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A rapid wet digestion method for plant analysis

Article January 1993
DOI: 10.1007/978-94-017-2496-8_1

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M.A.e. Fragoso and M.L. van Beusichem (eds.), Optimization 01 Plant Nutrition, 3-6, 1993.

1993 Kluwer Academic Publishers. Printed in Ihe Nelherlands.

PLSO IAOPN-056

A rapid wet digestion method for plant analysis

A. PEQUERUL, C. PREZ, P. MADERO, J. VAL and E. MONGE
Department of Plant Nutrition, Estacin Experimental de Aula Dei, es/e, Aptdo. 202, 50080
Zaragoza, Spain

Key words: AAS, AES, dry ashing, ICP, lucerne, macronutrients, Medicago sativa L., micronut
rients, plant analysis, wet digestion
Abstract

Analysis of nutrients in plant material requires previous digestion. Although a variety of digestion
methods are used, they are usually time-consuming procedures to digest and prepare the samples.
Calcination methods, using classical muffles furnaces, allow the treatment of a high number of samples
but the process requires at least 24-48 hours. Sulfuric acid based wet digestion methods, have generally
the inconvenient of low Fe and Al recoveries (Bowman, 1989), and the analysis is restricted to ICP. In
this work, total P, Ca, Mg, K, Fe, Mn, Zn, and Cu, in lucerne leaves (Medicago saliva L.) were
determined. The solutions for analysis were prepared by an improved wet digestion method (7-8 min)
based on the addition of hydrogen peroxide to the sample previously introduced in concentrated
HN0 3 , followed by modera te heating (100 oC). Ca, Mg, Fe, Mn, Zn and Cu were determined by AAS
and ICP, K, by AES, and P by colorimetry. Standard addition of iron in the leaf samples avoided the
interference of HN0 3 in AAS determination of Fe. The results are compared with the obtained by
classical dry calcination (muffle) and other wet methods.

Abbreviations: AAS = atomic absorption spectrometry; AES

inductively coupled plasma.

Introduction

Plant material analysis provides estimations of

levels of plant nutrients and is used as a tool to
investigate nutritional imbalances, physiological
disorders, etc. Nutrient analysis requires pre
liminary treatment of leaves and other plant
material. One of the analytical bottlenecks, is
the digestion of samples. The time required by
traditional dry ashing (Jones, 1984; Jones et al.,
1991; Pinta and DeWele, 1975) or wet digestion
(Jones, 1984; Jones et al., 1991) is quite long or
else a low number of samples can be processed
in case of using microwave ovens.
To determine cations in solution, the use of
ICP has obvious advantages over other methods,

atomic emission spectrometry; ICP

because at the temperature reached (above

6000 oC), complexes and/or organics that may
exit, are converted to elemental forms (Bow
man, 1989; Halvin and Soltanpour, 1980). How
ever, ICP is an expensive technique which is not
available in many laboratories.
A rapid and accurate wet digestion method is
presented for the preparation of samples for
nutrient analysis (P, Ca, Mg, K, Fe, Mn, Zn and
Cu) by AAS (equipment available in most plant
nutrition laboratories), as welJ as by ICP. The
method here proposed, is based on the oxidation
and solubilization of plant organic matter when
HzO z (33%) ~s directly added to the sample in
concentrated HN0 3 and heated during a short
time interval.

Pequerul el al.

Material and methods

Leaves of lucerne (Medicago saliva L.) were
sampled according to the general procedure used
for foliar diagnosis. In brief, they were washed
and dried for two days at 65 oC and ground to
pass through a 60 mesh screen.
Ca, Mg, Fe, Mn, Zn and Cu were determined
by AAS, K by AES (Pinta and c.I.!., 1973;
Pinta and DeWele, 1975) and P was analyzed by
molybdate-blue colorimetry (Jones, 1984).
The methods used to digest the samples were:

i) Dry ashing (Jones, 1984)

1 g of dry material was weighed in a high form
crucible and placed in a muffte furnace for 24
hours at 500C. Once cooled, it was wet with a
few drops of distilled water, and 4 mL of diluted
(1:1) HN0 3 (density 1.3g mL- 1) were carefully
added. The solution was evaporated to dryness
on a hot pi ate (lOO-120C), and set in the muffle
furnace during two hours. Once cooled, 10 mL
1
ofdiluted (1:1) HCI (density 1.18gmL- ) were
added and heated on the plate. The solution was
filtered, transferred to volume f1ask and diluted
with H 2 0 up to 25 mL.
Wet acid digestion procedures:
ii) HN0 3 / HCL0 4 digeslion (Jones, 1984)
5 mL of HN0 3 (70%) and 1.5 mL of HCI0 4
(60%) were added to 0.5 g of sample, and the
solution heated until the disappearance of the
brown fumes. It was then cooled, 5 mL of
diluted (1: 1) HCI (density 1.18 g mL -1) added,
and finally diluted with H 2 0 up to 25 mL solu
tion.
iii) Modified HN0 3 /HCL0 4 digeslion
The procedure was basically the same as previ
ous but modified in our laboratory by the addi
tion of 7 mL H 2 0 2 (33%).

iv) HN0 3 /H 2 0 2 wel digeslion (Jones et al.,

1991)
8 mL of HN0 3 were added to 0.5 g of sample
and let stand overnight. The solution was then
heated for one hour at 120C on hot plate, and
several additions of 4 mL 33% H 2 0 2 were made
until the digest was colorless. The residue was

taken to dryness at low heat (80C), cooJed and

1
diluted with (1:10) HCI (density 1.18g mL- ).

v) lmproved HN0 3 / H 2 0 2 digeslion

5 mL of HN0 3 were added to 0.5 g of sample in
a 250 mL dry f1ask and stirred. Thus, all the
material was wet. Then 4 mL of 33% H 2 0 2 were
carefully added in a well ventilated hood and
slightly stirred after the addition. It was heated
on a hot plate and a strong effervescence was
produced. When the brown fumes were less
dense (7-8 minutes), the solution was aJlowed to
cool. A slightly yellow dissolution and a small
white solid quantity in suspension stiJl remained.
The solution was filtered, washed with 5 mL of
(1:1) HCI (density 1.18g mL- 1 ) and diluted up
to 25 mL with distilled H 2 0.

Results
SeveraJ procedures were tested: a conventionaJ
wet digestion (ii); a method (iii) based on a
c1assical HN0 3 /HCJ0 4 wet procedure, modified
in our laboratory by the addition of 7 mL H 2 0 2 ;
a method (iv) reported by Jones et al. (1991) and
a method similar to previous (v) but using
different proportions of HN0 3 (5 mL) and H 2 0 2
(4 mL), also modiffiying the sequence of addition
of reagents. The time of digestion, and the
concentration in base to dry matter of macro and
microelements are shown in Table 1. The results
were compared with those obtained by tradition
al dry ashing (i) for organic matter destruction
(Jones, 1984), which were taken as a reference,
as this method is widely used in our laboratory
and a large data bank of analysis in many higher
plants, is available.
In digestions (iii) and (v), a small quantity of
white solid remained in suspension, indepen
dentJy of the amount of H 2 0 2 added, and did
not disappear either when the time of digestion
was increased or with the increased addition of
HN0 3 or HCI0 4 .
The concentrations of P, Ca, Mg, K, Mn, Zn
and Cu obtained by methods (ii), (ji), (iv) and
(v) were not significatively different from those
obtained by dry ashing (i). However, the values

A rapid wet digestion method lor plant analysis

Table l. Nutrient element concentrations following the different analytical methods used and the time required for digestion of
samples
Method

Time

( i)
(ji)
(iii)
(iv)
(v)

30 h
3h
30 min
24 h
7 min

0.31
0,31
0,33
0,32
0,32

Ca
=
=
=
=
=

0,00
0,01
0.01
0.02
0.00

1.38 =
1.15 =
1.26 =
1.35 =
1.31 =

Mg
0,02
0.05
0.01
0.10
0,05

0,26 =
0,24 =
0.27 =
0.26 =
0.28 =

0,00
0.01
0.00
0.01
0.01

3,32 =
2.63 =
2.85 =
3.33 =
3,34 =

0,02
0.13
0,30
0,02
0.02

Fe

Mn

Zn

Cu

188,81 = 1.63
105.00=4.16
111.33 = 1.17
173.67 = 1.23
114.19 = 3.45

19,50 = 0.21
18.00=4.16
16,83 = 1.01
18,63 = 0.10
19.68 = 0,65

47.11=0.62
53.17 = 6,61
59,50 = 1.04
61.35 = 2.03
52.35 = 4,41

6.30 =
5.42 =
5.58 =
6,25 =
6.15 =

0,09
0.22
0.08
0.04
0,07

i, ii, and iv methods were reported by lunes (1984 and 1991 respectively). Methods iii and v were developed in our laboratory.
Macroelements are given in % dry maller and microelements in mg kg -] dry matter. The results are the average of eight
replications = standard error.

of iron analyzed by the methods (ii), (iii), (v)

and (iv) were lower (60-70% and 91% respec
tively).
In order to investigate the low iron recovery in
wet method (v), known amounts of ron (100,
200, 300 and 400 fLg) were added to each sample,
corresponding to hypothetical iron concentra
tions in the plant of 200, 400, 600 and 800 mg
kg - J respectively, then the digestion of samples
(8 replications) was carried out (Table 2).
In all cases, when ron was added, an increase
in the concentration of this element over that
obtained by digestion without the addition was
observed. The optimum quantity estimated was
of 300 fLg of Fe per 0.5 g of sample, which gave a
recovery of 96% compared with the obtained by
the dry method.
The ICP analytical data were similar to those
obtained by AAS (resuits not shown).

Table 2, Effect of different standard additions of Fe on the

concentration uf the element in the plant sample
Method

Concentration

(i) Dryashing
(iv) Original
(v) Improved with iron addition

188,81 = 1,63
173,67 = 1.23

Iron added (mg kg ~])

200,00
400,00
600.00
800,00

1l4,19 =
127,00 =
181.50 =
182,50 =
18270 =

1.34
1.72
1.52
1.69
1.43

The results for all methuds are the average of eight

rcplications = standard error,

Discussion
Preliminary studies on the amounts of reagents,
specially hydrogen peroxide, and the adequate
time of reaction for the total destruction of
organic matter were necessary to select the most
adequate method.
In the preliminary assays the addition of
different amounts (7-13 mL) of hydrogen perox
ide to HCI0 4 /HNO) mixture (iii) promoted the
acceleration of the oxidation process, We ob
served that hydrogen peroxide did not inf1uence
the recovery of macro and microelements but the
time of digestion was considerably lower (Table
1) than in the original method (ii).
HNO)/HzO z has been previously used (Hal
vin and Soltanpour, 1980; Jones et aL, 1991),
without any improvement over other mixtures
used in wet methods. Although in the improved
method (v) a HN0 3 /H zO z mixture was used,
important changes were observed. In the previ
ous method (iv), colourless solutions were ob
tained after an extended digestion (several
hours), while in the improved method (v) slightly
yellow dissolutions and a small white solid
quantity remained in suspension. Except for the
case of iron, interferences in the recovery of the
elements were not detected (Table 1). It can be
thought that the low recovery of iron could be
assigned to precipitation of this element within
the white solid, but a very sensitive direct assay
of iron (color reaction with a,a'-dipyridyl) in the
precipitate was negative.
In method (v), the addition of hydrogen pero x
ide was accomplished in one step, in contrast to
several successive additions reported elsewhere

A rapid wet digestion method for plant analysis

(Jones et al., 1991). The amount of HzO z added

to accelerate the oxidation of organic matter,
was minimized, so that it did not inftuence the
recovery of the eJements (Table 1), and also, the
risk of losses through splashes and the use of a
hazardous material decrease.
The low iron concentration measured (Table
1) was due rather to the interferences in AAS
than to incomplete digestion, as both HN0 3
(methods iii and iv) and HCJ0 4 (method iii)
reduce the atomic absorption of iron at 248.33
nm. As usual iron concentrations in lucerne
leaves vary between 30 and 250 mg kg -\ (Jones
et al., 1991) and the values obtained by the dry
method were 180-190 rng kg -1, the optimum
quantity of ron added was estimated to be
300 .l-g in 0.5 g of sampte (Table 2), which
corresponded to a theoretical iron concentration
of 600 mg kg -1 (approximately 3 times the usual
concentration). The interference of HN0 3 was
thus minimized as the averages and standard
errors obtained for this method (iv) (182.50
1.69) compared with those obtained for dry
ashing (1) (188.81 1.63) were very close.
In the proposed method (v), a much more
reduced time of digestion (7-8 minutes) and a
subsequent filtering of the resulting solution is
sufficient to recover the macro and microele
ments Ca, Mg, K, Mn, Zn and Cu as well as P
(Table 2). Therefore, the improved method
proves to be rapid and accurate, and does not
show significant differences when compared to
others methods widely used.
The method is currently assayed in different
plant material, sugar beet and soybean (Ieaves,
roots) cultured in hydroponic conditions, and

apple, pear and peach tree Ieaves in field con

ditions.

Acknowledgements
The authors thank Mrs M A Garcia, C Lope and
Ms C Fustero for their excellent technical assis
tance. Work carried out under research project
CONAI-DGA: PCA-4/91.

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