Standards

• Stock fructose solution, 1000 pg ml-1 of fructose - dissolve 1.0000 g fructose in saturated benzoic acid solution and make up to 1 l in a volumetric flask and mix.

• Working fructose standards, 10-300 pg ml-1 of fructose - pipette 1, 5, 10, 20, 25 and 30 ml of the stock fructose solution into a series of 100-ml volumetric flasks, make up to the mark and mix. This gives standards containing 10, 50, 100, 200, 250 and 300 pg ml-1 of fructose.

Procedure. Weigh 0.1000 g freeze-dried plant material into a 250-ml wide-mouth high-density polyethylene screw-cap bottle. At the same time, weigh a sample for moisture determination. The square type bottles fit best the square box of the reciprocating shaker. Add 100 ml saturated benzoic acid solution and shake for 60 min. Note: For samples with < 25% WSC in DM, weigh 0.2 g sample. Filter through a Whatman No. 1, 15-cm filter paper, rejecting the first few millilitres, and save in polythene capped sample tubes. The colour is measured at 620 nm, and the flow diagram is given in Fig. 7.5.

Fig. 7.5. Flow diagram for determination of water soluble carbohydrate. * Black acid-resistant tubing.

Note: the jacketed mixing coil has 14 turns (Bran+Luebbe Part No. 114-022201), and all the PVC sleeving tubing collars (ID 3.2 mm, % in) which connect the fittings under pressure should be wired on (a couple of turns of wire with the ends twisted together with pliers). This is because the viscous anthrone reagent causes considerable back-pressure, and if a fitting becomes disconnected, all the bubbles expand and force out a hot spray of 76% sulphuric acid. It may also be wise to tape a polythene sheet over the manifold in order to prevent serious injury from hot acid. Switch on the heating bath approximately 90 min beforehand to allow it to reach 95°C, then the rest of the modules for the autoanalysis. Pour the anthrone reagent into a conical flask, and then immerse in a beaker of crushed ice. Water coolant is a 2-l bottle of tap water, which has previously been refrigerated, and the wash solution is saturated benzoic acid. Commence pumping reagents and load the standards into a sample tray. After 20-30 min pumping, analyse at a rate of 40 samples per hour, adjusting the sensitivity of the detection-readout to bring the baseline and top standards on scale. If the samples are all low, the sensitivity should be increased and appropriate lower standards used. After a baseline, analyse a tray of samples plus standards, and follow with an adequate baseline before analysing the second tray of samples and standards. Conclude by aspirating the wash solution to obtain a final baseline. The sugar content of plants can vary greatly, so high peaks often obscure low ones, and repeats are often necessary. To reduce this inconvenience, try to group low and high sugar samples together. Check that the acid resistant pump tubes have not started to 'snake' due to stretching after an hour or so, as they are more prone to this than the ordinary PVC tubes; increase the tension to compensate for this, possibly at the start.

Calculation. Draw a baseline on the chart under all the sample peaks by connecting the baseline from aspirating wash at the start, between trays and at the end. Read the concentration of the sample solutions by comparing the peak heights of the samples with the standards using a chart reader (see Chapter 1, 'Chart reader'). Divide the concentration in pg ml-1 of soluble carbohydrate in the sample solution by 10 to get the % water soluble carbohydrate in the freeze-dried sample. Multiply by 100/(100 - % moisture) to give the percentage water soluble carbohydrate in the sample DM.

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