HPLC Application for Simultaneous Separation of Amino Acids, Hydrophilic Acidic and Hydrophobic Neutral Compounds

Mixed-mode chromatography allows separating, in single run, compounds with vastly different properties. A method for separation of amino acids (cysteine, methylcysteine, cystine and dimethylcysteine) in the presence of carboxylic acid (benzoic) and hydrophobic neutral compounds was developed on Primesep 100 mixed-mode column. At lower pH ionization of carboxylic acids is suppressed. Amino acids are retained as basic compound based on reverse phase and cation exchange mechanisms. Carboxylic acids are retained on this column based on weak reverse phase mechanisms. Neutral compounds are retained by reverse phase mechanism as on any other column. Retention time of basic, zwitter-ionic and hydrophobic compound can be adjusted by manipulation of mobile phase composition. ELSD, UV or LC/MS detection can be used based on the properties of analytes and mobile phase selection.


Column Primesep 100, 4.6x150 mm, 5 µm, 100A
Mobile Phase MeCN/H2O
Buffer H2SO4
Flow Rate 1.0 ml/min
Detection UV, 210 nm


Class of Compounds Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid
Analyzing Compounds Cysteine, Methylcysteine, Cystine, Dimethylcysteine,  Benzoic acid, Toluene,

Application Analytes:
Benzoic Acid