Simultaneous Determination of Phosphate and Silicate in Waste Water by Sequential Injection Analysis F. Mas-Torres, A. Mun�oz, J. M. Estela and V. Cerd`a* Department of Chemistry, University of Balearic Islands, 07071-Palma de Mallorca, Spain A sequential injection analysis system for the simultaneous determination of phosphate and silicate in waste water is proposed. The method is based on the formation of yellow vanadomolybdophosphate and molybdosilicate, respectively, in addition to the use of large sample volumes.The mutual interference between both analytes was eliminated by selection of the appropriate acidity and by sample segmentation with oxalic acid. The calibration graph for phosphate and silicate is linear up to 12 mg l21 P and 30 mg l21 Si, respectively. The detection limits are 0.2 mg l21 P and 0.9 mg l21 Si. The method provides a throughput of 23 samples h21 with a relative standard deviation < 1.4% for phosphate and < 4% for silicate.The method was found to be suitable for the determination of these species in waste water samples. Keywords: Sequential injection analysis; simultaneous determination; phosphate; silicate; waste water The introduction of sequential injection analysis (SIA) by R°uöziöcka, and Marshall1 responded to the difficulties of implementing flow injection analysis (FIA) on an industrial scale. SIA has opened up new possibilities in flow techniques. Among the acknowledged advantages of SIA are the greater versatility of the manifold, thus avoiding the physical reconfiguration required in FIA systems when changing the chemical determination, and the considerable saving of reagents since a continuous consumption is not involved in SIA systems. In essence, SIA consists in the sequential aspiration of welldefined sample and reagent zones into a holding coil by means of a multi-position (selection) valve.The flow is then reversed and the entire contents of the holding coil are propelled towards the detector.Consequently, there is a considerable decrease in the sampling frequency in relation to the comparable FIA method. However, the favourable aspects of FIA provide a good source for the development of automated monitors. During the aspiration and propelling steps an interpenetration zone, necessary for the required reaction to be accomplished; is generated due to axial and radial dispersion which will depend on the volumes and concentrations of the reagents used together with the geometric conditions of the system.In order to provide sufficient robustness for industrial control process, the proponents of the technique recommend the use of sinusoidal flow piston pumps. Nevertheless, other more available options have been suggested such as the use of peristaltic pumps2 or titration burettes.3 A consequence of the increasing control demanded either in industrial processes or in the environmental field is the increase in the number of parameters to be determined for a particular sample, which has, therefore, led to greater interest in the development of multiparametric automated methods.FIA, in addition to coupling with other techniques such as HPLC or ICP-AES, offers several possibilities regarding the determination of two or more parameters. The most common design of multicomponent systems is based on the use of several detectors connected in parallel or in series.Other less used alternatives are those based on spectral resolution by means of multicomponent techniques and those based on the way in which the sample is introduced into the system. The latter possibility has given rise to the ‘sandwich’ technique, which consists in introducing a sample zone between two different reagent solutions.4,5 The sample volume should be sufficiently large to obtain two clearly differentiated peaks corresponding to the two sample/reagent mixing zones.Both possibilities can be implemented in an SIA system. G�omez et al.6 used multicomponent SIA for the simultaneous determination of calcium and magnesium in waters. On the other hand, Estela et al.7 carried out a study on the feasibility of the use of large sample volumes in SIA. Other workers, as has been reviewed by Robards et al.,8 have proposed several FIA methods for the simultaneous determination of phosphate and silicate with on-line column separation, 9–11 methods based on the different formation rates of the corresponding molybdate heteropolyacid12–14 or by using intermittent flows.15 In the present work, an SIA method is proposed by using large sample volumes for the simultaneous spectrophotometric determination of phosphate and silicate.The mutual interference was eliminated by adjusting the acid concentration and by segmenting the sample by the addition of oxalic acid. The established method was applied to the analysis of waste waters.Experimental Reagents All reagents were prepared from analytical-reagent grade chemicals (Merck, Darmstadt, Germany) and stored in polyethylene bottles, except for phosphate solutions which were stored in glass containers. Stock standard solutions of phosphate (50 mg l21 P) and silicate (1000 mg l21 Si) were prepared from KH2PO4 and Na2SiO3·5H2O, respectively. Working phosphate and silicate solutions were prepared daily by suitable dilution of the stock solutions. A 0.5 m ammonium molybdate solution was prepared from (NH4)6Mo7O24·4H2O.This solution was diluted 5-fold before use as a reagent solution (R1) for the determination of silicate. For phosphate determination, the vanadomolybdate reagent (R2) was prepared to contain 0.035 m ammonium molybdate and 3 31023 m ammonium vanadate in 0.65 m HCl. The carrier was 0.05 m HCl. A 5.6% oxalic acid solution (R3) was prepared by dissolving the solid in distilled water. Hydrochloric acid was added to the solution to obtain a final concentration of 0.18 m.A 0.002% Bromothymol Blue (BTB) solution in 0.01 m sodium tetraborate was used in preliminary studies of the system. Apparatus The sequential injection system depicted in Fig. 1(a) was constructed from the following components: a Crison (Alella, Spain) 738 titration autoburette with adjustable dispensing rate, Analyst, October 1997, Vol. 122 (1033–1038) 1033a laboratory-built electromechanically controlled Rheodyne (Cotati, CA, USA) 5011 six-port valve, a Gilson (Villiers le Bel, France) Sample Changer-22 autosampler and a HP-8452A diode-array spectrophotometer (Hewlett-Packard, Waldbronn, Germany) equipped with a 10 mm Hellma (Jamaica, NY, USA) flow-through cell (volume 18 ml).Data acquisition and device control were achieved using a PC-486 compatible computer. All the tubing connecting the different units was made of PTFE. The holding coil (HC) was 300 cm 3 1.5 mm id. All the remaining tubing was 0.86 mm id.The lengths of reaction coil 1 (RC1) and reaction coil 2 (RC2) were 3.5 m and 130 cm, respectively. Procedure Ports 1–6 were connected to R1, sample, R3, R2, detector and waste, respectively. The analytical procedures of the SIA system were controlled by DARRAY† version 2.0 software developed by the authors’ group. The protocol sequence is listed in Table 1 and the zone sequence is illustrated in Fig. 1(b). R1 (150 ml) was first aspirated into RC1 followed by the sample zone (800 ml) and the flow was then stopped to allow the reaction to take place.Next, R3 (75 ml), a further sample volume (1200 ml) and R2 (75 ml) were sequentially aspirated into RC1 and subsequently propelled towards the detector. The absorbance was measured at 616 nm during the BTB experiments and at 400 nm for the determination of both phosphate and silicate. Dual wavelengths were used to minimize Schlieren noise and the correction was made at 800 nm.Results and Discussion Preliminary Studies of the System As in other continuous-flow methods, SIA requires an overlap zone to achieve a significant reaction at the two reagent/sample interfaces. In addition, in order to determine two different analytes in the same sample volume, a sufficient separation between peaks is required, which can be attained by using a sufficiently large sample volume. Prior to optimization of the system, severxperiments were carried out by alternately using an indicator (BTB) as sample and reagent (100 ml at both sides of the sample), and registering the resulting overlap profiles in order to observe the influence of the flow rate, reaction tube diameter and sample volume on the abovementioned aspects.The flow rate during aspiration of the sample and flush sequences was investigated by using a RC1 of 0.86 mm id. The flow rate was varied between 3.6 and 9.1 ml min21. On maintaining the slowest propulsion rate, the corresponding overlap zones remained virtually constant with the sample aspiration rate.However, for a propulsion rate higher than 4.5 ml min21, the first registered peak was exceedingly narrow and, therefore, the inaccuracy involved in its detection was increased. A flow rate of 4 ml min21 was selected, for both aspiration and propelling purposes. The influence of the tube diameter of RC1 was studied between 0.56 and 1.5 mm id. Obviously, when the internal diameter of the tubing decreases, the length occupied by the same volume of liquid is then increased, and separation of the two sample/reagent interfaces is favoured. Seemingly, with smaller diameter tubing the sensitivity was slightly enhanced. Nevertheless, diameters smaller than 0.5 mm could not be used owing to excessive back-pressure produced in the flow by the liquid-driver used.The reaction coil diameter finally chosen was 0.86 mm id. Owing to the large sample volume, the † The software used in this work can be obtained on request from SCIWARE, Banco de Programas, Departament de Qu�ýmica, Universitat de les Illes Balears, E-07071 Palma de Mallorca, Spain.Fig. 1 Schematic diagram of the SIA system used for the simultaneous determination of phosphate and silicate. P, titration burette, 5 ml; V, six-port valve; HC, holding coil; RC1, reaction coil 1; RC2, reaction coil 2; R1–R3: reagents; S, sample; C, carrier; and D, detector. For details see text. Table 1 Protocol sequence of the SIA system for the simultaneous determination of phosphate and silicate Step Time/s Valve Burette* Sampler Description 1 Initialize Initialize Initial piston position 0 ml 2 7.65 6 D 2500 ml Place burette piston for subsequent steps 3 6 Next sample 4 6.6 2 A 1000 ml Aspirate sample for washing the sample line 5 6.6 6 D 2000 ml Dispense to waste 6 2.5 1 A 150 ml Aspirate R1 solution to RC1 7 12.0 2 A 800 ml Aspirate sample 8 10 2 Stop Stopped-flow for 10 s 9 1.25 3 A 75 ml Aspirate oxalic acid solution 10 18 2 A 1200 ml Aspirate sample 11 1.7 4 A 100 ml Aspirate R2 solution 12 62.1 5 D 3725 ml Dispense flow to the detector; acquire data 13 11.2 5 L 3400 ml Load the burette with carrier via its own two-way valve 14 5 5 D 1000 ml Dispense carrier to wash the line and adjust piston position for next cycle 15 Repeat from step 6, n replicates 16 Repeat from step 3, n samples * Aspirate; D, dispense; L, load burette movement. 1034 Analyst, October 1997, Vol. 122reagents undergo a larger path in RC1 than in RC2 (fixed at a smaller length); thus, the latter does not considerably affect the spatial resolution of the peaks. The diameter selected for RC2 was the same as that of RC1. The influence of the sample volume on spatial resolution was evaluated by injecting volumes from 500 to 2000 ml. As expected, peak separation was improved with an increase in the sample volume since the separation between the two sample/ reagent overlap zones was greater.However, owing to the higher dispersion of the reagent aspirated initially (R1), as a result of the larger distance travelled inside RC1, a widening of the second peak takes place. The increase in the peak amplitude corresponding to the reaction with R1, obtained by using a volume of 2000 ml, was approximately four times that obtained with a volume of 500 ml. The decrease in the height of the valley between both peaks is of the same order. Finally, a 2000 ml sample volume was chosen in order to achieve a good spatial resolution.The increase in the dispersion of R1 might be reflected in a decrease in sensitivity; thus, the possibility of introducing into the system an element which restrained this dispersion, such as a small volume of air or an organic compound immiscible in an aqueous medium, was considered. The use of an organic solvent (e.g., CCl4) gave rise to adherence to the walls of the PTFE tubing, which might have an influence on the reproducibility of the method; thus, no improvement was attained.Air bubbles can be used to avoid dispersion of a certain liquid zone. In order to restrain dispersion of R1 the air zone must be previously aspirated prior to any other reagent. Fig. 2 shows the influence of the signal corresponding to the second peak regarding the presence or absence of air. Probably owing to a build-up of pressure in the selection valve together with the inaccuracy of the burette used as a liquid-driver, the smallest volume of air that gave rise to reproducible results was 15 ml.The air zone could be eliminated by using porous tubular or planar membranes just before detection took place. Finally, it was decided not to use segmentation with air since the selection valve was not provided with sufficient ports; in addition implementation of a new valve would involve a more complex system. In any case, the final range obtained for the studied analytes was sufficiently sensitive for the analysis of waste water.Simultaneous Determination of Phosphate and Silicate The determination of phosphate and silicate is based on the formation of vanadomolybdophosphate in acidic medium. The aim of this work was to design a system which only allowed the reaction of one of the two species at each end of the sample zone, thus simplifying the treatment which would be involved in relation to multicomponent techniques. In order to achieve this objective, different acidic conditions were fixed at both ends of the sample, since silicate reacts more slowly than phosphate, the reaction being accelerated when the acidity is decreased.Because of this fact, together with the need for a higher sensitivity for phosphate, it was decided to determine phosphate in the front peak and silicate in the rear peak. Study of the effect of different parameters Reagent concentration. For the determination of silicate, it was initially decided to use a 0.15 m MoO4 22 solution in 0.5 m HCl; however, finally, the concentrations were decreased (0.1 m MoO4 22 in 0.4 m HCl) in order to reduce the precipitation of MoO3 and other condensed forms which take place in acidic medium.Owing to the persistence of precipitation, the following recommendations should be taken into account: daily calibrations, change of solutions every 2–3 d and washing with an NaOH solution after the work has been concluded. The reagent initially employed for the determination of phosphate was proposed in previous work:16 0.035 m MoO4 22, 2.5 31023 m VO32 and 0.5 m HCl.Under these conditions, and for a volume of 2000 ml a slight interference from silicate was observed; therefore, the acidity was increased to 0.8 m HCl. The lowest concentration possible with which the silicate interference was eliminated was selected (0.65 m) to avoid a larger decrease in the phosphate response. In order to compensate for the loss of response regarding phosphate, the concentration of molybdate was increased in the vanadomolybdate solution, however, only a parallel shift of the calibration graph towards higher values of the coordinates in the origin was obtained.If the acid concentration remains constant (0.65 m HCl), when the [H+]/[Mo] ratio decreases the silicate interference increases. The initial molybdate concentration was considered in further studies. An increase in the vanadate concentration slightly favoured an increase in phosphate sensitivity in addition to the linear range. For example, the correlation coefficient of the calibration graph up to 16 mg l21 P was 0.9951 for 2 3 1023 m VO32 and 0.9992 for 1022 m.However, owing to the absorption of vanadate in the yellow region, the presence of a peak just prior to the phosphate peak is observed, which is due to the excess of reagent that did not undergo reaion. The former peak increases with vanadate concentration and decreases with the concentration of acid in the reagent solution.A concentration of 3 31023 m VO32 was selected in this work. In order to eliminate the pre-peak of phosphate, the carrier solution (distilled water) was replaced by a slightly acidic solution. The lowest concentration that allowed the former peak to be eliminated was 0.05 m HCl. Aspirated reagent volume. The reagent volumes used so far were: 100 ml of R1, 2000 ml of sample and 50 ml of R2. As previously reported,7 volumes of R1 and R2 that allow the required resolution of the peaks would be Vm/10 and Vm/20, respectively, where Vm is the sample volume.These earlier workers7 recommended sample volumes larger than 1500 ml, and, as described under Preliminary Studies of the System, a sample volume of 2000 ml was selected, for which, according to the previous criterion, volumes of R1 and R2 of 200 and 100 ml, respectively should be used; such volumes are larger than those used so far.However, under our experimental conditions (volumes and reagent concentrations) silicate interfered with the determination of phosphate, its elimination being achieved by adjusting the volumes to 150 ml for R1 and 75 ml for R2. Elimination of phosphate interference in the determination of silicate. Phosphate interference in the determination of silicate (second peak) could not be eliminated by simply adjusting the Fig. 2 Effect of the insertion of an air zone prior to aspiration of reagents.(A) Without air; (B) with 15 ml of air. Analyst, October 1997, Vol. 122 1035volumes and concentrations of the reagents. Thus, under the previously established conditions, the contribution to the Si signal of a 10 mg l21 P solution corresponded to a concentration of 12 mg l21 Si. Phosphate interference is usually eliminated by decomposition of phosphomolybdic acid by means of oxalic acid.17 This can easily be implemented in FIA; however, in SIA it involves the insertion of an additional reagent, which may hinder the degree of mixing necessary for the reaction to take place.Aspiration of the oxalic acid solution (R3) could be considered prior to R1, the sequence thus being R3–R1–sample–R2. However, under these conditions formation of the silicomolybdic complex is hindered and, therefore, the reaction between R1 and the sample should take place prior to the addition of R3. The following conditions were considered: the same aspiration sequence as that used so far, propulsion of a certain volume (1.2–1.5 ml) towards the detector in such a way that the fraction for the determination of phosphate remained post-valve, flow halting, insertion of 150 ml of R3 and, finally, continuing with the determination of silicate.However, possibly because the mixing of the molybdate–sample with the oxalic acid was incomplete, decomposition of molybdophosphoric acid was not attained. A further sequential aspiration of all the reagents was considered, including R3, and further propelling towards the detector.Thus, R3 divides the sample zone into two, the aspiration sequence being as follows: R1–sample–R3–sample– R1. Several positions regarding the addition of oxalic acid were tested (Table 2) for a total sample volume of 2000 ml; phosphate interference was avoided when fragmentation of the sample corresponded to 800 and 1200 ml for Si and P, respectively. On varying the volume of R3 from 50 to 150 ml, the only effect observed was that of the persistence of phosphate interference when the volume was only 50 ml.In order to avoid this inconvenience, a volume of 75 ml was selected. The same problem arises when very dilute solutions of oxalic acid are used. The concentration of oxalic acid in 0.24 m HCl was varied from 1.4 to 5.6%. The phosphate signal (5 mg l21 P) remained constant and the interference of phosphate with the determination of silicate completely disappeared for concentrations higher than 3%. A concentration of 5.6% was selected for further experiments.Evaluation of the method Under the selected conditions, silicate interference was avoided in the first peak (phosphate) and vice versa, a good spatial resolution of both peaks was also obtained. Hence, in spite of the fragmentation of the sample zone, which might involve modification of the optimum experimental conditions, it was decided to assess whether the present SIA system met the required needs (detection limit, linear range, etc.).Linearity and accuracy. The performance of the SIA system for the simultaneous determination of phosphate and silicate by using large sample volumes is given in Table 3. The detection limit was calculated as three times the standard deviation of the blank for phosphate and three times the standard deviation of the noise level for silicate. A representative run illustrating the peaks obtained for standards and real samples is shown in Fig. 4. Accuracy. The accuracy of the proposed SIA method was evaluated by comparing the results for several synthetic samples with different phosphate and silicate ratios. The results shown in Table 4, were fairly good in all cases. The divergence becomes greater as the difference in concentrations between the two analytes increases and is also higher for very low phosphate concentrations. Interferences. The possible interference of several species for a mixture of phosphate and silicate at concentrations of 4 and 12 mg l21, respectively, was studied.The interference criterion established was 10% of the concentration value. The maximum concentration tested was 800 mg l21 for most of the species, except for arsenic, chromium(vi) and nitrite for which lower concentrations were considered (1, 5 and 20 mg l21, respectively), owing to the low levels of these species in urban waste water. The results obtained are summarized in Table 5.In addition to the concentration at which the considered species starts to interfere, it is indicated in parentheses whether the interference is either positive or negative. Waste water samples The proposed method was applied to the determination of phosphate and silicate in urban waste water. The waste water samples were filtered through a 0.45 mm filter prior to analysis; thus, only the soluble fraction was analysed. On attempting to analyse waste water samples, the precipitation of calcium oxalate made acidification of the oxalic acid solution necessary.Different HCl concentrations were tested between 0.06 and 0.24 Table 2 Effect of the position of oxalic acid (R3) regarding the interference of different phosphate solutions on the silicate peak* Height of the silicate peak/AU31023 Phosphate/ mg l21 P A† B‡ C§ D¶ 0 0 0 0 0 2 33 11 0 0 6 88 27 0 0 10 136 46 12 0 * R1: 0.1 m MoO4 22, R2: 0.035 m MoO4 22–3 3 1023 m VO32–0.65 m HCl.† A: 150 ml R1 + 2000 ml sample + 75 ml R2. ‡ B: 150 ml R1 + 1000 ml sample + 150 ml R3 + 1000 ml sample + 75 ml R2. § C: 150 ml R1 + 850 ml sample + 150 ml R3 + 1150 ml sample + 75 ml R2. ¶ D: 150 ml R1 + 750 ml sample + 150 ml R3 + 1250 ml sample + 75 ml R2. Table 3 Performance of the SIA system for the simultaneous determination of phosphate and silicate Parameter Phosphate Linear calibration range 0–12 mg l21 P Regression equation H(AU31023) = 48.2 + 41.53[mg l21 P] Correlation coefficient (r) 0.9982 Detection limit (3s) 0.2 mg l21 P RSD (n = 10) 1.38% (9 mg l21 P) Throughput Sample consumption (per sample) Reagent consumption (per sample) Silicate 0–36 mg l21 Si H(AU31023) = 3.7 + 7.43[mg l21 Si] 0.9980 0.9 mg l21 Si 3.87% (23.8 mg l21 Si) 23 samples h21 3.0 ml 0.150 ml molybdate solution 0.075 ml oxalic acid solution 0.075 ml molybdovanadate solution 1036 Analyst, October 1997, Vol. 122m. Within this range, the acidity does not affect the signal significantly since a variation in the blank results in a proportional variation in the sample. By means of several batch assays it was proved that, as the acidity increased, the formation of the precipitate was delayed.Hence, whereas precipitation was almost instantaneous with a concentration of 0.06 m HCl it was delayed for more than 2 min with 0.18 m HCl. The latter concentration was employed for working purposes after ascertaining its compatability with the SIA manifold used in this study.Table 6 compares the results for several waste water samples with those obtained by standard spectrophotometric methods18 based on the formation of vanadomolybdophosphoric and molybdosilicate. Fig. 3(a) and (b) shows that there is good correlation between the methods, with most points lying within the 95% confidence limits. As depicted in Fig. 4, in spite of the fact that the separation of the peaks is substantially worse in the samples than in the mixtures prepared from standard solutions, indicative of a matrix effect, integration of the peaks by using the peak height as an analytical signal was sufficient in order to Fig. 3 (a) Correlation between proposed SIA method and standard method for phosphate and (b) silicate in waste water. Table 6 Results of the determination of phosphate and silicate in waste waters Dilution mg l21 P mg l21 Si Sample factor No. Type* for SIA Batch SIA Error (%) Batch SIA Error (%) 1 E 1 7.3 7.7 +5.5 15.4 15.3 20.6 2 E 1 6.4 6.7 +4.7 17.2 17.1 20.6 3 I 1 7.4 7.5 +1.4 21.2 19.8 26.6 4 I 1 7.8 7.8 0 28.1 27.3 22.8 5 PS 1 7.7 7.8 +1.3 12.6 12.5 20.8 6 PS 1 5.7 5.7 0 19.4 19.5 +0.5 7 PS 1 8.2 8.3 +1.2 15.9 15.1 25.0 8 I 5 14.46 15.03 +3.9 90.53 84.05 27.2 9 I 5 12.76 12.85 +0.7 52.0 57.3 +10.2 10 E 2.5 8.15 7.66 +6.0 31.7 33.6 +6.0 11 I 2.5 9.6 9.55 20.5 31.3 35.4 +13.1 12 I 2.5 11.13 10.05 29.7 31.64 36.8 +16.3 13 I 2.5 16.95 16.67 21.7 29.85 30.8 +3.2 14 I 5 7.07 6.0 215.1 65.93 64.82 21.7 15 E 2.5 6.23 6.05 22.9 36.77 40.73 +10.8 16 E 2.5 9.69 10.03 +3.5 26.1 30.0 +14.9 17 I 2.5 5.67 5.48 23.4 31.34 31.9 +1.8 18 E 1 1.35 1.16 214 24.9 26.6 +6.8 19 I 2.5 7.31 7.41 1.4 35.9 36.1 +0.5 20 E 2.5 7.27 7.94 9.2 44.7 41.9 26.0 * I = Influent; E = Effluent; PS = primary settled.Table 4 Results of the analysis of several standard mixtures of silicate and phosphate Taken/ Taken/ Found/ Error Found/ Error mg l21 P mg l21 Si mg l21 P (%) mg l21 Si (%) 1 1.2 0.78 222.0 1.3 +8.3 1 5.95 0.95 25.0 6.2 +4.7 1 11.9 0.80 220.0 12.1 +1.7 1 23.8 1.2 +20.0 21.4 210.1 2 3.0 2.1 +5.0 3.1 +3.3 2 5.95 2.1 +5.0 5.91 20.7 2 11.9 2.3 +15.0 13.1 +10.1 3 11.9 3.3 +10.0 11.4 24.2 3 17.85 3.2 +6.7 16.6 27.0 6 5.95 6.6 +10.0 5.7 24.2 6 23.8 6.2 +3.3 20.2 215.1 6 35.7 6.7 +11.7 31.3 212.3 Table 5 Interference of several species with the determination of phosphate (4 mg l21 P) and silicate (12 mg l21 Si) Species* Phosphate Silicate Species* Phosphate Silicate Fe3+ 5 (+) 2 (+) S22 1 (2) 15 (+) Fe2+ 2 (2) 4 (+) NO22 > 20 > 20 AsV > 1 > 1 CO3 22 800 (+) 75 (+) CrVI > 5 > 5 SO4 22 800 (2) > 800 K+, Na+ 200 (2) 800 (+) Cl2 > 800 > 800 NH4 + 800 (2) > 800 NO32 > 800 800 (+) Mg2+ > 800 250 (+) * Concentrations in mg l21.Analyst, October 1997, Vol. 122 1037obtain results in good agreement with those of the classical method. Conclusions In the present work the use of large sample volumes has been applied to sequential injection systems for the simultaneous determination of phosphate and silicate, in such a way that each species is analysed at each end of the sample zone.Configuration of the system, which is totally automated, is very simple. Although the reagent consumption is small, the substantial amount of sample required limits the application of the method to those situations in which large amounts of samples are available, as with environmental monitoring.In spite of the difficulty arising from the mutual interference between the two analytes determined, the use of sample segmentation has led to satisfactory results in the analysis of urban waste water. Better results might be anticipated in addition to greater ease of implementation, if two analytes of totally different chemical behaviour were considered. A possible disadvantage regarding SIA methods is the slow analysis rate; however, in this work a throughput of 23 samples h21 was attained, which is sufficient for most applications. The authors thank the CICyT (Spanish Council for Research in Science and Technology) for financial support of this work as part of projects AMB94-0534 and AMB94-1033.References 1 R°uöziöcka, J., and Marshall, G. D., Anal. Chim. Acta, 1990, 237, 329. 2 Ivaska, A., and R°uöziöcka, J., Analyst, 1993, 118, 885. 3 Cladera, A., Tom�as, C., G�omez, E., Estela, J. M., and Cerd�a, V., Anal. Chim. Acta, 1995, 302, 297. 4 Alonso-Chamarro, J., Bartrol�ý, J., and Barber, R., Anal. Chim. Acta, 1992, 261, 219. 5 Araujo, A. N., Lima, J. L. F. C., Rangel, O. S. S., Alonso, J., Bartrol�ý, J., and Barber, R., Analyst, 1989, 114, 1465. 6 G�omez, E., Tom�as, C., Cladera, A., Estela, J. M., and Cerd`a, V., Analyst, 1995, 120, 1181. 7 Estela, J. M., Cladera, A., Mu�noz, A., and Cerd`a, V., Int. J. Environ. Anal. Chem., 1996, 64, 205. 8 Robards, K., McKelvie, I. D., Benson, R. L., Worsfold, P. J., Blundell, N.J., and Casey, H., Anal. Chim. Acta, 1994, 287 , 147. 9 Narusawa, Y., and Hashimoto, T., Chem. Lett., 1987, 1367. 10 Narusawa, Y., Anal. Chim. Acta, 1988, 204, 53. 11 Jones, P., Stanley, R., and Barnett, N., Anal. Chim. Acta, 1991, 249, 539. 12 Linares, P., Luque de Castro, M. D., and Valc�arcel, M., Talanta, 1986, 33, 889. 13 Mas, F., Estela, J. M., and Cerd`a, V., Int. J. Environ. Anal. Chem., 1991, 43, 71. 14 Kircher, C. C. and Crouch, S. R., Anal. Chem., 1983, 55, 248. 15 Jacintho, A. O., Kronka, E. A. M., Zagatto, E. A. G., Arruda, M. A. Z., and Ferreira, J. R., J. Flow Injection Anal.,1989, 6, 19. 16 Mu�noz, A., Mas Torres, F., Estela, J. M., and Cerd`a, V., Anal. Chim. Acta, in the press. 17 Chalmers, R. A., and Sinclair, A. G., Anal. Chim. Acta, 1966, 34, 412. 18 American Public Health Association, American Water Works Association, Water Pollution Control Federation, Standard Methods for the Examination of Water and Wastewater, American Public Health Association, 17th edn., 1989.Paper 7/01646H Received March 10, 1997 Accepted June 30, 1997 Fig. 4 Representative run for the simultaneous determination of phosphate and silicate by SIA. Concentration expressed as mg l21; S1 and S2, waste water samples. 1038 Analyst, October 1997, Vol. 122 Simultaneous Determination of Phosphate and Silicate in Waste Water by Sequential Injection Analysis F. Mas-Torres, A. Mun�oz, J. M. Estela and V. Cerd`a* Department of Chemistry, University of Balearic Islands, 07071-Palma de Mallorca, Spain A sequential injection analysis system for the simultaneous determination of phosphate and silicate in waste water is proposed.The method is based on the formation of yellow vanadomolybdophosphate and molybdosilicate, respectively, in addition to the use of large sample volumes. The mutual interference between both analytes was eliminated by selection of the appropriate acidity and by sample segmentation with oxalic acid.The calibration graph for phosphate and silicate is linear up to 12 mg l21 P and 30 mg l21 Si, respectively. The detection limits are 0.2 mg l21 P and 0.9 mg l21 Si. The method provides a throughput of 23 samples h21 with a relative standard deviation < 1.4% for phosphate and < 4% for silicate. The method was found to be suitable for the determination of these species in waste water samples. Keywords: Sequential injection analysis; simultaneous determination; phosphate; silicate; waste water The introduction of sequential injection analysis (SIA) by R°uöziöcka, and Marshall1 responded to the difficulties of implementing flow injection analysis (FIA) on an industrial scale.SIA has opened up new possibilities in flow techniques. Among the acknowledged advantages of SIA are the greater versatility of the manifold, thus avoiding the physical reconfiguration required in FIA systems when changing the chemical determination, and the considerable saving of reagents since a continuous consumption is not involved in SIA systems.In essence, SIA consists in the sequential aspiration of welldefined sample and reagent zones into a holding coil by means of a multi-position (selection) valve. The flow is then reversed and tthe holding coil are propelled towards the detector. Consequently, there is a considerable decrease in the sampling frequency in relation to the comparable FIA method.However, the favourable aspects of FIA provide a good source for the development of automated monitors. During the aspiration and propelling steps an interpenetration zone, necessary for the required reaction to be accomplished; is generated due to axial and radial dispersion which will depend on the volumes and concentrations of the reagents used together with the geometric conditions of the system. In order to provide sufficient robustness for industrial control process, the proponents of the technique recommend the use of sinusoidal flow piston pumps. Nevertheless, other more available options have been suggested such as the use of peristaltic pumps2 or titration burettes.3 A consequence of the increasing control demanded either in industrial processes or in the environmental field is the increase in the number of parameters to be determined for a particular sample, which has, therefore, led to greater interest in the development of multiparametric automated methods.FIA, in addition to coupling with other techniques such as HPLC or ICP-AES, offers several possibilities regarding the determination of two or more parameters. The most common design of multicomponent systems is based on the use of several detectors connected in parallel or in series. Other less used alternatives are those based on spectral resolution by means of multicomponent techniques and those based on the way in which the sample is introduced into the system.The latter possibility has given rise to the ‘sandwich’ technique, which consists in introducing a sample zone between two different reagent solutions.4,5 The sample volume should be sufficiently large to obtain two clearly differentiated peaks corresponding to the two sample/reagent mixing zones. Both possibilities can be implemented in an SIA system. G�omez et al.6 used multicomponent SIA for the simultaneous determination of calcium and magnesium in waters.On the other hand, Estela et al.7 carried out a study on the feasibility of the use of large sample volumes in SIA. Other workers, as has been reviewed by Robards et al.,8 have proposed several FIA methods for the simultaneous determination of phosphate and silicate with on-line column separation, 9–11 methods based on the different formation rates of the corresponding molybdate heteropolyacid12–14 or by using intermittent flows.15 In the present work, an SIA method is proposed by using large sample volumes for the simultaneous spectrophotometric determination of phosphate and silicate. The mutual interference was eliminated by adjusting the acid concentration and by segmenting the sample by the addition of oxalic acid.The established method was applied to the analysis of waste waters. Experimental Reagents All reagents were prepared from analytical-reagent grade chemicals (Merck, Darmstadt, Germany) and stored in polyethylene bottles, except for phosphate solutions which were stored in glass containers.Stock standard solutions of phosphate (50 mg l21 P) and silicate (1000 mg l21 Si) were prepared from KH2PO4 and Na2SiO3·5H2O, respectively. Working phosphate and silicate solutions were prepared daily by suitable dilution of the stock solutions. A 0.5 m ammonium molybdate solution was prepared from (NH4)6Mo7O24·4H2O. This solution was diluted 5-fold before use as a reagent solution (R1) for the determination of silicate. For phosphate determination, the vanadomolybdate reagent (R2) was prepared to contain 0.035 m ammonium molybdate and 3 31023 m ammonium vanadate in 0.65 m HCl.The carrier was 0.05 m HCl. A 5.6% oxalic acid solution (R3) was prepared by dissolving the solid in distilled water. Hydrochloric acid was added to the solution to obtain a final concentration of 0.18 m. A 0.002% Bromothymol Blue (BTB) solution in 0.01 m sodium tetraborate was used in preliminary studies of the system.Apparatus The sequential injection system depicted in Fig. 1(a) was constructed from the following components: a Crison (Alella, Spain) 738 titration autoburette with adjustable dispensing rate, Analyst, October 1997, Vol. 122 (1033–1038) 1033a laboratory-built electromechanically controlled Rheodyne (Cotati, CA, USA) 5011 six-port valve, a Gilson (Villiers le Bel, France) Sample Changer-22 autosampler and a HP-8452A diode-array spectrophotometer (Hewlett-Packard, Waldbronn, Germany) equipped with a 10 mm Hellma (Jamaica, NY, USA) flow-through cell (volume 18 ml).Data acquisition and device control were achieved using a PC-486 compatible computer. All the tubing connecting the different units was made of PTFE. The holding coil (HC) was 300 cm 3 1.5 mm id. All the remaining tubing was 0.86 mm id. The lengths of reaction coil 1 (RC1) and reaction coil 2 (RC2) were 3.5 m and 130 cm, respectively. Procedure Ports 1–6 were connected to R1, sample, R3, R2, detector and waste, respectively. The analytical procedures of the SIA system were controlled by DARRAY† version 2.0 software developed by the authors’ group.The protocol sequence is listed in Table 1 and the zone sequence is illustrated in Fig. 1(b). R1 (150 ml) was first aspirated into RC1 followed by the sample zone (800 ml) and the flow was then stopped to allow the reaction to take place. Next, R3 (75 ml), a further sample volume (1200 ml) and R2 (75 ml) were sequentially aspirated into RC1 and subsequently propelled towards the detector.The absorbance was measured at 616 nm during the BTB experiments and at 400 nm for the determination of both phosphate and silicate. Dual wavelengths were used to minimize Schlieren noise and the correction was made at 800 nm. Results and Discussion Preliminary Studies of the System As in other continuous-flow methods, SIA requires an overlap zone to achieve a significant reaction at the two reagent/sample interfaces.In addition, in order to determine two different analytes in the same sample volume, a sufficient separation between peaks is required, which can be attained by using a sufficiently large sample volume. Prior to optimization of the system, several experiments were carried out by alternately using an indicator (BTB) as sample and reagent (100 ml at both sides of the sample), and registering the resulting overlap profiles in order to observe the influence of the flow rate, reaction tube diameter and sample volume on the abovementioned aspects.The flow rate during aspiration of the sample and flush sequences was investigated by using a RC1 of 0.86 mm id. The flow rate was varied between 3.6 and 9.1 ml min21. On maintaining the slowest propulsion rate, the corresponding overlap zones remained virtually constant with the sample aspiration rate. However, for a propulsion rate higher than 4.5 ml min21, the first registered peak was exceedingly narrow and, therefore, the inaccuracy involved in its detection was increased.A flow rate of 4 ml min21 was selected, for both aspiration and propelling purposes. The influence of the tube diameter of RC1 was studied between 0.56 and 1.5 mm id. Obviously, when the internal diameter of the tubing decreases, the length occupied by the same volume of liquid is then increased, and separation of the two sample/reagent interfaces is favoured. Seemingly, with smaller diameter tubing the sensitivity was slightly enhanced. Nevertheless, diameters smaller than 0.5 mm could not be used owing to excessive back-pressure produced in the flow by the liquid-driver used.The reaction coil diameter finally chosen was 0.86 mm id. Owing to the large sample volume, the † The software used in this work can be obtained on request from SCIWARE, Banco de Programas, Departament de Qu�ýmica, Universitat de les Illes Balears, E-07071 Palma de Mallorca, Spain.Fig. 1 Schematic diagram of the SIA system used for the simultaneous determination of phosphate and silicate. P, titration burette, 5 ml; V, six-port valve; HC, holding coil; RC1, reaction coil 1; RC2, reaction coil 2; R1–R3: reagents; S, sample; C, carrier; and D, dtor. For details see text. Table 1 Protocol sequence of the SIA system for the simultaneous determination of phosphate and silicate Step Time/s Valve Burette* Sampler Description 1 Initialize Initialize Initial piston position 0 ml 2 7.65 6 D 2500 ml Place burette piston for subsequent steps 3 6 Next sample 4 6.6 2 A 1000 ml Aspirate sample for washing the sample line 5 6.6 6 D 2000 ml Dispense to waste 6 2.5 1 A 150 ml Aspirate R1 solution to RC1 7 12.0 2 A 800 ml Aspirate sample 8 10 2 Stop Stopped-flow for 10 s 9 1.25 3 A 75 ml Aspirate oxalic acid solution 10 18 2 A 1200 ml Aspirate sample 11 1.7 4 A 100 ml Aspirate R2 solution 12 62.1 5 D 3725 ml Dispense flow to the detector; acquire data 13 11.2 5 L 3400 ml Load the burette with carrier via its own two-way valve 14 5 5 D 1000 ml Dispense carrier to wash the line and adjust piston position for next cycle 15 Repeat from step 6, n replicates 16 Repeat from step 3, n samples * Aspirate; D, dispense; L, load burette movement. 1034 Analyst, October 1997, Vol. 122reagents undergo a larger path in RC1 than in RC2 (fixed at a smaller length); thus, the latter does not considerably affect the spatial resolution of the peaks.The diameter selected for RC2 was the same as that of RC1. The influence of the sample volume on spatial resolution was evaluated by injecting volumes from 500 to 2000 ml. As expected, peak separation was improved with an increase in the sample volume since the separation between the two sample/ reagent overlap zones was greater. However, owing to the higher dispersion of the reagent aspirated initially (R1), as a result of the larger distance travelled inside RC1, a widening of the second peak takes place.The increase in the peak amplitude corresponding to the reaction with R1, obtained by using a volume of 2000 ml, was approximately four times that obtained with a volume of 500 ml. The decrease in the height of the valley between both peaks is of the same order. Finally, a 2000 ml sample volume was chosen in order to achieve a good spatial resolution. The increase in the dispersion of R1 might be reflected in a decrease in sensitivity; thus, the possibility of introducing into the system an element which restrained this dispersion, such as a small volume of air or an organic compound immiscible in an aqueous medium, was considered. The use of an organic solvent (e.g., CCl4) gave rise to adherence to the walls of the PTFE tubing, which might have an influence on the reproducibility of the method; thus, no improvement was attained.Air bubbles can be used to avoid dispersion of a certain liquid zone.In order to restrain dispersion of R1 the air zone must be previously aspirated prior to any other reagent. Fig. 2 shows the influence of the signal corresponding to the second peak regarding the presence or absence of air. Probably owing to a build-up of pressure in the selection valve together with the inaccuracy of the burette used as a liquid-driver, the smallest volume of air that gave rise to reproducible results was 15 ml. The air zone could be eliminated by using porous tubular or planar membranes just before detection took place.Finally, it was decided not to use segmentation with air since the selection valve was not provided with sufficient ports; in addition implementation of a new valve would involve a more complex system. In any case, the final range obtained for the studied analytes was sufficiently sensitive for the analysis of waste water. Simultaneous Determination of Phosphate and Silicate The determination of phosphate and silicate is based on the formation of vanadomolybdophosphate in acidic medium.The aim of this work was to design a system which only allowed the reaction of one of the two species at each end of the sample zone, thus simplifying the treatment which would be involved in relation to multicomponent techniques. In order to achieve this objective, different acidic conditions were fixed at both ends of the sample, since silicate reacts more slowly than phosphate, the reaction being accelerated when the acidity is decreased.Because of this fact, together with the need for a higher sensitivity for phosphate, it was decided to determine phosphate in the front peak and silicate in the rear peak. Study of the effect of different parameters Reagent concentration. For the determination of silicate, it was initially decided to use a 0.15 m MoO4 22 solution in 0.5 m HCl; however, finally, the concentrations were decreased (0.1 m MoO4 22 in 0.4 m HCl) in order to reduce the precipitation of MoO3 and other condensed forms which take place in acidic medium.Owing to the persistence of precipitation, the following recommendations should be taken into account: daily calibrations, change of solutions every 2–3 d and washing with an NaOH solution after the work has been concluded. The reagent initially employed for the determination of phosphate was proposed in previous work:16 0.035 m MoO4 22, 2.5 31023 m VO32 and 0.5 m HCl. Under these conditions, and for a volume of 2000 ml a slight interference from silicate was observed; therefore, the acidity was increased to 0.8 m HCl.The lowest concentration possible with which the silicate interference was eliminated was selected (0.65 m) to avoid a larger decrease in the phosphate response. In order to compensate for the loss of response regarding phosphate, the concentration of molybdate was increased in the vanadomolybdate solution, however, only a parallel shift of the calibration graph towards higher values of the coordinates in the origin was obtained.If the acid concentration remains constant (0.65 m HCl), when the [H+]/[Mo] ratio decreases the silicate interference increases. The initial molybdate concentration was considered in further studies. An increase in the vanadate concentration slightly favoured an increase in phosphate sensitivity in addition to the linear range. For example, the correlation coefficient of the calibration graph up to 16 mg l21 P was 0.9951 for 2 3 1023 m VO32 and 0.9992 for 1022 m.However, owing to the absorption of vanadate in the yellow region, the presence of a peak just prior to the phosphate peak is observed, which is due to the excess of reagent that did not undergo reaction. The former peak increases with vanadate concentration and decreases with the concentration of acid in the reagent solution. A concentration of 3 31023 m VO32 was selected in this work. In order to eliminate the pre-peak of phosphate, the carrier solution (distilled water) was replaced by a slightly acidic solution.The lowest concentration that allowed the former peak to be eliminated was 0.05 m HCl. Aspirated reagent volume. The reagent volumes used so far were: 100 ml of R1, 2000 ml of sample and 50 ml of R2. As previously reported,7 volumes of R1 and R2 that allow the required resolution of the peaks would be Vm/10 and Vm/20, respectively, where Vm is the sample volume.These earlier workers7 recommended sample volumes larger than 1500 ml, and, as described under Preliminary Studies of the System, a sample volume of 2000 ml was selected, for which, according to the previous criterion, volumes of R1 and R2 of 200 and 100 ml, respectively should be used; such volumes are larger than those used so far. However, under our experimental conditions (volumes and reagent concentrations) silicate interfered with the determination of phosphate, its elimination being achieved by adjusting the volumes to 150 ml for R1 and 75 ml for R2.Elimination of phosphate interference in the determination of silicate. Phosphate interference in the determination of silicate (second peak) could not be eliminated by simply adjusting the Fig. 2 Effect of the insertion of an air zone prior to aspiration of reagents. (A) Without air; (B) with 15 ml of air. Analyst, October 1997, Vol. 122 1035volumes and concentrations of the reagents.Thus, under the previously established conditions, the contribution to the Si signal of a 10 mg l21 P solution corresponded to a concentration of 12 mg l21 Si. Phosphate interference is usually eliminated by decomposition of phosphomolybdic acid by means of oxalic acid.17 This can easily be implemented in FIA; however, in SIA it involves the insertion of an additional reagent, which may hinder the degree of mixing necessary for the reaction to take place. Aspiration of the oxalic acid solution (R3) could be considered prior to R1, the sequence thus being R3–R1–sample–R2.However, under these conditions formation of the silicomolybdic complex is hindered and, therefore, the reaction between R1 and the sample should take place prior to the addition of R3. The following conditions were considered: the same aspiration sequence as that used so far, propulsion of a certain volume (1.2–1.5 ml) towards the detector in such a way that the fraction for the determination of phosphate remained post-valve, flow halting, insertion of 150 ml of R3 and, finally, continuing with the determination of silicate.However, possibly because the mixing of the molybdate–sample with the oxalic acid was incomplete, decomposition of molybdophosphoric acid was not attained. A further sequential aspiration of all the reagents was considered, including R3, and further propelling towards the detector. Thus, R3 divides the sample zone into two, the aspiration sequence being as follows: R1–sample–R3–sample– R1.Several positions regarding the addition of oxalic acid were tested (Table 2) for a total sample volume of 2000 ml; phosphate interference was avoided when fragmentation of the sample corresponded to 800 and 1200 ml for Si and P, respectively. On varying the volume of R3 from 50 to 150 ml, the only effect observed was that of the persistence of phosphate interference when the volume was only 50 ml. In order to avoid this inconvenience, a volume of 75 ml was selected.The same problem arises when very dilute solutions of oxalic acid are used. The concentration of oxalic acid in 0.24 m HCl was varied from 1.4 to 5.6%. The phosphate signal (5 mg l21 P) remained constant and the interference of phosphate with the determination of silicate completely disappeared for concentrations higher than 3%. A concentration of 5.6% was selected for further experiments. Evaluation of the method Under the selected conditions, silicate interference was avoided in the first peak (phosphate) and vice versa, a good spatial resolution of both peaks was also obtained.Hence, in spite of the fragmentation of the sample zone, which might involve modification of the optimum experimental conditions, it was decided to assess whether the present SIA system met the required needs (detection limit, linear range, etc.). Linearity and accuracy. The performance of the SIA system for the simultaneous determination of phosphate and silicate by using large sample volumes is given in Table 3.The detection limit was calculated as three times the standard deviation of the blank for phosphate and three times the standard deviation of the noise level for silicate. A representative run illustrating the peaks obtained for standards and real samples is shown in Fig. 4. Accuracy. The accuracy of the proposed SIA method was evaluated by comparing the results for several synthetic samples with different phosphate and silicate ratios.The results shown in Table 4, were fairly good in all cases. The divergence becomes greater as the difference in concentrations between the two analytes increases and is also higher for very low phosphate concentrations. Interferences. The possible interference of several species for a mixture of phosphate and silicate at concentrations of 4 and 12 mg l21, respectively, was studied. The interference criterion established was 10% of the concentration value.The maximum concentration tested was 800 mg l21 for most of the species, except for arsenic, chromium(vi) and nitrite for which lower concentrations were considered (1, 5 and 20 mg l21, respectively), owing to the low levels of these species in urban waste water. The results obtained are summarized in Table 5. In addition to the concentration at which the considered species starts to interfere, it is indicated in parentheses whether the interference is either positive or negative.Waste water samples The proposed method was applied to the determination of phosphate and silicate in urban waste water. The waste water samples were filtered through a 0.45 mm filter prior to analysis; thus, only the soluble fraction was analysed. On attempting to analyse waste water samples, the precipitation of calcium oxalate made acidification of the oxalic acid solution necessary. Different HCl concentrations were tested between 0.06 and 0.24 Table 2 Effect of the position of oxalic acid (R3) regarding the interference of different phosphate solutions on the silicate peak* Height of the silicate peak/AU31023 Phosphate/ mg l21 P A† B‡ C§ D¶ 0 0 0 0 0 2 33 11 0 0 6 88 27 0 0 10 136 46 12 0 * R1: 0.1 m MoO4 22, R2: 0.035 m MoO4 22–3 3 1023 m VO32–0.65 m HCl.† A: 150 ml R1 + 2000 ml sample + 75 ml R2. ‡ B: 150 ml R1 + 1000 ml sample + 150 ml R3 + 1000 ml sample + 75 ml R2. § C: 150 ml R1 + 850 ml sample + 150 ml R3 + 1150 ml sample + 75 ml R2.¶ D: 150 ml R1 + 750 ml sample + 150 ml R3 + 1250 ml sample + 75 ml R2. Table 3 Performance of the SIA system for the simultaneous determination of phosphate and silicate Parameter Phosphate Linear calibration range 0–12 mg l21 P Regression equation H(AU31023) = 48.2 + 41.53[mg l21 P] Correlation coefficient (r) 0.9982 Detection limit (3s) 0.2 mg l21 P RSD (n = 10) 1.38% (9 mg l21 P) Throughput Sample consumption (per sample) Reagent consumption (per sample) Silicate 0–36 mg l21 Si H(AU31023) = 3.7 + 7.43[mg l21 Si] 0.9980 0.9 mg l21 Si 3.87% (23.8 mg l21 Si) 23 samples h21 3.0 ml 0.150 ml molybdate solution 0.075 ml oxalic acid solution 0.075 ml molybdovanadate solution 1036 Analyst, October 1997, Vol. 122m. Within this range, the acidity does not affect the signal significantly since a variation in the blank results in a proportional variation in the sample. By means of several batch assays it was proved that, as the acidity increased, the formation of the precipitate was delayed.Hence, whereas precipitation was almost instantaneous with a concentration of 0.06 m HCl it was delayed for more than 2 min with 0.18 m HCl. The latter concentration was employed for working purposes after ascertaining its compatability with the SIA manifold used in this study. Table 6 compares the results for several waste water samples with those obtained by standard spectrophotometric methods18 based on the formation of vanadomolybdophosphoric and molybdosilicate.Fig. 3(a) and (b) shows that there is good correlation between the methods, with most points lying within the 95% confidence limits. As depicted in Fig. 4, in spite of the fact that the separation of the peaks is substantially worse in the samples than in the mixtures prepared from standard solutions, indicative of a matrix effect, integration of the peaks by using the peak height as an analytical signal was sufficient in order to Fig. 3 (a) Correlation between proposed SIA method and standard method for phosphate and (b) silicate in waste water. Table 6 Results of the determination of phosphate and silicate in waste waters Dilution mg l21 P mg l21 Si Sample factor No. Type* for SIA Batch SIA Error (%) Batch SIA Error (%) 1 E 1 7.3 7.7 +5.5 15.4 15.3 20.6 2 E 1 6.4 6.7 +4.7 17.2 17.1 20.6 3 I 1 7.4 7.5 +1.4 21.2 19.8 26.6 4 I 1 7.8 7.8 0 28.1 27.3 22.8 5 PS 1 7.7 7.8 +1.3 12.6 12.5 20.8 6 PS 1 5.7 5.7 0 19.4 19.5 +0.5 7 PS 1 8.2 8.3 +1.2 15.9 15.1 25.0 8 I 5 14.46 15.03 +3.9 90.53 84.05 27.2 9 I 5 12.76 12.85 +0.7 52.0 57.3 +10.2 10 E 2.5 8.15 7.66 +6.0 31.7 33.6 +6.0 11 I 2.5 9.6 9.55 20.5 31.3 35.4 +13.1 12 I 2.5 11.13 10.05 29.7 31.64 36.8 +16.3 13 I 2.5 16.95 16.67 21.7 29.85 30.8 +3.2 14 I 5 7.07 6.0 215.1 65.93 64.82 21.7 15 E 2.5 6.23 6.05 22.9 36.77 40.73 +10.8 16 E 2.5 9.69 10.03 +3.5 26.1 30.0 +14.9 17 I 2.5 5.67 5.48 23.4 31.34 31.9 +1.8 18 E 1 1.35 1.16 214 24.9 26.6 +6.8 19 I 2.5 7.31 7.41 1.4 35.9 36.1 +0.5 20 E 2.5 7.27 7.94 9.2 44.7 41.9 26.0 * I = Influent; E = Effluent; PS = primary settled.Table 4 Results of the analysis of several standard mixtures of silicate and phosphate Taken/ Taken/ Found/ Error Found/ Error mg l21 P mg l21 Si mg l21 P (%) mg l21 Si (%) 1 1.2 0.78 222.0 1.3 +8.3 1 5.95 0.95 25.0 6.2 +4.7 1 11.9 0.80 220.0 12.1 +1.7 1 23.8 1.2 +20.0 21.4 210.1 2 3.0 2.1 +5.0 3.1 +3.3 2 5.95 2.1 +5.0 5.91 20.7 2 11.9 2.3 +15.0 13.1 +10.1 3 11.9 3.3 +10.0 11.4 24.2 3 17.85 3.2 +6.7 16.6 27.0 6 5.95 6.6 +10.0 5.7 24.2 6 23.8 6.2 +3.3 20.2 215.1 6 35.7 6.7 +11.7 31.3 212.3 Table 5 Interference of several species with the determination of phosphate (4 mg l21 P) and silicate (12 mg l21 Si) Species* Phosphate Silicate Species* Phosphate Silicate Fe3+ 5 (+) 2 (+) S22 1 (2) 15 (+) Fe2+ 2 (2) 4 (+) NO22 > 20 > 20 AsV > 1 > 1 CO3 22 800 (+) 75 (+) CrVI > 5 > 5 SO4 22 800 (2) > 800 K+, Na+ 200 (2) 800 (+) Cl2 > 800 > 800 NH4 + 800 (2) > 800 NO32 > 800 800 (+) Mg2+ > 800 250 (+) * Concentrations in mg l21.Analyst, October 1997, Vol. 122 1037obtain results in good agreement with those of the classical method. Conclusions In the present work the use of large sample volumes has been applied to sequential injection systems for the simultaneous determination of phosphate and silicate, in such a way that each species is analysed at each end of the sample zone. Configuration of the system, which is totally automated, is very simple. Although the reagent consumption is small, the substantial amount of sample required limits the application of the method to those situations in which large amounts of samples are available, as with environmental monitoring. In spite of the difficulty arising from the mutual interference between the two analytes determined, the use of sample segmentation has led to satisfactory results in the analysis of urban waste water. Better results might be anticipated in addition to greater ease of implementation, if two analytes of totally different chemical behaviour were considered. A possible disadvantage regarding SIA methods is the slow analysis rate; however, in this work a throughput of 23 samples h21 was attained, which is sufficient for most applications. The authors thank the CICyT (Spanish Council for Research in Science and Technology) for financial support of this work as part of projects AMB94-0534 and AMB94-1033. References 1 R°uöziöcka, J., and Marshall, G. D., Anal. Chim. Acta, 1990, 237, 329. 2 Ivaska, A., and R°uöziöcka, J., Analyst, 1993, 118, 885. 3 Cladera, A., Tom�as, C., G�omez, E., Estela, J. M., and Cerd�a, V., Anal. Chim. Acta, 1995, 302, 297. 4 Alonso-Chamarro, J., Bartrol�ý, J., and Barber, R., Anal. Chim. Acta, 1992, 261, 219. 5 Araujo, A. N., Lima, J. L. F. C., Rangel, O. S. S., Alonso, J., Bartrol�ý, J., and Barber, R., Analyst, 1989, 114, 1465. 6 G�omez, E., Tom�as, C., Cladera, A., Estela, J. M., and Cerd`a, V., Analyst, 1995, 120, 1181. 7 Estela, J. M., Cladera, A., Mu�noz, A., and Cerd`a, V., Int. J. Environ. Anal. Chem., 1996, 64, 205. 8 Robards, K., McKelvie, I. D., Benson, R. L., Worsfold, P. J., Blundell, N. J., and Casey, H., Anal. Chim. Acta, 1994, 287 , 147. 9 Narusawa, Y., and Hashimoto, T., Chem. Lett., 1987, 1367. 10 Narusawa, Y., Anal. Chim. Acta, 1988, 204, 53. 11 Jones, P., Stanley, R., and Barnett, N., Anal. Chim. Acta, 1991, 249, 539. 12 Linares, P., Luque de Castro, M. D., and Valc�arcel, M., Talanta, 1986, 33, 889. 13 Mas, F., Estela, J. M., and Cerd`a, V., Int. J. Environ. Anal. Chem., 1991, 43, 71. 14 Kircher, C. C. and Crouch, S. R., Anal. Chem., 1983, 55, 248. 15 Jacintho, A. O., Kronka, E. A. M., Zagatto, E. A. G., Arruda, M. A. Z., and Ferreira, J. R., J. Flow Injection Anal.,1989, 6, 19. 16 Mu�noz, A., Mas Torres, F., Estela, J. M., and Cerd`a, V., Anal. Chim. Acta, in the press. 17 Chalmers, R. A., and Sinclair, A. G., Anal. Chim. Acta, 1966, 34, 412. 18 American Public Health Association, American Water Works Association, Water Pollution Control Federation, Standard Methods for the Examination of Water and Wastewater, American Public Health Association, 17th edn., 1989. Paper 7/01646H Received March 10, 1997 Accepted June 30, 1997 Fig. 4 Representative run for the simultaneous determination of phosphate and silicate by SIA. Concentration expressed as mg l21; S1 and S2, waste water samples. 1038 Analyst, Oc