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M.A.e. Fragoso and M.L. van Beusichem (eds.), Optimization 01 Plant Nutrition, 3-6, 1993.
PLSO IAOPN-056
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.
Introduction
Pequerul el al.
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
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.
Concentration
(i) Dryashing
(iv) Original
(v) Improved with iron addition
188,81 = 1,63
173,67 = 1.23
1l4,19 =
127,00 =
181.50 =
182,50 =
18270 =
1.34
1.72
1.52
1.69
1.43
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
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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