2,3-Dihydroxybenzoic Acid

Dihydroxybenzoic acids are aromatic compounds consisting of a phenolic ring and a carboxylic acid. The six main compounds are structurally similar and are difficult to separate in reverse-phase HPLC. The can be separated by using a mixed-mode Newcrom B column with the mobile phase having either methanol (MeOH) or acetonitrile (ACN) as an organic modifier having different retention characteristics. Using the gradient of organic modifier, water and sulfuric acid (H2SO4) as buffer, dihydroxybenzoic acids can be separated and UV detected at 250nm.

Separation of hydrophilic isomers is very difficult when only one mechanism of retention, like reversed-phase, is available. Isomers have very similar hydrophobic properties. In case of mixed-mode chromatography, small difference in hydrophobic and ionic properties allows to separate isomers. Isomers of dihydroxybenzoic acids are separated on a Primesep D column with good selectivity and peak shape. This method can be used for separation of other hydrophilic acidic compounds as well as hydrophobic basic compound.

Primesep B offers better selectivity over traditional, reversed-phase C18 columns for separating regioisomers of aromatic dihydroxybenzoic acids (2,3-dihydroxybenzoic, 2,4-dihydroxybenzoic, 2,5-dihydroxybenzoic 3,4-dihydroxybenzoic, 3,5-dihydroxybenzoic acids) Primesep B combines reversed-phase and anion-exchange mechanism with a mass spec compatible mobile phase of water, acetonitrile (MeCN, ACN) and trifluoracetic acid (TFA).