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Boron determination
in plant tissue by the
azomethine H method
a a
T. Powell Gaines & G. Allen Mitchell
a
Department of Agronomy, University of
Georgia, Coastal Plain Experiment Station,
Tifton, GA, 31794
Published online: 11 Nov 2008.

To cite this article: T. Powell Gaines & G. Allen Mitchell (1979): Boron
determination in plant tissue by the azomethine H method, Communications in
Soil Science and Plant Analysis, 10:8, 1099-1108

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 COMMUN. IN SOIL SCIENCE AND PLANT ANALYSIS, 10(8), 1099-1108 (1979)

BORON DETERMINATION IN PLANT TISSUE BY THE AZOMETHINE H METHOD

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KEY WORDS : plant or leaf analysis, dry ashing

T. Powell Gaines and G. Allen Mitchell

Department of Agronomy
University of Georgia
Coastal Plain Experiment Station
Tifton, GA 31794

ABSTRACT

A c o l o r i m e t r i c method u s i n g the azomethine H r e a g e n t is

d e s c r i b e d for determining boron i n an a c i d e x t r a c t of p l a n t

t i s s u e ash. The method c o n s i s t e n t l y gave the c e r t i f i e d value of

33±3 ppm B for NBS orchard l e a f r e f e r e n c e m a t e r i a l No. 1571 with

3.2% c o e f f i c i e n t of v a r i a t i o n . Samples were dry ashed and the

ash e x t r a c t e d i n 10 ml 0.36 N H2SO4 f o r 1 h r . at room t e m p e r a t u r e .

Several a c i d i c e x t r a c t a n t s and e x t r a c t i n g c o n d i t i o n s were compared

which gave the c e r t i f i e d v a l u e , but involved h e a t i n g and readjust-

ing the f i n a l volume.

INTRODUCTION

Recent p u b l i c a t i o n s r e p o r t i n g the use of azomethine H as a

c o l o r reagent for determining boron i n d i c a t e t h a t t h i s reagent is

becoming widely accepted i n boron a n a l y s i s , and w i l l l i k e l y replace

1099

Copyright © 1979 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part
may be reproduced or transmitted in any form or by any means, electronic or mechanical, including
photocopying, microfilming, and recording, or by any information storage and retrieval system,
without permission in writing from the publisher.
 1100 GAINES AND MITCHELL

1 o
the older boron methods using quinalizarin , carmine and cur-

cumin^ which have been used extensively in the past. The

quinalizarin and carmine methods have the disadvantage of re-

quiring the use of concentrated sulfuric acid for color develop-

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ment. The curcumin procedure has several advantages over the

former two methods, but is susceptible to several interferences,

especially nitrates, if specific precautions are not observed.

Basson et. al. reported using azomethine H in an automated

procedure for determining boron in plant materials. A review by

Wolf of colorimetric methods for determining boron showed a

preference for using azomethine H based on the accuracy and

relative simplicity of the method. A subsequent report by Wolf"

offered improvements in his original azomethine H method.

Mazaheri? described a method for preparing azomethine H , although

the reagent is commercially available". Sippola and Ervio' re-

ported a preference for using azomethine H over the more trouble-

some carmine method, and Thorpe*" in a recent collaborative study

reported that the azomethine H method is accurate and precise for

all levels of boron in fertilizer. Technicon Industrial Systems

has a tentative automated method using azomethine H for deter-

mining boron in soils .

While many methods have used dry ashing as a means of sample

preparation for determining boron in plants, ash extracting

conditions have been inconsistent. In the quinalizarin method,

Berger and Truog 1 extracted plant ash in 0.36 N H2SO4. This

method was later adopted by the AOAC as the official boron

 BORON DETERMINATION BY AZOMETHINE H METHOD 1101

method 12 . Hatcher and Wilcox 2 extracted plant ash in 6 N HC1 for

30 minutes on a steam bath. Others have extracted plant ash with

0.1 N H C l 3 ' 9 ' 1 3 and 0.7 N H 2 S O 4 without heat.

The purpose of this study was to investigate dry ashing pro-

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cedures of sample preparation for determining boron in plant

tissue, and to develop a method of sample preparation that would

consistently give the certified value of 33 + 3 ppm boron for NBS

orchard leaves reference material No. 1571 using the azomethine

H colorimetric method.

MATERIALS AND METHODS

Apparatus

1. Muffle furnace.
2. Hot plate.
3. Colorimeter.

Reagents

1. Buffer-masking reagent: Dissolve 280 g ammonium

acetate, 20 g potassium acetate, 20 g tetrasodium
salt of EDTA and 8 g nitrilotriacetic acid in 400
ml water. Slowly add 125 ml acetic acid while
stirring. Heat if necessary to dissolve contents
and filter through Whatman #4 filter paper to remove
any undissolved residue.

2. Azomethine H reagent: Dissolve 0.9 g azomethine H

(Pierce Chemical Co., Rockford, IL 61105), and 2 g
ascorbic acid in water with gentle heating and
dilute to 100 ml volume. Reheat if solution is
turbid. Reagent will keep for 14 days if stored in
brown bottle under refrigeration.

3. Working solution: Add 20 ml azomethine H reagent to

80 ml buffer-masking reagent for approximately 12
samples including standards. Prepare fresh daily.

4. Boron stock solution: 100 ppm B. Dissolve 0.5716 g

boric acid in water and dilute to 1 L.
 1102 GAINES AND MITCHELL

5. Boron standards: Boron standards are made as follows

in 100 ml volumetric flask.

Boron
Cone. Stock 0.36 N H 2 S O 4 Final
ug B/ml Solution Volume
ml
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1.0 1.0 99 100

2.0 2.0 98 100
3.0 3.0 97 100
4.0 4.0 96 100
5.0 5.0 95 100

6. Dilute sulfuric acid (0.36 N ) : Dilute 10 ml

centrated H2SU4 to 1 L with water.

Sample Preparation

Weigh 1.0 g sample into 30 ml high-form porcelain crucible.

Muffle for 1 hr. at 600°C and cool. Wet the ash with 5 drops

water and then pipet 10 ml 0.36 N H2SO4 into the crucible. Let

stand at room temperature for 1 hr., stirring occasionally with

glass rod to break up ash. Filter through Whatman #1 filter paper.

Filtrate is ready for B determination.

Color Development

Pipet 4 ml filtrate into test tube. Add 4 ml buffer-masking

reagent and 1 ml azomethine H reagent or 5 ml working solution.

Mix thoroughly and allow color to develop for 1 hr. Measure

absorbance at 420 nm using a colorimeter. Determine concentra-

tion of sample from standard curve constructed by plotting

absorbance vs concentration of standards in ppm (Fig. 1 ) .

Calculations

10 ml
B ppm Sample = ug B/ml x Final Vol.
1 g Sample
 BORON DETERMINATION BY AZOMETHINE H METHOD 1103

1.0 r

0.8

0.6
O
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1
m
0.2

0
0 1.0 2.0 3.0 4.0 5.0
B CONCENTRATION (jugB/ml)

FIG. 1
Effect of time on color development of the boron-azomethine H
complex.

RESULTS AND DISCUSSION

Color reaches about maximum absorbance after 1 hr. and re-

mains fairly stable for 2 hrs. which supports the findings of

Sippola and Ervio . Good separation between absorbance and con-

centrations was observed even after 15 min. and the response was

linear. Although color continues to develop after 15 min., it

should be noted that if both standards and samples could be

measured exactly after 15 min., as could be done with an auto-

mated procedure using a time delay coil, a wide separation

between absorbance and concentration could be expected.

As previously stated, the purpose of this study was to

investigate various dry ashing procedures of sample preparation,

 1104 GAINES AND MITCHELL

including ash extractants, ashing and extracting conditions, so

that a B method could be developed that would consistently give

the certified value of 33 + 3 ppm B for orchard leaf reference

material No. Í571 using the azomethine H colorimetric method.

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The certified value was determined not by colorimetry, but by

isotope dilution mass spectrometry, nuclear track technique, and

optical emission spectroscopy .

Since some previous methods had extracted plant ash at room

temperature (22°C) with 10 ml 0.1 N H C 1 3 ' 9 ' 1 3 , this extracting

procedure was tried first (Table 1). The data show that 10 ml

0.1 N HCl did not extract sufficient B to fall within the certi-

fied range, whether the ash was extracted at room temperature for

TABLE 1

Effect of Ash Extracting Treatments Using Dll. HCl on B

Concentration of Orchard Leaf Standard*

Sample Treatment B
ppm
Orchard Leaf Std. 30 min. extraction in 10 ml 21
0.1 N HCl at 22°C
Orchard Leaf Std. Overnight extraction in 10 ml 22
0.1 N HCl at 22°C
Orchard Leaf Std. 1 hr. extraction in 10 ml 27
0.1 N HCl at 70°C
Orchard Leaf Std. 1 hr. extraction in 10 ml 32
0.5 N HCl at 70°C
Orchard Leaf Std. 1 hr. extraction in 10 ml 31
1.0 N HCl at 70°C
Orchard Leaf Std. Certified Value 33+3

*Average of 5 replicated analyses.

 BORON DETERMINATION BY AZOMETHINE H METHOD 1105

30 min. or overnight, or for 1 hr. at 70°C. On the assumption

that 0.1 N HCl was too weak an extractant, increased concentrations

of the acid were tried. The data show that orchard leaf ash

extracted in 10 ml 0.5 N HCl for 1 hr. at 70°C gave a value of

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32 ppm. Ash extracted in 10 ml 1.0 N HCl for 1 hr. at 70°C also

fell within the desired range, but was consistently lower than ash

extracted in 0.5 N HCl for 5 replicated analyses.

It should be noted that throughout this study when samples

were extracted with various acids, standards were made accordingly

to keep this parameter consistent with the samples. Also, when

samples were extracted for 1 hr. at 70°C, some evaporation

occurred. Consequently, the final volume had to be readjusted

to 10 ml with the experimental acid.

The data in Table 1 suggest that if dilute HCl is to be used

as the ash extractant, extracting the ash in 10 ml 0.5 N HCl for

1 hr. at 70°C is adequate. The final volume has to be readjusted

to 10 ml however, due to evaporation, and the standards should

also be made with 0.5 N HCl.

Elemental B is practically insoluble in water and does not

react with acids unless they are strong oxidizing agents like

nitric or sulfuric acid . While B in plants is probably in the

form of borates, a study was conducted to show the effect of

extracting orchard leaf ash with these two oxidative acids at 1%

concentration (10 ml acid per L ) . Orchard leaf ash extracted in

10 ml 0.36 N H2SO4 gave B values that fell within the certified

range, whether heated or not (Table 2 ) . Orchard leaf ash ex-

 1106 GAINES AND MITCHELL

TABLE 2

Effect of Various Acids as Ash Extractants on B Concentration of

Orchard Leaf Standard*

Sample Treatment B
ppm
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Orchard Leaf Std. 1 hr. extraction in 10 ml 31

0.36 N H2SO4 at 22°C
Orchard Leaf Std. 1 hr. extraction in 10 ml 32
0.36 N H2SO4 at 70°C
Orchard Leaf Std. 1 hr. extraction in 10 ml 29
0.16 N HN0 3 at 22°C
Orchard Leaf Std. 1 hr. extraction in 10 ml 31
0.16 N HNO3 at 70°C
Orchard Leaf Std. Certified Value 33+3

*Average of 5 replicated analyses.

tracted in 10 ml 0.16 N HNO3 also gave B values that fell within

the certified range when heated for 1 hr. at 70°C. Perhaps HNO3

slightly stronger than 0.16 N would enable B to be quantitatively

extracted from plant ash without heating.

Ash extractant procedures that gave B values within the certi-

fied range were subjected to a precision test to determine repeat-

ability in terms of °L coefficient of variation (CV) . All ash

extracting procedures described had a very low % CV, indicating

good precision can be expected from each of these extracting pro-

cedures (Table 3 ) .

Ashing the sample for 1 hr. at 600°C was found to be as

effective as ashing at 600°C for 2 or 4 hrs. Ashing with Ca(0H) 2

 BORON DETERMINATION BY AZOMETHINE H METHOD 1107

Table 3

Precision of Methods Using Various Ash Extractants on Orchard

Leaf Standard*

Method ppm CV.%

B
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1 hr. extraction in 10 ml 0.36 N H2SO4 at 22°C 31 + 1.0 3.2

1 hr. extraction in 10 ml 0.36 N H2SO4 at 70°C 32 + 0.7 2.2
1 hr. extraction in 10 ml 0.16 N HNO3 at 70°C 31 + 1.0 3.2
1 hr. extraction in 10 ml 0.5 N HCl at 70°C 32 + 0.7 2.2

*Based on 5 consecutive analyses.

is not necessary for most plant tissue, but probably should be

added in the analysis of seeds or other oily materials low in

bases to prevent B loss by volatilization 13.

The buffer used in the color development and reported by

Wolf adequately held all test solutions at a pH of 4.9. Boron

.does not exceed 50 ppm concentration in many plants, but for

samples > 50 ppm, the final volume should be diluted to 20 ml and

ug B/tnl readings from the standard curve are multiplied x 20.

Since extracting ash at room temperature involves less work,

results in this study suggest that extracting plant ash in 10 ml

0,36 N ^ S O ^ for 1 hr. at room temperature is adequate for the

quantitative determination of boron in plant tissue. If HCl or

HNO3 is to be used as the plant ash extractant, heating for 1 hr.

at 70°C with 10 ml of 0.5 N HCl or 0.16 N HNO3 will work just as

effectively, but final volume has to be readjusted to 10 ml.

 1108 GAINES AND MITCHELL

REFERENCES

1. Berger, K. C. and E. Truog. 1939. Ind. Eng. Chem., Anal.

Ed. 11:540.

2. Hatcher, J. T. and L. V. Wilcox. 1950. Anal. Chem. 22:567.

3. Dible, W. T., Truog, E. and K. C. Berger. 1954. Anal.

Downloaded by [University of Illinois at Urbana-Champaign] at 04:19 04 May 2013

Chem. 26:418.

4. Basson, W. D., Bohmer, R. G. and D. A. Stanton. 1969.

Analyst, Lond., 94:1135.

5. Wolf, B. 1971. Comm. Soil Sci. Plant Anal. 2:363.

6. Wolf, B. 1974. Comm. Soil Sci. Plant Anal. 5:39.

7. Mazaheri, A. 1976. Comm. Soil Sci. Plant Anal. 7:331.

8. Pierce Cehmical Co., Rockford, IL 61105.

9. Sippola, J. and R. Ervio. 1977. Finn. Chem. Lett., 138.

10. Thorpe, V. A. 1978. J. Assoc. Off. Anal. Chem. 61:894.

11. Technicon Industrial Method No. 517-77 A, 1977.

12. Official Methods of Analysis. 1970. 11th Ed. AOAC, Washing-

ton, D.C., sec. 3.080.

13. Greweling, T. 1976. Cornell Univ. Ag. Expt. Stn. Search 6

(8), 35 pp.

14. National Bureau of Standards, Certificate of Analysis,

Standard Reference Material 1571, Orchard Leaves, 1976.

15. Textbook of Chemistry. 1956. 2nd Ed., Ginn and Co., New
York p. 734.