|Molecular Weight||92.141 g/mol|
Primesep 100 separates a mixture of amino acids (tyrosine, phenylalanine), organic acids (benzoic acid, mandelic acid), amines (benzylamine, pyridine), and neutrals (benzonitrile, toluene) in one HPLC run by combining reversed-phase, cation-exchange, and polar interactions. The method is tunable and peak order can be changed significantly by adjusting acetonitrile and trifluoroacetic acid concentrations. The separation method uses a mobile phase mixture of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA) and compatible with UV, mass spec (LC/MS) and evaporative light scattering (ELSD) detection.
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.
Primesep 100 and Primesep 200 columns can be used as a universal column for analysis of wide range of compounds. These mixed-mode reversed-phase ion-exchange HPLC columns can provide a valuable alternative to traditional reversed-phase column. Amines, amino acids, quaternary amines, and various zwitter-ions can be analyzed along with hydrophobic compounds and organic and inorganic counter-ions. In this application, 8 compounds with different hydrophobic, hydrophilic, basic and acidic properties are separated based on their properties. Primesep 100 column is a mixed-mode HPLC column with a C12 carbon chain and carboxylic acid on the surface with pKa of 1. Primesep 200 column is a mixed-mode HPLC column with a C12 carbon chain and carboxylic acid on the surface with pKa of 2. These columns can be used with 100% organic (ACN) and 100% aqueous mobile phases. This HPLC method can be adopted as a generic and robust approach for analysis of acidic, basic and neutral compounds within the same run.
|Column||Obelisc R, 4.6x250 mm, 5 µm, 100A|
|Mobile Phase||MeCN/H2O - 35/65%|
|Buffer||AmAc 10 mM pH 4.0|
|Flow Rate||1.0 ml/min|
|Detection||UV, 250 nm|
|Class of Compounds||Drug, Acid, Bases, Neutral, Hydrophilic, Ionizable, Vitamin, Supplements, Amino acid|
|Analyzing Compounds||Amino acids|
Parabens are common preservatives in pharmaceutical and cosmetic industries. They are esters of p-hydroxybenzoic acid. Method for separation of methyl paraben, propyl paraben, benzonitrile and toluene was developed on a Obelisc R column. All four compounds are neutral and are retained by reverse-phase mechanism. In case of reversed-phase stationary phase, no effect of pH is observed. Retention time for all four compounds changes on an Obelisc R column when pH is changed. pH of the mobile phase affects ionization state of stationary phase. Obelisc R column has C12 carbon chain and carboxylic acid with pKa of 4. At lower pH (pH 2, TFA), carboxylic acid of stationary phase is not ionized and thus adds hydrophobicity to stationary phase. Obelisc R column can be used for analysis of basic, acidic and neutral compounds with suitable detection techniques - UV, ELSD, CAD, LC/MS.