Spectrophotometric Determination of Ethylenediaminetetraacetic Acid and Its Related Compounds with P-Carboxyphenylfluorone, Titanium(IV) and Hydrogen Peroxide1

ANALYTICAL SCIENCES DECEMBER 1998, VOL. 14 1157 1998 © The Japan Society for Analytical Chemistry

Notes Spectrophotometric Determination of Ethylenediaminetetraacetic Acid and Its Related Compounds with p-Carboxyphenylfluorone, Titanium(IV) and Hydrogen Peroxide1

Yoshikazu FUJITA, Itsuo MORI and Takako MATSUO

Osaka University of Pharmaceutical Sciences, Nasahara, Takatsuki, Osaka 569Ð1094, Japan

Keywords Ethylenediaminetetraacetic acid and its related compounds, spectrophotometry, p-carboxyphenylfluorone- titanium(IV) complex, hydrogen peroxide

Ethylenediaminetetraacetic acid (EDTA), an (polyethylene glycol-p-nonylphenylether, Nakarai aminopolycarboxylic acid, is widely used in industry, Tesque) and 0.8% Amphitol 24B (betaine lauryldimethyl- detergents and foods. Recently, environmental pollu- aminoacetate, Kao Chem.) in the final concentration. tion by EDTA is getting more and more serious due to A buffer solution of pH 5.5 was prepared by mixing a its strong affinity with highly toxic heavy metals and its 0.2 M disodium hydrogenphosphate solution and a 0.1 resistance to biodegradation. Thus, it is imperative that M citric acid solution. Reagent-grade chemicals were sensitive and selective means of analysis are available. used throughout. Pure water was prepared by purifying We have reported some sensitive spectrophotometric deionized water with a Milli-Q Labo system just before methods2,3 for the determination of hydrogen peroxide use. (H2O2) based on fading of a dye-titanium(IV) complex A Shimadzu spectrophotometer (Model UV-160) with in the presence of EDTA. We speculated that a method 1.0-cm matched silica cells was used for an absorbance which would utilize fading of a dye-titanium(IV) com- measurement. The pH measurements were made with a plex in the presence of H2O2 would serve as a sensitive Horiba (F-11) pH meter in combination with a calomel determination for EDTA. Here, we report on a spec- glass electrode. trophotometric method for the determination of EDTA and its related compounds based on fading of a p-car- Standard procedure boxyphenylfluorone (PCPF)-titanium(IV) complex in The following components were mixed in a 10-cm3 Ð6 the presence of H2O2. volumetric flask: a solution containing 1.0×10 Ð 6.0×10Ð6 M of EDTA and its related compounds, 0.5 cm3 of a 2.0×10Ð4 M titanium(IV) solution, 0.2 cm3 of a Ð2 3 Experimental 1.5×10 % H2O2 solution, 0.5 cm of the surfactant solution, 2.5 cm3 of the buffer solution and 0.4 cm3 of a Reagents and apparatus 1.0×10Ð3 M PCPF solution. The mixture was diluted to A stock solution (1.0×10Ð3 M, 1 M=1 mol dmÐ3) of 10 cm3 with water, transferred into a test tube, mixed EDTA was prepared by dissolving disodium ethylene- well and kept at 60ûC for 25 min. After the solution diaminetetraacetate (Dojindo Lab.) with water. had been cooled in water to room temperature, the dif- Working solutions were prepared by the dilution of the ference in the absorbance (∆A) between the resultant stock solution. Other EDTA-related compounds, solution and a reagent blank solution prepared under EDTA-metal chelates and EDTA derivatives, were also the same conditions was measured at 615 nm against obtained from Dojindo Lab. A titanium(IV) solution water. (2.0×10Ð4 M) was prepared from a stock solution (Wako Pure Chem. Co. Ltd., 1000 µg/cm3) by dilution with 0.1 M hydrochloric acid, as required. Solutions of all dyes, Results and Discussion which had been synthesized according to the method described in the literature4,5, were prepared in 1.0×10Ð3 Optimization of reaction variables M methanol solution containing one drop of hydrochlo- In preliminary experiments, the effect of dyes was Ð2 ric acid. A H2O2 solution (1.5×10 %) was prepared by studied by measuring the difference of absorbance (∆A) diluting a 30% hydrogen peroxide (Mitsubishi Gas between a dye-titanium(IV) and a dye-titanium(IV)- Chem. Co. Ltd.) with water. A surfactant solution was EDTA solutions in the presence of H2O2. The dyes obtained as a mixed solution of 0.4% Triton N-101 used were PCPF, o-sulfophenylfluorone (SPF), o- 1158 ANALYTICAL SCIENCES DECEMBER 1998, VOL. 14

Fig. 1 Structures of dyes studied

hydroxyhydroquinonephthalein (QP), vanillylfluorone (VF), salicylfluorone (SF), phenylfluorone (PF) and m- fluorophenylfluorone (MFPF). These structures are shown in Fig. 1. These dyes could be arranged in the Fig. 2 Absorption spectra of PCPF-Ti(IV) and PCPF solu- Ð5 Ð5 following order with respect to sensitivity: PCPF>QP, tions. [Ti(IV)]=1.0×10 M; [PCPF]=4.0×10 M; [H2O2] SPF>VF, SF>> PF, MFPF. =3.0×10Ð4%; [surfactant]=0.5 ml of the surfactant solution/10 The best pH range was from 5.2 to 5.9 by using 2.5 ml; pH=5.5. Curve 1: PCPF-Ti(IV) or PCPF-Ti(IV)-H2O2 cm3 of 0.2 M disodium hydrogenphosphate/0.1 M citric solutions. Curves 2, 3 and 4: PCPF-Ti(IV)-H2O2-EDTA solu- acid buffer solution. tions (EDTA concentration(M): curve 2=2.0×10Ð6; curve 3= × Ð6 × Ð6 The amount of PCPF was examined while maintain- 4.0 10 ; curve 4=6.0 10 ). Curve 5: PCPF or PCPF-H2O2 ing a fixed final concentration of titanium(IV)(1.0×10Ð5 solution. Reference: water. M). The use of 4.0×10Ð5 M PCPF in the final concentration was the best with regard to the reaction rate and stability of color development and in consideration of Calibration curves absorbance of a PCPF-titanium(IV) solution (a reagent A calibration curve for EDTA was constructed by the blank). standard procedure. A good linear relationship was In order to stabilize the color development and to observed over the 1.0×10Ð6 M Ð 6.0×10Ð6 M range of increase the sensitivity, the effects of different surfac- EDTA in the final volume of 10 cm3. This procedure is tants were examined: nonionic (Triton N-101, Tween about 3 Ð 20 times more sensitive than the other spec- 20, Brij 35, Triton X-100), anionic (sodium dodecylsul- trophotometric methods, such as bathocuproine-copper6, fate, perfluorooctanoic acid, sodium di(2-ethylhexyl)- Pyrocatechol Violet-bismuth7, phosphomolybdic acid8, sulfosuccinate), cationic (stearyltrimethylammonium 1,2-naphthoquinone-4-sulfonic acid phenylthiosemicar- chloride, cetylpyridinium chloride, cetyltrimethylam- bazone-bismuth9 and 4,7-diphenyl-1,10-phenanthro- monium chloride) and amphoteric (Amphitol 24B, line-disulfonic acid-iron10 methods, and is as sensitive Swanol AM-103, sodium N-lauroylsarcosine). The as HPLC methods.11 The relative standard deviation simultaneous use of Amphitol 24B with a nonionic sur- (RSD) for five runs of 2.0×10Ð6 M EDTA was 1.8%. factant, particularly Triton N-101, was the most effec- The calibration curves for EDTA related compounds tive. (EDTA-metal chelates and other aminopolycarboxylic 3 Ð2 When 0.1 Ð 2.0 cm of 1.5×10 % H2O2 was used, ∆A acids) were also constructed under the optimum condi- was found to remain constant. Thus, 0.2 cm3 of tions. EDTA-metal chelates and aminopolycarboxylic Ð2 1.5×10 % H2O2 solution was chosen as described in acids include EDTA-calcium(II), EDTA-zinc(II), the standard procedure. ∆A was considerably influ- EDTA-manganese(II), EDTA-cobalt(II), EDTA-Cu(II), enced by the mixing order. The best mixing order was EDTA-iron(III), trans-1,2-diaminocyclohexane- EDTA, titanium(IV), H2O2, surfactant, buffer solution N,N,N′,N′-tetraacetic acid (CyDTA), diethylenetri- and PCPF. amine-N,N,N′,N″,N″-pentaacetic acid (DTPA), N-(2- The color formation in this reaction system did not hydroxyethyl)ethylene-N,N′,N′-triacetic acid (EDTA- occur instantaneously at room temperature. Thus, the OH), nitrilotriacetic acid (NTA), 1,3-diamino-2- effects of incubation temperature and time were exam- hydroxypropane-N,N,N′,N′-tetraacetic acid (DPTA- ined at 40, 50, 60, 70 and 80ûC. A maximum and con- OH), ethylenediaminedi(o-hydroxyphenylacetic acid) stant ∆A value was obtained on heating at 60ûC for 25 (EDDHA) and glycolethylenediaminetetraacetic acid min, following by cooling to room temperature. The (GEDTA). The sensitivities of EDTA-copper(II) and ∆A value remained constant for at least 5 h after the GEDTA were remarkably low. EDTA-copper(II) was solution had been cooled to room temperature. Figure could be determined in the presence of thiosulfate ion 2 shows the absorption spectra of PCPF-titanium(IV) (5.0×10Ð4 M). EDTA-iron(III) could not be determined and PCPF solutions under the standard procedure. at all. The reason why EDTA-iron(III), even in the presence of a reductant, gives no response is probably ANALYTICAL SCIENCES DECEMBER 1998, VOL. 14 1159 that free iron(II) ion released from EDTA-iron(III) Table 1 Determination of EDTA in foods (µg/g) forms a deeply colored complex with PCPF. In conclu- Founda sion, though the present method is not free from the a a, b Sample RSD , Recovery , interference of coexisting aminopolycarboxylic acids, Proposed Other % % EDTA-metal chelates except EDTA-iron can be deter- method methodc mined as total amounts of EDTA. A 20.2 18.9 1.8 101.9 B 32.8 31.6 2.3 97.5 Effects of foreign substances C 54.3 —d 2.2 100.3 The effects of many foreign substances on the deter- D 132.3 —d 2.0 98.0 mination of EDTA were examined. Inorganic ions such E 32.0 32.8 2.2 101.2 as sodium, potassium, calcium, magnesium, cobalt, A, soft drink; B, salada dressing (emulsion type); C, canned zinc, tin, aluminum, chloride, sulfate, nitrate, iodide, mushroom; D, canned crab meat; E, canned green peas. thiosulfate, thiocyanate, cyanide and bromate ions did a. Average of 5 determinations. not noticeably affect the accuracy of the determination, b. EDTA taken 8.8 µg. even when these ions were present in large excess c. ref. 10. amounts compared with that of EDTA. A small amount d. Undetermined. of iron(III) and copper(II) ions caused decreases in ∆A. Interference from copper(II) ion could be overcome by the addition of thiosulfate ion (5.0×10Ð4 M) as a these viewpoints that the reaction mechanism involves masking agent. The effect of iron(III) ion could be the color reaction (the formation of Ti(PCPF)3) between decreased by a standard addition method. The presence residual titanium(IV) ion and PCPF after production of of most organic compounds, such as ascorbic acid, the EDTA-titanium(IV)-H2O2 (1:1:1) complex. thiamine, caffeine, glycine and glucose, interfered very little. References Application of this method to EDTA in foods The proposed method was applied to determine 1. This work was presented at the 116th Annual Meeting of EDTA in food products. The results are shown in Table the Pharmaceutical Society of Japan, Kanazawa, March 1. The overall recoveries of EDTA from each sample 1996. were within 97 Ð 102%, with RSD values of less than 2. Y. Fujita, I. Mori and M. Toyoda, Anal. Sci., 7, 327, 2.3%. (1991). 3. Y. Fujita, I. Mori, M. Toyoda and T. Matsuo, Anal. Sci., 10, 827 (1994). Composition and reaction mechanism 4. C. Lieberman, Chem. Ber., 34, 2299 (1901). The titanium(IV)-to-PCPF ratio determined by Job’s 5. H. Sano, Bull. Chem. Soc. Jpn., 55, 3649 (1958). method of continuous variation was 1:3 at the 6. T. Hamano, Y. Mitsuhashi, S. Yamamoto, Y. Matsuki, Y. experimental conditions, and no response of H2O2 to Tonogai, K. Nakamura and Y. Ito, Nippon Shokuhin Kogyo titanium(IV) was observed. Thus, the formed complex Gakkaishi, 34, 603 (1987). may be expressed as Ti(PCPF)3. On the other hand, 7. D. Honova, I. Nemcova and V. Suk, Talanta, 35, 803 titanium(IV):H2O2 and EDTA:[H2O2-titanium(IV)] (1988). were 1:1(at 390 nm) and 1:1(at 360 nm) by the molar 8. R. Parkash and R. Bansal, Anal. Lett., 23, 1159 (1990). ratio method, respectively. The experimental result was 9. A. T. Pilipenko and M. F. Tulyupa, Zh. Anal. Khim., 45, 965 (1990). in agreement with the literature value.12 Furthermore, 10. T. Hamano, Y. Mitsuhashi, N. Kojima and N. Aoki, we found that the formation reaction rate of the Analyst[London], 118, 909 (1993). titanium(IV)-PCPF complex was slow in compari- 11. M. Sillanpaa and M.-L. Sihvonen, Talanta, 44, 1487 son with that of the EDTA-titanium(IV)-H2O2 ternary (1997). complex. The order of adding the reagents is impor- 12. S. Musha and K. Ogawa, Nippon Kagaku Zasshi, 78, 1686 tant: H2O2 and titanium(IV) should be added to EDTA (1957). prior to the addition of PCPF. In addition, the apparent 13. Dojindo Laboratories Catalogue 20th Edition, p.430, 13 chelate formation constants (pKML) of EDTA-titani- Dojindo Laboratories, Kumamoto, 1996. um(IV)-H2O2, EDTA-iron(III), EDTA-copper(II), EDTA-zinc(II), EDTA-cobalt(II), EDTA-manganese(II) (Received May 14, 1998) and EDTA-calcium(II) were 20.43, 25.1, 18.80, 16.50, (Accepted July 29, 1998) 16.31, 14.04 and 10.96, respectively. It follows from