Automatic multicommutation flow system for wide range spectrophotometric calcium determination

Analytica Chimica Acta 366 (1998) 45±53

Automatic multicommutation ¯ow system for wide range spectrophotometric calcium determination FaÂbio R.P. Rocha1, PatrõÂcia B. Martelli2, Rejane M. Frizzarin, Boaventura F. Reis* Centro de Energia Nuclear na Agricultura, Universidade de SaÄo Paulo, PO Box 96-13400-970, Piracicaba, SP, Brazil Received 13 August 1997; received in revised form 20 October 1997; accepted 24 October 1997

Abstract An automatic ¯ow system based on multicommutation concept is proposed to widen the linear concentration range for spectrophotometric calcium determination. The ¯ow network was build up with three way solenoid valves to permit implementation of different sample processing conditions in order to achieve limited, medium and large dispersion degree without modi®cation of the manifold con®guration. Dilutions were carried out by changing both sampled volume and the analytical path length or applying zone sampling approach. The software was developed to control all steps of sample processing and to allow changing the manifold con®guration in order to obtain suitable sample dilution. This condition was attained with up to 3 trials. A linear response from 0.250 to 1000 mg lÿ1, and a detection limit of 7 mg lÿ1 (99.7% con®dence level) were achieved. The relative standard deviation was 0.83% (nˆ10) or better. The sampling rate was ca. 60 hÿ1 and 0.27 mg of the chromogenic reagent (3,30 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolphthalein) was consumed per determination. The procedure was applied to calcium determination in waters, plant materials, milk, antacid tablets, fertilizers and calcareous rocks. The results were in agreement with certi®ed values or with those obtained with ¯ame atomic absorption spectrophotometry at a 95% con®dence level. # 1998 Elsevier Science B.V. Keywords: Multicommutation; Automation; Zone sampling; Wide range determination; Flow injection spectrophotometry

1. Introduction Spectrophotometry is a widespread detection technique employed in association with ¯ow injection analysis. Despite the large use of this association, there are some limitations due to the narrow linear *Corresponding author. Fax: +55 19 4294610; e-mail: [email protected] 1 Departamento de QuõÂmica, Universidade Federal de SaÄo Carlos, SaÄo Carlos, Brazil. 2 Instituto de QuõÂmica de SaÄo Carlos, Universidade de SaÄo Paulo, SaÄo Carlos, Brazil. 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(97)00634-X

response range of the Beer's law. Therefore, time consuming sample dilution steps are often needed prior measurements. Otherwise, manifolds need to be changed to permit higher dilutions necessary for some samples which can be inadequate to others. Spectrophotometric calcium determination is affected by this limitation since this element is commonly found in a wide concentration range in different kinds of samples. Several ¯ow injection procedures have been used to promote dilutions exploiting the sample dispersion in the carrier stream. The practical way to change dispersion is to modify the sampled volume and the path

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F.R.P. Rocha et al. / Analytica Chimica Acta 366 (1998) 45±53

length [1]. This was ingeniously exploited to widen the linear response range for manganese determination, by injecting simultaneously different sample volumes that were dispersed in different path lengths [2]. This approach allowed obtaining several analytical curves corresponding to different dispersion degrees of the sample. Higher dilutions can be carried out by splitting zones [3,4], zone sampling [5], by using a mixing chamber [6] or by means of gradient dilution [7]. The combination of these techniques was proposed to achieve very high dilutions [8±10]. A comparison between manifolds employing splitting and mixing chambers for dilution in single and dual stages was performed by Garn et al. [8], attaining dispersion coef®cients as high as 106. The proposed system is complex and the manifold must be changed to allow different dispersion levels. A system to perform cascade dilutions [9] by combining sample splitting and merging streams was proposed to obtain dilutions factors from 100 to 500, permitting chloride determination in solutions as concentrated as 1.7 mol lÿ1. In addition to the system complexity, two peristaltic pumps were used. Fang et al. [10] employed a computer controlled stepper motor to select low sample volumes (0.7 ml). By using a mixing chamber, a 1000 fold sample dilution was achieved with a relative standard deviation lower than 2%. Despite the possibility to attain higher dilutions, the previously mentioned systems can be inadequate for determinations in sample lots with a wide concentration range of the analyte. The large dispersion could be excessive for some samples and the manifold con®guration should be modi®ed. This drawback can be overcome by employing automatic systems able to self adjustment to attain the necessary dilutions. This approach was adopted for manganese determination by trial measurements [11] and for metals determination by ¯ame atomic absorption spectrophotometry (FAAS) and ¯ame emission spectrometry [12]. These works exploited the alteration of both sample volume and analytical path length or zone sampling process, respectively. A feedback system able to adjust the maximum signal to previously de®ned thresholds was employed. Multicommutation is a novel approach in ¯ow analysis that can be implemented by using discrete commutation devices, such as three way solenoid valves. The approach was employed to perform binary

sampling [13], making feasible the reduction of the reagent consumption, sequential determinations [14,15], and sequential management of incompatible reagents in single line manifolds [15]. Additionally, this approach was employed to perform dilutions. A system analogous to zone sampling and splitting zones was proposed by ArauÂjo et al [16]. A three way solenoid valve was employed to permit the cleavage of the sample zone to determine creatinine in urine. Gine et al. [17] employed the binary sampling process to carry out up to 40 fold automated dilutions in inductively coupled plasma atomic emission spectrometry (ICP-AES) by changing the sample volumetric fractions. Discrete commutation devices permit to enhance system versatility, since several steps of the analytical procedure can be implemented independently. This feature can be exploited to achieve different sample dispersion degrees to allow widening the linear working range. In this work, discrete commutation devices were applied to develop an automatic spectrophotometric procedure for calcium determination in a wide concentration range. As chromogenic reagent, it was used 3,30 -bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolphthalein (CPC). Different sample dispersion levels were achieved by changing both analytical path and sampled volume, by splitting the sample zone or by employing zone sampling. The possibility of associating splitting and zone sampling in cascade was also investigated. The software was developed with a feedback structure in order to allow modi®cations in the operation conditions without changing the ¯ow network. The feasibility of the procedure was demonstrated for calcium determination in water, milk, plant materials, antacid tablet, fertilizers and calcareous rocks. 2. Experimental 2.1. Apparatus The ¯ow network was built up by assembling a set of three way solenoid valves (161T031-NResearch), mixing coils and transmission lines of polyethylene tubing (0.8 mm i.d.). Measurements were carried out with a 432 Femto spectrophotometer furnished with a

F.R.P. Rocha et al. / Analytica Chimica Acta 366 (1998) 45±53

10 mm optical path ¯ow cell. A 486 DX microcomputer with an electronic interface (Advantech PCL711S), running a software written in Quick BASIC 4.5, controlled commutation devices, performed data acquisition and processing. An Ismatec IPC-4 peristaltic pump with Tygon tubes was employed to propel the solutions. This device is equipped with an electronic interface that was employed to change the pumping rate through a serial port (RS-232) of the microcomputer. An electronic interface previously described [13] was used to switch the solenoid valves. The sampling step was synchronized with pumping pulsation by reading the peristaltic pump tachometer signal by means of the PCL-711S analog input. 2.2. Reagents and solutions All solutions were prepared with analytical reagent grade chemicals and distilled deionized water. As chromogenic reagent (R1), a 0.01% (w/v) CPC solution was used. This solution also contained 0.2% (w/v) 8-hydroxyquinoline to mask magnesium. A 0.5 mol lÿ1 ammonium/ammonia buffer solution, pH 10.5, containing 1.5% (v/v) triethanolamine (R2) was used to pH adjustment and to mask iron and aluminum. A 10.00 g lÿ1 calcium stock solution (0.012 mol lÿ1 HCl) was prepared from CaCO3. Reference solutions were prepared in three different media by appropriated dilutions of the stock with water (0.25 to 10.0 mg lÿ1), 0.25 mol lÿ1 HClO4 (10 to 500 mg lÿ1) or 0.25 mol lÿ1 HCl (500 to 1000 mg lÿ1) to match reference and sample solutions. Water samples were

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collected in polyethylene bottles and preserved by addition of 1.0 ml of concentrated nitric acid per liter. Milk and plant materials were mineralized employing nitric±perchloric digestion procedure as described elsewhere [18], using ca. 0.5 g of the sample and adjusting the ®nal volume to 50 ml with water. Calcareous rocks (dolomite and calcite), antacid tablets and fertilizers were prepared by extraction with 10 ml HCl 6.0 mol lÿ1 by heating for 30 min. The ®nal volume was adjusted with water to 250 ml. Results obtained with the automatic system were compared with certi®ed values or with those attained by FAAS, after batch dilution of the samples. A lanthanum nitrate solution was added to the samples to avoid chemical interferences. 2.3. Flow diagram and procedure The ¯ow diagram of the system is shown in the Fig. 1, where dashed lines represent the ¯ow paths when the valves are switched on. The sample volume was selected by simultaneously switching the valves V1 and V2 on. By modifying the switching time of these valves (0.2, 2.2, and 10.0 s) it was possible to insert sample volumes of 10, 110, and 500 ml, respectively. The valve V3 permitted both to perform zone sampling and to direct the sample zone towards path a or b. The stream through the ¯ow cell was maintained independently of the selected path (valve V3 switched on or off). The path b was selected to reduce the sample dispersion when the sample volume was 500 ml.

Fig. 1. Flow diagram of the system for automatic single stage dilutions. V1±V5 ± three way solenoid valves. B1±B3 ± coiled reactors (100, 200 and 125 cm, respectively). C ± carrier stream (H2O). S ± sample. R1 ± chromogenic reagent. R2 ± buffer solution pH 10.5. x ± confluence point. DET ± spectrophotometric detector (575 nm). W ± waste. Dashed lines represent the flow paths when the valves are switched on. a and b are the path ways selected when the valve V3 is switched off or on. Numbers between brackets represent flow rates in ml minÿ1.

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Zone sampling was carried out by selecting 10 ml sample volume and re-sampling a portion of the sample dispersed in B1. After injection, the valve V3 was switched on to direct the front portion of the sample zone towards waste (W) through path b. After a previously established time interval (splitting time, ts), the portion of the sample zone to be resampled was selected by switching the valve V3 off during 1 or 5 s (re-sampling time, tr). This allowed to select a sample aliquot that was maintained inside the coil B2. Regarding sample injection, the splitting times were 10.5, 15.5 or 19.0 s. The remaining rear portion of the sample zone into B1 was directed towards waste by switching the valve V3 on again. The discarded portions of the sample zone were directed towards waste without reagent addition, in order to avoid baseline perturbations. The reagents (R1 and R2) were added to the re-sampled aliquot by switching the valves V4 and V5 on when the sample zone was passing through the con¯uence point x. To save reagents, these valves were switched off after the maximum signal was achieved. The reaction was developed inside the coil B3 and the formed product was measured at 575 nm. Dilution in a single (zone sampling) and dual (associating splitting and zone sampling in cascade) stages were compared to attain the higher dispersion degrees. Single stage dilution was carried out as previously described. The splitting and zone sampling in cascade were implemented by using the manifold shown in the Fig. 2. This diagram is similar that showed in Fig. 1, but an additional solenoid valve (V6) was connected between the coil B2 and the

con¯uence point x. After selection of the sample aliquot, the valve V3 was switched on to direct the front of the sample zone dispersed in B1 toward waste. The remaining portion was dispersed inside the coil B2 and the front of the sample zone was again diverted to waste by switching the valve V6 on. After a selected time interval, the valve V6 was switched off and the remaining rear portion was dispersed in the coil B3. Reagents were introduced similarly as the procedure for dilution in a single stage. The software to control the ¯ow setup and to perform data acquisition and processing was developed after considering chemical reactions characteristics and the required dilutions. The ¯owchart of the software is shown in Fig. 3. All events were executed automatically, depending on the necessary dilution. The operation times of the valves (Table 1) were de®ned to permit limited, medium and large dispersion to cover a wide concentration range. A set of reference solutions was employed to obtain analytical curves corresponding to each sample processing condition. For samples, the ®rst measurement was made in the condition of medium dispersion (Table 1, condition c). The signal measured was compared to previously set threshold values and a feedback system allowed to change the sample processing condition. If the signal maximum surpassed the higher threshold level, the growing rate (
signal/
t considering rising time up to half of the signal maximum) of the transient signal was checked to decide the level of dispersion to be implemented in the next step. If the signal obtained in the ®rst measurement is lower than the minimum threshold level, the sample dispersion was reduced to

Fig. 2. Flow diagram of the system for automatic dual stage dilutions. V1±V6 ± three way solenoid valves. B1±B4 ± coiled reactors (100, 100, 100 and 125 cm, respectively). Dashed lines represent the flow paths when the valves are switched on. Other symbols are described in the legend of Fig. 1.

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Fig. 3. Flowchart of the software. Threshold values are informed as input variables and the parameters k and p are automatically estimated from the reference solutions measurements.

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Table 1 Sample processing conditions. The splitting time (ts) and the re-sampling time (tr) are defined in the text and in Fig. 4 Condition

Sample volume (ml)

ts (s)

tr (s)

Linear range (mg lÿ1)

Dispersion coefficient

a b c d e

500 110 10 10 10

Ð Ð 10.5 15.5 19.0

Ð Ð 5.0 1.0 1.0

0.250±2.50 1.00±10.0 10.0±100 100±500 250±1000

2.15 6.58 30.7 252 754

match the signal with the linear response range. Threshold values were informed as input variables. The parameters k and p were automatically estimated when the reference solutions were run. Samples were reprocessed until obtaining a suitable analytical signal which is attained with up to 3 trials. When a suitable dispersion degree was attained, the predetermined number of replicates was performed and the concentration was calculated by least square regression. 3. Results and discussion Initial experiments were carried out to establish the sample processing conditions to allow analyzing samples containing different calcium concentrations. In this sense, the effect of variation of the sampled volume and coiled reactors length, and the feasibility of using splitting or zone sampling approaches were evaluated. These strategies allowed modifying the sample dispersion providing feasibilities to implement different sample dilutions to widen the linear response of the procedure. The lower dispersion conditions were attained by inserting 500 or 110 ml sample volume and directing the sample zone towards the detector through path b, by switching valve V3 on (Fig. 1). The linear response range and dispersion coef®cients attained are shown in Table 1 (conditions a and b, respectively). A linear response up to 100 mg lÿ1 was obtained by reducing the sample volume to 10 ml and diverting the sample zone towards path a. To attain high dilutions, zone sampling and splitting zone approaches were compared considering the precision obtained. The splitting was performed by switching the valve V3 on, to direct the front of the sample zone towards waste through the path b (Fig. 1). The rear portion was transported towards detector

through path a. A sigmoidal attenuation of the signal was obtained by increasing the switching time of the valve V3. However, the precision was lessened (relative standard deviation>5%) for dispersion coef®cients higher than 100. A system associating splitting and zone sampling in cascade was evaluated to improve the precision. This was carried out with the ¯ow network shown in the Fig. 2. In spite of the high dilutions achieved the precision was unsatisfactory. Precision was improved by re-sampling an aliquot in the central portion of the dispersed sample zone. This was carried out by selecting different splitting times (ts) and re-sampling times (tr), such as indicated in the Fig. 4(a). By maintaining the re-sampling time

Fig. 4. Recorder outputs: (a) ± transient signal and indication of the splitting time (ts) and the re-sampling time (tr). I represents the instant of sample injection. (b) ± profile of the sample zone obtained by changing the splitting time from 9.0 to 23.5 s with increments of 0.5 s (re-sampling time maintained in 1 s). The arrows c, d and e represent the splitting times values adopted in the sample processing conditions c, d and e defined in Table 1. (c±e) ± repeatability study for solutions containing 100, 500 or 1000 mg Ca2‡ lÿ1 processed in the conditions c, d, or e (Table 1), respectively.

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of 1 s, the splitting time was varied from 9.0 to 23.5 s with increments of 0.5 s yielding the pro®le shown in Fig. 4(b). These results allowed selecting the suitable conditions to determine samples with higher calcium concentrations. For obtaining a linear response up to 500 or up to 1000 mg lÿ1, splitting times of 15.5 and 19.0 s were chosen, respectively. When ts was 10.5 s and the re-sampling time was increased to 5 s, a linear response from 10 to 100 mg lÿ1 was obtained. The selected ts values are indicated by the arrows c, d and e in Fig. 4(b). The linear response range and the dispersion coef®cients attained are also shown in Table 1. In the selected conditions, the repeatability of the results was evaluated (Fig. 4(c)±(e)) and the relative standard deviations were estimated as 0.70, 0.77 and 0.83% (nˆ10), for ts values of 10.5, 15.5, and 19.0 s, respectively. Without zone sampling, the relative standard deviation was 0.37% (nˆ10). Despite the high dilutions achieved, a good repeatability of results was obtained emphasizing the feasibility of the proposal. In the ®ve sample processing conditions adopted, the dispersion coef®cients varied from 2.15 to 754 (Table 1), making feasible a linear response range from 0.250 to 1000 mg lÿ1. Typical analytical curves are shown in Fig. 5.

Fig. 5. Typical analytical curves obtained in the adopted sample processing conditions (a±e), such as described in the Table 1. (a) and (b) ± upper x axis and (c±e) ± lower x axis.

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The system ran automatically based on the operational conditions indicated in Table 1. A set of reference solutions was used to obtain analytical curves in each of the ®ve selected conditions (Fig. 5). Initially, samples are processed under a medium dispersion condition (Table 1, condition c). The signal obtained is compared with pre set thresholds values, informed as input variables. The feedback system was allowed to decide if the attained dilution is adequate or if it needs to be changed. If the signal is higher than the upper threshold, the growing rate of the signal (
signal/
t in the portion corresponding to the front of the sample zone) is checked and compared with pre set values to decide if the sample should be processed in the condition d or e (see Table 1). Alternatively, if the signal is lower than the inferior threshold, the sample is processed again under conditions of limited dispersion (Table 1, condition a). If the signal obtained is too high, the sample is re-processed in the condition b. When a suitable signal is achieved, replicated measurements are performed and the concentration is determined by interpolation in the suitable analytical curve. For the chromogenic reaction selected, pH adjustment is essential to allow the complex formation and to avoid excessive dissociation of the CPC reagent, since the dissociated ligand presents absorption maximum in the same wavelength of the calcium complex. This was achieved by using a buffer solution with pH adjusted at 10.5. Interferences of magnesium and iron were suppressed by using 8-hydroxyquinoline and triethanolamine. The multicommutation approach permitted intermittent addition of reagents only in the sample zone and this allowed reducing the reagent consumption to 0.27 mg CPC, 0.53 mg 8-hydroxyquinoline and 4.5 mg triethanolamine per determination. Since the sample dilution was performed before reagents addition, an excess of the reagents in the sample zone was assured. The sampling rate was dependent on the sample processing condition; 42 or 68 determinations can be carried out per hour with and without applying zone sampling. By software the pump rotation was increased 3 times to reduce both the time to direct the rear portion of the sample zone to waste (through valve V3) and the washing time. With this strategy, the sample throughput was increased in 50%. Under the lowest dispersion condition, the detection

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limit was estimated in 7 mg lÿ1 at 99.7% con®dence level. The feasibility of the procedure was demonstrated by determining calcium in waters (Table 2), plant materials and milk (Table 3), fertilizers, calcareous rocks and antacid tablets (Table 4). Results were in agreement with certi®ed values or with those obtained by FAAS at the 95% con®dence level. The procedure is suitable for calcium determination in a large variety of matrices. In the samples analyzed, the concentration of the analyte varied from 0.181 (mineral water) to 850 mg lÿ1 (calcite) that emphasizes the wide range of application of the procedure. Use of discrete components permits to attain different Table 2 Mean values and uncertainties (nˆ3) for calcium determination in water samples Sample

Multicommutation (mg lÿ1)

FAAS (mg lÿ1)

Mineral Mineral River River River River River River

0.1810.001 6.230.01 3.550.01 7.310.01 2.840.03 8.630.06 6.150.04 8.060.05

0.1920.001a 6.200.08 3.660.06 7.320.02 2.960.02 8.520.04 6.070.10 8.180.10

a

Determined by ICP-AES.

Table 3 Mean values and uncertainties (nˆ3) for calcium determination in plant and milk powder digests Sample

Multicommutation (mg/g)

Reference method (mg/g)

Soya flour Corn leaves Heartwood Heartwood Heartwood Apple leaves Tomato leaves Hay powder Milk powder

1.910.04 10.50.05 4.120.06 7.760.07 6.200.06 15.40.1 50.01.1 21.30.2 11.30.7

1.840.06a 10.70.0a 4.080.01a 7.820.07a 6.110.01a 15.260.15b 50.50.9b 21.60.6c 12.870.32c

a

Flame atomic absorption spectrophotometry. Standard reference material ± National Institute of Standards and Technology. c Standard reference material ± International Atomic Energy Agency. b

Table 4 Mean values and uncertainties (nˆ3) for calcium determination in fertilizers, calcareous rocks and antacid tablet samples Sample

Multicommutation (%, w/w)

FAAS (%, w/w)

Fertilizer Fertilizer Dolomite Calcite Antacid tablet

18.40.3 12.10.1 23.00.1 33.70.1 19.30.2

18.40.2 12.90.1 22.20.1 33.70.1 20.00.1

operation modes by changing the sample processing without altering the ¯ow network. The automation of the procedure allows eliminating the multistep dilutions necessary to determine calcium in samples with higher concentrations and to employ the same ¯ow set up independently of the sample to be measured. Another pro®table characteristic is the low reagents consumption provided by the intermittent addition. As can be deduced from Fig. 4, the system would permit to attain dispersion coef®cients as higher as 1000 by increasing the splitting time in the zone sample procedure. Acknowledgements The authors are grateful to E.A.G. Zagatto and J.A. NoÂbrega for critical comments and to A.R.A. Nogueira for discussions and for supplying calcareous rocks samples. FAPESP, FINEP/PRONEX, CAPES and CNPq are thanked for ®nancial support. References [1] J. Ruzicka, E.H. Hansen, Flow Injection Analysis, 2nd ed., Wiley, New York, 1988. [2] E.A.G. Zagatto, M.F. GineÂ, E.A.N. Fernandes, B.F. Reis, F.J. Krug, Anal. Chim. Acta 173 (1985) 289. [3] J. Ruzicka, J.W.B. Stewart, E.A.G. Zagatto, Anal. Chim. Acta 81 (1976) 387. [4] G.D. Clark, J. Ruzicka, G.D. Christian, Anal. Chem. 61 (1989) 1773. [5] B.F. Reis, A.O. Jacintho, J. Mortatti, F.J. Krug, E.A.G. Zagatto, H. Bergamin F, L.C.R. Pessenda, Anal. Chim. Acta 123 (1981) 221. [6] K.K. Stewart, A.G. Rosenfeld, Anal. Chem. 54 (1982) 2368. [7] S. Olsen, J. Ruzicka, E.H. Hansen, Anal. Chim. Acta 136 (1982) 101.

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[14] P.B. Martelli, B.F. Reis, E.A.M. Kronka, H. Bergamin F, M. Korn, E.A.G. Zagatto, J.L.F.C Lima, A.N. Araujo, Anal. Chim. Acta 308 (1995) 397. [15] E.A.M. Kronka, B.F. Reis, M. Korn, H. Bergamin F, Anal. Chim. Acta 334 (1996) 287. [16] A.N. ArauÂjo, J.L.F.C. Lima, B.F. Reis, E.A.G. Zagatto, Anal. Chim. Acta 310 (1995) 447. [17] M.F. GineÂ, A.P. Packer, T. Blanco, B.F. Reis, Anal. Chim. Acta 323 (1996) 47. [18] F.J. Krug, H. Bergamin F, E.A.G. Zagatto, S.S. Joergensen, Analyst 102 (1977) 503.