{
  "url": "https://openlegislation.uk/eu-regulation-2009-152/annex-III/division-F/division-5/division-5.4",
  "json_url": "https://openlegislation.uk/eu-regulation-2009-152/annex-III/division-F/division-5/division-5.4.json",
  "legislation": "eur/2009/152",
  "legislation_title": "Commission Regulation (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis for the official control of feed (Text with EEA relevance)",
  "legislation_url": "https://openlegislation.uk/eu-regulation-2009-152",
  "path": "annex/III/division/F/division/5/division/5.4",
  "kind": "division",
  "number": "5.4",
  "heading": "Chromatography",
  "text": "Before chromatography bring the extract (5.2) or hydrolysate (5.3.4) to room temperature. Shake the mixture and filter a suitable amount through a 0,22 μm membrane filter (4.5). The resulting clear solution is subjected to ion exchange chromatography, using an amino acid analyser (4.9).\nThe injection may be performed manually or automatically. It is important that the same quantity of solution ± 0,5 % is added to the column for the analysis of standards and samples except when an internal standard is used, and that the sodium:amino acid ratios in the standard and sample solutions are as similar as is practicable.\nIn general the frequency of calibration runs depends on the stability of the ninhydrin reagent and the analytical system. The standard or sample is diluted with citrate buffer (3.24) to give a peak area of the standard of 30 %-200 % of the sample amino acid peak area.\nThe chromatography of amino acids will vary slightly according to the type of analyser employed and resin used. The chosen system must be capable of separating the amino acids from each other and from the ninhydrin-positive materials. In the range of operation the chromatographic system must give a linear response to changes in the amounts of amino acids added to the column.\nDuring the chromatography step the valley:peak height ratios mentioned below apply, when an equimolar solution (of the amino acids being determined) is analysed. This equimolar solution must contain at least 30 % of the maximum load of each amino acid which can be accurately measured with the amino acid analyser system (4.9).\nFor separation of threonine-serine the valley:peak height ratio of the lower of the two overlapping amino acids on the chromatogram must not exceed 2:10. (if only cyst(e)ine, methionine, threonine and lysine are determined, insufficient separation from adjoining peaks will adversely influence the determination). For all other amino acids the separation must be better than 1:10.\nThe system must ensure that lysine is separated from ‘lysine artifacts’ and ornithine.",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eur/2009/152/annex/III/division/F/division/5/division/5.4",
    "retrieved_at": "2026-11-01T14:51:38.610873+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."
}