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ผ่านทาง scsb-PlantAnalysis.

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

3.1 Concentrated sulfuric acid, 18 M.
3.2 Standardized hydrochloric acid, (0.01 M) or sulfuric acid, 0.005 M.
3.3 Salt/Catalyst mixture – Mix 200 g of K2SO4, 20 g of cupric sulfate pentahydrate (CuSO4 @
5H2O) and 2 g of Se. The K2SO4 and/or CuSO4 @ 5H2O may need to be ground using a mortar
and pestle if the crystals are too large. It is important that the salt and catalyst be well mixed.
3.4 Sodium hydroxide solution, 10 M – Add 1600 g of NaOH to 2 L of carbon dioxide (CO2)-free
deionized water in a 4-L narrow neck polypropylene container. Stopper and cool the solution
before bringing it to a volume of 4 L. Protect the solution from contamination by
atmospheric CO2 with an ascarite guard tube at the air inlet.
3.5 Mixed indicator – Dissolve 0.01 g of bromocresol green and 0.07 g of methyl red in 100 mL
of ethyl alcohol (90%).
3.6 Boric acid indicator solution, 4% – Dissolve 40 g of H3BO3 in 800 mL of boiling deionized
water in a 2-L volumetric flask. Cool the solution and dilute to 1900 mL. Add 40 mL of the
mixed indicator solution. Carefully adjust the pH of this mixture with 0.1 M NaOH and 0.05
M H2SO4 until the solution turns a reddish purple color (pH 5.0) and then bring the solution to
2 L volume.
4. Procedure
4.1 The Kjeldahl method for determination of total N has been the subject of many modifications
according to the type of material to be analyzed. For instance, some plant materials do not
require a digestion temperature as high as that recommended in paragraph 4.4 for complete
digestion. The user may desire to alter digestion times, temperatures, or reagent proportions
for a specific application. Useful references for this purpose are Nelson and Sommers (1973,
1980) and Schuman et al. (1973).
4.2 Place sample (weight depends on N content) or standard in Kjeldahl digestion tube and add
1.1 g of salt/catalyst mixture.
4.3 Digest blanks containing only reagents with each set of samples.
4.4 Add 3 mL of concentrated H2SO4. Slowly heat to 200oC. Once the frothing has subsided,
bring the temperature up to 350 to 375oC and heat until the digest clears. Digest at 350 to
375oC for an additional 35 minutes to 1 hour past clearing.
4.5 Cool the digest and add 20 mL of deionized water. If solidification has occurred within the
digest, it is important to mix the tube contents using a vortex mixer to dissolve the solid.
4.6 Add 5 mL of H3BO3 indicator solution to a 50-mL flask and place the flask under the
condenser with the condenser tube below the surface of the indicator solution.
4.7 Add 20 mL of 10 M NaOH to the digested sample. Immediately transfer the tube to the
Kjeldahl distillation apparatus and begin distillation. Collect distillate until the level in the
H3BO3 flask has reached approximately 35 mL (usually 12 minutes).

ผ่านทาง scsb-PlantAnalysis.

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

4.8 Titrate the NH3 distilled into the H3BO3 solution using standard 0.01 M HCl or 0.005 M
H2SO4. The end point is reached when the solution goes to a pink color.
5. Calibration and Standards
5.1 To verify that the digestion procedure is sufficient to digest organic N in the plant sample,
primary N standards should be digested with each set of samples. Primary standards should
be of a known N content, high purity, and non-hygroscopic for ease of storage. An effective
series of standard reference materials (available from Hach Co., Loveland, CO) are some
derivatives of para-toluene sulfonate (PTSA); NH4-PTSA (requires no digestion), glycine-
PTSA (relatively easy to digest), and nicotinic acid-PTSA (very difficult to digest).
Reference standards are also provided by the National Institute of Standards and Technology,
Office of Standard Reference Materials, Gaithersburg, MD.
6. Calculations
6.1 %N = (T – B) x N x 1.401 where: T = mL of sample titrated
g sample B = mL of blank titrated
N = acid normality
7. Remarks
7.1 Samples should contain about 1 mg of N (no more than 5 mg). The sensitivity of the
procedure depends upon a number of factors, including weight of the sample, strength of the
acid, and accuracy of titration.
7.2 If the ratio of acid to salt is low at the end of the digestion step, a significant amount of NH3
can be volatilized during the digestion process (Bremner and Mulvaney, 1982). Other
situations which may cause N loss during digestion and should be avoided are: localized
heating in the digestion flask (temperatures above 410oC), and the use of 30% hydrogen
peroxide (H2O2) as an oxidant.
7.3 The time required for digestion will be affected by the catalyst, temperature, and type of plant
tissue. It is important to allow an equal length of additional time after the sample clears.
Jones et. al (1991) reported that as much as 10% of the organic N may not yet be converted to
NH4
+ at clearing.
7.4 Homogeneity of the sample is very important for greatest precision. For best results, dried
tissue should pass a 40-mesh sieve. In cases where sample size is less than 0.25 g, special
care should be taken to insure sample homogeneity (Jones et al., 1991).
7.5 Digested samples may be stored for several days provided samples are covered and placed in
a cool area.
7.6 Samples distilled into the H3BO3 solution should be titrated within a short time to avoid
absorption of atmospheric CO2.

ผ่านทาง scsb-PlantAnalysis.

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

8. References
Bremner, J.M., and C.S. Mulvaney. 1982. Nitrogen – Total. In A.L. Page et al. (ed.) Methods of soil
analysis. Part 2. 2nd ed. Agronomy 9:595-624.
Jones, J.B., Jr., B. Wolf, and H.A. Mills. 1991. Plant analysis handbook. p. 30-34. Micro-Macro
Pub., Athens, GA.
Nelson, D.W., and L.E. Sommers. 1973. Determination of total nitrogen in plant material. Agron. J.
65:109-112.
Nelson, D.W., and L.E. Sommers. 1980. Total nitrogen analysis of soil and plant tissues. J. Assoc.
Off. Anal. Chem. 63:770-779.
Schuman, G.E., M.A. Stanley, and D. Knudsen. 1973. Automated total nitrogen analysis of soil and
plant samples. Soil Sci. Soc. Am. Proc. 37:480-481.

ผ่านทาง scsb-PlantAnalysis.

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

Determination of Nitrogen in Plant Tissue Using
Continuous Flow, Segmented Stream Autoanalyzer
R. A. Isaac and W. C. Johnson, Jr.*
1. Principle of the Method
1.1 This is an instrumental method which involves delivering a liquid digest to an analytical
cartridge by means of a peristaltic pump. The sample is then combined with reagents and air
bubbles in a continuous moving stream, ultimately producing a color which is specific for the
analyte in the sample. The stream then flows to a photometer where the color intensity is
converted to an electronic signal and displayed on a chart recorder.
1.2 Plant tissue samples may be digested by either AOAC method 976.06G (Helrich, 1990) or the
method reported by Isaac and Johnson (1976) for the conversion of nitrogen compounds to
ammonia.
1.3 Sample digests are analyzed for ammonia on a continuous flow, segmented stream,
autoanalyzer, utilizing the Berthelot reaction.
1.4 Ammonia reacts with sodium phenoxide in the presence of sodium hypochlorite to form a
green colored complex.
1.5 Potassium sodium tartrate is added to the sample stream in order to prevent the precipitation
of heavy metal hydroxides.
2. Apparatus
2.1 Alpkem RFA300 system equipped with an ammonia cartridge, or a Technicon AutoAnalyzer
II system equipped with an ammonia cartridge, or any other equivalent continuous flow,
segmented stream autoanalyzer system.
3. Reagents
3.1 Potassium sodium tartrate.
3.2 Sodium hydroxide.
3.3 Disodium ethylenediamine tetraacetate.
3.4 Phenol.
1Professor, Analytical Chemistry and Extension Chemist, University of Georgia, Cooperative Extension Service,
Athens, GA 30605.

ผ่านทาง scsb-PlantAnalysis.

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

3.5 Sodium hypochlorite.
3.6 Ammonium sulfate.
4. Autoanalyzer So lutions
4.1 Potassium sodium tartrate solution – Dissolve 50 g potassium sodium tartrate, 28 g sodium
hydroxide, and 5 g disodium ethylenediamine tetraacetate in 1 L deionized water.
4.2 Sodium phenoxide solution – Dissolve 200 g sodium hydroxide in 500 mL deionized water,
let cool and slowly add 276 g phenol. Dilute to 1 L with deionized water and store in a dark
polyethylene bottle.
4.3 Sodium hypochlorite solution – Use a commercial grade bleach such as Chlorox (5.25%
NaOCl) without dilution.
5. Standards
5.1 2500 mg N L-1 – Weigh 11.79 g (NH4)2SO4 into a 1-L volumetric flask, dissolve and dilute to
volume with deionized water.
5.2 Working standards – To 50-mL volumetric flasks containing 20 mL deionized water and an
equivalent volume of digestion reagent, pipet 1, 2, 3, 4, 5, and 6 mL of 2500 mg L-1 N
standard. Allow to cool and dilute to volume with deionized water. When using a 250 mg
sample and diluted to 50 mL volume, these standards are equivalent to 1, 2, 3, 4, 5, and 6% N,
respectively.
6. Procedure
6.1 Turn on all system modules and engage the peristaltic pump.
6.2 Pump reagents through the system for 10 minutes before beginning analysis of samples and
standards.
6.3 Before analysis of samples and standards, inspect the system and verify consistent air bubble
patterns throughout the system and also verify that the stream is flowing smoothly.
6.4 Begin analysis of samples and standards.
6.5 When analysis is complete, place reagent lines in water and allow the system to run for 10
minutes.

ผ่านทาง scsb-PlantAnalysis.

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

7. Remarks
7.1 Nitrogen concentrations in plant tissue samples may be determined in a range of 0.10 to 6.00
%.
7.2 Isaac and Johnson (1976) reported an average difference of 0.02% N between an
AutoAnalyzer nitrogen method and AOAC method 2.049 (Horwitz, 1975) on eight plant
tissue samples.
7.3 Analysis time per sample is 60 seconds.
8. References
Helrich, K., ed. 1990. Official methods of analysis of the association of official analytical chemists.
15th Edition. pp 73-74.
Isaac, R. A., and Johnson, W. C. 1976. Determination of total nitrogen in plant tissue using a block
digestor. J. of the AOAC. 59:98-100.
Horwitz, W., ed. 1975. Official methods of analysis of the association of official analytical chemists.
12th Edition. pp 15-16.

ผ่านทาง scsb-PlantAnalysis.

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

Determination of Total Nitrogen in
Plant Tissue by Combustion
C. R. Campbell*
1. Principle of the Method
1.1 Modern elemental organic analysis (EOA) has evolved from the work of a number of
scientists and is based on measurements of one or more physical properties of oxidation gases
including pressure, light adsorption, or thermal and electrical conductivity (Pella, 1990).
Major obstacles of incompatibility between the combustion process and gas chromatography
separation of combustion products were overcome with the dynamic flash combustion
principle first introduced in 1973 (Pella and Colombo, 1973).
1.2 Flash combustion is achieved by dropping an organic sample into a quartz reactor (1030oC) in
which the helium carrier gas is enriched with oxygen. The quartz reactor consists of a layer
of chromic oxide (Cr2O3) and cobaltous-cobaltic oxide (Co3O4) coated with silver to
compliment oxidation and adsorb halogens and sulfur oxides, respectively.
1.3 The combustion gases are reduced in a copper column (650oC). Water is then removed using
a trap containing magnesium perchlorate [Mg(ClO4)2] or molecular sieve (3Å). Carbon
dioxide is removed in a second trap containing ascarite, Na2O, or LiOH.
1.4 For quantific ation, the nitrogen gas is carried to a chromatographic column (Porapak QS 2/m)
and then to a thermal conductivity detector. The resulting signal is passed to an electronic
recorder and integrator.
2. Apparatus
2.1 Sartorius 4504MP8 microbalance or equivalent.
2.2 Carlo Erba NA 1500 automatic N analyzer or equivalent.
2.3 Unisys PW 500 or equivalent IBM compatible computer.
2.4 Eager 100 Software for weight collection, instrument control, and integration.
2.5 Sperry 115 printer or equivalent.
2.6 Desiccator.
1Chief-Plant/Waste/Solution Advisory Section Agronomic Division, North Carolina Department of Agriculture,
Raleigh, NC 27611.

ผ่านทาง scsb-PlantAnalysis.

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

3. Reagents
3.1 Chromic oxide (Cr2O3).
3.2 Silvered cobaltous-cobaltic oxide (Co3O4/Ag layer).
3.3 Quartz wool.
3.4 Quartz turnings (SiO2).
3.5 Reduction copper (Cu).
3.6 Magnesium perchlorate [Mg(ClO4)2] or molecular sieve 3Å.
3.7 Tin capsules (Conroy 4001 or equivalent).
3.8 Na2O, LiOH, or ascarite.
3.9 Helium (99.996% purity).
3.10 Air (breathing quality).
3.11 Oxygen (99.996% purity).
3.12 NBS standard plant material with certified N value.
3.13 Acidanilide.
4. Procedure
4.1 Prepare and install columns for combustion and reduction chambers.
4.2 Prepare and install traps for H2O and CO2.
4.3 Check gas flow rates.
4.4 Preheat combustion and reduction chambers and turn on filament heat.
4.5 Check instrument settings for carrier gas flow and oxygen injection.
4.6 Run 3 to 4 conditioning samples.
4.7 Standardize for linear regression model using 4 to 5 acidanilide samples of varying weights to
cover the working range of nitrogen concentration in unknowns.
4.8 Weigh internal checks and unknowns.
4.9 Place internal checks and unknowns in autosampler and activate analytical procedure.
4.10 Capture data electronically or record results manually.

ผ่านทาง scsb-PlantAnalysis.

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

5 Remarks
5.1 Total nitrogen concentrations in organic materials can be determined over a range of 0.01 to
100.00%.
5.2 Results are equal or superior to those of traditional Kjeldahl procedures (Sweeney, 1989;
Bellomonte et al., 1987; Colombo and Giazzi, 1982). Accuracy varies with N concentration
and ranges from “10% deviation at 0.01% to “0.4% deviation at 50.0%. Reproducibility is
better than “0.1% of absolute value.
5.3 For best results, samples must be ground to pass at least a 1-mm screen size and thoroughly
mixed. Finer grinds improve precision and accuracy. A suitable grind is one which results in
a relative standard deviation (RSD) # 2.0% for ten successive determinations (Sweeney,
1989).
5.4 Sample size can range from 3 to 30 mg. Research does not indicate that sample size limits
accuracy and precision (Colombo and Giazzi, 1982) as long as sample preparation is
sufficient.
5.5 Samples must be weighed to 0.001 g to ensure accuracy and precision. Large sample
quantities are not necessary provided the sample has been properly homogenized and
blended. (Colombo and Giazzi, 1982).
5.6 Analysis time is one sample per 160 seconds.
5.7 Routine maintenance is approximately one half hour per day. One operator can run 100 to
150 samples per average work day.
6. References
Bellomonte, G., A. Constantini, and S. Giammarioli. 1987. Comparison of modified automatic
dumas method and the traditional Kjeldahl method for nitrogen determination in infant food. J.
Assoc. Off. Anal. Chem. 70:227-229.
Colombo, B., and G. Giazzi. 1982. Total automatic nitrogen determination. Am. Lab. 14:38-45.
Pella, E., and B. Colombo. 1973. Study of carbon, hydrogen, and nitrogen determination by
combustion-gas chromatography. Mikrochim. Acts. 697-719.
Pella, E. 1990. Elemental organic analysis. Part 1: Historical developments. Am. Lab. 22:116-125.
Sweeney, R. A. 1989. Generic combustion method for determination of crude protein in feeds:
Collaborative study. J. Assoc. Off. Anal. Chem. 72:770-774.

ผ่านทาง scsb-PlantAnalysis.

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

Determination of Nitrate Nitrogen in
Plant Samples by Selective Ion Electrode
W. H. Baker and T. L. Thompson*
1. Principle of the Method
1.1 The nitrate (NO3) electrode contains an internal reference solution in contact with a porous
plastic organophilic membrane. This membrane acts as a selective nitrate ion (NO3
-)
exchanger. When the membrane is exposed to NO3
-, a potential develops across the
membrane. This potential, E, is measured against a constant reference electrode potential, Eo.
1.2 The magnitude of E is dependent on the NO3 activity (ANO3) and can be described as the sum
of several individual potentials by the empirical equation:
E = Eo + [ S x log (ANO3 + EKai) ] + EJ [eq. 1]
where the potential across the liquid junction, EJ, represents the mobility of the cation and
anion between the outer sample solution and the reference electrode filling solution, the slope,
S (59.16 mV at 25oC), embodies the Faraday and gas constants, the valence of the ion, and the
solution temperature, and because the NO3 electrode is not completely specific, the selectivity
ratio, EKai, in principle, encompasses the error resulting from ions that are sensed by the
electrode falsely as NO3
-. The components between brackets in equation 1 form the basis of
the Nernst equation which describes the potential across the nitrate electrode membrane.
Assuming that EJ and temperature are constant between samples and there are no interfering
ions, the change in potential will be a direct linear function of ANO3:
E = Eo + [ S x log(ANO3) ] [eq. 2]
1.3 The relationship between ANO3 and concentration of NO3 (CNO3) is a composite function of
the solution ion concentration and their respective valences. This relationship is expressed as
ionic strength, I:
I = 1/2ECi Zi
2 [eq. 3]
where Ci is the concentration in moles L-1 and Zi is the valence of ion i.
1.4 In pure, dilute solutions ANO3 closely approximates CNO3. At higher ionic strengths, ANO3 will
decrease with respect to CNO3 due to the interaction of ions of opposite charge. The ratio of
ion activity to its concentration is termed the activity coefficient (gi):
gNO3 = ANO3 / CNO3. [eq. 4]
1.5 To avoid problems with standard and sample solutions having different ionic strengths, a
1Research Assistant Professor, Soil Test and Research Laboratory, University of Arkansas, Marianna, AR, and
Research Associate, Dept. of Agronomy, Iowa State University, Ames, IA.

ผ่านทาง scsb-PlantAnalysis.

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background salt solution (ionic strength adjuster, or ISA) of high ionic strength relative to
CNO3 can be added. This results in a reasonably constant gNO3 making ANO3 directly
proportional to CNO3 and stable between standard and sample solutions.
2. Apparatus
2.1 Nitrate electrode.
2.2 Double junction reference electrode (fill outer chamber with 0.02 M (NH4)2SO4 solution).
2.3 Specific ion meter or a pH/millivolt (mV) meter with readability to 0.1 mV.
3. Reagents and Solutions
3.1 Reference electrode filling solution – 0.02 M (NH4)2SO4. Add 2.64 g of (NH4)2SO4 in a 1-L
volumetric flask and fill to volume with deionized water. Note, chloride has a high selectivity
constant and significant contamination can occur if KCl is used as the internal filling solution.
3.2 Preservative solution – dissolve 0.1 g of phenylmercuric acetate in 20 mL of dioxane in a 100-
mL volumetric flask. Fill to volume with deionized water. Note, this solution is very toxic
and should not contact the skin.
3.3 500 mg nitrate-N L-1 stock solution – add 3.609 g of oven dried KNO3 into a 1-L volumetric
flask. Bring to volume with deionized water.
3.4 2 M ammonium sulfate ISA – add 26.4 g of (NH4)2SO4 to a 100-mL volumetric flask and fill
to volume with deionized water.
3.5 0.025 M aluminum sulfate + 10 mg nitrate-N L-1 extracting solution – add 32 g of Al2(SO4)3,
40 mL of 500 mg NO3-N L-1 stock solution, and 2 mL of preservative solution into a 2-L
volumetric flask. Bring to volume with deionized water.
3.6 40 mg nitrate-N L-1 standard – add 16 g of Al2(SO4)3, 80 mL of 500 mg NO3-N L-1 stock
solution, and 1 mL of preservative to a 1-L volumetric flask. Bring to volume with deionized
water.
4. Procedure
4.1 Weight out 0.10 g of dried plant tissue that has been ground to pass through a 20-mesh sieve.

ผ่านทาง scsb-PlantAnalysis.

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4.2 Add 30 mL of Al2(SO4)3 extracting solution and shake for 15 minutes. If deionized water is
used as the extracting solution, omit Al2(SO4)3 in the NO3 standard described in sections 3.5
and 3.6 and use 2 mL of the (NH4)2SO4 ISA solution for every 100 mL of standard and
sample solution.
4.3 Record meter reading.
5. Calibration and Standards
5.1 Standards are made up in the 0.025 M Al2(SO4)3 background solution. It is important that the
concentration of the samples be within the range of the standards. The concentration is
determined by comparison to the standards. For most potentials between 1 and 40 mg NO3-N
L-1, suitable results can be obtained using the 40 mg NO3-N L-1 solution in section 3.6 as the
high standard and using the 0.025 M Al2(SO4)3 + 10 mg NO3-N L-1 extracting solution in
section 3.5 as the low solution. The 0.025 M Al2(SO4)3 + 10 mg NO3-N L-1 extracting
solution should be set to read zero to subtract out the 10 mg NO3-N L-1 background in the
sample solutions.
5.2 The purpose of using 0.025 M Al2(SO4)3 as an extractant is to acidify the sample to a pH near
3 and provide a constant ionic strength background (Baker and Smith, 1969; Carlson and
Keeney, 1971). The Al in solution complexes organic acids and the lower solution pH
suppresses their ionization.
5.3 The extraction solution described in section 3.5 contains 10 mg NO3-N L-1 to maintain a
linear calibration curve, depress the effect of interfering anions, and shorten the electrode
equilibration time (Baker and Smith, 1969). Errors will increase rapidly as sample NO3
becomes small compared with the background NO3 in the extracting solution.
6. Calculations
6.1 The sample meter concentration reading should be multiplied by 300 to correct for dilution.
7. Remarks
7.1 The NO3 electrode has a working range of 0.14 to 1400 mg NO3-N L-1. Deviation from linear
calibration curves will begin at NO3-N concentrations below 1.40 mg L-1. Above 140 mg
NO3-N L-1, effects due to differential cation and anion mobilities at the reference electrode
interface can significantly bias ANO3 measurement.
7.2 Additionally, some salts may become fixed into the electrode membrane at high salt
concentrations resulting in poor performance.
7.3 Serious interference occurs in the presence of perchlorate (HClO4
-), chloride (Cl-), phosphate
(H2PO4
-, HPO4
2-, HPO4
3-), and nitrite ions (NO2
-). In plant tissue, Cl- is the main concern.
Orion Research, Inc. (1986) offers a more complete list of common interfering ions and their
respective selectivity constants.
7.4 Electrode measurements reproducible to +2% can be obtained using a well calibrated

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instrument for solutions in the range of 14 mg NO3-N L-1 with temperature fluctuations
within +1oC (Orion Research, Inc., 1986).
7.5 Plant concentrations of most interfering ions will be too low to adversely affect the
determination of NO3-N. However, where Cl- is a concern the inclusion of a buffer solution
containing 0.01 M Ag2SO4 in the extracting solution can be used to precipitate Cl- (Mills,
1980).
7.6 All samples should be analyzed within half a day of extraction to ensure measurement
uniformity and avoid any bacterial degradation of NO3 in the sample.
7.7 The NO3 electrode should soak in a 0.01 M NO3 solution between each analysis period.
8. References
Orion Research, Inc. 1986. Model 93-07 nitrate electrode instruction manual. Boston, MA.
Mills, H.A. 1980. Nitrogen specific ion electrodes for soil, plant, and water analysis. J. Assoc. Off.
Anal. Chem. 63:797-801.
Baker, A.S., and R. Smith. 1969. Extracting solution for potentiometric determination of nitrate in
plant tissue. J. Agric. Food Chem. 17:1284-1287.
Carlson, R.M., and D.R. Keeney. 1971. Specific ion electrodes: Techniques and uses in soil, plant,
and water analysis. p. 39-63. In L.M. Walsh (ed.) Instrumental methods for analysis of soils and plant
tissue. Soil Sci. Soc. Am., Madison, WI.

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Determination of Phosphorus in Plant
Tissue by Colorimetry
K. P. Moore *
1. Principle of the Method
1.1 An acidified solution of ammonium molybdate containing ascorbic acid and antimony is
added to a digested plant tissue sample. The phosphate in the plant tissue sample reacts with
the acidified ammonium molybdate to form an ammonium molydiphosphate complex. A
blue colored solution is generated from the reduction of the ammonium molydiphosphate
complex by ascorbic acid. The intensity of the blue color is proportional to the amount of
molybdophosphorus present. Antimony potassium tartrate accelerates the color development
and stabilizes the color for several hours.
1.2 The amount of light absorbed by the solution at 660 nm is measured with a
spectrophotometer.
1.3 The procedure is described by Murphy and Riley (1962) and Watanabe and Olsen (1965).
2. Apparatus
2.1 Analytical balance.
2.2 150-mL beakers or 50-mL porcelain crucibles.
2.3 Wet oxidation hot plate and hood or furnace.
2.4 1-L and 2-L volumetric flasks.
2.5 100-mL volumetric flasks.
2.6 1:100 dilutor dispenser.
2.7 Test tubes for color development.
2.8 Visible spectrophotometer with 1-cm light path. Spectrophotometers equipped with a
flowcell accessory can be used to read samples directly from the color development test
tubes.
2.9 Pipettes for making reagents, standards, and any appropriate dilutions.
1Supervisor, Plant & Feed Analysis Lab, Agricultural Service Laboratory, Clemson University, Clemson, SC.

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3. Reagents
3.1 Nitric acid (HNO3) – for wet ashing.
3.2 Perchloric acid (HClO4) – for wet ashing.
3.3 0.1 M hydrochloric acid (HCl) – for dry ashing.
3.4 Acid molybdate stock solution – In a 2-L volumetric flask, dissolve 125 g ammonium
molybdate [(NH4)6Mo7O24@4H2O] in 400 mL distilled water by heating to 60oC. Allow to
cool, then dissolve 2.9 g antimony potassium tartrate [K(SbO)C4H4O6@1/2H2O] in the
molybdate solution. Place the flask in an ice bath and slowly add 1500 mL concentrated
sulfuric acid (H2SO4). Cool the mixture in the ice bath and slowly dilute to volume with
distilled water. Store in a brown bottle at 4 oC.
3.5 Ascorbic acid stock solution – Dissolve 211.2 g ascorbic acid (C6H8O6) in 1500 mL distilled
water and dilute to 2 L with distilled water. Store in a brown bottle at 4 oC.
3.6 Working solution (prepare fresh daily) – Add 20 mL of the acid molybdate stock solution and
10 mL of the ascorbic acid stock solution to 800 mL distilled water, then dilute to 1 L with
distilled water.
3.7 Standards – To make 1,000 ppm phosphorus stock solution, dissolve 4.3937 g of dried
monopotassium phosphate (KH2PO4) in distilled water then dilute to 1 L. To make 20, 40,
60, and 80 mg P L-1 standards, pipet 2, 4, 6, and 8 mL, respectively, of the 1000 mg L-1 stock
solution into separate 100-mL volumetric flasks and dilute to volume with distilled water.
4. Procedure
4.1 Digestion – Weigh 1.0000 g + 0.0005 g of dried and ground plant tissue into 150-mL beakers
or 50-mL porcelain crucibles. Digest samples using the wet oxidation procedure or the dry
ashing procedure, respectively. Quantitatively transfer samples into 100-mL volumetric
flasks and dilute with distilled water.
4.2 Color Development – Using a dilutor-dispenser, dilute the samples and the 20, 40, 60, and 80
mg P L-1 standards 1:100 with the working solution. Allow color to develop for at least 30
minutes before reading. Read the concentration at 660 nm with a visible spectrophotometer.
5. Calibration and Standards
5.1 To calibrate the spectrophotometer for routine analysis, use the working solution (see 3.6) as
the blank and the developed 0.80 mg P L-1 standard to establish the slope of the line. To
check for linearity, read the developed 0.20, 0.40, and 0.60 mg P L-1 standards. If the sample
concentration lies above the linear working range, dilute the samples appropriately.



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