Abstract
The physical separation of iodoamino acids has been studied in the past by means of paper and column chromatography and electrophoresis(l,2). Despite its reputation as a highly useful analytical tool in organic and biologic chemistry, few papers describing the separation of thyroid hormones by 2-dimensional TLC have appeared (3,4,5). In addition, the use of 2 solvents in the same direction to effect separations has been described (6).
This communication describes a 2-dimensional method of TLC for use in the separation and quantitative recovery of radio labeled iodoamino acids and iodide and its application to analysis of rat thyroid hydrolysates.
Methods and materials. Solutions of thy-roxine (T4) 3:3′: 5 triiodothyronine (T3), 3:5 diiodotyrosine (DIT), 3-monoiodotyrosine (MIT), 3:5 diiodothyronine, sodium iodide, tetraiodothyroacetic acid (TETRAC) and triiodothyroacetic acid (TRIAC) were prepared by dissolving each compound in metha-nol cone, ammonium hydroxide (99:1) so that 1 microliter of each solution contained the equivalent of 1 microgram of iodide. Preparation of the solutions of the individual 131-1 labeled compounds (T4, T3, DIT, MIT and Nal) was accomplished by the addition of each to methanokconc. ammonium hydroxide (99:1).
The absorbent was prepared by suspending 30 g of cellulose powder (Whatman C-41) in 75 cc of distilled water which was then slurried for 5 minutes with a magnetic stirrer.
The Desaga Apparatus was utilized to prepare chromatoplates (20 cm X 20 cm) of 0.35 mm thickness. Prior to chromatography the plates were air-dried over night. Drying the plates in a desiccator or pre-heating offered no advantages. For chromatography of the unlabeled iodinated compounds, a known amount of a mixture of the individual compounds was applied to the chromatoplate as a spot less than 5 mm in diameter at the lower left corner 2 cm from each edge using a micro-syringe under a stream of air at room temperature.
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