The isolation of a toxic substance from agenized wheat flour / by P.N. Campbell and T.S. Work and E. Mellanby.

  • Campbell, P. N. (Peter Nelson)
Date:
[1951?]
Catalogue details

Licence: In copyright

Credit: The isolation of a toxic substance from agenized wheat flour / by P.N. Campbell and T.S. Work and E. Mellanby. Source: Wellcome Collection.

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    Table 1. Stages in the isolation of toxic substance from wheat flour Toxic Total N No. of Process dose available doses Gluten — 220 g. — Treated with 11 g. NC13 1-6 g. N 220 g. 140 After enzymic digestion (A) 1-5 g. N Approx. 150 g. 101 After dialysis against distilled water (B) 1-5 g. N 125 g. 83 After hydrolysis with cone. HC1 (C) 2-3 g. N 118 g. 50* After electrodialysis 700 mg. N 35 g. — After removal of crystalline material ( D) 500 mg. N 25 g. 50 After removal of aromatic amino-acids on charcoal (E) 460 mg. N 23 g. — After fractionation on Zeo-Karbf — — — Total of all active fractions 170 mg. N 5-5 g. 32 After removal of crystalline material 130 mg. N 4g. 32 Most active fraction from column 96 mg. N 2-2 g. 23 After removal of crystalline material (F) 56 mg. N 1-3 g. 23 After fractionation on paper column{ (G) 4 mg. N 64 mg. 16 Crystallization from aqueous ethanol 3 mg. solid —. 12 * Acid hydrolysis + electrodialysis caused a loss of one-third of the toxic substance. This is assumed to occur during acid hydrolysis during which stage there is little loss of N, thus the toxic dose goes up. t Tor fractionation from Zeo-Karb column, see Table 3. t For fractionation from paper column, see Table 4. volume of 4 1. The most satisfactory membranes were pre¬ pared by treatment of animal parchment (plain skins from , Witherby and Co., 326 High Holborn, London, W.C. 1) with 10% (w/v) formaldehyde for 5 hr. at 20°. These membranes ad great mechanical strength and electro-endosmosis was inimal. The centre compartment of the box was fitted with mechanical stirrer and the outer walls of the electrode ompartments (platinum electrodes) were cooled (for a review if electrodialysis methods cf. Svensson, 1948). The hydro- ysate (3-4 1.; 30-40 g. N) was poured into the centre com- lartment of the box and the end compartments were filled vith distilled water. The electrodes were then connected to 230 V., d.c. supply main with a 100Cl variable resistance in re circuit. The current rose rapidly to 2*5-2-8 amp., the pH l^f the anode compartment fell and that of the cathode com- artment rose. The duration of the dialysis depended on the mount of N initially added to the centre compartment and the membranes, but for 40 g. N there was usually a fall in rrent to about 1-8 amp. in 24 hr. At the end of this time e contents of the end compartments were withdrawn (all it about 200 ml.), and were replaced with distilled water, ithin the next 4-8 hr. the current fell to 0-4 amp. and then mained steady. At this point electrodialysis was con- ered to be complete. Table 2. Distribution of nitrogen during electro - dialysis of acid hydrolysate (Results expressed as g. N.) Compartment ._A__ an values for four runs centage of total N Cathode 9-6 33 Centre 5-0 17 Anode 14-2 50 While the major part of the toxic substance remained in centre compartment during dialysis a small amount elled to the cathode compartment so that the contents he latter were collected, concentrated under reduced sure and re-electrodialysed together with sufficient of the e solution to bring the pH to 5-5. The anode solution was not toxic. The total amount of N in the combined neutral fractions from the electrodialysis was 35 g., the toxic dose containing 700 mg. N. The combined neutral fractions were concentrated under reduced pressure on a water bath until crystallization took place. The crystalline material was removed by filtration and the concentration repeated until no further crystallization took place. By this means the total N in the solution was reduced to 25 g. and the toxic dose to the equivalent of 500 mg. N (fraction D, Table 1). The dis¬ tribution of N during four electrodialyses is shown in Table 2. Removal of aromatic amino-acids with charcoal The aromatic amino-acids were removed by adsorption on activated charcoal. The charcoal was first treated with acetic acid according to the method of Tiselius, Drake & Hagdahl (1947) and then used as described by Schramm & Primosigh (1943). The filtrate (D) was treated in batches such that each batch contained approximately 4 g. N. Each batch was first diluted with 5% (v/v) acetic acid until the concen¬ tration of N was 1 mg. N/3 ml. Preliminary experiments on a pilot scale had shown that less than 20 % of the N was in the form of aromatic amino-acids and this figure was used as a basis for the calculation of the amount of charcoal required ; 1 g. of charcoal being required to adsorb 1 mg. of aromatic amino-acid N. The required amount of charcoal in a sus¬ pension in 5 % acetic acid was poured into a Buchner funnel and washed with further quantities of 5 % acetic acid. When the charcoal was nearly dry the solution of amino-acids was added and the effluent collected at a rate of approximately 5 l./hr. The charcoal was then washed with a volume of 5 % acetic acid equivalent to 25 ml./g. of charcoal present, at a rate of approximately 10 l./hr. The effluent and washings were combined. The total amount of charcoal required to treat the 25 g. of N was 5 kg. and the total effluent amounted to 200 1. (fraction E). Fractionation on Zeo-Karb 215 column The effluent (E) from the charcoal was divided into three parts (approx. 10 g. N each) for fractionation (Partridge, 1949) on a cation-exchange column of Zeo-Karb 215 (obtain-