• Acid digestion unit - see Chapter 4 for details of aluminium blocks and hotplate.
• Autoanalysis modules for segmented flow system - sampler, pump, manifold, colorimeter (with 640 nm interference filters) or spectrophotometer with 10-mm flowcell, and chart-recorder or computer readout.
• Digestion test tubes - 150 x 16 mm diameter, heavy wall (BS 3218) borosil-icate glass rimless type; (Fisher Cat. No. TES-674-150S). These should have graduation lines inscribed at 5 ml and 10 ml positions.
• Tongs, stainless steel.
• Acid-digest solution, 0.4% selenium in sulphuric acid (approximately 98% m/m H2SO4) - wear PPE. See Chapter 4 for full details of making this highly corrosive solution, and note the safety precautions.
• Acid-wash solution - wear PPE. Add 250 ml sulphuric acid (approximately 98% m/m H2SO4) to 250 ml water slowly with stirring. Allow to cool, then make up to 500 ml with water and carefully invert to mix.
• Sodium hypochlorite solution - dilute 800 ml of sodium hypochlorite solution (Merck, 12% w/v available chlorine) to 2 l with water.
• Sodium phenate solution - dissolve 259 g of sodium hydroxide in approximately 1 l of water and allow to cool. Add 315 ml of 80% m/m phenol solution (Fisher) slowly with stirring. Cool and make up to 2 l; store in a refrigerator.
• Stock standard solution, 2000 |jg N ml-1, 200 |jg P ml-1, 1600 |jg K ml-1, (400 |jg Ca ml-1) - omit the Ca if it is unlikely to be required, so as to avoid the precipitation of calcium sulphate in the diluted standards. This combined standard solution can be used for the autoanalysis of P and K, and also provides a similar matrix to the sample digests. Each reagent should be dried at 102°C for 1 h and cooled in a desiccator before weighing. Dissolve 1.3745 g potassium chloride, 0.4393 g potassium dihydro-gen phosphate, 4.7162 g ammonium sulphate, (and 0.5000 g calcium carbonate), in sulphuric acid (approximately 98% m/m H2SO4) and make up to 500 ml with sulphuric acid.
• Working standards - using a pipette filler, and allowing the pipette to drain thoroughly, measure 5, 10, 15, 20 and 25 ml stock standard into a series of 100-ml beakers, followed by 45, 40, 35, 30 and 25 ml sulphuric acid (approximately 98% m/m H2SO4) respectively. Carefully transfer, with rinsing, to a series of 150-ml beakers containing approximately 40 ml water, stir slowly to mix, and allow to cool. Transfer, with rinsing, to a series of 100-ml volumetric flasks numbered 1-5, make up to the mark with water, firmly stopper, and invert carefully to mix. The standards will contain as follows: No. 1, 100 |jg N ml-1, 10 |jg P ml-1, 80 |jg K ml-1, (and 20 |jg Ca ml-1); No. 2, 200 |jg N ml-1, 20 |jg P ml-1, 160 |jg K ml-1, (and 40 |jg Ca No. 3, 300 |jg N ml-1, 30 |jg P ml-1, 240 |jg K ml-1, (and 60 |jg Ca No. 4, 400 |jg N ml-1, 40 |jg P ml-1, 320 |jg K ml-1, (and 80 |jg Ca No. 5, 500 |jg N ml-1, 50 |jg P ml-1, 400 |jg K ml-1, (and 100 |jg Ca Higher standards up to 1000 pg N ml-1, etc., can be similarly made ml-1) ml-1) ml-1) ml-1)
Procedure (digestion). For full details see Chapter 4, 'Acid digestion procedure'; a summary is given here. Weigh exactly 0.1000 g oven-dry plant material, ground to 1 mm, into the digestion tube. If the sample is freeze-dried, immediately weigh an extra portion for dry matter determination. A stock sample kept as a control could also be weighed. Note: wear PPE for the following stages. Carefully dispense 5 ml of the acid-digest solution into the tube, avoiding too rapid a stream of acid, which may cause loss of sample or spillage of acid. Switch on the fume cupboard extractor-scrubber unit. Using stainless steel tongs, load the samples in batches of about ten into the heating block, which should be within 20°C of the ultimate digestion temperature of 310°C. Remove any tubes in danger of frothing over, and replace after approximately 10 min when frothing has subsided. After about an hour, clean the inside wall of the tubes with a length of 4-mm glass rod, reintroducing any particles back into the acid.
After the digestion period of 4.25 h, remove the tubes to the stainless steel racks and allow to cool in the fume cupboard. Make up to the 5-ml mark with sulphuric acid (approximately 98% m/m H2SO4) using a dropping bottle. Next, carefully add water to the 10-ml mark, forming two layers. Starting at the junction of the layers, slowly oscillate a 4-mm glass rod, flattened into a disc at one end, until the two layers are thoroughly mixed; allow the rod to drain completely by touching the wall of the tube. Do this for all the tubes in the rack, then partially immerse in cold water to cool the tubes. Again make up to the 10-ml mark with water and mix with the rod. If there is to be a delay in analysing the solutions, they should be stoppered to prevent absorption of atmospheric moisture. If calcium is to be determined in the diluted solutions, and it is present at ^0.4% Ca in DM, they should be analysed immediately after dilution because CaSO4 will precipitate. It will not precipitate from the undiluted solution.
Procedure (autoanalysis). The flow diagram is shown in Fig. 7.3. Note 1: the mixing of the sample solution in 50% sulphuric acid with the highly alkaline phenate may cause turbulence, which breaks up the segmenting bubbles, resulting in an irregular bubble pattern. To avoid this, connect a right-angle bend immediately after the horizontal A1 connector, insert a 70-mm length of straight glass transmission tubing and allow it to slope downwards at 45°. Add a right-angle bend followed by a 100-mm length of glass transmission tubing and slope this upwards at about 25°, then insert a U-bend to meet the connection to the double mixing coil. The bends can be varied to suit the arrangement on the platter, and the slope of the straight
Single mixing coil <
Single mixing coil <
Colorimeter/ S/photometer 640 nm
Sampler 40 h-1 ratio 2:1 sample:wash Pump coil
Colorimeter/ S/photometer 640 nm
To chart recorder
To sampler wash
From sampler wash (if required)
sample phenate water air phenate
Fig. 7.3. Flow diagram for determination of nitrogen in plant digest solutions in
tubes, which affect the mixing dynamics of the reagent solutions of differing densities and viscosities, fine tuned to give the optimum bubble pattern. Note 2: the stainless steel sample probe is liable to corrode in 50% sulphuric acid. This probe should be raised in the sampler probe holder and merely used to support the polythene capillary, which is attached to it by means of a couple of rings sliced from the end of some PVC transmission tubing (ID 1.6 mm, 1/6 in). The lower ring should be positioned so that it is just above the liquid level in the wash and sample cups, which prevents cross-contamination. If two polythene capillaries are attached to it, the tips should be held apart and one raised a few millimetres above the other so that a large droplet is not held between them; this would prevent the formation of a small bubble separating sample from wash, and result in a loss of valleys between the peaks.
Switch on the autoanalyser modules and allow pumping of reagents for 30 min to flex the tubes. Carefully pour the sample digest solution from the digestion tube into the sample cup. Use 8.5-ml industrial polystyrene cups or similar size if more than one determination is required on the same sample solution. Load the first sampler tray in the sequence of low to high standards. It is recommended that the lowest standard is in duplicate, and the first peak rejected. After the highest standard, aspirate acid-wash solution for about 5 min to ensure the baseline is reached. Load further trays with about 32 sample cups followed by five standards. The remaining three spare cup positions can be used for higher standards or repeats. Again aspirate acid-wash solution to obtain a baseline between trays; this will enable a correction for any baseline drift to be made. If possible, keep samples of similar analyte concentrations together to avoid interference between adjacent low and high peaks. The sampling rate is set at 40 h-1 with a sample:wash ratio of 2:1. When using a chart-recorder, record the sample number on every tenth peak and label the standard peaks; this makes reading the charts easier and enables the identification of problem peaks so that a repeat can be inserted.
Calculation. Draw a baseline under all the peaks by connecting the baselines obtained at the start, between the tray changes, and at the end. Draw a standard curve using a chart reader (see Chapter 1, 'Chart reader'). If exactly 0.1000 g sample was taken, then divide the concentration corresponding to the sample peak, in pg N ml-1, by 100 to give the % total N in DM. Multiply by 6.25 to obtain the % crude protein in DM (but see the exceptions in Discussion 7.7).
If y g sample was taken, then (pg N ml-1) x 0.1/100y = % N in DM.
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