Plant and Soil 192: 115–118, 1997. 115c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

A sensitive and convenient assay for boron in plant using chromotropic acidand HPLC

Toru Matoh, Ryo Akaike and Masaru KobayashiPlant Nutrition Laboratory, Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University,Kyoto 606-01, Japan�

Received 3 October 1996. Accepted in revised form 3 May 1997

Key words: boron, boron determination, chromotropic acid, high performance liquid chromatography,plant material

Abstract

A sensitive and convenient assay for boron (B) in plant material is reported. The assay involves extraction of B fromplant material with dry ashing or 0.5 M HCl, formation of a B-chromotropic acid (1,8-dihydroxynaphthalene-3,6-disulfonic acid) complex in an aqueous solution, and determination of the complex on HPLC. Reliability of theassay was evaluated by analyzing a standard reference sample and satisfactory results were obtained. The proposedassay is applicable to dried plant material ccntaining 0.1 to 1 �g B, which is equivalent to a batch of 20 mg driedplant material containing B of 5 to 50 mg kg�1.

Abbreviations: CA - chromotropic acid, TBA - tetrabutylammonium bromide.

Introduction

Boron (B) was found to be an essential micronutri-ent for higher plants at the beginning of this century(for review, see Loomis and Durst, 1992), however,its primary function remains unclear. It is important toquantitate the amount of B in cells and cellular com-partments to elucidate its physiological function.Manyspectrophotometric assays using curcumin (Dible etal., 1954), chromotropic acid (Kuemmel and Mellon,1957) and Azomethin H (Wolf, 1974) have been usedsatisfactory for plant material. Recently B in higherplant cell walls is revealed to bind to a particular pec-tic polysaccharide (Matoh et al., 1993; Kobayashi etal., 1996). To trace B in intracellular compartments, asmaller sample size is more convenient, because lessconsumption of the sample. Therefore, a microscalemethod is mandatory. The B assay using chromotropicacid and HPLC, proposed by Motomizu et al. (1983),is especially suitable for the purpose.

The merits of CA as an assay reagent for B areas follows; water does not interfere with the complexformation, the pH range for the complex formation is

� FAX No: +81 75 7536128. E-mail: matoh@kais.kyoto-u.ac.jp

wide, the complex formed is very stable and the reac-tion time is short (Motomizu et al., 1983). However, itis reported that the peak heights are affected when con-centrations of contaminating salts increase (Motomizuet al., 1983; Zou et al., 1988). When plant materi-al is subjected to dry ashing, supplement of Ca(OH)2

is recommended to prevent loss of B (Gopal, 1969).Also, contaminating salts may reduce the column lifeof HPLC. Therefore, to make this assay multifaceted,clean up of the digest with a cation-exchange resin isintroduced and reported.

Materials and methods

Reagents and apparatus

A standard reference material (tomato leaves 1573a,the certified concentration is 33.3�0.7 mg B kg�1

dried plant material) was obtained from Nation-al Institute of Standards and Technology, Gaithers-burg, MD 20899, USA. Chromotropic acid (CA)was purchased from Dojindo Laboratories (Kumamo-to Techno-Research Park, Kumamoto 861-22, Japan).

ICPC: PIPS No.: 140361 BIO2KAPplso6607.tex; 12/08/1997; 10:56; v.7; p.1

116

Tetrabutylammonium bromide (TBA) was purchasedfrom Wako Junyaku Co. Shimadzu LC-6A HPLCequipped with a UV detector (Shimadzu SPD-6A) wasused throughout this study.

Boron extraction from plant material with dry ashing

A batch of ca. 20 mg of the tomato leaf powder wasweighed into a porcelain crucible and a 100 �L sus-pension of 10 mg Ca(OH)2 in water was added anddried on a hot plate. The plant material in a cruciblewas ashed at 550 �C for 2 h in an oven. A cruciblewithout plant material but Ca(OH)2 was served as ablank. Solutions containing known amounts of B up to1.0 �g were treated similarly. After crucibles cooleddown, the content was suspended in 1 mL of 0.5 MHCl and sonicated 2 s at a minimum power with aprobe-type sonicator (Microson Ultrasonic Cell Dis-rupter, Heat Systems Inc., Farmingdale, N.Y., USA) tohelp complete dispersion. The content was transferredto a polyethylene-made centrifuge tube (1.5 mL) inwhich a ca. 100 mg batch of the air-dried Amberlite IR120 resin (H+ form, 100 - 200 mesh) had been placed.The tubes were vortexed and the resin was allowed tosettle down. A 100 �L aliquot of the supernatant wastaken into another centrifuge tube (1.5 mL). Hundred�L of 0.5 M, NaOH was added to neutralize acidityand filled up to 1 mL with water.

Boron extraction from plant materials with 0.5 M HCl

Boron could be extracted completely from plant mate-rial with 0.5 M HCl (Yoshida and Yoshida, 1965). Abatch of 20 mg of the tomato leaf powder was weighedinto a polyethylene-made centrifuge tube (1.5 mL) and1 mL of 0.5 M HCl was added, vortexed and leftovernight. After centrifugation at 5,000 g for 10 min,a 0.1 mL-aliquot of the supernatant was transferred toanother centrifuge tube and neutralized with 0.1 mL of0.5 M NaOH, then filled up to 1 mL with water.

Determination of B with chromotropic acid and HPLC

To the 1 mL sample solution, 50�L of the TBA reagent(1.61 g TBA was dissolved in 5 mL of 1 M sodiumacetate buffer, pH 4.8) and 50 �L of the CA reagent(150 mg CA and 186 mg of EDTA-2Na was dissolvedin 5 mL of water) were added, vortexed and left morethan 10 min at room temperature. An aliquot of 20�L of the reaction mixture was injected into an HPLCat a flow rate of 1 mL min�1 and the effluent was

Figure 1. Typical chromatogram for a B-chromotropic acid complexon HPLC. The arrow denotes the eluting position of the complex.The Y axis is an arbitrary unit of OD 350 nm.

monitored at an wavelength of 350 nm (Motomizu etal., 1983). The HPLC is fitted with a silica-based C18

reverse-phase column (6�150 mm, AQ-312, YMC,Karasuma-Oike, Kyoto 604). The eluant was preparedas follows: A solution containing 3.5 g TBA, 6.06 gTris and 33 g of 1 M HCl was mixed with 550 mLmethanol and filled up to 1 L with water.

Results and discussion

Figure 1 shows typical chromatogram for the B-CAcomplex. The peak for the complex was well separatedfrom that of CA. A calibration curve for B is presentedin Figure 2. Although the maximum B level in thefinal reaction mixture (1.1 mL) was 1.0 �g under theproposed assay, the linearity was held at least up to10.0 �g B in the final reactant (data not presented).From the calibration curve, a minimum requirementof B in the final reactant of 1.1 mL was ca. 0.1 �g B.That is, if the B concentration of the sample is morethan 5 mg B kg�1 dried plant material, 20 mg of thematerial is enough for the analysis. There is still a roomto improve the lower detection limit, such as increasein the sample amounts subjected to ashing and increase

plso6607.tex; 12/08/1997; 10:56; v.7; p.2

117

Figure 2. A calibration curve for B with the chromotropic acidassay. Values on the abscissa denote the amount of B (�g) in the finalreactant (1.1 mL) and each has three determinations. The equationof the regression line was y = 861000 x + 1154, r2 = 0.9999.

Table 1. Determination of B levels of the standard ref-erence material, tomato leaves, with various samplesizes. The certified concentration of B was 33.3�0.7mg B kg�1 dried plant material

Sample size Determined B (sample number)

(mg) (mg B kg�1 dried plant material)

10 32.1�0.66 (4)

20 32.7�0.30 (32)

50 33.3�0.38 (3)

100 32.0�0.57 (4)

in the amount of the HCl extract applied to the colordevelopment with CA from 0.1 mL to 0.5 mL.

The certified concentration of the B in the standardreference material, tomato leaves 1573a, is 33.3�0.7mg B kg�1 dried plant material. The concentrationsdetermined by the proposed method using dry ashingfor sample decomposition was 32.7�0.30 mg B kg�1,an average value � standard deviation of 32 determi-nations when a batch of 20 mg of the plant sample wassubjected to analysis. Acceptable values were obtainedwhen various amounts of the tomato leaf powder wereused (Table 1). These results indicate that the modi-fied assay is satisfactory for B determination in plantmaterial.

When B was extracted with 0.5 M HCl from thetomato leaves, an average value � standard devia-tion of 10 determinations were 33.4�0.46 mg B kg�1.Direct extraction of B with 0.5 M HCl was also satisfac-tory under the proposed conditions. However, since the

HCl extract may contain interfering organic material,the conditions for the assay using 0.5 M HCl extractionshould be examined further carefully.

The major modification of the original method(Motomizu et al., 1983) was supplement of Ca(OH)2

for dry ashing of plant material (Gopal, 1969) andclean up the digest with a cation-exchange resin. Theresin is useful to remove not only Ca ions but alsocontaminating cations originate from plant materials.As the formation of the B-CA complex was not affect-ed by NaCl up to 0.5 M (Motomizu et al., 1983) theresin-treated acidic solution could be neutralized withNaOH. When the cation-exchange resin was omittedfrom the proposed procedure, the B content was alwaysunderestimated. This was due to the higher pH of thereaction mixture than 4.8, may be due to the consump-tion of HCl by the supplemented Ca(OH)2 to dry ash-ing.

The inductively coupled plasma emission spec-troscopy (ICP-ES) has been introduced for B deter-mination of plant materials, however, the instrumentis not always available due to its high budget require-ment. Therefore, we consider the proposed method ismore suitable for many laboratories. Using the pro-posed method with an auto injector (Shimadze SIL-10A), 5.2 samples can be analyzed every hour. Recent-ly, use of an anion-exchange column (Zou et al., 1988)and of fluorimetric detection of the B-CA (Motomizuet al., 1991) has been suggested to increase further thesensitivity.

Acknowledgements

Part of this work was supported by a Grant-in-Aid(no 08660073) from the Ministry of Education, Sci-ence and Culture to T M. The authors are grateful toProfessor J Sekiya, Plant Nutrition Laboratory, KyotoUniversity, for his encouragement and useful discus-sion.

References

Dible W T, Truog E and Berger K C 1954 Boron determination insoils and plants simplified curcumin procedure. Anal. Chem. 26,418–421.

Gopal N H 1969 Need for Ca(OH)2 in boron determination in plantvegetative tissue and oilseeds. J. Agric. Food Chem. 17, 1146–1147.

plso6607.tex; 12/08/1997; 10:56; v.7; p.3

118

Kobayashi M, Matoh T and Azuma J 1996 Two chains of rhamno-galacturonan II are cross-linked by borate-diol ester bonds inhigher plant cell walls. Plant Physiol. 110, 1017–1020.

Kuemmel D F and Mellon M G 1957 Ultraviolet absorptiometricdetermination of boron in aqueous medium using chromotropicacid. Anal. Chem. 29, 378–382.

Loomis W D and Durst R W 1992 Chemistry and biology of boron.BioFactors 3, 229–239.

Matoh T, Ishigaki K, Ohno K and Azuma J 1993 Isolation andcharacterization of a boron-polysaccharide complex from radishroots. Plant Cell Physiol. 34, 639–642.

Motomizu S, Sawatani I, Oshima M and Toei K 1983 Deter-mination of boron by ion-pair liquid chromatography with1,8-dihydroxynaphthalene-3,6-disulfonic acid. Anal. Chem. 55,1629–1631.

Motomizu S, Oshima M and Jun Z 1991 Fluorimetric determinationof boron with chromotropic acid by flow-injection analysis. Anal.Chim. Acta 251, 269–274.

Wolf B 1974 Improvement of the Azomethine-H method for thedetermination of boron. Commun. Soil Sci. Plant Anal. 5, 39–44.

Yoshida Y and Yoshida S 1965 A rapid method for the determinationof boron in plant material using extraction with 0.5 M HCl (inJapanese). Jpn. J. Soil Sci. Plant Nutr. 36, 45–48

Zou J, Oshima M and Motomizu S 1988 Determination of boron withchromotropic acid by high-performance liquid chromatography.Analyst 113, 1631–1634

Section editor: A C Borstlap

plso6607.tex; 12/08/1997; 10:56; v.7; p.4