Agronomic Services - Plant Tissue Analysis Methods
Introduction
The NCDA&CS Plant Tissue Analysis Lab performs the following analyses based on standard industry methods:
- Nitrogen (N) and carbon (C)
- Phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), iron (Fe), manganese (Mn), zinc (Zn), copper (Cu), boron (B), molybdenum (Mo), sodium (Na), aluminum (Al), arsenic (As), cadmium (Cd) chromium (Cr), nickel (Ni), lead (Pb) and selenium (Se)
- Nitrate-nitrogen (NO3-N) and chloride (Cl-)
Minimum Sample Masses
No matter how precise and accurate an analytical method is, meaningful results are only as good as the sample itself. The most significant cause of poor statistical results in plant analysis is due to imprecise sample collection and preparation rather than analytical measurement.
Clients should endeavor to submit the recommended amount of plant material (Table 1). Research samples automatically receive quality control rechecks on 10% of their samples at no additional charge, but only if the recommended masses are submitted. Also, human error occasionally leads to the need to re-analyze a sample. Without sufficient material, this results in data loss for the researcher.
Where recommended amounts are not possible, please note the minimum dried, ground plant material required to perform each analysis.
Sample Test | Minimum mass | Recommended mass | Analytical Method | Reference |
|---|---|---|---|---|
N, C | 20 mg | 50 – 100 mg | Oxygen combustion (Dumas method) | AOAC 972.43; Campbell 1992 |
| P, K, Ca, Mg, S, Fe, Mn, Zn, Cu, B, Na, Al | 0.20 g | 0.5 g | Acid digestion; ICP-OES | Donohue and Aho 1992; EPA 200.7 |
| Mo, As, Cd, Cr, Ni, Pb, Se | 0.50 g | 1.0 g | Acid digestion; ICP-OES | Donohue and Aho 1992; EPA 200.7 |
NO3-N | 0.10 g | 0.25 g | Acetic acid extraction; Flow Injection Analysis | Miller 1998; EPA 353.2; FIA NO3-W-1-1 |
Cl- | 0.10 g | 0.25 g | Acetic acid extraction; Flow Injection Analysis | Miller 1998; EPA 325.2; FIA Cl-W-1-1 |
Methods
Upon receipt, samples are examined for condition (e.g., mold, inadequate mass for analysis) and correct plant part. Depending upon the crop, sufficiency ranges may be based on leaf blade only, petiole only or the whole leaf (blade plus petiole). Where sufficiency ranges are based on the leaf blade only or petiole only, it is recommended that the petiole be detached in the field so that nutrients are not moving from the petiole into the leaf blade tissue during shipment to the lab. If the client has not done so for crops where it is recommended to detach petioles, the lab detaches the petioles upon receipt.
Prior to homogenization of plant material by grinding, samples are dried overnight (12–24 hr) at 80oC. Each sample is then processed through a stainless-steel grinder with a 20-mesh (1 mm) screen (Campbell and Plank 1992). Most samples are ground on a cutting-grinding mill (IKA Works, Inc.; Wilmington, NC), but large volume and/or coarse samples are ground on a Fritsch Pulverisette 19 cutting mill (Fritsch USA, Inc. Idar-Oberstein, Germany). Small mass samples (< 5 g, wet weight basis) are ground on a tube mill without a screen (IKA Tube Mill 100; IKA Works, Inc.; Wilmington, NC).
The dried, ground plant material is stored at room temperature in a 7-dram plastic snap cap vial (~26 cm3) until analysis. Research samples are stored for one calendar year and grower samples for six weeks from date of sample receipt.
Total nitrogen and total carbon are determined by oxygen combustion gas chromatography with subsequent quantification by thermal conductivity detector (AOAC 1990b; Campbell 1992). Total nitrogen is analyzed using a Thermo Scientific FlashSmart EA Combustion Nitrogen/Protein Analyzer (CE Elantech Instruments; Lakewood, NJ) on a 49-51 mg aliquot of dried, ground plant material. For samples also requiring Total C, Total N and Total C are determined using a Thermofinnigan Flash EA1112 (CE Elantech Instruments; Lakewood, NJ) on an 8-10 mg aliquot of dried, ground plant material. Nitrogen content is analyzed as part of the standard analysis. Carbon is measured only by request. Results are reported in % on a dry weight basis.
Total concentrations of P, K, Ca, Mg, S, Fe, Mn, Zn, Cu, B, Na, Al, Ni, Cd, Pb, As, Cr, Se, and Mo are determined with Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) (Spectro Arcos EOP and Arcos II EOP, Spectro Analytical: A Division of Ametek; Mahwah, NJ) (Donohue and Aho 1992; adapted USEPA 2001), after closed-vessel nitric acid (HNO3) digestion in a microwave digestion system (MARS 6 Microwaves; CEM Corp.; Matthews, NC).
For most elements, a 0.5 g dried/ground aliquot is digested in 10 mL 15.6N HNO3. When heavy metals (Ni, Cd, Pb, As, Cr, Se) or Mo are requested, a 1.0 g aliquot of dried/ground sample is digested in 15 mL or 10 mL HNO3, respectively. Samples are digested for 30 minutes at 200°C in a microwave (Plant Materials method; CEM), and the digested sample volume is brought to 50 mL with deionized water and then filtered through pre-folded Advantec #2 filter paper (Folded Filter Paper, Albuquerque, NM). Elements are measured at the wavelengths listed in the detailed Plant Tissue Analysis Methods guide.
Nitrate-nitrogen (NO3-N) is extracted from plant tissue with 2% acetic acid (25 mL) on a 0.25 g dried/ground aliquot of sample (Miller 1998). The extract is filtered, and NO3-N is determined by cadmium reduction, where nitrate is reduced to nitrite with copperized cadmium, under alkaline conditions. The NO2-N concentration (that originally present plus reduced nitrate) is determined by diazotizing with sulfanilamide and coupling with N-(1-Naphthyl) ethylenediamine dihydrochloride to form a magenta-colored azo dye which is measured at 520 and 600 nm (USEPA 1993; FIA NO3-W-1-1).
NO3-N is quantified by flow injection analysis (FIAlyzer-1000, FIA Lab; Seattle, WA). Nitrate-nitrogen (NO3-N) and nitrite-nitrogen (NO2-N) are reported as NO3-N in mg kg-1on a dry-weight basis. Nitrate-nitrogen is analyzed on all strawberry and cotton samples and on other crops by request.
Chloride is extracted from plant tissue with 2% acetic acid (25 mL) on a 0.25 g dried, ground aliquot of sample (Miller 1998). The extract is filtered and then used for chloride determination by the thiocyanate displacement method by the formation of soluble mercuric chloride. The liberated thiocyanate forms a red colored complex with ferric iron ions also present in solution, which is measured at 480 nm (Standard Methods; FIA CL-W-1-1).
Cl is quantified by flow injection analysis (FIAlyzer-1000, FIA Lab; Seattle, WA). Results are expressed in mg kg-1 on a dry-weight basis.
Association of Official Analytical Chemists. (1990). AOAC 972.43: Microchemical determination of carbon, hydrogen, and nitrogen. In: Official methods of analysis. Volume 1. 15th ed.
Campbell CR. (1992). Determination of total nitrogen in plant tissue by combustion. In:
Campbell CR, Plank CO. (1992). Sample preparation. In:
Donohue SJ, Aho DW. (1992). Determination of P, K, Ca, Mg, Mn, Fe, Al, B, Cu, and Zn in plant tissue by inductively coupled plasma (ICP) emission spectroscopy. In:
FIA Lab. CL-W-1-1. Method for Chloride Determination by Ferric Thiocyanate, Version 7.
FIA Lab. NO3-W-1-1. Method for Nitrate Determination, Version 2.
Miller, R.O. (1998). “Extractable Chloride, Nitrate, Orthophosphate, Potassium, and Sulfate-Sulfur in Plant Tissue: 2% Acetic Acid Extraction,” Handbook of Reference Methods for Plant Analysis, p.115-118.
United States Environmental Protection Agency. (2001). Method 200.7: Trace elements in water, solids, and biosolids by inductively coupled plasma–atomic spectrometry, revision 4.4.
United States Environmental Protection Agency. (1993). Method 353.2, Revision 2: Methods for Determination of Nitrate-Nitrite Nitrogen by Automated Colorimetry.
United States Environmental Protection Agency. (1978). Method 325.2: Methods for the Chemical Analysis of Water and Wastes. Chloride (Colorimetric, Automated Ferricyanide AAII).
Permissions
Researchers with a Cooperative Research Project Agreement (CRPA) with NCDA&CS Agronomic Services are permitted to include these method descriptions, as written here, in journal publications.