HPLC Separation of Isomers of Amino Salicylic Acid

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Condition 1

Column Newcrom A, 4.6x50 mm, 5 µm, 100A
Mobile Phase MeCN/H2O - 20/80%
Buffer H3PO4 - 0.5%
Flow Rate 0.5 ml/min
Detection UV 275 nm
  4-Aminosalicylic acid (PAS) is an antibiotic used in treatment of tuberculosis. It is a polar amino acid with limited retention on traditional C18 (reversed-phase) columns. Other isomers of aminosalicylic acid exist, but the main impurity in PAS is 5-Aminosalicylic acid, which also serves as anti-inflammatory drug. Both compounds are isomers with similar empirical structure and properties. These two isomers were separated on a Primesep 100 column with UV, ELSD and LC/MS compatible mobile phase. Method can be used a generic approach for separation of isomers of basic and zwitter ionic compounds. Isomers are retained and separated based on reversed-phase and cation-exchange properties. Retention time is controlled by the amount of acetonitrile, buffer concentration and buffer pH. Buffer pH is affecting ionization of these two compounds and thus serves as a powerful tool to adjust selectivity of separation.

Condition 2

Column Newcrom A, 4.6x150 mm, 5 µm, 100A
Mobile Phase MeCN/H2O - 50/50%
Buffer AmFm pH 3.0- 40 mM
Flow Rate 1.0 ml/min
Detection UV 256 nm,  MS-compatible mobile phase


Class of Compounds Acids
Analyzing Compounds 5-aminosalicylic acid, 4-aminosalicylic acid
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Application Analytes:
4-Aminosalicylic Acid
5-Aminosalicylic Acid

Application Detection:
UV Detection