6533b827fe1ef96bd1286dce

RESEARCH PRODUCT

Extraktionschromatographische trennung der freien uroporphyrinisomere i und iii und deren simultane quantitative bestimmung

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Abstract Separation and simultaneous quantitative determination of the free uroporphyrin isomers I and III by means of extraction chromatography Separation and quantitative determination of the uroporphyrin isomers I and III in the acid from can be performed simultaneously in the partition system tri-n-butylphosphate/I N hydrochloric acid using columns with a large number of theoretical plates (N) = 300-450). The eluent is passed through a flow cuvette and the transmission is recorded continuously. The transmission peaks of the isomers are digitized and transformed into extinction values. By integrating the extinction peaks, the amount of the uroporphyrins can be evaluated from the corresponding peak areas. Using the mathematical procedure described the time necessary for separation (tEl.) can be shortened appreciably. This method allows the decomposition of complex elution peaks even at low distances of the peak centres (Δχp/ T 50 = 1.5-2.0, depending on the mass ratio) and unfavourable mass ratio (0.06 ≦ UP-I/UP-III ≦ 16_. Under suitable conditions (length of the column: L8 = 120 cm; temperature of the column: T8 = 15-30°; concentration of the eluent: cHCl = 1 N) complete analysis of both isomers can be achieved in about 20–30 h. The quantitative determination can be performed up to approx. 1 μg with an accuracy of 10% (95% deviation). No appreciable losses of the uroporphyrins occur (R = 99 ± 5%) even under extreme conditions (T8 = 60°; cHCl = 3N).