{
  "url": "https://openlegislation.uk/feedingstuffs-zootechnical-products-regulations-1999/schedule-5/part-II/paragraph-5",
  "json_url": "https://openlegislation.uk/feedingstuffs-zootechnical-products-regulations-1999/schedule-5/part-II/paragraph-5.json",
  "legislation": "uksi/1999/1871",
  "legislation_title": "The Feedingstuffs (Zootechnical Products) Regulations 1999",
  "legislation_url": "https://openlegislation.uk/feedingstuffs-zootechnical-products-regulations-1999",
  "path": "schedule/5/part/II/paragraph/5",
  "kind": "paragraph",
  "number": "5",
  "heading": null,
  "text": "PROCEDURE\n5.1 Extraction of meticlorpindol\nWeigh, to the nearest 0.001g, approximately 50g of the finely divided and mixed sample, or a suitable amount expected to contain about 12mg of meticlorpindol, transfer to a 500ml graduated flask, and add 400ml of ammoniacal methanol solution (3.7). Place a magnetic stirring bar in the flask and stir the mixture on a magnetic stirrer for 20 minutes. Remove the stirring bar from the flask, dilute to the mark with ammoniacal methanol solution (3.7), mix the contents well, and set aside for 20 to 30 minutes.\n5.2 Purification\n5.2.1 Aluminium oxide column: For each column required weigh approximately 25g of aluminium oxide (3.1) into an aluminium foil dish and place in an oven at 105±5°C for 1 hour. Remove the dish from the oven and cool to room temperature in a desiccator. Make a slurry of the aluminium oxide with 25ml of ammoniacal methanol solution (3.7) and filter on a Buchner funnel. Wash the aluminium oxide with methanol (3.5) until the washings are neutral. Form a slurry of the aluminium oxide with 50ml of methanol (3.5) and pour the slurry into the column (4.1). Allow the methanol to drip through the column. Place a plug of glass wool lightly on top of the aluminium oxide and then wash with 25ml of methanol (3.5). Do not allow the liquid in the column to fall below the top of the aluminium oxide. Discard the eluate.\n5.2.2 Anion exchange column: Form a slurry in acetic acid (3.4) of sufficient resin (3.3) to fill the columns required. Filter on a Buchner funnel, wash the resin with twice its own volume of acetic acid (3.4) and then with aqueous methanol (3.6) until the washings are neutral. Form a slurry of a resin with aqueous methanol (3.6) and add sufficient to a column (4.2) to give a resin bed 20 to 30 mm deep after settling. Place a small plug of glass wool on top of the resin and wash the column with two 13ml portions of aqueous methanol (3.6). Do not allow the liquid level in the column to fall below the top of the resin. Discard the eluate.\n5.2.3 Chromatographic procedure: By pipette transfer 10.0ml of the extract of the feed sample (5.1) directly onto an aluminium oxide column and at the same time transfer the same volume of ammoniacal methanol solution (3.7) directly onto a second aluminium oxide column (reagent blank). Allow the solutions to drain to the top of the aluminium oxide and then wash each column with three 12ml portions of aqueous methanol (3.6), allowing the liquid to drain to the top of the aluminium oxide each time. Let all the eluate from each column drain directly into separate ion-exchange columns, and then remove the aluminium oxide columns. Allow the liquid to drain to the top of the ion-exchange resin and then wash each column with four 13ml portions of aqueous methanol (3.6). Discard the eluates.\nElute each column with two 10ml and then one 4ml portions of acetic acid (3.4). Collect the eluates from each column in separate 25ml graduated flasks and dilute the contents of each to the mark with acetic acid (3.4).\n5.3 Determination\nRecord the absorption spectrum of the sample extract between 350 and 245nm in 10mm silica cells with the reagent blank solution (5.2.3) as reference. Measure the absorbance of the sample extract at 267nm above a baseline obtained by drawing a line through the absorbance at 327 and 297nm and extending it through 267nm.\n(Note: Background absorption due to the feed approaches a linear function that can be described by the points on the curve at 296 and 327nm. Occasionally this is not the case, as can be detected by absorption peaks in the region between 350 and 297nm).\nDetermine the concentration of meticlorpindol in the sample by reference to the calibration curve (5.4).\n5.4 Calibration curve\nBy pipette transfer 1, 5, 7.5, 10, 12.5 and 15ml portions of meticlorpindol standard solution (3.8) to separate 250ml graduated flasks. Dilute the contents of each flask to the mark with acetic acid (3.4). Record the absorption spectra of these solutions in 10mm silica cells between 350 and 245nm with acetic acid (3.4) as reference. Construct a calibration curve using the absorbances at 267nm as ordinates and the corresponding concentrations of meticlorpindol in μg per ml as abscissae.",
  "extent": null,
  "status": null,
  "version": "current",
  "annotations": [],
  "outstanding_effects": [
    {
      "type": "amended",
      "affecting": "uksi/2000/1686",
      "affecting_title": "The Feedingstuffs (Zootechnical Products) and Medicated Feedingstuffs (Amendment) Regulations 2000"
    }
  ],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/uksi/1999/1871/schedule/5/part/II/paragraph/5",
    "retrieved_at": "2026-10-29T10:20:25.118862+00:00"
  },
  "licence": "Open Government Licence v3.0",
  "licence_url": "https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/",
  "attribution": "Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives."
}