Nitrate and watersoluble carbohydrate

The same extractant is used for both nitrate and water-soluble carbohydrate (WSC) determinations, however the ratio of sample to extractant is different. The herbage may be oven-dried for nitrate, but must be freeze-dried for WSC determination. The extractant is saturated benzoic acid solution. Benzoic acid is sparingly soluble in cold water, and the solution is made by adding an excess quantity to deionized water at ambient temperature in a blender, which is then switched on for about a minute. It is filtered through a Whatman No. 4 paper into a storage container fitted with a tap. If the ambient temperature should fall several degrees, it is possible for some crystals to separate out. These would make little quantitative difference, but might block the sample capillary probe or tubing. If this is thought likely, the containers for samples and standards should be warmed and shaken gently to redissolve the crystals. The benzoic acid acts as a preservative, allowing the storage of sample extracts at room temperature almost indefinitely, so they can be analysed at a convenient time. It has been noticed, however, that the concentration of nitrite (as opposed to nitrate, which is stable) decreases to zero after a day or so. Methods estimating nitrite, therefore, must use an extractant such as water, followed by immediate analysis.

Nitrate

The autoanalysis method was developed at the WPBS and modified by using benzoic acid extractant solution. It is based on the method of Follett and Ratcliff (1963), which was itself based on that of Grace and Mirna (1957). It relies on the reduction of nitrate to nitrite by adding the sample solution to an ammonium chloride buffer (pH 7.5) containing EDTA disodium salt and copper sulphate and passing through a glass tube containing cadmium filings which become copper-plated. The nitrite immediately reacts with sulphanil-amide to form a diazo salt which couples with 8-aminonaphthalene-2-sul-phonic acid (Cleve's acid) to form an orange acid azo dye which is measured at 470 nm on a spectrophotometer.

Another method used for nitrate determination on dried and milled herbage employs the nitrate selective electrode. One of the first published methods was that of Paul and Carlson (1968). Other anions, especially chloride, can interfere. These authors removed chloride with silver resin, but Barker et al. (1971) omitted the resin because it tended to foul the electrode and cause excessive drift. Normally the Cl-:NO3- ratio is so low as not to interfere, but saline precipitation from coastal plots could affect this. The method was further modified to allow storage of extracts for up to 64 h by adding a preservative of phenyl-mercuric acetate and dioxane, both very toxic (Baker and Smith, 1969). This paper mentions the need to change the electrode's membrane, filling solution and liquid ion exchanger every 2 months to minimize chloride interference. It is easy to overlook electrode maintenance between batches of nitrate analyses, and this can lead to errors and sluggish performance.

The method was extended from plants to include soils and waters by Milham et al. (1970). They point out that nitrate reductase activity in fresh plant samples often causes a rapid decline in nitrate content, so samples collected from remote sites should be frozen in dry ice. A trace of chloroform was used to protect soil and water samples before freezing. We are now more aware of the harmful effects of chloroform inhalation and suggest immediate freezing without preservative and analysis within a few days as a safer alternative - especially with student projects.

One drawback with selective ion electrodes is their slow response at low concentrations of analyte, perhaps below 2 mg l-1 NO3-N. It can take several minutes to equilibrate, and slow drifting can give a measure of uncertainty as to the equilibration point. If this cannot be remedied by reducing the dilution factor, an alternative method should be sought. They are also sensitive to changes in temperature, in excess of 1°C being significant. Mechanically driven magnetic stirrers get warm, therefore electronic ones are preferable.

Water soluble carbohydrate

This is basically the anthrone method of Yemm and Willis (1954) which was developed at the WPBS for use with an autoanalyser (Thomas, 1977) and modified by using benzoic acid extractant solution. The extract is reacted with anthrone in 76% sulphuric acid. Heating to 95°C develops the green colour which is measured at 620 nm. Fructose, sucrose and inulin give the colour at room temperature, but heating is necessary for glucose, maltose, fucose and rhamnose to react (Van Handel, 1967). Fructose and glucose are hydrol-ysed by hot sulphuric acid to 5-hydroxymethyl-furfural which reacts with anthrone to give 10-{5-(anthron-10-ylmethyl)-2-furfurylidene} anthrone, which couples with brown resin by-products to give the colour (Hoermann, 1968).

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