Anal. Chem. 1996, 68, 1394-1400
Development of a Sequential Microtitration System Llorenc¸ Alerm and Jordi Bartrolı´*
Grup de Sensors & Biosensors, Departament de Quı´mica, Universitat Auto` noma de Barcelona, 08193 Bellaterra, Spain
The design and construction of a sequential microtitration system are reported in the present work. The combination of the sequential injection analysis technique and a reaction chamber is the basis of this novel system. The reaction chamber features an integrated sensing assembly with miniature all-solid-state sensors. This setup is operated easily using titrant and sample solutions in the microliter range, much lower than the volumes employed in automatic titration systems. Furthermore, a separate reaction vessel for each sample is not required in the system reported here. The proposed setup titrates using sequential injections and does not need calibration procedures, as often happens with other pseudotitration systems adapted to flow systems. The sequential microtitration system has been applied to a wide range of acidbase, complexometric, precipitation, and redox titrations. Accuracy and reproducibility of the method proposed are comparable to those obtained in batch titrations. The typical analysis time is around 5 min. The time employed in a cleaning operation or in a sample change procedure is under 2 min. Volumetric analytical procedures experienced great progress in the 1970s, thanks to the application of microprocessors to chemical analysis.1-4 The introduction of the piston buret had a great impact on titration automation, especially when used with step motors. A commercial microprocessor can control a piston buret driven by a step motor with great precision and reproducibility.5 Currently, automatic titration systems are widely available. However, they cannot be considered microscale instruments. Several alternatives have been suggested when samples smaller than about 100 µL are to be titrated.6,7 Diffusional microtitration8 and microflow systems9 have been developed, where titrant and sample volumes are kept to a minimum, and where a discrete reactor is used. (1) Betteridge, D.; Dagless, E. L.; David, P.; Deans, D. R.; Penketh, G. E.; Shawcross, P. Analyst 1976, 101, 409-420. (2) Christiansen, T. F.; Busch, J. E.; Krogh, S. C. Anal. Chem. 1976, 48, 10511056. (3) Pehrsson, L.; Ingman, F.; Johansson, A. Talanta 1976, 23, 769-788. (4) Pehrsson, L.; Ingman, F.; Johansson, A. Talanta 1976, 24, 79-90. (5) Dahmen, E. A. M. F. Electroanalysis. Theory and Application in Aqueous and Non-Aqueous Media, and in Automated Chemical Control; Techniques and Instrumentation in Analytical Chemistry 7; Elsevier: Amsterdam, 1989; pp 1-28. (6) Kolthoff, I. M., Elving, Ph. J., Eds. Treatise on Analytical Chemistry; Wiley-Interscience: New York, 1975; Part I, Vol. 11; Part II, Vol. 11. (7) To ¨lg, G., Svehla, G., Eds. Elemental Analysis with Minute Samples; Wilson and Wilson’s Comprehensive Analytical Chemistry 3; Elsevier: Amsterdam, 1975; Chapter 1, pp 81-87. (8) Gratzl, M.; Yi, C. Anal. Chem. 1993, 65, 2085-2088. (9) Sagara, F.; Kobayashi, T.; Tajima, T.; Ijyuin, H.; Yoshida, I.; Ishii, D.; Ueno, K. Anal. Chim. Acta 1992, 261, 505-508.
1394 Analytical Chemistry, Vol. 68, No. 8, April 15, 1996
Flow injection analysis (FIA) and air-segmented continuous flow analysis (SCFA) allow the present practice of microscale automated solution analysis.10,11 Flow injection systems actually deal with pseudotitrations. Ruzicka et al. presented a new approach to continuous flow titration based on studies of dispersion patterns.12 Nagy et al. described a novel continuous titration technique with triangle-programmed reagent addition.13 According to the IUPAC definition,14 and considering the arguments put forward by Pardue and Fields,15 these techniques cannot be classified as titration methods. Flow injection analysis is based on the fact that sample dispersion is constant and reproducible; therefore, continuous flow titration has to be considered as a gradient technique.16 The outstanding feature of the more common automated flow titration techniques is the use of a mixing chamber10,11 and the variation of titrant flow rate.17-19 Injected solutions of different, but known, concentrations yield a concentration gradient. In the second case, concentration gradients can be described by establishing flow velocity models. The use of an automatic buret in continuous flow automatic titrations has been reported.20,21 Recently, a flow injection analysis technique based on flow coulometric titrations has been described, using a gradient chamber, a reagent generation chamber, and a detector flow cell integrated into a single unit.22,23 A single line FIA manifold and a manifold featuring sequential injection (SI) with sinusoidal flow24 were used. In both cases, a sample is injected into a flow system containing a gradient chamber. All the titration techniques mentioned above need calibration procedures in order to obtain results that are comparable to those arising from classical titration approach. This is because of the (10) Ruzicka, J. Flow Injection Analysis, 2nd ed.; Chemical Analysis 62. A series of Monographs on Analytical Chemistry and Its Application; Wiley-Interscience: New York, 1988; pp 1-19. (11) Karlberg, B.; Pacey, G. E. Flow Injection Analysis. A Practical Guide; Techniques and Instrumentation in Analytical Chemistry 10; Elsevier: Amsterdam, 1989; pp 1-28. (12) Ruzicka, J.; Hansen, E. H.; Mosbaek, H. Anal. Chim. Acta 1977, 92, 235249. (13) Nagy, G.; Fehe´r, Z. S.; To´th, K.; Pungor, E. Anal. Chim. Acta 1977, 91, 87-106. (14) Irving, H. M. N. H.; Freiser, H.; West, T. S. IUPAC Compendium of Analytical Nomenclature, Definitive Rules 1977; Pergamon: Oxford, 1978; p. 41. (15) Pardue, H. L.; Fields, B. Anal. Chim. Acta 1981, 124, 39-79. (16) Jordan, J. M.; Hoke, S. H.; Pardue, H. L. Anal. Chim. Acta 1993, 272, 115134. (17) Fehe´r, Z.; Nagy, G.; Slezsa´k, I.; To´th, K.; Pungor, E. Anal. Chim. Acta 1993, 273, 521-530. (18) Marcos, J.; Rı´os, A.; Valca´rcel, M. Anal. Chim. Acta 1992, 261, 489-503. (19) Fuhrmann, B.; Spohn, U. Anal. Chim. Acta 1993, 282, 397-406. (20) Bartrolı´, J.; Alerm, Ll. Anal. Chim. Acta 1992, 269, 29-34. (21) Bartrolı´, J.; Alerm, Ll.; Garcı´a-Raurich, J.; Masip, J. Quı´m. Anal. 1994, 13, 31-35. (22) Taylor, R. H.; Ruzicka, J.; Christian, G. D. Talanta 1992, 39, 285-292. (23) Taylor, R. H.; Rotermund, J.; Christian, G. D.; Ruzicka, J. Talanta 1994, 41, 31-38. (24) Taylor, R. H.; Winbo, C.; Christian, G. D.; Ruzicka, J. Talanta 1992, 39, 789-794. 0003-2700/96/0368-1394$12.00/0
© 1996 American Chemical Society
Figure 1. Diagram of the sequential microtitration system. PP, piston pump; PB, piston buret, MTC microtitration cell; V1, six-port distribution valve; V2 and V3, two-port on/off valves; holding coil, PTFE tubing, 1.5 mm i.d., length 650 mm; R1, NH4Cl/NH3 (pH 10) buffer solution in EDTA titrations, 0.01 M HNO3 in precipitation titrations, and 1:50 H2SO4 in redox titrations.
dispersion in a flow system. Flow titrations without calibration runs are possible if known volumes of sample and titrant remain invariant under flow conditions.25 Sequential injection analysis (SIA) uses injection techniques that are more rugged for process control applications.26-29 Dasgupta et al. proposed the use of a reaction chamber with an integrated sensor system in the preparation of an automated microbatch analyzer.30 The design and construction of a sequential microtitration system are reported in the present work. The combination of the SIA technique and a reaction chamber featuring an integrated sensing assembly is the basis of this sequential microtitration system. The system uses a titration microchamber with the miniature sensor system placed inside. Sequential injection uses a six-port distribution valve. Precisely measured volumes of air (as a bubble spacer), sample solution, reference electrolyte solution, and reagent solution are aspirated into a holding coil (HC) by means of a piston pump through this valve. Afterward, the valve is switched to the titration chamber position. In the next step, the flow is reversed so that the stacked zones are propelled through the valve and into the titration cell. The combination of two on/off valves, one above and the other below the cell, allows for the retention of the sample volume inside the microtitration chamber. The air bubble produces a dispersionfree transport of the sample to the titration cell. An automatic buret adds the titrant to the sample in the reaction chamber. The titration curve is obtained from the change of the signal after each addition of titrant solution. According to the standard mathematical procedure, an equivalence volume is calculated from (25) Bartrolı´, J.; Alerm, Ll. Anal. Lett. 1995, 28 (8), 1483-1497. (26) Ruzicka, J. Anal. Chim. Acta 1992, 261, 3-10. (27) Ivaska, A.; Ruzicka, J. Analyst 1993, 118, 885-889. (28) Christian, G. D. Analyst 1994, 119, 2309-2314. (29) Cladera, A.; Toma`s, C.; Go´mez, E.; Estela, J. M.; Cerda`, V. Anal. Chim. Acta 1995, 302, 297-308. (30) Sweileh, J. A.; Dasgupta, P. K. Anal. Chim. Acta 1988, 214, 107-120.
this curve which reveals the concentration of the sample without the need for doing calibration procedures. The precision and reproducibility of this microtitration method yield quick results comparable to those obtained from batch titration methods. In addition to its inherent swiftness, the sequential microtitration system proposed here does not require a separate reactor vessel for each sample, and it works with very small volumes. The time needed for an analysis is around 5 min. A cleaning cycle or a sample change operation takes under 2 min. EXPERIMENTAL SECTION Reagents. Analytical-grade chemicals and distilled water were used. The pH-sensitive plastic membrane was prepared using tridodecylamine (TDDA; Hydrogen Ionophore I, Fluka 95292), bis(2-ethylhexyl) sebacate (BEES; Fluka 84818) as plasticizer, potassium tetrakis(4-chlorophenyl)borate (KTPB; Fluka 60591), and poly(vinyl chloride) (PVC; Fluka 81392). The membrane sensitive to the calcium ion was prepared using bis[4-(1,1,3,3tetramethylbutyl)phenyl]phosphate calcium salt (Fluka 15180), dioctyl phenylphosphonate (Fluka 12584), and PVC. The preparation of both membranes will be detailed later, in the section where the fabrication of the titration cells is described. The solid conductive internal reference used in the electrodes was prepared from an epoxy resin mixture (Araldite M and hardener HY5162 in a 1:0.4 weight ratio, both from Ciba-Geigy) and graphite. The powdered graphite, with a particle size of 50 µm (Merck 4206), was mixed with the epoxy resin in a 1:1 weight ratio. Platinum wire (0.5 mm diameter, Goodfellow PT005145) was used in the redox titration cell. Silver wire (0.5 mm diameter, Goodfellow AG005150) anodically coated with silver chloride was used in all three microtitration cells as a reference electrode. Instrumentation. The sequential microtitration manifold is shown in Figure 1. It was constructed using a piston pump (Crison MicroBU 2030, syringe volume 2.500 ( 0.010 mL single Analytical Chemistry, Vol. 68, No. 8, April 15, 1996
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injection precision) controlled with a personal computer (Toshiba T1000, Model PA7027E, Toshiba Corp.). This setup provided forward and reverse fluid movements in the sequential system. An automatic buret (Metrohm Dosimat 665) featuring a 1 mL syringe ((3 µL single injection precision, (1 µL reproducibility) was used to introduce the titrant into the titration chamber. A six-port distribution valve (Rheodyne 5012) selected flow direction. Two two-way on/off valves (Omnifit 1101) allowed the retention and evacuation of samples in the titration chamber and were also used in the cleaning of the cell after each titration. The flow system was assembled using Teflon tubing (1.5 mm i.d., Omnifit 3011). The holding coil had a volume of ∼1.15 mL (650 mm length, 1.5 mm i.d.). Potentiometric measurements were performed with a (1 mV accuracy multivoltmeter (Crison MicropH 2002). Preparation of Titration Cells. Three cells with a built-in miniature sensor were designed, constructed, and optimized for the development and evaluation of the sequential microtitration system for in situ monitoring of the sample/titrant mixtures. The cells were optimized hydrodynamically and with regard to their assembly and construction. Hydrodynamic optimization involved the minimization of diffusion effects, facilitation of effective cleaning, elimination of cross contamination of the samples, reduction of the volume needed to cover the electrodes, and minimization of overpressures. Sensor devices, already optimized were built according to the recommendations found in the literature.31,32 The dimensions of the cells allowed for the introduction of the sensing assembly and the magnetic stirrer. Figure 2A is a schematic representation of the titration cell used for acid-base, complexometric, and precipitation titrations. For acid-base titrations, the pH sensitive membrane31 was prepared using 1.5 mg of TDDA (ionophore) mixed with 100.0 mg of BEES (plasticizer), 1.0 mg of KTPB (as an interferencesuppressing additive), and 50.0 mg of PVC (as the polymeric matrix). This mixture was dissolved in 3 mL of tetrahydrofuran (Merck 9731). The membrane was cast from this cocktail on the conductive epoxy-graphite resin. The miniature reference system was a thin (0.5 mm diameter) silver wire coated anodically with silver chloride. For the complexometric titrations, the membrane32 in the calcium ion and water-hardness sensor was prepared using 4.5 mg of bis[4-(1,1,3,3-tetramethylbutyl)phenyl]phosphate calcium salt (ionophore) mixed with 64.8 mg of dioctyl phenylphosphonate (plasticizer) and 30.7 mg of PVC (polymeric matrix) dissolved in 1 mL of tetrahydrofuran. The membrane was cast from this cocktail on the conductive epoxy-graphite resin. The miniature reference system was a thin (0.5 mm diameter) silver wire coated anodically with silver chloride. Chloride ion concentration was measured using precipitation titration. In this instance, the Ag/AgCl wire was used as the sensing electrode, and the pH electrode was used as a reference electrode. This is the principle employed in the Titrodes sensors commercialized by Metrohm.33 (31) Alegret, S.; Garcı´a-Raurich, J.; Iba´n ˜ez-Porcel, C.; Martı´nez-Fa´bregas, E.; Martorell, D. Quı´m. Anal. 1994, 13, 176-181. (32) Alonso, J.; Bartrolı´, J.; Lima, J. L. F. C.; Machado, A. A. S. C. Anal. Chim. Acta 1986, 179, 503-508. (33) Titration now even simpler with the new Titrodes. Metrohm Ion Analysis 08-93, Titrodes, 6-0430.100 Silver Titrode and 6.0431.100 Platinum Titrode; Metrohm Ltd.: CH-9101 Herisau, Switzerland.
1396 Analytical Chemistry, Vol. 68, No. 8, April 15, 1996
Figure 2. Schematic representation of titration cells. Cells were made in methacrylate, and their external dimensions were 45 mm × 35 mm × 37 mm (length × width × height). The internal volume of the titration chamber was ∼1200 µL (11 mm diameter × 12 mm height). 1, sample input; 2, air output; 3, waste output; 4, titrant input; R, Ag/AgCl reference electrode; C, electrical connector; M, magnetic stirrer. (A) Acid-base, complexometric, and precipitation microtitration cell. E, ion-selective membrane; G, epoxy-graphite solid internal reference. (B) Redox microtitration cell. Pt, platinum electrode.
Figure 2B shows the redox titration cell. The sensing electrode was a thin platinum wire, and the reference electrode was a Ag/AgCl wire as described above. In all the cells, the miniature sensing assemblies were integrated and placed inside the chambers. All cells were connected to the flow system by four mouths. Mouths 1 and 4 (Figure 2) were used as inputs to introduce volumes of liquid from the holding coil and titrant volumes, respectively. Mouth 2 was an output used to draw air out of the system, using an on/off valve. Waste solution was evacuated through mouth 3, also controlled by an on/off valve. A Tefloncovered magnetic stirrer (6 mm long, 3 mm diameter) inside each cell accelerated the mixing of the sample and the titrant. Cells were made of methacrylate, and their external dimensions were 45 mm × 35 mm × 37 mm (length × width × height). The
internal volume of the titration chamber was 1200 µL (∼11 mm diameter × 12 mm height). The mouths labeled 1, 2, and 3 had a diameter of 1 mm, and that of mouth 4 was 0.5 mm. The reason for the diminished diameter in mouth 4 was to reduce titrant diffusion during the titration and washing processes. The reduction of the diameter helped diminish titrant diffusion but did not eliminate it altogether. Commercially available automatic titration systems adopt this strategy. Titrant concentration losses were prevented between samples because of the titration chamber was maintained empty and clean between titrations. The overall result was that titrant diffusion was negligible, as seen in the precision of measurements detailed below in the Results and Discussion sections. All connections were made using standard FIA connectors. Procedure. The titration sequence comprised the following steps: (1) aspiration of 100 µL of air as a spacer, (2) aspiration of 200 µL of reference electrolyte, (3) aspiration of sample solution, (4) aspiration of reagent solution, (5)dispensation of a volume larger than the aspirated volumes (by 300 µL) to the titration chamber. The distribution valve changed position in each of the steps accordingly. The dispensed liquid was kept in the titration chamber with the upper on/off valve open (air valve) and the lower on/off valve closed (waste valve). The Ag/AgCl reference electrode placed in the chambers did not have an internal reference solution. Therefore, the sample had to contain a high and invariant chloride ion concentration. A 0.1 M NaCl solution was introduced as “reference electrolyte”. The presence of this electrolyte helped maintain a stable reference potential. Once the sample had been prepared inside the chamber, it was titrated by several additions of titrant solution, measuring the voltage after each addition. A titration curve was obtained with the experimental points [Vtitrant (mL) vs E (mV)]. The equivalence volume was obtained from the first and second differentials. The piston pump impelled distilled water into the system to evacuate the titrated sample and to wash the system. The upper valve (air valve) was kept closed, while the lower valve (waste valve) was open. This wash cycle was repeated for three times using 7.5 mL of distilled water. Afterwards, 1500 µL of air was aspirated. Only 1000 µL of this air was dispensed to the chamber, so all liquid was evacuated from the chamber. The distilled water left in the syringe of the piston pump was eliminated via the waste channel in the distribution valve. After these steps, the system was ready for the next titration cycle. To perform a change of sample, distilled water was used to wash the aspiration tube of the distribution valve. All liquids were evacuated from the aspiration tube using air. A new sample volume was drawn into this tube, leaving a small surplus in the holding coil. The holding coil was washed with distilled water, which was discarded through the waste channel of the distribution valve. The time needed for a measurement cycle is less than 5 min. The time needed for an analysis cycle depends on the reactions involved (kinetics) and the response time of the sensor. In the present study, we chose simple, fast, and quantitative reactions (typical of titration processes) and fast-responding sensors. If slower reactions or slower sensors were involved, analysis time would increase. This increment would not pose a problem, since the titration process is controlled by the response of the sensor. A new titrant addition is performed when the sensor signal is
Table 1. Correlation between the Piston Pump Steps and the Titrated Sample Volumesa PP steps
titrant Veq (µL)
sample Vcalc (µL)
mean Vcalc (µL)
RSD (%)
25 25 25
61.08 62.17 61.51
24.823 25.266 24.997
25.03
0.88
50 50 50
123.33 124.02 122.96
50.121 50.402 49.969
50.16
0.44
100 100 100
245.74 245.41 247.73
99.868 99.734 100.675
100.09
0.51
150 150 150
37.03 37.72 37.10
149.507 152.299 149.801
150.54
1.02
200 200 200
49.02 49.92 49.74
197.931 201.553 200.812
200.10
0.95
a Sample volumes are calculated by V calc (µL) ) [NaOH]batch Veq(µL)/[CH3COOH]batch. Sample solutions are acetic acid [0.110 04 M (0.10), 0.011 076 M (0.02)], and titrant solutions are sodium hydroxide [0.044 72 M (0.12)].
Table 2. Repeatability Study of the Sequential Microtitration System at Different Concentrationsa sample (mol/L)
sample V (µL)
titrant Veq (µL)
mean %RSD
100 100 100
108.47 107.31 107.72
107.8 (0.56)
0.111 1 0.109 9 0.110 3
0.110 45 (0.56)
100 100 100
112.35 112.28 112.47
112.4 (0.09)
0.011 15 0.011 14 0.011 16
0.011 147 (0.09)
200 200 200
45.08 45.76 45.10
0.001 089 0.001 105 0.001 089
0.001 095 (0.85)
45.31 (0.85)
mean (RSD, %)
a Sample solutions are acetic acid [0.110 06 M (0.04), 0.011 076 M (0.02), 0.001079 M (0.75)] and titrant solutions are sodium hydroxide [0.102 43 M (0.09), 0.009 92 M (0.14), 0.004 831 M (0.36)].
stable (or within a predetermined range, (1 mV). The sampling and washing sequence is controlled by the titrator, and a new washing and sampling cycle will begin only when a titration cycle has been completed. Hence, slower titration processes will yield longer analysis times, but the instrument will still function satisfactorily without modifying the hardware or the software. RESULTS AND DISCUSSION Validation of the Sequential Microtitration System. Acidbase titrations were done in all the validation procedures. A titration cell featuring a pH ISE was included in the flow system shown in Figure 1. The proposed system was evaluated by comparing the volume introduced to the titration chamber and the movements of the piston pump. Acetic acid and sodium hydroxide solutions were used as sample and titrant, respectively. Both solutions were standardized using discontinuous titrations. Table 1 summarizes the results obtained by comparing sample volumes and the steps aspirated into the holding coil. It is shown that a match exists between the titrated volume of the sample and the volume aspirated by the piston pump. Analytical Chemistry, Vol. 68, No. 8, April 15, 1996
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Figure 3. Titration curves obtained with the sequential microtitration system. (A) Titration of different acids with NaOH. Strong acid (3): sample, 0.1247 M HCl, 100 µL; titrant, 0.2187 M NaOH, 57.0 µL. Weak acid (b): sample, 0.001 079 M CH3COOH, 200 µL; titrant, 0.004 831 M NaOH, 45.31 µL. Polyprotonic acid (1): sample, 0.1018 M H3PO4, 100 µL; titrant, 0.2187 M NaOH, 46.3 µL. (B) Titration of different bases with HCl. Weak base (b): sample, 0.1407 M NH3, 100 µL; titrant, 0.2086 M HCl, 67.2 µL. Dibasic base (3): sample, 0.0944 M Na2CO3, 100 µL; titrant, 0.2086 M HCl, 45.2 µL. (C) Titration of calcium and total hardness with EDTA. Standard of Ca2+ (b): sample, 406.0 mg/L Ca2+, 100 µL; titrant, 0.008 79 M EDTA, 114.0 µL. Freshwater hardness (3): sample, freshwater, 173 mg/L CaCO3, 200 µL; titrant, 0.008 79 M EDTA, 39.8 µL. (D) Titration of chloride with silver nitrate. Standard of Cl- (b): sample, 360.32 mg/L Cl-, 100 µL; titrant, 0.010 26 M AgNO3, 99.6 µL. Freshwater (3): sample, freshwater, 68.63 mg/L Cl-, 250 µL; titrant, 0.010 26 M AgNO3, 43.6 µL. (E) Redox titration. Standard of Cr2O72- (b): sample, 0.1000 equiv/L Cr2O72-, 100 µL; titrant, 0.0985 M Fe(II), 101.5 µL. Standard of H2O2 (3): sample, 0.2212 wt % H2O2, 50 µL; titrant, 0.0200 M MnO4-, 65.5 µL. 1398
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Table 3. Application of the Sequential Microtitration System batch titration n
sample
sequential titration
titrant
sample concn (%RSD) 0.124 7 (0.08)b 0.020 16 (0.10)b 0.110 06 (0.04)b 0.011 076 (0.03)b 0.100 23 (0.04)b 0.101 83 (0.06)b 0.140 7 (0.36)b 0.023 0(0.20)b 0.094 4 (0.32)b
sample (µL)
titrant equiv vol (µL)
Acid-Base Titrations 100 100 100 100 100 100 100 200 100
3 3 3 3 3 3 3 3 3
HCl HCl CH3COOH CH3COOH C6H5KO4 H3PO4 NH3 B4O7 Na2CO3
NaOH NaOH NaOH NaOH NaOH NaOH HCl HCl HCl
3 3 3
Ca2+ Ca2+/Mg2+(10/1) freshwater
EDTA EDTA EDTA
Complexometric Titrations 416.4 (1.30);c 406.0 (0.91)d 100 181.2 (0.57);c 181.9 (0.73)d 200 172.0 (0.72);c 173.0 (0.36)d 200
3 3
NaCl freshwater
AgNO3 AgNO3
360.32 (0.10)d 63.83 (0.16)d
3 3 3
Cr2O72H2O2 H2O2
Fe(II) MnO4MnO4-
0.1000 (0.41)e 0.2212 (0.83)f 2.325 (0.29)f
57.0 44.8 107.8 112.3 45.7 46.3 67.2 21.8 45.2
sample concn 0.124 6 (0.64)b 0.020 1 (0.99)b 0.110 4 (0.54) 0.011 147 (0.09)b 0.100 0(0.31)b 0.101 4 (0.98)b 0.140 1 (0.29)b 0.022 8 (1.30)b 0.094 2 (0.42)b
rel errora (%) 0.08 0.30 0.31 0.64 0.23 0.42 0.43 0.87 0.21
114.0 40.8 39.8
406.9 (160)d 181.7 (1.65)d 173.1 (1.21)d
0.22 0.11 0.06
Precipitation Titrations 100 250
99.6 43.6
362.60 (0.70)d 63.46 (0.72)d
0.63 0.59
Redox Titrations 100 50 15
101.5 65.5 205.3
0.0999 (0.80)e 0.2228 (0.20)f 2.328 (3.05)f
0.14 0.76 0.13
a The percentile relative error between batch sample concentration and sequential sample concentration. b Potentiometric, in mol/L. c Colorimetric, in mg/L. d Potentiometric, in mg/L. e Potentiometric, in equiv/L. f In wt %.
When a linear regression of these results was performed [sample volume calculated (µL) vs piston pump steps], the regression coefficients obtained were n ) 5, intercept ) 0.07 (0.19 SD) µL, slope ) 1.001 (0.002 SD), r2 ) 0.999 997. As can be seen, the confidence interval (P ) 0.05) encompassed 0 as intercept and a slope of 1. This shows that the aspirated sample volume was not affected by constant or proportional errors introduced by the piston pump steps. When Student’s t data paired test analysis was performed, results were (n ) 5, P ) 0.05), t(calculated) ) 2.014, t(tabulated) ) 2.776. Both statistical methods showed that the results are comparable. These results show that the volume introduced into the titration chamber can be derived by observing the movements of the piston pump. Repeatability studies were done by titrating at different concentrations. Sodium hydroxide and acetic acid solutions were prepared and standardized by discontinuous titrations and then applied to the proposed sequential system. Table 2 shows the results of the repeatability studies using three different concentrations. Standard deviation values stayed below 1% for concentrations between 1 mM and 0.1 M. Furthermore, mean concentration values were comparable to the results obtained using batch titrations. Finally, carry-over between samples was studied. Performance would be improved by assessing the extent of carry-over problems from one sample to the next. Incorporation of the wash stream was necessary. The effectiveness of this stream to remove carryover from one sample to the next is shown by running interaction test patterns of samples containing low (L) and high (H) concentrations of analytes. The procedure involves injecting a sample of low concentration and finding its concentration experimentally (L1). The system is washed, and a high-concentration sample is injected and analyzed. The system is washed for a second time, and another low-concentration sample is injected and its concentration assessed experimentally (L2). The concentrations found for L1 and L2 should not vary. The interaction, which
can be computed as
I% ) 100 × (L2 - L1)/H
(1)
indicates the fractional contribution made by H to L2, and will be zero if carryover is absent. Hydrochloric acid solutions were prepared and standardized using sodium hydroxide with the sequential system. The solutions used were 0.020 10 M (0.51 % RSD; L) and 2.2905 M (0.02 % RSD; H) HCl. A titration was performed using 100 µL of the diluted solution. A concentration of 0.020 0375 M (L1) was found. A new titration was done with 100 µL of the concentrated solution, and a concentration of 2.290 612 M (H) was found. When the sample was again of a low concentration, a concentration of 0.020 0738 M (L2) was found. Applying eq 1, a value of I% ) 0.001 58 was calculated. As shown, the values of L1 and L2 were comparable, and both were within the confidence interval found in the standardization of the diluted solution. The small size of the interaction test value is proof that carry-over between samples is negligible. Application and Evaluation of the Microtitration System. All the solutions used in the application and evaluation of the sequential microtitration system were previously standardized in batch using the automatic titrator as recommended in the official guidelines.34,35 Throughout the present work, the results obtained with the microtitration system were compared with the results of an automatic and discontinuous titrator, using standard volumetric methods for each analyte.34,35 Acid-base titrations were done with the cell depicted in Figure 2A, using a hydrogen ion-sensitive membrane. Weak, strong, and polyprotonic acids were analyzed. Figure 3A shows the resulting titration curves. Weak monobasic and dibasic bases were also (34) Howell Furman, N., Ed. Standard Methods of Chemical Analysis, 6th ed.; VanNostrand Co.: Princeton, NJ, 1968. (35) Midgley, D.; Torrance, K. Potentiometric Water Analysis, 2nd ed.; 1991; John Wiley & Sons: Chichester, U.K., 1991.
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analyzed (Figure 3B). In all sequential microtitrations, 200 µL of 0.1 M NaCl was used as the reference electrolyte. The cell described in Figure 2A, featuring a calcium ionsensitive membrane, was used in the sequential system for EDTA titrations. Natural and synthetic water samples were assayed for calcium ion concentration and total hardness. The results obtained with the sequential system were compared with results obtained using discontinuous potentiometric titrations featuring a calcium ion ISE (Crison 15 2203000 2) and discontinuous colorimetric titrations. The end point was determined using Eriochrome Black T solution and Murexide indicator. In all the sequential titrations additions of 100 µL of buffer solution (NH4Cl/NH3, pH 10) and 200 µL of 0.1 M NaCl as the reference electrolyte were done. Figure 3C shows a titration curve obtained with the sequential titration system. The cell shown in Figure 2A was used for chloride titrations by precipitation. In this cell, a pH-sensitive sensor was used as a reference, and a Ag/AgCl wire was used as the sensor. At first, 50 µL of 0.01 M HNO3 was added to obtain a stable signal, but later it was found that this was not necessary. Figure 3D shows some of the titration curves obtained. The pH-sensitive electrode acts here as a reference electrode. The results obtained were matched to the results obtained with discontinuous titrations. To realize redox titrations, the cell shown in Figure 2B was installed in the microtitration sequential system. A silver/silver chloride wire was used as a reference electrode, and a platinum wire was used as the sensor. An addition of 50 µL of a 1:50 H2SO4 solution and 200 µL of 0.1 M NaCl as reference electrolyte was made for all sequential titrations. Figure 3E shows some of the titration curves that were obtained. Table 3 summarizes the information obtained upon the application of the sequential microtitration system. All results provided by the sequential microtitration system were compared with the results obtained for the same samples using the automatic
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titration system as recommended in official guidelines. The relative errors found after comparing these sets of results are less than 1%. The results provided by the sequential microtitration system show standard deviations below 2%. There is a good correspondence between the results obtained using sequential microtitrations and those from discontinuous titrations. These results show that the sequential microtitration system is comparable to commercially available automatic titrators, with the added advantage of smaller sample volumes and smaller titrant volumes. Additionally, there is no need for a separate reactor for each sample. CONCLUSIONS The sequential microtitration system has been applied to a wide range of acid-base, complexometric, precipitation, and redox titrations. The method is fast, uses tiny amounts of reagents, and is practical, reliable, and reproducible. An additional advantage of the approach presented here is that no calibration is needed in order to evaluate the results, as opposed to many published flowthrough titration systems. The sequential microtitration system can be applied in clinical and pharmaceutical applications, where small sample volumes and low reagent use are needed. Biochemical applications also come to mind when the features of the sequential microtitrator, featuring a single chamber with the sensor assembly, are examined. For kinetics studies of incubation, immunoassay, and other applications where reactions are slow and require the presence of several reagents, systems based on stopped-FIA and SFA techniques may be replaced. Received for review April 11, 1995. Accepted November 8, 1995.X AC950361A X
Abstract published in Advance ACS Abstracts, January 1, 1996.
