Continuous sample recirculation in an opened-loop multicommutated flow system

Analytica Chimica Acta 377 (1998) 103±110

Continuous sample recirculation in an opened-loop multicommutated ¯ow system Rui A.S. Lapaa,*, Jose L.F.C. Limaa, Boaventura F. Reisb, JoaÄo L.M. Santosa a

CEQUP/Departamento de QuõÂmica-Fisica, Faculdade de FarmaÂcia, Universidade do Porto, Rua AnõÂbal Cunha, 164, 4050 Porto, Portugal b Centro de Energia Nuclear na Agricultura, Universidade de SaÄo Paulo, Piracicaba SP, Brazil Received 27 April 1998; received in revised form 14 July 1998; accepted 9 August 1998

Abstract A continuous circulating multicommutated ¯ow system based on an opened-loop con®guration is proposed. The sample is inserted into the ¯ow system and continuously recirculated until a suitable dilution level is attained, which is permanently surveyed by including the detector in the circular reactor. A continuous removal of the highly dispersed front and trailing zones of the sample plug improves dilution ef®ciency. The chromogenic reagent used as carrier solution is permanently renewed, which contributes to a constant renewal of the reaction zone increasing the reaction rate by reducing its dependence on the dispersion at the sample/reagent interface. The developed methodology was tested in the determination of chloride in parenteral solutions by the formation of the iron (III) thiocyanate complex with expansion of the linear range of determination up to 10 000 mg lÿ1. Results were reproducible (RSD <1.8%) and in agreement with those obtained by the conventional procedure. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Opened loop; Multicommutated ¯ow system; Sample recirculation; Dilution; Chloride determination

1. Introduction Continuous ¯ow techniques are based on the insertion of a sample plug into a continuous carrier stream of an appropriate composition, which is then transported toward a suitable detector [1] and from there to waste. Typically, a ®xed-length reaction coil is used and a single analytical signal is attained with height or area related to the analyte concentration. These essential and valuable aspects of the ¯ow methodologies have also some major limitations: an ineffective control of dispersion and the placement of the detector at a *Corresponding author. Fax: +351-2-2004427; e-mail: [email protected]

de®ned site where it behaves as a static component. Consequently, the dilution level attained is ®xed and a single data point per determination is obtained. Several solutions were proposed to circumvent these problems, like the utilisation of closed loop con®gurations, multidetectors placed in serial or parallel, relocation of the detector in the ¯ow manifold or exploitation of reversal ¯ow. Recirculation of the sample plug was accomplished by using ¯ow manifolds with a closed loop con®guration, mainly for continuous solvent extraction or kinetic measurements. Ruzicka et al. [2] proposed an in-line concentration system by continuous extraction into an organic solvent in a closed loop whereas Iob and Mottola [3] and Roehring et al. [4] developed closed

0003-2670/98/$ ± see front matter # 1998 Elsevier Science B.V. All rights reserved. PII: S0003-2670(98)00561-3

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loop ¯ow systems for enzyme recycling. ValcaÂrcel et al. [5] proposed a closed system for kinetic measurements and sample dilution and a similar approach for iterative detection and simultaneous determination of iron (III) and cobalt (II) [6]. Other applications of closed loop systems include the utilisation of micro-columns for continuous enzymatic oxidation [7] and analysis of SO2 in air by direct intercalation of an air sample into a liquid carrier solution [8]. Parallel monitoring was ®rst proposed by Stewart and Ruzicka [9]. Multisite detection was achieved by using a single detector, which was relocated at different sites of the ¯ow manifold [10±12]. Townsend and Crouch [13] proposed both a ¯ow reversal and a ¯ow recycling system for multiple sampling points from a single reaction plug. A general review on multiple peak recording [14] and two comprehensive reviews dealing with applications of closed loop con®gurations in pharmaceutical determinations [15] and ¯ow-injection atomic spectrometry [16] were also presented. A chemical reaction in a continuous ¯ow system depends on the mutual dispersion at the interface sample/reagent. When highly concentrated samples with an excess of analyte are involved, the reaction proceeds with the consumption of the reagent at the reaction zone, yielding analytical signals that are not directly related to concentration. A typical solution is the adjustment of concentration by sample dilution. A similar result would be obtained by continuously renewing the reagent solution yielding both a continuous consumption of analyte and an improved sample dilution. If the reagent renewals were monitored, in a multidetection process, the analytical signals would continuously decrease until a linear concentration range was reached. Development of multicommutated ¯ow systems [17,18] based on the utilisation of three-way solenoid valves enabled the implementation of highly versatile ¯ow networks that combine easily automated procedures with a high ¯ow control and a multiple sample processing. The feasibility of a continuous circulating multicommutated ¯ow system based on an opened loop con®guration and a continuous renewal of the carrier solution is demonstrated in this paper. As an application, the determination of chloride in parenteral solutions through the formation of the iron (III) thio-

cyanate complex after displacement of thiocyanate by chloride [19,20] was selected. 2. Experimental 2.1. Reagents All chemicals were of analytical reagent grade and doubly deionised water (conductivity <0.1 mS cmÿ1) was used throughout. The chromogenic reagent (37.1 mmol lÿ1 iron (III) sulphate and 1.90 mmol lÿ1 mercury (II) thiocyanate) was weekly prepared by dissolving 0.626 g Hg(SCN)2 in 150 ml of ethanol followed by the addition of 140 ml of 1.4 mol lÿ1 nitric acid. An amount of 14.85 g Fe2(SO4)3 was dissolved in 600 ml of deionised water with heating. After cooling the iron solution to room temperature the two solutions were mixed and the ®nal volume completed to 1000 ml. A 1% chloride stock standard solution was prepared by dissolving the required amount of NaCl, after drying at 1108C during at least 24 h, in 0.014 mol lÿ1 nitric acid and completing the volume to 500 cm3 with the same acid solution. Working standards were daily prepared by appropriate dilutions of the above solution with 0.014 mol lÿ1 nitric acid. 2.2. Apparatus For the absorbance measurements, at 480 nm, a LaboMed model Spectro 22RS spectrophotometer equipped with a 15 ml inner volume ¯ow-cell was used. The ¯ow manifold comprised a set of three-way solenoid valves (161 T031, NResearch), ¯ow lines and reaction coils made from 0.8 mm i.d. PTFE tubing. Home-made end-®ttings, connectors and con¯uence points were also used. The solutions were aspirated by means of an A-99 Razel syringe equipped with a 50 ml syringe. A home-made power driver based on an ULN 2003 integrated circuit was used to operate the solenoid valves and the syringe. For data acquisition and for control of the circulating system a PC-LABCard model PCL-818L interface card from Advantech and a 486DX based microcomputer were used. The software was developed in Visual Basic 3.0 (Microsoft) and permitted to control the syringe and solenoid valves

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and to process the data acquired from the spectrophotometer. During system optimisation the analytical signals were recorded in a model BD111 Kipp and Zonen recorder.

always kept in position 1 except during sample insertion when it was switched to position 2. Initially V1, V2 and V5 were in position 1 while V3 and V4 were in position 2. In this position, the carrier solution, which was the chromogenic reagent, was inserted in the circular manifold and ¯owed through the detector placed at reactor L3, to waste. The sample was inserted in the circular system (phase A in Fig. 2) by actuation of V5 to position 2, during a pre-set time interval (ts), ®lling L1 and being aspirated through V4. When V5 was switched back to position 1 (phase B in Fig. 2), the carrier solution pushed the sample plug toward the detector in L3 for ®rst detection. After a second pre-set time interval (td) V1 and V2 were switched to position 2 while V3 and V4 were placed in position 1. With this positioning (phase C in Fig. 2) the carrier solution was now inserted through valve V3 carrying the sample zone until L1 before ¯owing to waste through V2. After a third pre-set time interval (tc) V1 and V2 were again actuated to position 2 while V3 and V4 were actuated to position 1 (phase B in Fig. 2), the sample zone was sent again to L3 and the second detection took place. Afterwards, phase B and C were successively repeated at the same time interval (tc), the sample zone was continuously recirculated and successive detections occurred. The length of the global reaction coil considered as the sum of all individual coils forming the circular system (L1±L4), was 60 cm. L3 had a 30 cm length, which was the minimum length required for the inclusion of the spectrophotometer ¯ow-cell. To obtain a symmetric system ensuring that the successively recirculated sample zone had the same volume (discarding the volume of the ¯ow-cell) L1 had as well 30 cm length. To obtain the analytical curve, a calibration procedure was performed involving the insertion of a set of standard solutions, which were recirculated employing the same time intervals used for the samples.

2.3. Circulating flow manifold

2.4. Reference method

The ¯ow network was designed with ®ve three-way solenoid valves (Fig. 1). Valves V1±V4 were arranged in a circular-like array, linked by reactors L1±L4, and their combined actuation constituted the circular manifold. Valve V5 was connected to valve V1 and was responsible for sample insertion. This valve was

In order to assess the accuracy of the results obtained by the developed system, determination of chloride in parenteral solutions was carried out by potentiometric titration according to the British Pharmacopoeia [22]. Samples were titrated with silver (I) using a home-made [21] silver sensitive electrode with

Fig. 1. Flow diagram of the sample recirculating flow manifold: V1, V2, V3, V4 and V5 ± three-way solenoid valves, D ± detector, P ± syringe, L1, L2, L3 and L4 ± reaction coils, Q ± flow rate (0.5 ml minÿ1), S ± sample, I ± reagent solution (carrier solution), C ± confluence points.

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Fig. 2. Schematic diagram of the sample zone recirculation: A ± sample inserted in the recirculating system into L1 through valve 5, being aspirated through valve 4; B ± sample zone transported into L3 by the carrier solution inserted through valve 5, being aspirated through valve 4; C ± sample zone transported back into L1 by the carrier solution now inserted through valve 3, being aspirated through valve 2. Afterwards, B and C are successively repeated.

an homogeneous crystalline membrane as detector to monitor the concentration of titrant. The silver (I) solution used as titrant was a 0.1 M silver (I) nitrate solution with a concentration potentiometrically standardised against a sodium chloride standard solution. Titrations were performed in acidic medium, using HNO3. 3. Results and discussion A noteworthy aspect associated to a continuous sample recirculating system is the easily adaptable length of the reaction (recirculation) coil through which the sample zone would ¯ow till detection, which would be automatically selected (in terms of number of cycles) according to the reaction development and/or analytical signal. This approach presents some advantageous features like the possibility of continuous dilution of the sample plug, enabling an automatic expansion of the conventional working range of analyte concentration. 3.1. System dimensioning The developed ¯ow methodology is essentially based on four three-way injection valves (V1±V4)

(Fig. 1) linked by reaction coils in such a circularlike arrangement that the system has two inlets and two outlets, placed at opposite sites in an opened loop con®guration. The carrier solution entering the circular system through V1 may leave it at V2 or ¯ow further and leave the system at V4 (phase B in Fig. 2). A similar situation occurs when the carrier solution enters through V3 and leaves through V4 or V2 (phase C in Fig. 2). A ®fth valve linked to V1 ensures a timebased sample insertion. As a result, the sample plug travels around the analytical pathway transported successively by the carrier entering through V1 or the carrier entering through V3. The detector may be placed at any site inside the circular system allowing a continuous monitoring of the sample zone, or outside, allowing a single detection. When the detector is placed inside the circular system, a multipeak recording is obtained (Fig. 3). A microcomputer ensures an automatic control of the sample position by means of a time-based approach. The recirculated sample volume is independent of the initially inserted volume and depends exclusively on the internal volume of the coils L1 (placed between V1 and V2) and L3 (placed between V3 and V4). In fact, during phase B the carrier solution transports the entire content of L1, which is dispersed inside L3, while an opposite situation occurs during phase C,

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Fig. 3. Typical multipeak detection obtained with a 2000 mg lÿ1 standard solution.

when the content of L3 is dispersed inside L1. In an extreme situation the largest sample volume that could be initially inserted would correspond to the internal volume of L1. Any value higher than this would ¯ow to waste through V2. Additionally, as far as highly concentrated samples are concerned, the ®rst peaks would have no analytical value, even for low sample volumes, in as much as they would be outside the linear concentration range. Nevertheless, low sample volumes should be used since the number of cycles required to attain an analytical signal within the linear working range increased with sample volume. Evaluation of the circulating system performance was accomplished throughout its application to the determination of chloride in parenteral solutions, by means of the formation of the iron (III) thiocyanate complex and spectrophotometric detection. For this task, sample insertion times from 2 to 4 s, corresponding to sample volumes of about 16±32 ml, for a ¯ow rate of 0.5 ml minÿ1, were adequate. Utilisation of a binary sampling approach [17] by alternating insertion of very small segments of sample and reagent did not

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result in an evident improvement of the reaction development. The length of L2 and L4 had a great in¯uence on the peak pro®le: during phase B, when the sample zone was carried out from L1 till L3, the dispersed trailing portion of the sample plug remained in L2. During phase C, when the sample zone was carried back to L3, the content of L2 was not affected because it did not participate in the recirculation process. However, when a new phase B occurred, the same trailing portion that remained in L2 became the front portion affecting the analytical signal or even originating a second low height peak if the concentration was suf®ciently high. A similar situation occurred during phase C when the trailing portion of the sample plug remained in L4. Therefore L2 and L4 were as short as possible (1 cm length), what was achieved by attaching directly valves V1±V4 and V2±V3. By almost eliminating L2 and L4 a valuable feature of the circular system was put in evidence: it was possible to recirculate any selected zone of the sample plug, which would depend exclusively on the recirculation time used to synchronise the valves. In the present work and with the purpose of showing all the system capabilities, it was decided to recirculate the central zone, which was the most concentrated one, thus obtaining a high number of detections (peaks) although a low dilution. For this reason the optimisation of valves synchronisation was based on the attainment of the highest peak height, which affected the sample throughput since a large number of recirculations increased the time for sample analysis. Nevertheless, a highly concentrated sample could be determined with only two detections providing that in the two recirculations involved only a small portion of the initial sample plug is recirculated, which will undergo a high level dilution. Hence, the circular system worked as a selective zone sampling [23] device in which the sampled aliquot depended on the coil length and synchronisation time. Any portion of the sample plug not considered to be of analytical interest could be discarded during each recirculation and the dilution level attained would only depend on the distance travelled by the sample and the sample zones successively discarded. By using very short reaction coils, which could be easily implemented if the conventional spectrophotometer was replaced by, for example, an optical-®bre spectrophotometer or in

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other situations by a tubular potentiometric detector, increased portions of the sample could be removed with an improvement of the dilution ef®ciency. A ¯ow rate of 0.5 ml minÿ1 allowed a convenient dispersion of the sample plug yielding a sample concentration pro®le that was suitable to withdraw the sample zone. Higher ¯ow rates, although increasing the sampling rate, resulted in lower dispersion and more pronounced concentration gradients, specially at the trailing edge of the sample plug where the zone removal was more evident. This led to a decrease of the reproducibility of the zone sampling process. With this ¯ow rate a high reproducibility of the recirculation process was achieved. Considering that the global recirculation time was 50 s (25 s phase B plus 25 s phase C), peak residence times between 49.4 and 50.4 s were obtained (RSD <0.96%, nˆ50). 3.2. Operating scheme of the valves The optimisation of the valves timing was based on evaluation of three time intervals: sample insertion (ts), ®rst detection (td) and continuous recirculation (tc). For sample insertion, periods from 2 to 4 s were selected. First detection determines the beginning of the recirculation process. After sample insertion, the maximum analytical signal was obtained at about 25 s, while after 60 s the signal almost vanished. At 50 s the signal showed a continuous decrease although not very pronounced making it suitable for the beginning of recirculation at phase C. This assumption is supported by the fact that, with a detector positioned at the middle of the reactor, at 50 s the central zone had already passed the detector and the front edge had already gone to waste. Moreover, since the concentration gradient was not very pronounced, any timing error on the zone sampling process would be attenuated and its effect on reproducibility would be minimised. The temporisation of phase C was accomplished by evaluating increasing time intervals. It was veri®ed that a tc of 25 s corresponded to the highest analytical signal. These results were con®rmed by evaluating tc with a td of 30 s (after maximum detection): starting with a tc of 1 s the analytical signal decreased till 21 s, increased up to 44 s and then decreased again. These results showed that with an interval of up to 21 s the amount of sample inside L3 that was carried out to L1 was not suf®cient and the

analytical signal was due to the trailing portion of the initial sample insertion that remained in L1. A similar result was obtained with tdˆ40 s showing a decrease till 11 s, followed by an increase till 34 s and a further decrease. With tdˆ50 s the signal increased immediately until 25 s. As it could be perceived the highest analytical signal was obtained with a sum of tc and td of around 75 s. A time interval of 25 s for phase B (after ®rst detection) was the one that enabled to attain the highest analytical signal. This interval is shorter than the one used for ®rst detection because the distance covered by the sample plug was correspondingly shorter. 3.3. Analytical characteristics An interesting feature of the proposed system is the continuous renewal of the reagent solution, when used as carrier, and therefore of the sample/reagent interface. During each phase of the recirculation process, the dispersed reaction zone is partially removed and replaced by a new interface, which creates a new reaction zone. This renewal occurs both at the trailing and at the front edge of the sample plug. In fact, when the sample plug is carried from L1 to L3, the last portion of the carrier solution remains intact in L1 and meets the front portion of the sample plug when it returns to L1. Thus the successive detections are not considered as the monitoring of the same reaction with different dispersion levels but as the result of an entirely new reaction. On the other hand, one could perceive the renewal of the reagent solution as a continuous addition of reagent to the same sample plug which might be exploited, for instance, as a titration strategy. The continuous addition of reagent could be particularly advantageous when low solubility reagents are concerned as it is the case of Hg(SCN)2. Considering that the recirculation process is accomplished in a reproducible way, the analytical signal for a particular detection might be interpolated in a calibration curve obtained with distinct standard solutions for the same detection. Moreover, by using the same set of standards and with a single calibration step it is possible to obtain several calibration curves, each one with a given dilution level. Hence, during sample analysis the distinct analytical signals obtained from the consecutive detections could be interpolated in the

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corresponding calibration curves, allowing the attainment of several and comparable concentration measurements of the same sample insertion, which could be used for an accuracy assessment [24]. Nevertheless, an interesting perspective that is in the process of evaluation is the possibility to assemble all sample detections in a single sample curve, whose pro®le (typically sigmoidal) might be used to assess sample concentration. The number of cycles required to dilute a standard solution to a concentration level that was almost not detectable depended on the initial sample concentration. A 100.0 mg lÿ1 chloride standard was resolved with four detections, corresponding to four consecutively lower peaks. A 2000 mg lÿ1 standard was resolved with eight detections (Fig. 3): the ®rst two peaks were comparable and the remaining were successively decreasing. A 10.0 g lÿ1 chloride standard required 12 detections in a sigmoidal pro®le: ®ve initial peaks almost identical at the highest absorbance value and seven consecutively decreasing peaks. Due to the speci®c number of detections shown by each standard solution, each detection presented a particular analytical range of application (Table 1). Analytical curves of the type A ˆ xC ‡ b where A is the absorbance reading and C the concentration value were established for distinct detections. The values for the slope (x), intercept (b) and correlation coef®cients are summarised in Table 1. The relative standard deviation (RSD) was lower than 1.8% (nˆ5). 3.4. Analysis of parenteral solutions In order to evaluate the proposed ¯ow system the determination of chloride was carried out in parenteral

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solutions of distinct composition and distinct chloride concentration. The results obtained with the developed methodology when compared with those obtained by the conventional method presented a relative deviation from ÿ5.6% to 3.2%. The analytical system was very stable and baseline drift was not observed. The results are summarised in Table 2. 4. Conclusions A continuous circulating ¯ow manifold with an opened loop con®guration was developed. The sample travelled through a reaction coil delineated in a circular-like shape, whose length was permanently adjusted by the number of cycles completed, being continuously diluted by dispersion. Removal of selected sample zones, both at the front and trailing edge of the sample plug, increased the dilution ef®ciency while contribute to a permanent renewal of the sample/reagent interface. The results showed that the analytical system could be a valuable strategy to attain high dilution levels, which would extend the analytical range of application. The multidetection process accomplished in the circular ¯ow could be an advantageous feature enabling a continuous monitoring of the sample zone. The recirculating ¯ow system is easily operated and can by fully automated by using, for example, optic or conductimetric sensors that monitor the sample plug position and controlled the valves, timing. A shortcoming of the closed-loop con®gurations, which was the placement of a peristaltic pump inside the recirculating loop affecting negatively the sample plug pro®le, was eliminated. Additionally, the accumula-

Table 1 Analytical figures of merit of the developed methodology for distinct detections Detection number

2 3 4 5 6 7 8

Analytical range (mg lÿ1)

0±500 0±500 0±1000 400±4000 400±10 000 1000±10 000 1500±10 000

Analytical curve Slope10ÿ4

Intercept10ÿ2

R2

6.5 3.9 1.9 0.90 0.40 0.27 0.17

0.63 ÿ0.32 0.46 1.1 2.9 0.89 0.45

0.996 0.996 0.992 0.999 0.996 0.995 0.998

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Table 2 Determination of chloride in parenteral solutions Sample

Soro FisioloÂgico Soro 210 Ionosteril Ionstreil Lact Glucosteril 5% Neodextril 70 a

Concentration determined (mg lÿ1) Reference procedure

Developed methodology

RDa (%)

5460 1803 3860 3870 939 5514

5440 1847 3773 3834 886 5691

ÿ0.37 2.4 ÿ2.3 ÿ0.67 ÿ5.6 3.2

Relative deviation, expressed in percentage, of the developed methodology regarding the reference procedure.

tion of air bubbles within the recirculating loop was reduced since they are purged during the removal of the dispersed sample zones. The amount of information provided by multidetection on the circular system could make it very useful to adapt conventional kinetic determinations to continuous ¯ow systems. More complex reactions could also be implemented by including an extra valve in the recirculating loop for each reagent required. The relatively large inner volume of the spectrophotometric ¯ow cell may be overcame in such situations as when tubular potentiometric detectors with very small inner volumes are used, which make them suitable and attractive detectors for application in sample recirculating ¯ow methodologies. Acknowledgements The authors are grateful to the consortium project FAPESP/JNICT for ®nancial support in the researchers' exchange. The Portuguese authors are grateful to JNICT for ®nancial support under the project PBIC/P/ QUI/2165/95. One of us (JLMS) thanks PRODEP for a Ph.D. grant. References [1] J. Ruzicka, E.H. Hansen, Flow Injection Analysis, 2nd ed., Wiley, New York, 1988, p. 15. [2] R.H. Atallah, J. Ruzicka, G.D. Christian, Anal. Chem. 59 (1987) 2909. [3] A. Iob, H.A. Mottola, Anal. Chem. 52 (1980) 2332. [4] P. Roehring, C.M. Wolff, J.P. Schwing, Anal. Chim. Acta 153 (1983) 181.

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