Aciddigestion of plant materials

The original method for the determination of nitrogen by sulphuric acid-digestion was published by Kjeldahl in 1883 and fully described by Burns (1984). Many modifications have since been made with various catalysts and acid mixtures.

The digestions can be carried out in up to 40-place multiple heating units using specialized glassware which is commercially available; some suppliers are listed below: Digestion systems:

Buchi Labortechnik AG: Gerhardt UK Ltd: Foss (Digestor 2000 System): Distillation systems:

Buchi Labortechnik AG: Foss (Kjeltec® 2300 Analyzer Unit): Gerhardt UK Ltd We have devised a method enabling the digestion of up to 152 samples at a time, and with the wearing of some essential personal protective equipment (PPE), it has proved successful for over 32 years (Faithfull, 1969).

Acid-digestion unit

The major expense is the hotplate, which has to have a sufficiently large working surface area and be able to sustain a temperature of 310°C. A suitable hotplate is the Gerhardt HC 63, nominal voltage 400 VAC, 4800 W, working area 650 x 300 mm, and a maximum temperature of 400°C ± 5°C. In the UK this is available from:

C. Gerhardt UK Ltd, Unit 5, Avonbury Court, County Road, Brackley, Northants. NN13 7AX. Tel. +44 (0) 1280 706772; Fax. +44 (0) 1280 706088 Other suitable hotplates are available from S & J Juniper & Co.: On the centre of the work surface are positioned two aluminium blocks, 440 x 100 x 100 mm (w x d x h), with the bottom surface machined flat to ensure good thermal contact with the hotplate. These are each drilled with 17 mm diameter holes to a depth of 86 mm and arranged in four rows of 19 holes. Thus each block accommodates 76 digestion tubes. These tubes are 150 mm long and 16 mm diameter, heavy wall (BS 3218) borosilicate glass rimless type; they are supplied by Fisher as TES-674-150S. The exposed areas of the work surface may be covered with a heat-resistant insulating material.

The whole unit is accommodated in a fume cupboard fitted with a scrubber unit to remove the acidic fumes before emission to the atmosphere. The constructional materials of the fume cupboard should be able to withstand the heat radiated from the hotplate and heating blocks. A digestion tube containing a 350°C thermometer with the bulb embedded in a 2-cm layer of sand occupies one hole in each block. The hotplate can be connected to the power source via a time-switch, which can be set to come on approximately 1 hour before commencement of work; this saves valuable time lost waiting for it to warm up.

Acid-digestion procedure

The acid used for the Kjeldahl digest is analytical quality concentrated sulphuric acid which contains 4 g l-1 selenium. This is prepared by heating a 250-ml portion of acid with 4 g selenium powder (Aldrich 20,965-1, 100 mesh) in a 1-l beaker on a hotplate in a fume cupboard, carefully stirring with a glass rod until dissolved to form a green solution (protective gloves, and safety spectacles or visor must be worn at all times when handling concentrated acids). After cooling, the solution is poured via a funnel into a glass storage bottle, and the balance of 750 ml acid added. Note: a dust mask should be worn when weighing selenium as it is easily absorbed by the lungs and is a possible teratogen. The beakers should be removed from the hotplate and left to cool in the fume cupboard after placing a watch glass over the top of the beaker. After cooling, the solution is poured via a funnel into a bottle or reservoir fitted with a bottle-top dispenser adjusted to 5 ml. All components with which the solution comes into contact must be resistant to concentrated sulphuric acid. Warning: the solution is highly corrosive and even when cold rapidly dissolves cellulosic materials. Wipe up any drips immediately with a wad of tissue and soak with plenty of running water before disposal. This is to both protect personnel involved in waste disposal and to prevent spontaneous combustion. Acid on the skin should be flooded with water for 1 min and medical advice sought for any blisters or burns; contaminated clothing should be removed and washed before reuse.

Exactly 0.1000 g milled plant sample is weighed into a glass weighing funnel and transferred to the numbered digestion tubes with the aid of a small paintbrush. The digestion tubes are held in stainless steel racks and either stoppered or covered with sheets of paper until ready for digestion. The tubes should have been previously marked with two scratch lines around the outside at the levels of 5 ml and 10 ml. The acid is dispensed carefully into each tube; if it is admitted too rapidly, fine sample powder as well as acid may be ejected from the tube. A few tubes at a time are loaded into the blocks. Some types of sample are prone to frothing, and if this occurs, it is easier to remove a few tubes and allow them to cool in their racks, rather than risk some frothing right over before they can be removed.

The most tedious aspect of the procedure is, after about an hour, to run a thin (4 mm diameter) glass rod vertically around the inside of the digestion tube in a downward spiralling motion in order to reintroduce any sample particles back into the acid. PPE must be worn for this. The temperature must not exceed 320°C because sulphuric acid boils at 330°C, which could cause injury; the two thermometers should be checked before the cleaning operation. (Note: the normal Kjeldahl procedure uses a salt such as sodium sulphate to raise the boiling point of the acid.) The samples are allowed to digest for a total of 4.25 h, when they are removed with stainless steel tongs and allowed to cool in their racks. The acid level is then adjusted dropwise with concentrated sulphuric acid to the 5-ml mark to replace any lost as fumes. Deionized water is then slowly added from a wash bottle, directing the jet down the side of the tube, up to the 10-ml mark so as to form two layers. Note: normally the safe way is to add concentrated sulphuric acid to water, especially when contained in a beaker - this is to prevent violent boiling. This does not happen here because of the restricted surface area and the formation of separate layers. The two layers are mixed by slowly oscillating a thin glass rod with one end flattened to form an 8-10 mm disc. Mixing should start from the junction of the layers, slowly working towards the top and bottom. The solution will contract after cooling, so the level must be again adjusted to the 10-ml mark and mixed with the rod. This final adjustment to 10 ml is best done immediately before analysis otherwise the tubes will need to be stoppered to avoid absorption of atmospheric moisture.

The advantages of this digestion technique are the large number of samples that can be processed at one time, the simple and cheap glassware involved, and the fact that the digest may be used for the subsequent determination of not only nitrogen, but calcium, magnesium, potassium, sodium, phosphorus and iron.

Summary of the indophenol blue colorimetric determination of nitrogen

To determine the nitrogen content of herbage and soils by autoanalysis, one must first carry out a Kjeldahl digest in concentrated sulphuric acid with selenium (0.4% w/v) catalyst; this converts protein nitrogen to ammonium nitrogen, as shown in Fig. 4.1. The density of the blue colour is proportional to the nitrogen content. It is measured using a spectrophotometer at a wavelength of 640 nm and the height of the peaks on a chart-recorder compared with those of known standards to obtain the nitrogen content of the original material. Protein content = %N x 6.25.

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