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, 1/ 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.

Method 8.2. Determination of moisture in silage

See Chapter 4, 'Water content in silage'. The subjection of silage to oven drying causes loss of volatile components as well as water, resulting in overestimated moisture content. One way of minimizing this effect is to distil the fresh silage in the presence of toluene. Titration of the acidic distillate with 0.1 M NaOH enables a correction to be made for the volume of the volatile acids. A suitable method is given in MAFF/ADAS (1986, pp. 85-87) where about 70 g silage plus 400 ml toluene is heated in a 1-l round-bottomed flask, the distillate collecting in a specially made Dean and Stark receiver. The modified procedure is described below and in Faithfull (1998); it uses only 10 g samples, which are distilled with 100 ml toluene from a 250-ml flask. The aqueous distillate is collected in a standard Quickfit® Dean and Stark receiver fitted with a Rotaflo® stopcock permitting easy release of the water before titration. The apparatus is shown in Fig. 8.1, and captions refer to Quickfit® Part Nos.

Reagents.

• Phenolphthalein indicator, 0.1% (m/v) - dissolve 0.1 g phenolphthalein in

100 ml 95% ethanol.

C1/12

C1/12

WE 6/23/10T

FR 250/5S

Fig. 8.1. Modified Dean and Stark apparatus for small silage samples.

FR 250/5S

Fig. 8.1. Modified Dean and Stark apparatus for small silage samples.

Procedure.

distillation. Weigh 10.0 g fresh or frozen silage into a beaker. Immediately replace the rest of the sample into the deep-freeze to prevent loss of volatiles. Transfer to a 250-ml round-bottomed flask, supported on a cork or rubber ring. Add 100 ml toluene, place in a heating mantle of the correct size, and connect to the 10-ml Dean and Stark pattern receiver/condenser assembly. Turn the energy controller on full to bring to boiling, then adjust to give a steady boil. After 20 min, and then after 5-min intervals, record the volume of aqueous phase in the receiver until two identical values are obtained. Dislodge any droplets adhering to walls with a glass rod or gentle rocking. Switch off the heater and allow the receiver to cool before recording the final volume. Put a black card behind the receiver to show the meniscus clearly. Run off most of the aqueous phase into a 50-ml beaker. Pipette 5 ml of the aqueous phase into a 50-ml conical flask for titration to determine the acid content.

titration. Add 20 ml ethanol to the 5 ml aqueous phase and 5 drops phe-nolphthalein indicator. Pipette into the flask 10 ml 0.05 M sodium hydroxide and titrate with the same solution from a 10-ml burette after noting the initial volume. Note: Read the burette to 0.02 ml accuracy. Titrate to the first permanent pink colour; this fades due to CO2 from the air, so don't delay. Add the 10 ml initially pipetted into the flask to the volume delivered from the burette to obtain the total titre (probably in the range 13-18 ml).

Calculation. The dry matter (g kg-1) is calculated according to the formula:

110,

DM = dry matter g kg-1 m = mass of silage sample (g)

V = volume of aqueous distillate f = factor (0.00555)

t = titre 998 = acid:water density correction

Microsoft® Excel Program. It is usual to use PC software such as Microsoft Excel to perform repetitive calculations such as the above. This enables a tabular printout and the generation of charts. The layout of the data sheet is shown in Fig. 8.2. The first four columns of data are entered directly. The fifth column is a correction of the titration using the actual strength of the sodium hydroxide used compared to what the result would have been if the concentration had been exactly 0.05 M. Thus, the equation to be entered in the cell to convert the previous column's value is in our case:

where G2 is the previous column's cell, 0.048 is the molarity of the acid used, and 0.05 is the molarity to which the titre is being corrected.

Sample No. Pit 1/

Mass of Silage(M)

Volume of Water (V)

Vol. ~.05M NaOH

Vol. .05M NaOH (T)

DM of Silage g/kg-1

Moisture in Silage %

1

10.00

7.70

21.30

20.45

240.26

75.97

2

10.00

7.25

19.02

18.26

283.78

71.62

3

10.00

7.30

19.48

18.70

279.02

72.10

4

10.00

7.25

19.76

18.97

284.07

71.59

5

10.00

7.25

22.50

21.60

285.12

71.49

6

10.00

7.40

17.20

16.51

268.25

73.18

7

10.00

7.70

18.70

17.95

239.20

76.08

8

10.00

7.50

14.26

13.69

257.19

74.28

9

10.00

7.60

14.70

14.11

247.46

75.25

10

10.00

7.35

16.64

15.97

272.97

72.70

11

10.00

7.20

15.68

15.05

287.44

71.26

12

10.00

7.60

18.68

17.93

249.07

75.09

13

10.00

7.90

17.66

16.95

219.00

78.10

14

10.00

7.70

15.66

15.03

237.95

76.20

15

10.00

7.50

16.92

16.24

258.25

74.18

16

10.00

7.15

17.94

17.22

293.25

70.67

17

10.00

7.50

15.16

14.55

257.55

74.25

18

10.00

7.45

14.64

14.05

262.29

73.77

19

10.00

7.65

14.72

14.13

242.52

75.75

20

10.00

7.80

16.38

15.72

228.35

77.16

21

10.00

7.45

15.20

14.59

262.51

73.75

22

10.00

7.75

16.08

15.44

233.18

76.68

23

10.00

7.60

13.54

13.00

246.99

75.30

24

10.00

7.30

15.00

14.40

277.28

72.27

Penglais

-irst Cut,

First Sampling

DM (dry matter g/kg)

38 cm

76 cm

114 cm

152 cm

190 cm

228 cm

Sampling

1

240.26

283.78

279.02

284.07

285.12

268.25

Position

2

239.20

257.19

247.46

272.97

287.44

249.07

3

219.00

237.95

258.25

293.25

257.55

262.79

4

242.52

228.35

262.51

233.18

246.99

277.28

Moisture%

38 cm

76 cm

114 cm

152 cm

190 cm

228 cm

Sampling

1

75.97

71.62

72.10

71.59

71.49

73.18

Position

2

76.08

74.28

75.25

72.70

71.26

75.09

3

78.10

76.20

74.18

70.67

74.25

73.77

4

75.75

77.16

73.75

76.68

75.30

72.27

305.90

299.26

295.28

291.64

292.30

294.31

38 cm

76 cm

114 cm

152 cm

190 cm

228 cm

Av. Moisture%

76.48

74.82

73.82

72.91

73.08

73.58

Fig. 8.2. Microsoft® Excel data sheet for moisture in silage from the Penglais third-cut, first sampling.

The next column converts the various data to dry matter of silage g kg-1. The formula to be entered in the first cell of this column is:

where E2 is the cell referring to the volume of aqueous distillate and I2 is the corrected volume of 0.05 M sodium hydroxide.

There are also other tables on the data sheet that allow the easier production of charts. They refer to DM and moisture at each depth and sampling position, also the average moisture for each depth.

Interpretation. The interpretation of the effects of moisture in silage is notoriously difficult. Moisture is essential for the proliferation of desirable microorganisms, but an excess will encourage the growth of undesirable types (Woolford, 1984). The DM range most favourable to the silage fermentation has been suggested as 200-250 g kg-1 and the optimum about 240 g kg-1.

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