-through cell was as shown in Figure 1, except that the membrane was removed, so that a mixing chamber was formed in front of the sensing end of the single fiber optic cable. Two different lines were used for the transport of the reagents (a solution of luminol in 0.10 M Na2C03-KOH (pH = 12) and a solution of 0.045% H202)which were merged in a confluence point right ahead of the flow cell. The flow rate measured after the confluence point was 0.9 mL/min. A water stream (3.1 mL/min) was used as carrier, and the samples were aqueous solutions of cobalt(I1). The injection volume was 100 pL in all experiments. Calibration graphs were constructed by plotting maximum emission intensity vs cobalt(I1) concentration. Determination of Ammonia by Gas Diffusion. In order to select the best membrane for the determination of ammonia, the manifold described in Figure 4 was used. In this way, an aqueous solution of ammonium chloride was merged with M NaOH
ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992
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P
Reflecting surface
A 0.15
I I
8
A 3
0.18
/
0.15
0.10 0.12 0.09
0.05 0.06
0.03
0
+-.
0.00 0
Time
2
4
6
8
1
0
1
2
BTB/ppm Figure 2. (A) Spectrophotometricmeasurements of a dye solution at 620 nm. C, carrier solution (0.75 mL/min); P, peristaltic pump; I, injection valve (loop volume: 100 pL); W, waste; LS, light source; D, detector. (B) Response for 10 ppm BTB with 1-, 2-, and 3-mm spacers. (C) Peak height and light path length: numbers on lines indicate spacer thickness in millimeters.
-
mV
0.12
-
- 0.30
0.0s
-
- 0.20
0.04
-
- 0.10
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1.8
1.2 0-
0 Time
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0 4 -
Time
m
e 3. Determination of Co(I1) by chemiluminescence. The sample was injected into water which entered the acceptor stream side of the cell (Figure 1). H202and luminol solutions were merged and then entered the donor stream side of the cell. Emission versus time profiles obtained at different pH values of luminol solution (numbers beside the curves indicate the pH values).
and pumped along the donor side of the membrane, while the M BTB, pH = 6.5) was flowing along the acceptor stream opposite membrane side. At a selected time, the flow on both sides of the membrane was stopped and the variation of absorbance with time at 620 nm was recorded. The calibration graph for ammonium determination was obtained by using the manifold described in Figure 4, except that the sample was injected into a carrier stream that merged with the reagent stream. The same acceptor stream was used as for the previous experiment; the
Figure 4. Gasdiffusion measurements. Sample and reagent streams merged and entered the donor side of the cell (Figure 1). The reagent stream entered the acceptor side of the cell. (A) Curves 1 and 2, 1.22 X lo-, M NH,CI (for details, see text). (B) Curves a-f, 4.88 X lo4 M NH,CI. Membranes used: a, 0.2-pm Teflon membrane; b, 1-pm Teflon membrane; c, Celgard 2402; d, Celgard 2400; e, Celgard 2500; f, “plumber” Teflon tape.
sample (200 pL), an aqueous solution of ammonium chloride, was injected in a carrier stream (deionized water). The ammonia generated after merging with M NaOH diffused through the Celgard membrane into the acceptor stream which was stationary. After the sample zone had passed (60 s after the injection), the acceptor stream was pumped away in order to renew the reagent before a new injection cycle. Determination of Hypochlorite by Chemiluminescence-Gas Diffusion. The chemiluminescencegasdiffusion flow system used for the determination of hypochlorite was as described in Figure 5 using the configuration of the flow-through cell as shown in Figure 1, with a Celgard 2500 porous hydrophobic membrane. Hypochlorite samples were injected into a carrier stream of 0.1 M hydrochloric acid flowing at 1 mL/min while the acceptor stream (luminol, pH = 12.3) was stopped in the cell. The light
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+Time Flgurr 5. Determinatlon of hypochlorite by gas dlffuslon-chemilumlnescence. The flow cell configuration was the same as depicted In F W e 1 but wlth a nonbifurcated fiber optlc cable. The sample was Injected Into HCI solution (donor side). Luminol was pumped through the acceptor side. (A) Emission-time profiles obtained for carrier sdutbn Row rates of 0.5, 0.7, 1.0, 1.5, and 2.5 W m i n (cuves in order from lett to while the reagent was stopped in the w. (B) ~ o f i l e g for constant car& solution Row rate (2.5 W m i n ) and 0, 0.5, 1.0, 1.5, and 2.5 mL/min reagent solution flow rates (peaks In order from top
w)
to bottom).
emission was continuously recorded and peak heights were measured. When the recorded signal declined back to the baseline level, the reagent flow was restarted for 60 s at 0.5 mL/min in order to renew the acceptor solution in the cell before the next injection cycle. Determination of Phenol. For the experiments without injection of the sample, an aqueous solution of the phenol is pumped continuouslythrough on one of the sides of the Celgard membrane, while the stream resulting from merging the 4-aminoantipyrine and the oxidant (peroxodisulfat.e).is pumped through on the other side of the membrane (1mL/min flow rate for all feed streams); at a selected time, the pump is stopped and the variation of abeorbance at 500 nm is recorded for 3 min. For the experiments with injection, the sample (200rL) is injected in the carrier (donor) stream (deionized water, 1mL/min) while the acceptor stream is stopped; the increase of absorbance is recorded and, after 60 s, the reagent flow is restarted (0.5 mL/min) for 60 s to renew the acceptor before the injection of a new sample.
RESULTS AND DISCUSSION Spectrophotometric Measurements. The simplest application of the proposed flow cell is its use as a spectrophotometric unit (Figure 2). The inlet and outlet in the lower part of the cell were omitted and a white reflecting surface (a piece of polymeric material) was placed opposite to the optical cable. The resulting chamber has a path length of 2d (d being the spacer thickness) since the light beam traversed the absorbing solution twice. The flow cell was tested by injecting serially diluted solutions of bromothymol blue in a single line manifold using three different spacer thicknesses. The shape of the peaks obtained and the calibration graphs constructed are shown in Figure 2, parta B and C, respectively. The path lengths for 1-, 2-, and 3-mm spacers are 2,4, and 6 mm, and the corresponding volumes are 10,20, and 30 pL, respectively (slots 1mm wide and 10 mm long). The slope of the calibration graph increases in proportion to the spacer thickness, the ratio being 1:1.82.7; the difference from the theoretical 1:2:3 sequence can be attributed to the fact that the spacers used do not have exactly the same thickness. The correlation coefficient for the leashqwes fittjng was 0.9999for the three calibration graphs. The relative standard deviation for ten injections of 5 ppm BTB sample with the two spacer cell was 0.6%. The sensitivity of this cell relative to a conventional cuvette will necessarily be smaller due to the shorter path length. Us& two cell spacers, the cell thickness is 1.6 mm. Since the reflected light travels through the path twice, the effective path length is 3.2 mm, and so the relative sensitivity should be 0.32 of that using a l-cm cuvette cell in a spectrophotometer. Absorbance measurements of a 1X lo+ M bromthymol
blue solution flowing through the cell with two spacers and in a l-cm cuvette were 0.20 and 0.60, respectively, a ratio of 0.33. It should be noted that when the cell is operated in the FIA mode, the sample will be diluted via dispersion, but on the other hand, a short transient signal is detected that may lead to improved detectability. Even for this simple use of the cell, the advantages are evident; the module serves as an inexpensive variable-pathlengbh spectrophotometricflow cell in which path lengths of up to 1 cm, if needed, could be obtained without undue increase of the cell volume (50 pL for a 5-mm spacer with 1mm-wide and 10-mm-long slot) thus without detriment of the sensitivity compared with regular spectrophotometric flow cells. For concentrated samples, as often encountered in process control where FIA is having growing applications, the possibility of using path lengths well below 1 mm can be considered. pH Optosensing. Measurements of pH were made with a cell configured as for the spectrophotometricmeasurements (Figure 2), but instead of the white reflecting surface, a section of colorpHast indicator strip (Merck, Cat. No. 9590) was placed in the flow-through cell to face the end of the optical cable. Two important variables in pH optosensing are the sample volume and the concentration of the acid or base in the carrier stream; if the sample volume is too small, a titration effect will occur and the signal obtained for a certain pH value will depend on the concentration of the carrier solution. On the other hand, if the concentration of the carrier solution is too low, the peak width will increase and large volumes of carrier will be needed to shift the indicator to the original form, thus reducing sample throughput. The experiments were carried out by using a sample volume of 600 p L that provides an undiluted zone in the middle of the plug with lo4 M HC1 as carrier. The experimental points obtained for a series of MacIlvaine buffer solutions covering a pH range from 6.0 to 8.0 were compared with the theoretical curve (absorbance of the alkaline form of the indicator as a function of pH) for an indicator with pK, = 7.0. The good fit of the experimental points to the theoretical line proves the excellent performance of the system and conforms with the observations reported in previous w ~ r k s . ' ~ J ~ J ~ Chemiluminescence. The transient nature of light emission generated by chemiluminescence requries a cell configuration that allow detection of the emitted light at the shortest possible delay after the chemiluminescent reaction has been initiated. Since chemiluminescent emission varies with time, the amount of light detected will correspond to that portion of the emission profile that occurs during the time interval spent in the detection cell. In a typical FIA-chemiluminescence manifold the flow cell is placed in front of the detector. Various studies of chemiluminescence flow cell and flow system designs have been described?l-%the aim being to minimize the loss of light emission while the sample is on the way between the point where the reaction is initiated and the detector. Such a lase of signal becomes significant for fast reactionswhere chemiluminescenceis generated within a short time span and the detection must occur immediately after reagent mixing. The use of the sandwich flow cell for monitoring of chemiluminescence was first tested by measuring cobalt(I1) by reaction with luminol and hydrogen peroxide, and then furthermore for the more complex determination of hypochlorite. Luminol (3-aminophthalhydrazide) reacts with hydrogen peroxide in the presence of a catalyst, producing luminescence. Different catalysts, such as ferricyanide or heavy metals, are needed to assist this r e a c t i ~ n . ~ ~ . ~ ~
ANALYTICAL CHEMISTRY, VOL. 84, NO. 8, APRIL 15, 1992
The determination of cobalt(I1) by measuring the Co(II)-HzOz-luminol chemilumineecencehas been carried out with the manifold described in Figure 3. The sandwich cell provides the advantage that analyte and reagents mix where the detection takes place, and therefore there is no light emission loss since the intensity-time profile can be recorded right from the beginning and the emitted light is integrated by the detector throughout the entire length of the sample zone. The injection of the sample into a separate carrier offers the additional advantage of providing a baseline caused by background emission from the reagents. With this arrangement, the typical emission versus time profiles obtained for the system cobalt(I1)-hydrogen peroxide-luminol are shown in Figure 3 as a function of the pH value of the luminol solution. The effect of changing the flow rate of the carrier solution in the range 1.0-4.0 mL/min was investigated, yielding a maximum intensity for flow rate values higher than 3.0 mL/min. Increasing the reagent flow rate led to decrease of the light intensity recorded, since light emission took place downstream from the detector. Under the experimental conditions described in the procedure, the calibration' curve of peak height versus Co(I1) concentration is linear from 5 to 400 ppb (0.5-40 ng injected) Co(II), fitting the equation y = -0.195 + 0.349~(r2 = 0.999). Deviations from linearity were found at higher concentrations. The coefficient of variation was 4.1% for five measurements of 40 ppb Co(I1). Gas Diffusion. Gas diffusion, which has been widely used in flow injection,1°J5 comprises two consecutive steps which take place in different parts of the manifold: separation in the diffusion module, and the detection in the flow cell. This approach leads to dilution of the sample by dispersion and to the loes of the kinetic information encoded in the separation process. Theory. The theory of gas diffusion as used in flow injection systems has been developed by van der Linden%by using the tank-in-series model for the general case in which donor and acceptor stream are continuously flowing. However, since it is useful to stop the acceptor stream and sometimes even both streams in order to increase the sensitivity, it is appropriate to consider a simplified model, derived here for systems in which the acceptor stream is stopped, the donor stream being either flowing or stopped. If both streams are stopped and the volumes at both sides of the membrane are the same and small enough to assume homogeneous concentration of species at any time dC,/dt = K(Cd - c,) and
c, = c d o - cd where Cd and c, are the concentrations of the diffusing species in the donor and acceptor streams at time t , c d o is the initial concentration in the donor stream, and K is a constant that includes the diffusion process across the membrane. dC,/dt = dCd/dt thus -dCd/(2Cd - cdo) = K d t giving by integration C, = f/zcdo(l(1) As could be expected, eq 1 predicts C, = 0 for t = 0 and C, = Cd0/2 for t = 03. If the diffusing species undergoes a chemical reaction in the acceptor side, taking place rapidly and with a high formation constant, C, = 0 and
927
dC,/dt = KCd
C, = cdo(l - e-Kt)
(2)
where C, is the concentration of the reaction product in the acceptor side. Equation 2 predicts C, = 0 for t = 0 and C, = cdofor t = OD. Equations 1and 2 are applicable for stop-flow operation. They predict the maximum concentration in the acceptor would be c, = cdo/2 if there is no reaction in the acceptor side or C, = c d o is there is a chemical process that consumes the species going through the membrane. The value of Cdo can be derived from the originally injection concentration of the d y t e through the dispersion coefficient D in the usual way (cdo= Co/D). If the acceptor stream is stopped and the sample solution is continuously pumped through the other side of the membrane at a flow rate high enough to make the renewal of species much faster than the transport across the membrane, Cd is a constant and equals c d o
e
By integration Equation 3 predicts C, = 0 for t = 0 and C, = cdofor t = (without any chemical reaction),the same result as for when the sample was stopped and a chemical reaction takes place. If there is a chemical reaction on the side of the acceptor stream 03
This is the only case in which a preconcentration is possible since chemical transformation of the analyte into a desirable species is a force driving analyte molecules through the membrane. Determination of Ammonia. A series of experiments in which several membranes were tested by using experimental conditions that fit the requirements for eq 2 was performed. Figure 4 describes the manifold used and shows the curves obtained. An aqueous solution of ammonium chloride was merged with a basic solution M NaOH) and pumped through one of the sides of the microporous membrane while a solution of BTB (pH = 6.5) was pumped through the acceptor side; a t a selected time the flow was stopped and the resulting kinetic curve was recorded (A = 620 nm). The curves (a-f in Figure 4B) fit the shape predicted by eq 2. The differences in the increase of signal with time for different membranes reflect the different pore sizes, percentage porosity, and membrane thickness. The different behavior predicted by eqs 2 and 4, respectively, is in good agreement with respective curves 1and 2 in Figure 4A in which the sample was stopped (curve 1) or flowing (curve 2) while the acceptor stream was stopped. For long stop-flow times, a deviation from the straight line (curve 2) is observed in the recording corresponding to the experiment in which the sample was constantly flowing, due to the saturation of the chemical indicator involved. The experiment depicted in curve 2 was repeated for three different donor flow rates (0.5, 1.0, and 1.5 mL/min), yielding exacting the same recording (superimposed in the figure), once again in agreement with eq 4 in which the predicted concentration of the reaction product in the acceptor side is independent of the donor flow rate for values that provide a renewal of the species faster than the transport rate across the membrane. The calibration graph was constructed as described in the Experimental Section. The signal was found to be linear with concentration up to 11ppm. The calculated detection limit
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 8, APRIL 15, 1992
(2 X noise) is 0.4 ppm, and the relative standard deviation for 10 injections of a 4.4 ppm sample was 2.1 % . Determination of Hypochlorite by Chemiluminescencffias Diffusion. The use of a separation step prior to reaction to produce a detectable product is the usual way to avoid interferences and matrix effects. Integration of separation, reaction, and detection processes in a single flow cell using a membrane as a barrier not only reduces interferences but also allows the use of samples and reagents with different chemical or physical properties such as pH, density, or viscogity (under such conditions, the detection in a FIA system is impossible without previous homogenization). The application of microporous membranes to chemiluminescence analysis as a way to keep the reagent and sample matrix separated has been previously r e p ~ r t e d . Both ~ ~ . ~analyte or reagent can diffuse across the membrane and then react to yield a measurable signal. Diffusion through the membrane can be easily enforced by adjusting an applied pressure gradient, the upper limit of which is set by the structural rigidity of the membraneVz7 We have investigated the incorporation of a microporous membrane to perform a gas-diffusionstep prior to the chemiluminescence measurement of hypochlorite with luminol in alkaline media. In this approach, a microporous hydrophobic membrane, Celgard 2500, is placed between the spacers in the flow cell to separate the reagent zone from the sample flow stream. This design permits one to optimize the volume in the chemiluminescence flow cell. The assay is based on the reaction between hypochlorite and hydrochloric acid to generate chlorine: OC1- + HC1- Clz OH-
+
Samples of hypochlorite are injected in a carrier stream of hydrochloric acid to yield chlorine. When the analyte stream reaches the flow cell, the Clz diffuses through the membrane into the luminol alkaline solution and the chemiluminescent reaction occurs in the other side of the cell where the optical fiber is placed. The kinetic curve obtained, the intensity versus time profile reflects the kinetics of the chemiluminescence reaction and of the gas-diffusion process. The flow rates of reagent and sample solutions have been studied, and the different intensity-time profies obtained are shown in the Figure 5. The sample flow rate (Figure 5A) was varied between 0.5 and 2.5 mL/min while the reagent solution was kept stationary in the cell until the light emission decayed to the baseline level. The observed emission intensity decreased at high sample flow ratea due to the reduced residence time within the flow cell relative to the diffusion rate. The influence of reagent flow rate is shown in Figure 5B, and, as can be seen, high reagent flow rates had a negative effect on the intensity recorded, as was expected. The control of both reagent and sample flow rate is a versatile way to modify the sensitivity of the system. An additional 2-fold increase in sensitivity is obtained when both the reagent and analyte solutions are captured within the cell by stopping the flow. The calibration curve was obtained as described in the Procedure. The plot of log intensity versus log OC1- concentration was found to be linear from 19 to 159 ppm of hypochlorite and fitted the equation y = -0.752 + 5.516%(P = 0.998). Determination of Phenol by Membrane Separation. Membrane separation can be performed by using homogeneoua silicone membranes (Figure 6A); in this situation donor and acceptor streams can be either organic or aqueous solutions and a double extraction process (donor stream-membrane, membraneacceptor stream) takes place, Le., separation is governed by solubility (extraction) and diffusion in the membrane. The extraction process can also be performed by using a microporous hydrophobic membrane (Figure 6B); at
"pi
Acceptor
Donor
Air
Acceptor (aqueous)
Donor (aqueous)
Figure 6. (A) Separation through a homogeneous silicone membrane: donor and acceptor can be either organlc or aqueous solutions. (B) Separatbn through a microporous hydrophobic membrane when one of the two phases Is an organic solution whlch wets the pores. (C) Separation through a microporous hydrophoblc membrane when the phases at both sMes are aqueous solutbns: the transport across the membrane lmplles a step In whlch the analyte has to diffuse across the air fllllng the pores.
least one of the two phases involved must wet the membrane pores in order to get contact between donor and acceptor streams. When the analyte transfer takes place between two aqueous phases separated by a microporous hydrophobic membrane (Figure 6C), a layer of air filling the pores of the membrane avoids any contact between the two liquid phases and the separation process is based on the diffusion of the analyte (vapor form) across the gas layer in between the two liquid phases. The performance of the new sandwich cell for achieving an aqueous-aqueous matrix exchange combined with analyte enrichment and in situ sensing of the d y t e transfer has been teated for the determination of phenol, using the Celgard 2500 microporous membrane. Phenol has a substantial vapor pressure, allowing its rapid diffusion through the membrane. The cell configuration proposed here not only fits the requirements of any separation process, namely, to recover as much as possible of the desired phase and to maintain the concentration profile of the analyte, Le., to prevent further sample dispersion during the separation, but also allows in situ monitoring of analyte transfer, opening up new perspectives for differential kinetic approaches based on separation or separation-reaction rate measurements. The reaction between phenol and 4-aminoantipyrine in the presence of an oxidant to form an intensely red-colored compound is well described in the literaturem as a useful way for the spectrophotometric determination of phenol.30 The adaptation of the 4-aminoantipyrine method to flow injection analysis has been reported?l but in order to avoid the interference from aromatic amines, a solvent-extractionstep was required. The separation of phenol through the Celgard 2500 microporous membrane into an acceptor which contains 4-AAP and an oxidant in basic medium permits its determination, by measuring the absorbance at 500 nm, in a clean bulk matrix. The driving force that creates the concentration gradient is the displacement of the acid base equilibrium to the phenolate form in the basic acceptor, followed by reaction with the 4-AAP reagent in the presence of peroxodisulfate.
ANALYTICAL CHEMISTRY, VOL. 04, NO. 8, APRIL 15, 1992
Two different manifolds were used as described in Procedures; in both cases the 4-aminoantipyrine and oxidant streams were merged immediately before the reaction-detection cell because of the long-term instability of the mixture. In the first configuration, the sample solution is pumped along one of the sides of the membrane, while the reagent stream is pumped along the other side; the flow through all lines is stopped, and the increase of absorbance is recorded. The kinetic curve obtained is a convolution of both the separation process and the reaction with 4-AAP; different membranes were tested, and the results were similar to those obtained for the previously described experiments with ammonia gas diffusion. In the second approach, the sample solution was injected in a carrier stream, deionized water, while the acceptor stream was stopped on the other side of the membrane. The flow of the reagent stream is restarted for a short period of time after each injection to renew the reagent; therefore, the recordings thus obtained are peak shaped,the decay of the signal being due to the removal of the reaction product when the reagent stream flow is restarted. The peak height was found to be linear with the phenol concentration up to 400 ppm, and the correlation coefficient for the least-squares fitting was 0.999. Preconcentration of phenol by a membrane procedure can be also performed by using a nonporous silicone membrane, which offers the advantages of robustness since samples containing particulate matter will not cause clogging, and in same cases, better selectivity is attained because of the additional equilibrium (solubility of the species in the membrane) involved in the process. On the other hand, diffusion is more rapid with the Celgard membrane due to gaseous diffusion of the phenol vapor through the air gap. Membrane Fouling. A series of ammonia measurements was performed as described above and in Figure 4 (1X lo4 M NH4C1,Celgard 2500 membrane) in which the ammonium chloride was dissolved in a fermentation bath medium.34 Injections were made every 5 min for a 24-h period, and there was no appreciable change in the response due to membrane fouling observed. However, it should be noted that, as with all measurements with gas-diffusion membranes, the pores will probably be fouled by high levels of surfactants. CONCLUSIONS The obvious feature of the sandwich cell is its versatility. The same device can be used to perform spectrophotometric measurementa, separation, preconcentration, and their combinations. This simplifies and lowers the cost of flow injection systems which now routinely include complex, cumbersome, and expensive modules for solvent extraction, gas diffusion, or dialysis. An important feature of the sandwich cell is that it provides additional kinetic information: reaction rates, separation rates, gas-diffusionrates, or combined separation-reaction kinetics. This kinetic information enriches the overall information gathered from a single analyte injection. Seen from a practical viewpoint, the sandwich cell is robust and easy to construct, reconfigure, clean, and repair. Use of
929
spacers with thin and narrow channels prevents entrapment of air bubbles and transmission/reflectance of light through a path perpendicular to the flowing stream reduces the influence of refractive index. It is therefore believed that the cell is well suitable for both laboratory and process control applications. So far, the sandwich cell has been used only for applications employing wavelengths within the visible zone of the spectrum; however, the use of quartz optical fibers will allow UV detection and will broaden the field of applications to fluorometry. Finally, it should be pointed out that the concept of integrated reaction-detection is fully compatible with the novel technique of sequential i n j e ~ t i o n which ~ ~ . ~ will ~ be further enhanced by the use of the sandwich fiber optic flow cell. ACKNOWLEDGMENT The financial support of J.L.P.P. by the DGICYT (Spain) is gratefully acknowledged. REFERENCES (1) Ruzicka, J.; Hansen, E. H. Fbw Injection AM~YS~S, 1st ed.;John Wiley and Sons, Inc.: New York, 1981. (2) Lacy, N.; Christian, G. D.; Ruzicka, J. Anal. Chem. 1990, 62. 1482- 1490. (3) Fernindez-Band, B.; Uzaro, F.; Luque de Castro, M. D.; Vaidrcei, M. Anal. Chlm. Acta 1990, 229, 177-182. (4) Val&rcel, M.; Luque de Castro, M. D. Analyst 1990, 775, 699-703. (5) Luque de Castro, M. D.; Vaidrcei, M. Trends Anal. Chem. 1991. 70, 114-121. (6) Liu, H.; Yu, J. C.; Blndra, D. S.; Givens, R. S.; Wilson, G. S. AMI. Chem. 1991, 63, 666-669. (7) Smart, R. B. Anal. Let?. lB81, 74, 189-195. (8) Piiosof. D.; Nleman, T. A. A M I . Chem. 1982, 54, 1698-1701. (9) Ruzicka. J.; Hansen, E. H. Anal. Chlm. Acta 1979, 706, 207-224. (10) Ruzicka, J.; Hansen. E. H. AMI. Chlm. Acta 1985, 773, 3-21. (11) Hungerfwd. J. M.; Christian, G. D.; Ruzlcka, J. AMI. Chem. 1985, 57, 1794- 1798. Ruzicka, J.; Singer. R. GBF Monograph Ser. 1987, 70, 103-112. Woods, B. A.; Ruzicka, J.; Christian, 0.D.; Charison, R. J. Anal. Chem. 1988, 58, 2496-2502. Yerian, T. D.; Christian, G. D.; Ruzicka, J. Analyst 1986, 7 7 7 , 865-873. Jeppensen. M. T.; Hansen, E. H. A M I . Chim. Acta 1988, 274, 147-159. Ruzicka, J.; Arndail, A. Anal. Chlm. Acta 1989, 276. 243-245. Uzaro, F.; Luque de Castro, M. D.; Vaidrcei. M. Anal. Chim. Acta 1988, 274, 217-227. Ruzicka, J.; Hansen, E. H. Anal. Chlm. Acta 1988, 274, 1-27. Ruzicka, J.; Christian, 0.D. Anal. Chlm. Acta 1990, 234, 31-40. Perrin, D. D.; Dempsey, B. Buffers for pH end Metal Ion Control; Chapman and Hail: New York, 1987. Steig, S.; Nieman, T. A. AMI. Chem. 1978, 50, 401-404. Burguera. J. L.; Townshend, A. Roc. Anal. Dlv. Chem. Soc. 1979, 76, 263-264. Muiiln, J. L.; Seitz, W. R. Anal. Chem. 1984. 58, 1048-1050. Burguera, J. L.; Townshend, A.; Greenfield, S. Anal. Chkn. Acta 1980, 774, 209-214. Seitz, W. R. CrH. Rev. AMI. Chem. 1981, 73,1-58. Van der Llnden. W. E. Anal. Chkn. Acta 1983, 757, 359-369. Nau. V.; Nieman, T. Anal. Chem. 1979. 57, 424-428. Freeman, T. M.; Seitz, W. R. Anal. Chem. 1981, 53, 98-102. Emerson, E. J. J . Org. Chem. 1843, 6,417-428. Ochynski, F. W. Analyst 1880, 65, 278-281. Moliar, J.; Martin, M. Fresenlus’ 2. Anal. Chem. 1988, 329, 728-731. Ruzlcka. J.; Marshall. G. D. AMI. Chim. Acta 1890, 237, 329-343. Ruricka, J.; Marshall, G. D.; Christian, G. D. AMI. Chem. 1990, 62, 1861-1886. Chung. S.; Wen, X.; Viholm, K.; DeBang, M.; Christian, G.; Ruzicka, J. Anal. Chim. Acta 1991, 249, 77-85.
RECEIVED for review September 27,1991. Accepted January 17, 1992.
