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Soil Science and Plant Nutrition

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Rapid, Micro-Methods to Estimate Plant Silicon

Content by Dilute Hydrofluoric Acid Extraction and
Spectrometric Molybdenum Method
a a b c
Kimio Saito , Akiko Yamamoto , Tongmin Sa & Masahiko Saigusa
a
Miyagi Prefecture Furukawa Agricultural Experimental Station , Furukawa, 989–6227,
Japan
b
Department of Agricultural Chemistry , Chungbok National University , Cheongju,
361–763, Korea
c
Experimental Farm, Graduate School of Agricultural Science , Tohoku University ,
Naruko, Miyagi, 989–6711, Japan
Published online: 17 Dec 2010.

To cite this article: Kimio Saito , Akiko Yamamoto , Tongmin Sa & Masahiko Saigusa (2005) Rapid, Micro-Methods to
Estimate Plant Silicon Content by Dilute Hydrofluoric Acid Extraction and Spectrometric Molybdenum Method, Soil Science
and Plant Nutrition, 51:1, 29-36, DOI: 10.1111/j.1747-0765.2005.tb00003.x

To link to this article: http://dx.doi.org/10.1111/j.1747-0765.2005.tb00003.x

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 Soil Sci. Plant Nuti:, 5 1 (l), 29-36, 2005 29

Rapid, Micro-Methods to Estimate Plant Silicon Content by Dilute

Hydrofluoric Acid Extraction and Spectrometric
Molybdenum Method
I. Silicon in Rice Plants and Molybdenum Yellow Method

Kimio Saito, Akiko Yamamoto, Tongmin Sa*, and Masahiko Saigusa**

Miyagi Prefecture Furukawa Agricultural Experimental Station, Furukawa, 989-6227 Japan; *Department of
Agricultural Chemistry, Chungbok National University, Cheongju, 361- 763 Korea; and
**Esperimental Farm, Graduate School of Agricultural Science,
Tohoku University, Naruko, Miyagi, 989-671 1 Japan

Received February 25,2004; accepted in revised form September 27, 2004

Downloaded by [University of New England] at 14:02 04 January 2015

By treating 0.6 g DW of a plant sample directly with 10 ml of a dilute hydrofluoric acid solu-
tion (HF solution, 1.6 M €IF-0.6 M HCl), all the silica in plant (as much 88 160 mg SiO,) was
dissolved within 1 h. After dilution of the extract with 40 mL of distilled water, the silica in
the extract was measured by the spectrometric molybdenum yellow method. The molybde-
num yellow method, in which silica in 0.1 mL of the diluted extract can be determined in 8
min, is well suited to rapid, micro-estimations of the silica content in plants. In the micro-
modification, the size of the plant sample was reduced to 100 mg DW. The analytical proce-
dure was simple, and the analytical time was less than 2 h. The method can save much labor
and time, compared with the gravimetric analysis. The dissolution with HF solution and the
molybdenum yellow method were also applied to the measurement of the content of silica
separated by acid digestion of rice plants. Excellent agreement in the silica measurement of
rice plants was confirmed among the direct extraction method, the gravimetric method and
the digestion-separation-dissolution method. In the molybdenum yellow method, the addi-
tion of boric acid enabled to mask the interference of hydrofluoric acid, and the least
amount of citric acid required for the elimination of phosphorus interference was proposed.
In conclusion in this report, recommended methods for the rapid estimation of the silica
content in rice plants were presented.
Keg Words: hydrofluoric acid, molybdenum, rice plant, silicon, spectrometric method.

Beneficial effects of silicon on rice plants such as
increase of the resistance to rice blast disease, of water
efficiency and of photosynthetic rate have been reported
(Okuda and Takahasi 1962; Agarie et al. 1998). The role
of silicon in the physiology of rice plants as well as
gramineous plants, however, needs to be elucidated with
the accumulation of data on silicon nutrition of plants.
The current gravimetric analysis for the measurement of
the silicon content of plants requires more than 1 g dry
matter of plant sample and more than 17 h for digestion,
drying, filtration, dehydration, and weighing. The meth-
od by acid digestion and dissolution with boiling
Na$O, suspension, followed by colorimetric determina-
tions (Nayar et al. 1975), can be applied for 100mg of
plant sample, but requires 24 h until the determination is
completed. In the autoclave-induced digestion method
(Elliot and Snyder 1991), in which plant tissues are
digested with H,O, and NaOH and the amount of dis-
solved silicon is measured using colorimetric methods,
the analysis is more rapid. However, the number of sam-
ples for the digestion may be limited by the autoclave
capacity.
Hydrofluoric acid, volatilizing silica as SiF, at high
concentrations, is well known for silica corrosiveness.
The silicon in plants is present in the form of hydrous
amorphous silica, so-called phytolith, which is deposit-
ed in plant tissues, and is expected to be dissolved rapid-
ly with a diluted hydrofluoric acid solution even under
ambient conditions.
In the present study, the spectrometric molybdenum
yellow method was selected for the determination of the
silicon content because of its rapidity, simplicity, and
 30 K. SAITO et al.
appropriate sensitivity. The method was improved to
eliminate the interference of hydrofluoric acid and P-
molybdenum yellow. Quenching of the hydrofluoric
acid activity to dissolve silica and the stability of molyb-
denum yellow were also examined to increase the preci-
sion in the analysis. Then, the extraction method with
diluted hydrofluoric acid solutions was applied to devel-
op rapid and simple methods for the estimation of the
silica content in rice plants. To determine the extraction
conditions, the effect of the concentration of hydrofluor-
ic acid on the dissolution of silica in plant was studied.
Subsequently, soils, which may contaminate plant sam-
ples, were also examined for dissolution in the diluted
hydrofluoric acid solution.
Downloaded by [University of New England] at 14:02 04 January 2015
MATERIALS AND METHODS
Preparation of diluted hydrofluoric acid
solution. In dealing with a solution containing hydro-
fluoric acid, the use of plastic ware is compulsory. The
extraction solution consisting of 1.5 M HF-0.6 M HCl
solution (HF solution) was prepared under a ventilated
hood by combining 1 v of concentrated hydrochloric
acid (36%), 1 v of concentrated hydrofluoric acid
(49%), and 18 v of distilled water. For the experiment
on the effect of the concentration of hydrofluoric acid on
silica dissolution, solutions with 2-3 M HF were pre-
pared. Although these solutions, thus diluted, are not
volatile, contact with the skin should be avoided. The
waste solution containing hydrofluoric acid can be treat-
ed with CaCO, to remove fluoride as CaF precipitates.
Preparation of silica standards in HF solu-
tion. Amorphous silica, prepared by wet ashing from
5 g of Na,Si03.9H,0, was pulverized, and completely
dehydrated by heating at 950'C for 2 h. The standards in
the 0.3 M HF-0.12 M HCl solution (3,000, 1,500. 750,
300, and 0 mg L-' SiO,) were prepared in plastic bottles
by dissolving the amorphous silica (300, 150, 75, 30,
and 0 mg) with 20 mL of the HF solution for about 3 h
and finally diluting with 80mL of distilled water to
100 mL.
Preparation of reagents for spectrometric
determination of silicon. The molybdenum yellow
method was derived from the molybdenum blue method
(Weaver et al. 1968) with some modifications, like the
addition of boric acid to mask the fluoride interference.
1) Silicon standard solution with 50 mg L-' Si was
prepared by the dilution of 1,000 mg L-' Si standard,
with the addition of 2.5 mmol L-' HC1 to adjust the pH
of the solution in the range between 2 and 4. 2) 0.5 M B
solution and 0.1 M B solution were prepared using
H,BO,, and stored in plastic bottles. 3) 0.5 M Mo (stock
Mo) solution was prepared from N%Mo04.2H20 and,
stored in a polypropylene bottle. 4)0.8 M H,S04-0.5 M
B (stock H2S0,) solution was prepared by dissolving
H,BO, and adding concentrated H,S04. 5 ) 0.25 M Mo-
0.4 M H,S04-0.25 M B (working Mo) solution was pre-
pared, before use, by combining 1 v of the stock H,SO,
and 1 v of the stock Mo. The working Mo solution had a
H,SO, / Mo molar ratio of 1.6 and was stable for about
1 month at 5°C. 6) 0.25 M Mo-0.4 M H,S04 was pre-
pared from the stock Mo and 0.8 M H,S04 solution. 7)
0.5 M citric acid (stock citric) solution was prepared
with 250 mg L-'of benzoic acid as antiseptic. 8) 0.1 M
citric acid (working citric) solution was prepared from
0.5 M citric acid. 9) 1 M tartaric acid was prepared. For
the preparation of all the reagents described above, dis-
tilled water was used.
Spectrometric measurement of silicon by
molybdenum yellow for the solutions contain-
ing hydrofluoric acid. Micropipettes with plastic
tips and plastic tubes 20 mL in volume, with or without
a 1O-mL volume mark, were used. An aliquot containing
3-30 pg of SiO, was transferred to the plastic tubes.
Two mL of the 0.1 M B solution and 2.0 mL of the Mo
working solution were added, and the content was
mixed. The solution was allowed to stand for 1-3 rnin
for the formation of molybdenum yellow to be complet-
ed. Then, 4.0 mL of the working 0.1 M citric solution
was added. After making the volume of the content to
10 mL with distilled water, the absorbance at 400 nm
was measured with a spectrophotometerequipped with a
sipper inlet unit from 5 min to 10 min after citric acid
addition.
B/Mo ratio to suppress HF interference with
molybdenum yellow. The reactions between Mo and
Si at three levels of B/Mo molar ratio were obtained as
follows: 2 mL of a 80 mg L-' SiO, solution containing
0 4 . 3 M HF was prepared in plastic tubes, and 1 mL of a
0.5 M B solution or 1 mL of distilled water was com-
bined. Then, 2 mL of the working Mo solution, with or
without 0.25 M B, was mixed to start the reaction of
molybdenum yellow formation. The absorbance at 400
nm was measured with a spectrophotometer 2 min after
the start.
Suppression of HF activity to dissolve silica
by inhibitory chemicals. The addition of H,BO,,
AlCl,, H,Mo04 (substituted by a 0.25 M Mo-0.4 M
H,SO, solution) or NaOH was tested to suppress the HF
activity. In a plastic tube, 1 mL of the HF solution was
combined with the solution containing inhibitory chemi-
cals, and was diluted to 10 mL with distilled water. The
composite solution (0.2mL) at various molar ratios of
chemicals to HF was put into a 20 mL borosilicate glass
tube (Pyrex), and was allowed to dissolve silica for 10
min at 30°C with shaking at 2 min intervals. The amount
of silica dissolved from the glass tubes was measured by
 Rapid, Micro-Methods to Estimate Plant Silicon
the molybdenum yellow method.
Inhibition of P-molybdenum by citric acid or
tartaric acid. In a plastic tube, 0.5 mL of a 100 mg
L-’ P solution, 0.2 mL of a 0.3 M HF-0.12 M HCl solu-
tion, and 2 mL of a 0.5 M B solution were combined.
The content was reacted with 2 mL of the working Mo
solution to produce a P-molybdenum complex. Various
amounts of a 0.5 M citric acid solution or a 1 M tartaric
acid solution were mixed. After making the volume of
the solution to 10mL with distilled water, the absor-
bance of the solution at 400 nm was measured.
Changes in the absorbance of molybdenum
yellow with time. Molybdenum yellow was formed
by combining 0.2 mL of a 1,500 mg L-’ SiO, standard
solution (in 0.3 M HF-0.12 M HCl), with 2 mL of the
0.1 M B solution and 2 mL of the Mo working solution.
Downloaded by [University of New England] at 14:02 04 January 2015
After the addition of 4 mL of 0.1 M citric acid or 4 mL
of distilled water, the changes in the absorbance at 400
nm were monitored at 25°C.
Plant materials for HF extraction. Plant sam-
ples examined consisted of leaves, stems, and husks of
rice plants (Oryza sutivu L.), collected at the vegetative
growth stage, full heading stage and maturity stage.
Culms of wheat (Triticum aestivum L.) or barley (Hor-
deum vulgare L.) were collected at the maturity stage.
The rice plants were grown in paddy fields located at the
Furukawa Agricultural Experimental Station (FAES)
and at the Kawatabi Field Research Center of Tohoku
University. Wheat and barley were grown in the rota-
tional field of the FAES. The plant samples were dried
at 80°C for more than 24 h, and were milled using
Wiley’s grinder. Some samples of rice husks were not
milled.
Soil materials for the dissolution in the HF
solution. The soil samples, which were examined for
the dissolution with the HF solution, were obtained from
paddy fields located at the Kawatabi Field Research
Center of Tohoku University (KT soil, Melanaquands),
and from paddy fields located at the FAES (FK soil, Flu-
vaquents), both in Miyagi Prefecture, Japan. The air-dry
soils, < 2 mm, after removal of organic matter, were
subjected to the DCB treatment (Mehra et al. 1970). The
fractions of clay ( < 2 pm), silt (2-20 km), and fine sand
(0.02-0.2 mm) were separated by a sedimentation meth-
od using Stoke’s Law.
Effect of the amount of hydrofluoric acid on
the dissolution of plant silica. To avoid leak
hazards during the extraction of silica, occasional stir-
ring was preferable to mechanical stirring. Samples
(0.5 g) of rice husks or rice stems were subjected to this
experiment. Silica in plant samples was dissolved by
immersion for 1 h in 10mL of hydrofluoric acid solu-
tions with 0.3-3 M HF. The content of dissolved silica
was measured by the molybdenum yellow method.
31
Dissolution of plant silica at different HF
concentrations. Plant samples consisted of 0.2 g
DW of rice husk (RH) and 0.5g DW of wheat culm
(WC). The solutions with 0.15-1.5 M HF concentrations
were prepared by combining 10 mL of the HF solution
with 0-90 mL of distilled water. The plant samples were
immersed in these HF solutions for various periods of
time at a constant temperature with stirring every 10
min. The content of dissolved silica was measured by
the molybdenum yellow method.
Analysis of silica by gravimetric method
after dry ashing or wet ashing. In the wet ashing
procedure, 1-0.5 g of plant sample was digested with
H,SO,-H,O, (Mizuno and Minami 1980), with final
heating for 1 h under the fume of sulfuric acid to pro-
mote the precipitation of silica. Dry ashing of 2 g of
plant sample followed the procedure of Takahashi
(1 957). In either method, the residual silica was separat-
ed by filtration. The silica on a filter paper was trans-
ferred to a porcelain crucible, heated at 105°C for 2 h to
remove water, heated at 300°C for 1 h to bum the filter
paper, and finally heated at 550°C for 2 h. After silica
was cooled in a desiccator, the weight of “crude silica”
(containing possible impurities) was determined.
Dissolution of silica from soil in the HF solu-
tion. Samples included the clay fraction (Clay), silt
fraction (Silt), fine sand fraction (Fine sand) of Kawa-
tabi Melanaquands (KT) and Furukawa Fluvaquents
(FK) soils. The soil sample, 50- 100 mg DW, was react-
ed with 10 mL of the HF solution with occasional stir-
ring at 30°C. The amount of dissolved silica was
determined by the molybdenum yellow method.
Analysis of silica by digestion-separation-
dissolution method. The silica on filter paper, pre-
pared from the digestion of 0.5 g of plant sample, was
placed in a polyethylene bag, and was dissolved with
10 mL of the HF solution for more than 3 h. The content
was made to the weight of 50.5 g (50-mL volume) with
distilled water on a balance using the tare weight of the
dry filter paper and the bag. The silica content in the
extract was determined by the molybdenum yellow
method.
RESULTS AND DISCUSSION
Effect of boric acid on masking of HF interfer-
ence with Si-molybdenum yellow
Hydrofluoric acid, which displays a high affinity to
molybdenum, interferes with the silicon molybdenum
reaction. With the increase in the interference, first, the
rate of reaction decreases, then the formation of Si-
molybdenum yellow is inhibited. The interference of HF
was inferred from the decrease in absorbance which was
 32
60
I'
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
Molar ratio of HF/Mo
Fig. 1. Boric acid addition to suppress HF interference in the
molybdenum yellow method. Under the following reaction con-
ditions: 32 mg L-' S O z , 0.1 M Mo concentration, HF/Mo=O-
1.3, and B/Mo=O, 1, 2, the absorbance at 400nm was mea-
Downloaded by [University of New England] at 14:02 04 January 2015
sured 2 min after the onset of the molybdenum yellow reaction.
%absorbance denotes the absorbance relative to that without HF
(=loo%).
measured at 2 min after the onset of the reaction. Such
changes were shown in Fig. 1, in which the interference
was expressed by the %absorbance relative to the absor-
bance without interference. The effects of boric acid
addition on the reduction of the hydrofluoric acid inter-
ference were indicated by the changes in the %absor-
bance at various molar ratios of HF / Mo. Without boric
acid, the %absorbance abruptly decreased when the HF /
Mo ratio exceeded 0.1. With B / Mo= 1, the range of
%absorbance= 100 expanded to HF / Mo=0.3. With B /
Mo=2,the range had almost no limit. Since the value of
the HF / Mo ratio is usually 0.06, hardly exceeding 0.2,
the B / Mo= 1.4 condition was recommended for the
molybdenum yellow method. A similar analysis of the
interference of acid and base indicated that the addition
of acid or base was permissible in the range of H+/
Mo 5 1 or OH-/ Mo S 0.5, respectively.
Use of several chemicals to quench the hydro-
fluoric acid activity to dissolve silica
Glassware silica is strongly dissolved by the dilute
hydrofluoric acid solution, even with a small amount.
Such ability of hydrofluoric acid needs to be controlled
to avoid hazards, though it is advantageous for the
development of a rapid method of estimating the silica
content. For this purpose, several chemicals were exam-
ined using grass for silica dissolution. In Fig. 2, the
effects of H,BO,, AICI,, H,MoO,, and NaOH on the
reduction of HF activity are depicted. When NaOH was
added, silica dissolution decreased with the increase in
the OH-/HF ratio. The dissolution ceased at OH- /
HF= 1.4, i.e. neutralization point (not at OH- / HF= 1,
because the HF solution contained HCl). These results
indicated that hydrofluoric acid loses the ability of silica
K. SAITO et al.
40
M 30
a
d
-P
p 20
v)
aN
0
lJY 10
I
I 0 0.3 0.6 0.9 1.2 1.5
Molar ratio of inhibitory element relative to HF
1.8
Fig. 2. Effect of B, Al, Mo, and OH--ion on the suppression
of HF activity to dissolve silica. The 0.15 M HF-0.06 M HCI
solutions treated with various amounts of the inhibitors (H,BO,,
AICI,, H2Mo0,, or NaOH), 0.2 mL, reacted with borosilicate
glass tubes (Pyrex) for 10 min. Amount of silica dissolved: p,g
per glass tube.
dissolution as HF is ionized to F- ion. When H,BO,,
AlCl,, or H,MoO, was added, the HF activity decreased
with the increase in the amount of chemicals, and was
completely lost with a molar ratio of chemicals to HF
above 0.3. The result indicated that a relatively small
amount of the chemical quenched the activity of hydro-
fluoric acid, suggesting that the metal element of the
chemicals was coordinated with 3-6 F- ions. It is appar-
ent that the dissolution of glassware is unlikely with far
excess of B / HF or Mo / HF molar ratios in the molyb-
denum yellow method. The result also confirmed that
the addition of B is effective in reducing the HF interfer-
ence with molybdenum yellow formation, in which B
and Mo compete for F.
Citrate to molybdenum ratio to mask the P
interference and absorbance decrease
Plants contain as much as 15 g kg-' of phosphorus,
some of which is extracted in the HF solution. In the
determination of the silicon content by the molybdenum
yellow method, it is necessary to prevent the interfer-
ence of P, since phosphorus forms yellow-colored
molybdenum complexes (P-molybdenum yellow) giving
a false positive increase in the absorbance. The P-
molybdenum yellow is almost instantly decomposed by
the addition of a chelating oxy acid, such as tartaric acid
or citric acid. The minimum amount of oxy acid should
be added, since the absorbance of molybdenum yellow
(Si-molybdenum yellow) decreases with time, and the
decrease is accelerated by the oxy acid. In Fig. 3, the
absorbance by the P-molybdenum yellow (at a P con-
centration of 5 mg L-') is plotted as a function of the
molar ratio of the oxy acid to molybdenum. The absor-
bance decreased with the increase in the molar ratio,
indicating the decomposition of the P-molybdenum yel-
low. The decomposition was completed at the ratio of
 Rapid, Micro-Methods to Estimate Plant Silicon 33

lslble 1. Effects of the amount of hydrofluoric acid and of sample pretreatments on the dissolution of plant silica.
Dissolution methods and conditions
Direct dissolution from plants" Digestion and
a b C d e separationb
Plant sample weight (g) 0.5 0.5 0.5 0.5 0.5 0.5
Solution volume (mL) 10 10 10 10 10 10
Concentration of HF (M) 0.3 0.6 1.5 2.0 3.0 1.5
~~
Amount of HF (mmol) 3 6 15 20 30 15
Plant sample Treatment Silica dissolved from plant mg SiO,
Rice husk Non treat 24 76 107 107 109 105
Rice husk Wiley milled 106 106 104 105
Rice husk Pulvelized 109 106 107 108
Rice stem Wiley milled 29 53 60 59 60 58 ~~~~

"In the direct dissolution, silica is dissolved by immersing 0.5 g of a plant sample for I h in 10 mL of HF solutions with different rnol
of HF. the digestion and separation, 0.5 g of a plant sample was digested, and the residual silica was separated and dissolved in
10 mL of 1.5 M HF solution. The amount of dissolved silica was determined by the molybdenum yellow method.
Downloaded by [University of New England] at 14:02 04 January 2015

0.20
y = -0.05 )I + 100

0.15 I
y = -0.1 x + 100

..
0.10 0without citric acid
u ,
C with citric acid
E 0.05
(molar ratio of citric acid to Mo is 0.8)

B 11.
9 70

0.00 L- .I .. I
0 20 40 60 80 100
Time after the onset min
120 140 160

0.0 0.5 1.0 1.5 2.0 2.5

Molar ratio of oxy acid relative to Mo
3- Inhibition of P-molYbdenum Yellow by citric acid Or
tartaric acid. P-molybdenum yellow complex, at 5 mg kg-' P,
decomposed by the addition of citric acid or tartaric acid. The
absorbance of remaining P-molybdenum yellow is plotted.
citric acid / Mo 2 0.8, and tartaric acid / Mo 2 2. Since
the P concentration is at most 0.5 mg L-' in the determi-
nation of the HF extract of rice plant with the molybde-
num yellow method, the minimum addition of 0.8 mol
of citric acid/Mo was employed. Figure 4 shows the
decrease of absorbance of Si-molybdenum yellow with
0.8 mol of citric acid / Mo at a temperature of 25"C,
together with the decrease of absorbance in the absence
of citric acid. The rate of the decrease with citric acid
was 1% per 10 min, while the decrease without citric
acid, the spontaneous decrease, was 1% per 20 min. In
order to obtain the precision of *0.3%, the lag time to
be allowed from the addition of citric acid to the mea-
surement of absorbance at 25°C was considered to be
2 3 min.
Effect of the amount and concentration of HF
on the dissolution of plant silica
In the hydrofluoric acid solution, silica is rapidly dis-
Fig. 4. Decrease in the absorbance of molybdenum yellow
with time at 2YC, with and without citric acid addition.
%absorbance denotes the absorbance at 400 nm relative to the
initial absorbance (= 100%).
solved according to the following equation: SO,+ 6HF
= 2H++SiF,2-+2H20, although in neutral to acidic
aqueous solutions, silica is slowly dissolved as H,SiO,
with limited solubility not exceeding 2 mmol L-'. It is
shown in Table 1 that the amount of silica dissolved
from 0.5 g of rice sample was limited by the amount of
HE With less than 15 mmol of HF, the dissolution of sil-
ica from the rice samples increased approximately at the
rate of 1 rnol of SiO, to 6 mol of HF, following the sto-
ichiometry of the reaction between SiOz and HF.The
result indicated that the amount of extracted silica was
limited by the insufficient amount of hydrofluoric acid.
With as much as 15 mmol of hydrofluoric acid, the max-
imum dissolution was attained, indicating the complete
dissolution of the silica in 0.5 g of plant sample.
For the recommended method of extraction of 0.5 g of
rice plant sample, we suggest the use of 10mL of the
HF solution (1.5 M HF-0.6 M HCl), which can dissolve
150 mg of silica. So far, we have not found rice plant
materials containing more than 120 mg in 0.5 g DW of
 34
180 1
Y $
-c.a la
- .+. .RH in 0.15 MHF.100 ml
+-RH in 0.3 MHF, 50 ml
120 +-RH in 0.75 MHF 20 ml
E +RH in 1.5 MHF. 10 ml
..+. .WC in 0.15 MHF.100 ml
4 - W C in 0.3 MHF. 50 ml
+WC in 1.5 MHF .I0rnl
+RH in 1.5 MHF. 18%
a - ~ " " " '
0 10 20 30 4 50 60 70 80 90 100 110
Dissolution time, min
Fig. 5. Dissolution of plant silica at different HF concentra-
tions. Plant samples: 0.2 g of rice husk (RH) and 0.5 g of wheat
culm (WC). HF solutions: 10 mL of HF solutions, diluted with
0, 10, 40, and 90mL of distilled water. Temperature: 30°C,
Downloaded by [University of New England] at 14:02 04 January 2015
except for RH dissolved at 18°C in 1.5 M HF.
SiO,. Hydrochloric acid was added in the HF solution to
counteract the bases derived from plant materials, since
bases are considered to reduce the hydrofluoric acid
concentration and thus the dissolution of silica. Table 1
also shows that the maximum amount of silica measured
by the direct extraction from rice plants was in agree-
ment with the silica content measured with the diges-
tion-separation-dissolution method. Regardless of
whether the rice husks were pulverized, the dissolution
was not appreciably affected, suggesting the rapid diffu-
sion of HF into the plant tissues and silica deposits.
So far, the data indicated that the silica dissolution in
plant was completed within I h when 10 mL of 1.5 M
HF was used. Questions arose about whether 50 mL of
0.3 M HF solution can be applied in the extraction, and
how the dissolution rate is affected by the decrease in
the HF concentration. The effect of the HF concentra-
tion on the dissolution of silica is presented in Fig. 5 .
The silica content of the wheat culm was low and the
dissolution was completed within 30 min regardless of
the HF concentration. The silica content of rice husks
was high, and the dissolution rate depended on the HF
concentration. The silica dissolution of rice husks took
20 rnin with 1.5 M HF, 60 rnin with 0.3 M HF, and 100
min with 0.15 M HF at 30°C. The results suggested that
a higher activity of HF was required to dissolve the firm
silica deposited on rice husks. Even at the concentration
of 1.5 M HF, silica dissolution from rice husks was
affected by the temperature, and the completion of dis-
solution took 60min at 18°C. It is concluded that the
concentration of 1.5 M HF was essential to complete the
dissolution of silica in rice plant materials within 30 min
at 3032, and within 1 h at 18°C.
K. SAlTO et al.
I 200 , YXl.977X-0.60
FO.982
3
0
160
i
A leaf sheath
1
120 0 40 80 120 160 200
-'
SiOz mg kg determined by dry-ashing
gravimetric method
Fig. 6. Determination of the content of rice plant silica by
the HF extraction-molybdenum yellow method, compared with
that by the dry-ashing gravimetric method. Size of the plant
sample: 0.1 g DW for the extraction method, and 2 g DW for
the gravimetric method.
Comparison between direct extraction and
gravimetric method
In Fig. 6, the content of silica of rice plants deter-
mined by HF extraction and the molybdenum yellow
method using 0.1 g DW of sample is compared with that
determined by dry-ashing and the gravimetric method
using 2 g DW of sample. The rice plant samples in this
experiment consisted of leaf blades and leaf sheaths of
various rice plants at the vegetative growth stage and at
the maturity stage. It was shown that the silica contents
determined by the two methods were highly correlated
@<0.001), with an intercept of -0.60 and a slope of
0.977, and a value of 0.982 for the correlation coeffi-
cient I-.The slightly higher silica contents by the gravi-
metric method may be due to incomplete dehydration or
to impurities which often occurs in dry ashing. As for
the analytical time and labor, the 10-stepped procedure
of the HF extraction and molybdenum yellow method
was completed within 2 h, although dry-ashing and the
gravimetric method required approximately 17 h and
close watch on the individual steps, particularly during
the evaporation process. Even though some attention to
the HF solution is necessary, the HF extraction method,
which does not require any special utensils, enables to
save time and labor.
Dissolution of silica from soils: a possible
cause of contamination
Dissolution of soil materials in the HF solution was
examined. It is considered that the error caused by soil
contamination depends on how specific to plant silica
the HF dissolution is. It is shown in Fig. 7 that, for clay
and silt materials, 500 mg g-' DW of silica was dis-
solved within 1 h, suggesting the presence of easily sol-
uble silicate materials in the fine fractions of soils.
 Rapid, Micro-Methods to Estimate Plant Silicon
I of the solution volume). The silica, separated on filter
0 30 60 90 120 150
Dissolution time min
-0-Clay KT
-- e-
&Clay FK
W - Silt KT
Silt FK
+Fine sand KT
&Fine sand FK
Fig. 7. Dissolution of silica from soil fractions in the HF
solution. Samples consisted of the clay fraction (Clay), silt frac-
Downloaded by [University of New England] at 14:02 04 January 2015
tion (Silt), fine sand fraction (Fine sand), separated from Kawa-
tabi soil (KT), and Furukawa soil (FK). Dry weight of soils: 50-
I 0 0 mg. Soil fractions were reacted with 10 mL of the HF solu-
tion at 30°C. Amount of dissolved SiO, mg kg-': determined by
the molybdenum yellow method.
Dissolution of silica from the fine sand fraction was
slow and was not completed in 1 h. Quartz sand is near-
ly insoluble in the HF solution (data is not shown). The
results showed that the HF solution method was not spe-
cific to plant silica; the finer were the soil components,
the more silica was dissolved in the HF solution. Care
for avoiding contamination from soils, especially of the
soil fine fraction, is necessary for the determination of
the true content of plant silica.
Recommended procedure for direct extraction
of silica from rice plant with dilute fluoric acid
solution
Use plastic ware (polypropylene, polyethylene etc.),
which can be soaked in 0.1% HF solution to become
free of silicon contamination. The extraction solution is
a 1.5 M HF-0.6 M HCI solution (HF solution), as
described in MATERIALS AND METHODS, and is handled
using pipettes with plastic tips. Dried rice plant samples
are milled or cut to small pieces. Place 0.5 g DW of
plant sample in a 50 mL plastic bottle. Add 10 mL of the
HF solution, stopper the bottle, stir the content of the
bottle to immerse the plant tissues in the HF solution,
and let it stand for 30 min at 30°C (1 h at 18°C) with
occasional stirring (roughly every 10 min). Add 40 mL
of distilled water, stir to homogenize the content, and let
plant materials settle for 20 min. The aliquot (0.1 mL) of
the clear supernatant is taken for the spectrometric
determination with the molybdenum yellow method.
Using smaller utensils, micro-estimation of silicon is
possible in a similar manner: for example, immersing
35
100 mg of plant samples in 2 mL of the HF solution for
1 h, and then, diluting with 8 mL of distilled water.
Recommended procedure for digestion-separa-
tion-dissolution
Prepare polyethylene bags and filter papers from the
same lots (reserve one set for tarring in the adjustment
paper by digestion of 0.5 g DW of plant sample, as
described in MATERIALS AND METHODS, is put into a
polyethylene bag together with the filter paper. Add
10 mL of the HF solution, knead the bag to disperse the
silica, and let the silica dissolve for more than 3 h.
Dilute to 50 mL by the addition of distilled water until
the weight of the solution reaches 50.5 g on the tarred
balance. The content is homogenized with stirring, and
the clear supernatant is taken for the determination of
silica with molybdenum yellow.
Recommended spectrometric determination
with molybdenum yellow
The reagents and the silicon standard solutions in
hydrofluoric acid are described in MATERIALS AND
METHODS. The working Mo solution is prepared prior to
use. Plastic tubes (20-mL volume) and micropipettes
with plastic tips are used. Each aliquot, 0.100mL, of
standards of 0-3,000mgL-' SO,, or of the sample
extract solutions is transferred to a plastic tube. Add
2.0 mL of the 0.1 M B solution, rinsing any droplets of
the extract solution inside the tubes. Add 2.0 mL of the
Mo working solution and mix the contents. Allow 3 min
(not more than 15 min) for the formation of molybde-
num yellow to be completed, and then, add 4.0mL of
the 0.1 M citric acid solution, stopper the tube and mix
the content. Measure the absorbance at 400nm with a
spectrophotometer, from 4 to 10min after the addition
of citric acid.
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