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Plant Analysis Reference Procedures,ix ธันวาคม 15, 2010

ผ่านทาง scsb-PlantAnalysis.

The publication can be downloaded from Plant Analysis Reference Procedures for the Southern Region of the United States.

Preface
Plant analysis has evolved into one of the important tools in crop production. It is a process in which
plant samples are collected from a plant at a specific time during the growing season and analyzed in a
laboratory for various essential nutrients. Nutrients of primary interest are nitrogen (N), phosphorus (P),
potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), manganese (Mn), molybdenum (Mo), iron (Fe),
boron (B), copper (Cu) and zinc (Zn). The plant analysis process also includes an evaluation of the analytical
data to determine whether an element is low, sufficient, or high and finally the formulation of a
recommendation.
Each step in the plant analysis process is equally important. In the laboratory phase many different
procedures are involved which include decontamination, drying, grinding, weighing, ashing, and analysis for
11 to 12 essential nutrients. Consequently, performing a plant analysis involves the use of a variety of
laboratory instruments.
Several laboratories in the Southern Region of the United States offer plant analysis services to
researchers and growers. This bulletin contains reference procedures commonly used by laboratories in the
region. Procedures were selected on the basis of their accuracy and precision as well as their popularity and
acceptance by workers in the area of plant analysis. These procedures also provide a reference for laboratories
to exchange samples to evaluate current plant analysis procedures or to implement new ones.
C. Owen Plank
Editor
Acknowledgements
Appreciation is expressed to all the Southern Extension and Research Activities members who
contributed to this bulletin and to each experiment station representative who submitted procedures.
Appreciation is also expressed to Dr. George Kriz, Administrative Advisor, for his leadership and sincere
support of this work group.
Editorial Committee
C. O. Plank, Editor
C. R. Campbell
F. R. Cox
V. W. Case
E. A. Hanlon
C. C. Mitchell
H. J. Savoy, Jr.
State agricultural experiment stations and extension services are equal opportunity/affirmative action agencies.

 

Plant Analysis Reference Procedures,x

ผ่านทาง scsb-PlantAnalysis.

The publication can be downloaded from Plant Analysis Reference Procedures for the Southern Region of the United States.

Members of the Southern Extension and
Research Activities Information Exchange
Group-6-Soil Test and Plant Analysis
1991
Administrative Advisor – G. J. Kriz, Associate Director, Agricultural Experiment Station, North
Carolina State University
Alabama C. E. Evans (Rep), C. C. Mitchell
Arkansas W. E. Sabbe (Rep), W. H. Baker, N. Miller, C. Snyder
Florida E. A. Hanlon (Rep), G. Kidder
Georgia C. O. Plank (Rep), R. A. Isaac, M. E. Sumner
Kentucky W. O. Thom (Rep), V. Case, D. Kirkland
Louisiana J. Kovar (Rep), J. Holder
Mississippi W. Houston (Rep), K. Crouse
North Carolina F. R. Cox (Rep), M. R. Tucker, C. R. Campbell
Oklahoma E. Allen (Rep), G. V. Johnson
Puerto Rico O. Muniz-Torres (Rep)
South Carolina R. M. Lippert (Rep), C. L. Parks
Tennessee G. Lessman (Rep), J. J. Jared, H. J. Savoy
Texas L. Unruh (Rep), H. D. Pennington
Virginia S. J. Donohue (Rep)
Virgin Islands E. Craft (Rep)

 

Plant Analysis Reference Procedures,p1

ผ่านทาง scsb-PlantAnalysis.

The publication can be downloaded from Plant Analysis Reference Procedures for the Southern Region of the United States.

Sample Preparation
C. R. Campbell and C. O. Plank*
Sample preparation is critical in obtaining accurate analytical data and reliable interpretation of plant
analysis results. Proven procedures must be followed during decontamination, drying, particle-size reduction,
storage, and organic matter destruction. Each of these preparatory procedures provide opportunities to
enhance the accuracy and reliability of the analytical results.
A. Decontamination
1. Principle
1.1 Plant materials must be clean and free of extraneous substances including soil and dust
particles, and foliar spray residues that may influence analytical results. Generally, the
elements most affected by soil and dust particles are Fe, Al, Si, and Mn. Foliar nutrient spray
and fungicide residues can affect several elements and should be taken into account in the
decontamination process and when evaluating the analytical results. The decontamination
process must be thorough while still preserving sample integrity. Therefore,
decontamination procedures involving washing and rinsing should only be used for fresh,
fully-turgid plant samples.
2. Reagents and Apparatus
2.1 Deionized water.
2.2 0.1 to 0.3% detergent solution (non-phosphate).
2.3 Medium-stiff nylon bristle brush.
2.4 Plastic containers suitable for washing and rinsing tissue samples.
3. Procedure
3.1 Examine fresh plant tissue samples to determine physical condition and extent of
contamination. Unless leaf tissue is visibly coated with foreign substances, decontamination
is usually not required except when Fe (Wallace et al., 1982) Al, Si, or Mn are to be
determined (Jones and Case, 1990).
1Chief-Plant/Waste/Solution Advisory Section, Agronomic Division, North Carolina Department of Agriculture,
Raleigh, NC 27611 and Extension Agronomist, Soil Testing and Plant Analysis, Cooperative Extension Service, The
University of Georgia, Athens, GA 30602.

 

Plant Analysis Reference Procedures,p2

ผ่านทาง scsb-PlantAnalysis.

The publication can be downloaded from Plant Analysis Reference Procedures for the Southern Region of the United States.

3.2 When Al, Si, Mn, and Fe are not of primary interest, plant leaves should be brushed briskly to
remove visible soil and dust particles.
3.3 When plant samples show visible residues from spray applications and when Al, Si, Fe
(Wallace et al., 1982), and Mn are elements of interest, leaves should be washed in a 0.1 to
0.3% detergent solution (Ashby, 1969 and Wallace et al., 1980) followed by rinsing in
deionized water. The wash and rinse periods should be as short as possible (Sonneveld and
Van Dijk, 1982) to avoid danger of N, B, K, and Cl leaching from the tissue (Bhan et al.,
1959).
3.4 After decontamination, samples should be dried immediately to stabilize the tissue and stop
enzymatic reactions.
4. Remarks
4.1 When proper sampling techniques have been utilized, decontamination should be minimized.
4.2 Decontamination is generally not necessary where tissue has been exposed to frequent rainfall
and/or not exposed to nutrient or fungicidal sprays (Jones et al., 1991). Small plants that have
been splattered with soil are the exception to this rule.
4.3 Excessive washing is likely worse than no decontamination since soluble elements, including
B, K, and N, are likely to leach from the tissue.
4.4 Samples should be dipped quickly in the wash and rinse solutions. Sonneveld and van Dijk
(1982) recommended a time of 15 seconds.
4.5 Relatively high concentrations of Al (>100 mg kg-1), Fe (>100 mg kg-1), and Si (>1.0%) are
strong indicators of contamination (Jones et al., 1991). Titanium (Ti) has also been suggested
as an indicator of soil or dust contamination (Cherney and Robinson, 1982).
B. Drying
1. Principle
1.1 Water is removed from plant tisssue to stop enzymatic reactions and to stabilize the sample.
Removal of combined water also facilitates particle size reduction, homogenization, and
weighing.
2. Apparatus
2.1 Forced-air oven equivalent to Blue M Model POM-166E.

 

Plant Analysis Reference Procedures,p3

ผ่านทาง scsb-PlantAnalysis.

The publication can be downloaded from Plant Analysis Reference Procedures for the Southern Region of the United States.

3. Procedure
3.1 Separate or loosen tissue samples and place in paper containers.
3.2 Place containers in forced-air oven and dry at 80oC for 12 to 24 hours. NOTE: The original
condition and sample size will affect drying time.
4. Remarks
4.1 Drying times longer than 24 hours may be required depending on the type and number of
plant samples in the dryer.
4.2 Drying at temperatures under 80oC may not remove all combined water (Jones et al., 1991)
and may result in poor homogenization and incorrect analytical results.
4.3 Drying temperatures above 80oC may result in thermal decomposition and reduction in dry
weight (Jones et al., 1991).
4.4 Enzymes present in plant tissue are rendered inactive at temperatures above 60oC (Tauber,
1949). As a result, air drying may not stabilize samples and prevent enzymatic
decomposition. Samples should, therefore, be properly dried as soon after taking the sample
as possible.
4.5 Quick drying of a limited number of samples can be accomplished using a microwave oven
provided the samples are turned often and the drying process is closely monitored (Carlier
and van Hee, 1971; Shuman and Rauzi, 1981; and Jones et al., 1991).
4.6 If samples absorb significant amounts of moisture during grinding, additional drying may be
required prior to weighing for analysis. Drying time required will vary. Dry to constant
weight by making periodic weighings.
C. Particle-Size Reduction
1. Principle
1.1 Plant tissue samples are reduced to 0.5- to 1.0-mm particle size to ensure homogeneity and to
facilitate organic matter destruction.
2. Apparatus
2.1 Standard and intermediate Wiley-type mills equipped with 20-, 40-, and 60-mesh screens and
stainless steel contact points.
2.2 Cyclotec or equivalent high-speed grinder.
2.3 Medium bristle brush.

 

Plant Analysis Reference Procedures,p4

ผ่านทาง scsb-PlantAnalysis.

The publication can be downloaded from Plant Analysis Reference Procedures for the Southern Region of the United States.

2.4 Vacuum system.
3. Procedure
3.1 After drying, samples should be ground to pass a 1.0-mm screen (20 mesh) using the
appropriate Wiley Mill. A 20-mesh sieve is adequate if the sample aliquot to be assayed is
>0.5 g. However, if the sample aliquot to be assayed is <0.5 g, a 40-mesh screen should be
utilized (Jones and Case, 1990).
3.2 After grinding, the sample should be thoroughly mixed and a 5- to 8-g aliquot withdrawn for
analyses and storage.
3.3 Using a brush or vacuum system, clean the grinding apparatus after grinding each sample.
4. Remarks
4.1 Uniform grinding and mixing are critical in obtaining accurate analytical results.
4.2 Exercise care when grinding very small samples or plant material that is pubescent,
deliquescent, or that has a fibrous texture. These samples are difficult to grind in Wiley mills
and the operator should allow sufficient time for the sample to pass through the screen to
ensure homogeneity. In these instances, experience has shown that Cyclotec or equivalent
high-speed grinders are preferable.
4.3 Most mechanical mills contribute some contamination of the sample with one or more
elements (Hood et al., 1944). The extent of contamination depends on condition of the mill
and exposure time (Jones and Case, 1990). Grier (1966) recommended use of stainless steel
for cutting and sieving surfaces to minimize contamination.
4.4 Routine maintenance should be performed on mills to ensure optimum operating conditions.
Cutting knives or blades should be maintained in sharp condition and in adjustment.
D. Storage
1. Principle
1.1 After particle size reduction and homogenization, samples should be stored in conditions that
will minimize deterioration and maintain sample integrity for weighing and follow-up
analytical work.
2. Apparatus
2.1 Airtight plastic storage containers.
2.2 Storage cabinet located in cool, dark, moisture-free environment

 

Plant Analysis Reference Procedures,p5

ผ่านทาง scsb-PlantAnalysis.

The publication can be downloaded from Plant Analysis Reference Procedures for the Southern Region of the United States.

2.3 Refrigerator.
3. Procedure
3.1 After grinding and homogenization, a representative subsample is taken from the ground
plant material for analyses and storage. The sample should be placed in a container that can
be securely sealed.
3.2 Containers should then be placed in a cool, dry place for storage.
3.3 For long term storage, ground samples should be thoroughly dried, sealed, and placed under
refrigerated conditions (4oC) until the required analysis can be completed.
4. Remarks
4.1 If samples are placed in a cool (4oC), dark, dry environment, storage life is indefinite (Jones
et al., 1991).
4.2 Coin envelopes can also be used for sample storage, however, somewhat greater care must be
exercised in handling to prevent absorption of moisture. Collecting the ground sample in the
envelope and immediately placing into a desiccator cabinet or desiccator will minimize
moisture absorption.
E. Organic Matter Destruction – Dry Ashing
Plant tissue samples previously dried, ground, and weighed are prepared for elemental analysis
through decomposition or destruction of organic matter. Extensive work has been done to evaluate published
methods and to develop new and improved procedures. The best overviews on organic matter destruction are
found in books by Gorsuch (1970) and Bock (1978) and in the review articles by Tolg (1974) and Gorsuch
(1976). Two commonly used methods of organic matter destruction include dry ashing (high temperature
combustion) and wet ashing (acid digestion) (Jones et al., 1991). Both methods are based on the oxidation of
organic matter through the use of heat and/or acids.
1. Principle
1.1 Dry ashing is conducted in a muffle furnace at temperatures of 500 to 550oC for 4 to 8 hours.
For tissue high in carbohydrates and oils, ashing aids (Horwitz, 1980) may be required to
achieve complete decomposition of organic matter. After ashing, the vessel is removed,
cooled, and the ash is dissolved in nitric (HNO3) and/or hydrochloric (HCl) acid. The vessel
is filled to volume and diluted as needed to meet range requirements of the analytical
instrument.
2. Reagents and Apparatus

 

Plant Analysis Reference Procedures,p6

ผ่านทาง scsb-PlantAnalysis.

The publication can be downloaded from Plant Analysis Reference Procedures for the Southern Region of the United States.

2.1 Muffle furnace with dual time and temperature control.
2.2 Fume hood.
2.3 Hot plate.
2.4 Porcelain or quartz crucibles, 30 mL.
2.5 Pyrex beakers, 50 mL.
2.6 Deionized water.
2.7 Hydrochloric acid (HCl), concentrated.
2.8 Nitric acid (HNO3), concentrated.
2.9 Sulfuric acid (H2SO4), 10%.
2.10 Magnesium nitrate [Mg (NO3)2 @ 6H2O], 7%.
2.11 Dilute aqua regia (300 mL HCl and 100 mL HNO3 in 1 L deionized H2O).
3. Procedure
3.1 Weigh 0.5 to 1.0 g of dried (80oC) plant material which has been ground (0.5 to 1.0 mm) and
thoroughly homogenized, into a high-form 30-mL porcelain or quartz crucible or 100-mL
pyrex beaker.
3.2 Place samples into a cool muffle furnace.
3.3 Set temperature control of the furnace to allow a gradual increase (2 hours) to the ashing
temperature (500 to 550oC) and maintain for 4 to 8 hours.
3.4 Turn furnace off, open door, and allow samples to cool.
3.5 Check the ash to determine extent of destruction. If a clean white ash is obtained, proceed
with step 3.9. If a clean white ash is not obtained, repeat step 3.1.
3.6 Moisten the tissue with concentrated HNO3.
3.7 Place the container on a hot plate and evaporate the HNO3 from the sample. Make sure the
residue is completely free of moisture before placing it into the muffle furnace.
3.8 Remove the container from the hot plate and repeat steps 3.2, 3.3, and 3.4 above.
3.9 Depending upon subsequent analytical procedures, the ash can be solubilized using the
appropriate acid and/or mixture of acids.

 

Plant Analysis Reference Procedures,p7

ผ่านทาง scsb-PlantAnalysis.

The publication can be downloaded from Plant Analysis Reference Procedures for the Southern Region of the United States.

4. Remarks
4.1 Critical factors in dry ashing procedures include selection of ashing vessel, sample number,
placement in furnace, ashing temperature, time, selection of acid to solubilize the ash, and
final volume (Jones and Case, 1990). The analyst has less latitude in choosing an ashing
temperature (Baker et al., 1964; Gorsuch, 1959, 1970, 1976; Isaac and Jones, 1972) than in
selecting the other parameters of the procedure. Placement of vessels and ashing time are
dependent on the type and number of samples. Selection of an ashing vessel, the solubilizing
acid, and final volume are dependent on the elements of interest and subsequent analytical
procedures. A number of combinations of these factors have been used successfully.
4.2 If a clean white ash remains after muffling, oxidation is complete and ashing aids are not
required.
4.3 Plant materials with high sugar or oil content (highly carbonaceous) may require an ashing
aid. Aids commonly used are 10% H2SO4, concentrated HNO3, and 7% Mg(NO3)2@6H2O
solutions. The latter is recommended when the tissue is to be assayed for S as SO4
2-.
Gorsuch (1970), Horwitz (1980), and Jones et al. (1991) provide details on the use of these
oxidizing aids.
4.4 Dry ashing is not recommended for plant materials that are high in Si. Dry ashing these
materials results in low micronutrient values, especially Zn.
4.5 Dry ashing results in lower Fe and Al values than wet ashing (Jones and Case, 1990).
F. Organic Matter Destruction-Wet Ashing
1. Principle
1.1 Wet digestion involves the destruction of organic matter through the use of both heat and
acids. Acids that have been used in these procedures include H2SO4, HNO3, and HClO4,
either alone or in combination. Hydrogen peroxide (H2O2) is also used to enhance reaction
speed and complete digestion. Most laboratories have eliminated the use of HClO4 due to
risk of explosion. Safety regulations require specially designed hoods where HClO4 is
utilized. Hot plates or digestion blocks are utilized to maintain temperatures of 80 to 125oC.
After digestion is complete and the sample is cooled, the vessel is filled to volume and
dilutions are made to meet analytical requirements.
2. Reagents and Apparatus
2.1 Hot plate.
2.2 Block digester.
2.3 Fume hood.
2.4 Nitric acid (HNO3), concentrated.

 

Plant Analysis Reference Procedures,p8

ผ่านทาง scsb-PlantAnalysis.

The publication can be downloaded from Plant Analysis Reference Procedures for the Southern Region of the United States.

2.5 Sulfuric acid (H2SO4), concentrated.
2.6 Hydrogen peroxide (H2O2), 30%.
2.7 200-mL tall-form beakers or digestion tubes.
2.8 Deionized water.
3. Procedure
3.1 Weigh 0.5 to 1.0 g of dried (80oC) plant material that has been ground (0.5 to 1.0 mm) and
thoroughly homogenized and place in a tall-form beaker or digestion tube.
3.2 Add 5.0 mL concentrated HNO3 and cover beaker with watch glass or place a funnel in the
mouth of digestion tube and allow to stand overnight or until frothing subsides.
3.3 Place covered beaker on hot plate or digestion tube into block digester and heat at 125oC for 1
hour. (Where elemental analysis is by ICP, the digestion time can be extended to 4 hours and
steps 3.5 and 3.6 omitted).
3.4 Remove beaker or digestion tube and allow to cool.
3.5 Add 1 to 2 mL 30% H2O2 and digest at the same temperature. Repeat heating and 30% H2O2
additions until digest is clear. Add additional HNO3 as needed to maintain a wet digest.
3.6 After sample digest is clear, remove watch glass or funnel and lower temperature to 80oC.
Continue heating until near dryness. The residue should be clear or white if digestion is
complete.
3.7 Add dilute HNO3, HCl, or a combination of the two acids and deionized water to dissolve
digest residue and bring sample to final volume depending upon requirements of subsequent
analytical procedures.
4. Remarks
4.1 Critical factors in wet digestion procedures include selection of the digestion vessel,
temperature and its control, time, the digestion mixture, and final volume. Selection of a
digestion vessel is dependent on the elements of interest and the heat source. Digestion
blocks have been developed (Tucker, 1974; Gallaher et al., 1975) and used successfully.
They shorten digestion time and allow very uniform temperature control. Time and
temperature are interrelated and are dependent on the digestion mixture. A number of ashing
mixtures have been utilized and include those reported by: Jones et al. (1991), Jones and
Case (1990), Wolf (1982), Parkenson and Allen (1975), Cresser and Parsons (1979), Zasoski
and Buran (1977), Adler and Wilcox (1985), Halvin and Soltanpour (1980), Zarcinas et al.
(1987), and Huang and Schulte (1985). Wet digestion procedures generally require greater
analyst supervision and intervention than dry procedures.
4.2 Nitric acid (HNO3) is used in most wet oxidation procedures. The addition of H2SO4 is used
to raise digestion temperature while HClO4 or 30% H2O2 are used to increase speed of

 

 
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