Method 81 Determination of ammoniumN in silage

A distillation method is given in MAFF/ADAS (1986, pp. 168-169), but a selective ion electrode method will be described below.

Reagents.

• Alkali reagent (disodium EDTA, 0.1 M + sodium hydroxide, 2 M) - weigh 80 g NaOH and 37.2 g disodium EDTA into a beaker and dissolve in ammonia-free water. Transfer to a 1-l volumetric flask, and when cool make up to the mark and mix.

• Ammonia-free water - add about 6 ml sulphuric acid (approx. 98% m/m H2SO4) to 2 l water and distil off sufficient ammonia-free water, topping up the boiling container with more water as necessary.

• Stock standard solution, 1000 pg ml-1 of ammonium-N - dissolve 0.955 g NH4Cl (previously dried at 102°C for 1 h) in ammonia-free water and make up to 250 ml and mix.

• Working standard solutions, 0-200 pg ml-1 of ammonium-N - pipette 0, 1, 2.5, 5, 10, 15 and 20 ml of the stock standard into a series of 100-ml volumetric flasks to obtain standards of 0, 10, 25, 50, 100, 150 and 200 pg ml-1 of ammonium-N. Note, solutions of standards and samples should be equilibrated to the same (room) temperature before measurement.

© 2002 CAB International. Methods in Agricultural Chemical Analysis: a Practical

Handbook (N.T. Faithfull)

Procedure. Weigh approx. 20 g fresh silage into a 250-ml wide-mouth high-density polyethylene screw-cap bottle. Add 100 ml ammonia-free water and shake for 1 h. Filter through a 150-mm Whatman No. 1 filter paper. Pipette 20 ml of the extract into a 50-ml beaker containing a magnetic stirring bar and add 2 ml of the alkali reagent. Immediately stir for exactly 1 min and insert the ammonia electrode. (Note: if the electrode has been stored in 0.1 M NH4Cl solution, it should be rinsed thoroughly before use.) Very low readings may take several minutes to stabilize. Repeat the determination using standards made up using ammonium chloride. Compare sample readings with the standard curve to determine the concentration of ammonium-N in the extract solutions. Note: the standard curve is prepared by plotting on semilog graph paper (e.g. Chartwell Graph Data Ref. 5231), which is Log 3 Cycles for the shorter x-axis (concentration) and mm, V> and 1 cm for the y-axis (mV). The greater the concentration, the greater the negativity in mV; it may therefore be better for the y-axis to go from zero at the bottom to -200 mV at the top of the axis. The sensitivity is quoted as about 56 ±3 mV per decade (= one log cycle, e.g. from 10 to 100 pg ml-1 ammonium-N) at 25°C. The graph is nearly a straight line, or slightly concave. A plot on ordinary graph paper is very convex and difficult to read accurately.

Calculation. Let the pg ml-1 ammonium-N be y, then this is 100y pg 100 ml-1, or y/10 mg 100 ml-1 of extract solution per 20 g fresh silage. Therefore the weight of ammonium-N per 100 g fresh silage is: (100/20 ) x y/10 mg, or 0.5y/1000 % ammonium-N, which is y/2000 % ammonium-N.

This must next be expressed in terms of DM. If the moisture content is m, then this becomes:

Finally, this is conventionally expressed as a percentage of the total N % in DM. The final expression for ammonium-N as a percentage of total-N in DM is therefore:

y/2000 x (100/100 - m) x (100/total-N), which reduces to

A typical sample gave a reading of -148 mV, equivalent to 45.5 pg ml-1 ammonium-N. The moisture content was 66.46%, and the total-N was 1.83% in DM. Substitution in the above equation gives: 45.5 x 5/(100 -66.46)(1.83) = 227.5/61.378 = 3.71% ammonium-N as a percentage of total-N in DM.

Interpretation. The ammonium-N expressed as a percentage of total-N should not exceed about 11% for a good fermentation. Levels over 15% reduce palatability and can reduce voluntary intake.

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