Hendrik Poorter, Yvonne de Jong-Van Berkel
Hendrik Poorter IBG-2, Forschungszentrum Jülich
Yvonne de Jong-Van Berkel, Ecophysiology of Plants, Faculty of Science, Utrecht University
Combustion of dried plant material in a muffle furnace oxidizes the organic matter. Minerals remain, as well as oxides of NO3– and organic acids. Upon cooling these oxides turn into carbonates. The carbonates can be determined acidimetrically and this value is called the ash alkalinity. When the nitrate content of the same sample is determined as well, the organic acid content and mineral content can be calculated.
- Sodium hydroxide, 0.05 M
- Hydrochloric acid, 0.05 M
- Methyl red in 96 % ethanol (indicator)
- Porcelain crucibles
- Muffle furnace
- pH meter
- Vials of 20-25 ml
Determination ash content
- Ensure instructions for use of the muffle furnace are carefully followed
- Weigh the porcelain crucibles
- Weigh about 100 mg of dried plant material in the crucibles
- Put the crucibles in a muffle furnace (combustion oven) at a temperature of 550∘C for 6 h
- Let the crucibles cool down in a desiccator
- Weigh the crucibles to determine the ash content
Determination ash alkalinity
- Preparation of 0.05 M HCI: add 1.25 ml of 12 M HCl (= 37% HCl) to ∓100 ml deionized water and fill up to 300 ml
- Preparation of 0.05 M NaOH: dissolve 1 gram NaOH in deionized water and fill up to 500 ml.
(If you are not able to weigh out exactly 1 g NaOH, you have to convert the M of NaOH to a precise value by titration against HCl)
- Add known weight of ash to a vial with 10 ml of 0.05 N HCl. Rinse the crucible with 3 ml deionized water (It is possible to store the samples at 4∘C)
- Boil the solution for max 30 secs to remove the carbonate
- Cool down to room temperature
- Add 2-3 droplets of methyl red (as an indicator)
- The ash alkalinity is determined by a titration of the acid with 0.05 N NaOH; the colour changes from red to yellow at pH 5.5-6.0
The more alkaline the ash, the less NaOH you need to add to bring back the pH to 6 after addition of the acid:
The mineral fraction equals the ash fraction, after subtracting the weight of carbonate (result of the oxidation of both organic acids and nitrate), and adding the weight of nitrate determined separately:
The amount of organic acids can be calculated by correcting ash alkalinity for the oxides originating from nitrate:
AA = Ash alkalinity (eq g-1)
Ash = Ash content (mg g-1)
OA = Organic Acids (meq g-1)
Min = Minerals (mg g-1)
M = Molarity NaOH/HCl
Neq = Nitrate (meq g-1)
Nw = Nitrate (mg g-1)
Notes and troubleshooting tips
- Also include some blanks in the ash alkalinity determination to check whether the molarity of HCl and NaOH are exactly equal
- By determining the exact composition of the organic acid fraction, it is possible to convert organic acid content from meq g-1 to mg g-1. You may use 1 eq. = 62.5 g as a first approximation (determined on grasses grown in hydroponics).
- Ash: 10 – 200 mg g-1 (DW) (Poorter & Bergkotte 1992)
- Ash Alkalinity: 0.5 – 3 meq g-1 (DW)
- Minerals: 30 – 170 mg g-1, much lower in woody stems (Poorter & Villar 1997)
Dijkshoorn, W. 1969. De ionenbalans en de groei van de plant. Stikstof 6: 26-33.
Jungk, A. 1968. Die Alkalität der Pflanzenasche als Mass für den Kationenüberschuss in der Pflanze. Pflanzenernähr. Bodenk. 120: 99-105.
Poorter, H. & Bergkotte, M. 1992. Chemical composition of 24 wild species differing in relative growth rate. Plant Cell Environ. 15: 221-229.
Poorter, H & Villar, R. 1997. The fate of acquired carbon in plants: chemical composition and construction costs. In: Plant Resource Allocation. Academic Press, pp 30-72.