ANALYTICAL SCIENCES VOL. 7 SUPPLEMENT 1991 811

CHARACTERIZATION OF OXALIC ACID IN VEGETABLES

YUUKO ISHII and KAZUYOSHI TAKIYAMA

Department of Home Science, Mukogawa Women's University. Ikebiraki-cho, Nishinomiya 663, Japan

Abstract - Vegetables containe oxalic acid. A part of it is fixed as calcium oxalate. Oxalic acid and calcium also exist as free ions. The free and total oxalic acids were determined by the extractions in boiling water and in 0.1 M HCI. The free and combined oxalic acids in several kinds of vegetables were determined. Cacium oxalate crystals isolated from some vegetables were observed.

Key words oxalic acid in vegetable, free oxalic acid, combined oxa~i ac cid, calcium oxalate, calcium oxalate monohydrate,

Vegetables contains more or less oxalic acid, which is produced by metabolism in cytoplasm[1] The most of oxalic acid is fixed as calcium oxalte to protect protoplasm from toxic action of it [2] Vegetables containing relatively a large quantity of oxalic acid were boiled in water before cooking to remove the most of free oxalic acid as in the case of Spinach. The oxalic acid contained in food materials was determined by KMnO4 titration of calcium oxalate or chromatographic methods after extraction in water [3,4]. Calcium oxalate crystals in the vegetable have been studied by mainly optical microscopy. In the present investigation, the free oxalic acid in vegetables was extracted in boiling water and the total oxalic acid was extracted in 0.1 M HC1, followed by the determination with an ion chromatography. Calcium oxalate crystals were isolated from several vegetables and the properties of them were investigated. A part of results has been published [5,6] The determination of calcium oxalate, the morphology of calcium oxalate crystals in several vegetables and the characterization of oxalic acid are discussed in this paper.

EXPERIMENTAL

Instruments A determination of oxalic acid was carried out with an ion chormatograph (IC) DIONEX 2000i/SP. Calcium oxalate crystals were observed with a scanning electron microscope (SEM), MIM 101 produced by Akashi Ltd. X-ray diffraction (XRD) analysis was carded out with an X-ray diffractometer JEX-7E produced by JEOL. Materials and pretreatment Spinach, Chard, Bamboo shoot, Garland chrysanthemum, Cabbage, Chinese cabbage, Saltwort, Soybean sprout, Zuiki, Apple, Strawberry, Goose-foot (grass) , Begonia (grass) , etc. were used for the investigation. They were mainly obtained at the market. The sample vegetables were washed with tap water and distilled water followed by wiping with paper towels. The vegetables were cut into small pieces with a stainless-steel knife and mixed thoroughly Extraction in boiling water Ten g of the sample vegetable were wrapped with gauze and transferred into 400 ml of boiling water in a 500 ml three-neck flask attached to a condenser and a thermometer. After one to 120 min of boiling, about 20 ml of the solution was removed and cooled in a test tube immersed in ice water. The solution was filtered with a membrane filter (pore size of 0.45 ,i m produced by Advantec Toyo) .

Extraction in 0.1 M HC1 Ten g of the sample vegetable were placed in 400 ml of 0.1 M HCI and the solution was stirred continuously. After one to 24 h, about 20 ml of the solution was removed and treated as described above. Determination of oxalate by ion chromatography The sample solution was filteredwith a MOLCUT=L Jfilter for removing protein, produced by MILIPORE FILTER Co.) and injected IC combined with an anoin exchange column. The

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concentration of oxalate (ppm) in the sample solution was determined by measuring the peak height of ion chromatogram using a calibration curve. From the concentration determined, the amount of oxalate (mg) in 400 ml of the solution at the time of taking 20 ml from the solution was obtained and then the amount of oxalate (mg) extracted from 100 g of vegetable sample was calculated. Determination of calcium by AA Concentration of calcium (ppm) in the sample solution was determined by AA [7] The amount of calcium (mg) extracted from 100 g of sample vegetable was calculated. Isolation and analysis of calcium oxalate crystals from vegetable Sample vegetable was cut into small pieces and pulverized Iii water with a food blender. The mixture was poured into a beaker and stood for 30 min followed by filtering through gauze. Calcium oxalate crystals and small fragments of cellulose passed through gauze to the filterate. The residue on the gauze was squirted with water to fall down crystals into the filtrate. Many small particles sank in the filtrate in 30 min. Upper part of the filtrate was drained off and white particles remained in the beaker were separated from cellulose fragments by washing carefully The crystals were carefully gathered into an another beaker. Dried particles were stored in a small sample bottle. The crystals were observed with SEM and analyzed with XRD. Contents of calcium and oxalate in the crystals were analyzed by EDTA and KMnO4 titrations after dissolution of the crystals in hot 3 M sulfuric acid and the content of water of crystallization was calculated.

RESULTS AND DISCUSSION

Extraction of oxalic acid and calcium ion in boiling water The changes of amounts of oxa ate and caesium ions extracted in ng water from 100 g of Spinach are shown in Fig. 1. In the first stage of boiling (20 min) the amounts of ions extracted rapidly increased; in the second stage of boiling (20 to 60 men), the amounts of ions extracted became maximum and maintained

at almost constant values. In the last stage of boiling the amounts of oxalate and calcium ions were slightly increased. Then amount of oxalate and calcium extracted from Spinach in boiling water increased with the immersion time and reached constant values on and after 20 min of immersion. It is therefore concluded that free oxalate and calcium ions in tissue of Spinach were completely extracted in boiling water for 20 min. Free oxalate and calcium ions in leaves of vegetables except Spinach were also estimated to be extracted in boiling water after imersion for 20 min. Extraction of oxalic acid and calcium ion in 0.1 M HC1 The changes of amounts oxa ate and calc am ons extracte in 1 HC1 from 100 g of Spinach are shown in Fig. 1. The amounts of ions in the extract increased with the immersion time and reached constant values after 4 to 5 h. Calcium oxalate crystals in Spinach disappeared after 4 to 5 h of immersion in 0.1 M HCl, based on observation with an optical microscope equipped with a sharp colored polarizing filter X-FRC (produced by Nikon) . It was considered that all of the free oxalic acid and oxalic acid combined with calcium could be extracted in 0.1 M HC1 after 5 h of immersion. Then, the quantity of oxalic acid in HC1 after immersion for one night was considered to be the total oxalic acid in Spinach. Determination of calcium oxalate in vegetables The free oxalic acid in sample vegetabTecan be extracted in boiling water by immersion for 20 min, while the total oxalic acid can be extracted in 0.1 M HC1 by immersion for one night of more than 5 h. The amount of calcium oxalate in the sample leaf (CaOx mg) can be calculated from the difference between the quantity of the

total oxalic acid (Tox mg) and that of the free oxalic acid (Fox mg) in 100 g of the sample, on the supposition that oxalic acid combined only with calcium[8] according to the following equation.

CaOx = (Tox - Fox) x (CaC2 04.2H2 0/CZ 04 ) where CaC2 O4 2H2 O and C204 were formula weights of-calcium oxalate dehydrate and oxalate. CaC2O4.2H2O was used for Spinach, Begonia and Begonia Lucerna and CaC2O4•H20 was used for other vegetables. The present method was applied to determine the contents of free oxalic acid and calcium oxalate in several vegetables as shown in Table 1. On the other hand, the amount of calcium oxalate crystals remained in Spinach after boiling for various hours was determined as follows: the difference between the amount of total oxalic acid and the amount of oxalic acid extracted in boiling water after immersion for various hours corresponded to the amount of combined oxalic acid remained in Spinach. Then the change of amount of calcium oxalate dihydrate remained in Spinach, which corresponded to the combined oxalic acid, after immersion in boiling water for vaious hours was obtained as shown in Fig. 1, The quantity of calcium oxalate decreased, which was more pronounced after 4 h. After 8 hours' boiling, about 26 % of calcium oxalate disappeared. Morphological analysis of calcium oxalate crystals isolated from vegetables As shown in Table i ,-only a few kind of vegetable contained a Iarge quantity

ANALYTICAL SCIENCES VOL. ? SUPPLEMENT 1991 813

of calcium oxalate such as Spinach, Saltwort, Garland chrysanthemum, etc. Calcium oxalate crystals could be isolated from Spinach, Saltwort and Garland chrysanthemum. Calcium oxalate crystals isolated from Spinach are shown in Figs. 2 a, b, c, and d. Flat octahedron and particles, whose shapes were derived from octahedron, were morphologically similar with those of calcium oxalate dehydrate precipitated in aqueous solution [9], The aggregate particle shown in Fig. 2 c was constructed by octahedron or diamond crystals and that shown in Fig. 2 d was constructed by longish hexagonal crystals, which were the same shape with calcium oxalate monohydrate precipitated in aqueous solution. Then crystals shown in Figs. 2 a, b and c were decided as calcium oxalate dihydrate and that shown in Fig. 2 d was decided as calcium oxalate monohydrate. Calcium oxalate crystals isolated from Begonia were only composed of octahedral particles and they were confirmed as calcium oxalate dihydrate by XRD. On the other hand calcium oxalate crystals isolated from Goose-foot were only composed of aggregate particles, whose constituent crystals were longish hexagonal crystals, and they were confirmed as calcium oxalate monohydrate by XRD. Then the estimation of the crystals isolated from Spinach shown above was reasonable. XRD showed that calcium oxalate crystals isolated from Spinach were generally a mixture of monohydrate and dihydrate. Calcium oxalate crystals isolated from Saltwort were composed of aggregate particles, which contained longish hexagonal plate crystals as shown in Fig. 3, and the particles were estimated as calcium oxalate monohydrate. Crystals isolated from Garland chrysanthemum were composed of three kinds of particles as shown in Fig. 4. A elliptical-shaped crystal was seemed to be calcium oxalate monohydrate and a flat octahedral crystal was seemed to be calcium oxalate dihydrate. A grain particle was seemed to be silica particle, which had a similar shape with that isolated from cabbage [10] The relation between the chemical composition of calcium oxalate crystals and the sort of vegetables (generally plants) might be relate with the contents of ions in cells of plants. The free ions in leaves of several plants were determined by the extraction after immersion of them in boiling water for 20 min [11]. It seems that calcium oxalate dihydrate was produced in the presence of relatively a large quantity of oxalic acid and citrate ion. The very low pH was also related to produce calcium oxalate dihydrate. On the other hand calcium oxalate monohydrate was seemed to be produced in the presence of succinate. Single and aggregate particles were found in Spinach, but the most plants contained aggergate particles. The aggregate was not simple collection of single crystals, but component crystals grew to all directions from the center of aggregate particle. Generally single crystal grew from the reagents of relatively low concentration and the dendrite particle grew from the reagents of a little higher concentration [12] The aggregate particle seemed to be grown by the dendrite growth. The total amounts of oxalic acid and calcium in cells of plants were determined and it was found that the single crystals such as octahedron or needle crystals, which were found in Spinach, Begonia or Aloe, seeme to be formed from relatively diluted solution of oxalate and calcium and aggregate particles were formed from a little higher concentrated solution of oxalate and calcium in cells.

Table 1 Contents of oxalic acid and calcium oxalate in plants (mg per 100 g of plants)

REFERENCES

1. H. H. Zenk, Ber. dtsch. Bet. Ges ., 80, 573 (1967)

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2. M. H. Zenk, C. R. Stocking, M. G. Harbour and T. L. Rost, Botany, An Introduction to Plant Biology, John Wiley, New York, 1982.

3. T. Kohara, T. Suzuki and H. Iwano, Shokuhinbunseki Handbook (Handbook of Food Analysis, in Japanese), Kenpakusha, Tokyo, 1984.

4. Japan Shokuhin Kogyokai, Shokuhinbunsekiho (Food Analysis, in Japanese), Korin, Tokyo, 1984. 5. Y. Ishii, Anal. Sci., 7, 263 (1991). 6. Y. Ishii and K. Takiyama, Bunseki Kagaku, 39, 145 (1990). 7. Y. Hyodoh, K. Yokofujita, Y. Ishii and K. Takiyama, Anal. Sci., 1, 151

(1985) . 8. K. Fuwa, Seitai to Jukinzoku (Living Body and Heavy Metals, in Japanese), Kodansha, Tokyo 1981. 9. Y. Ishii, Nippon Kagaku Kaishi, (1991) 63. 10. Y. Ishii and K. Takiyama, unpublished data. 11. Y. Ishii, Chemistry Express, 5, 213 (1990). 12. E. Suito and K. Takiyama, Bull. Chem. Soc. Japan, 27, 121 (1954).

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