HPLC Separation of Dihydroxybenzoic Acid

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.


Column Primesep D, 4.6x150 mm, 5 µm, 100A
Mobile Phase MeCN
Buffer TFA
Flow Rate 1.0 ml/min
Detection UV, 250 nm


Class of Compounds Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements
Analyzing Compounds 3.4-dihydroxybenzoic acid, 3.5-dihydroxybenzoic acid,2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 2,3-dihydroxybenzoic acid

Application Analytes:

2,3-Dihydroxybenzoic Acid
2,4-Dihydroxybenzoic Acid
2,5-Dihydroxybenzoic Acid
3,4-Dihydroxybenzoic Acid
3,5-Dihydroxybenzoic Acid