2,5-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.

Application Notes: Dihydroxybenzoic acids are polar acidic compounds containing two hydroxyl groups as well as a carboxylic acid. Isomers of dihydroxybenzoic acids are very polar with varying degrees of acidity. While the isomers can be analyzed by mixed-mode chromatography on Primesep D column, the presence of hydroxyls and carboxylic acid fragments also make these compounds good candidates for using the new hydrogen bonding columns for analysis. Since all these compounds have the same amount of hydroxyls they are separated based on accessibility of this hydroxyl group. Retention time can be adjusted by the amount of ACN, MeOh and concentration of additives (formic acid, ammonium formate, triethylamine, etc.). Our method is fully compatible with LC/MS and prep chromatography and can be used for other hydroxy and carboxylic acids containing compounds..

Application Columns: SHARC 1, 3.2×100 mm, 5 um, 100A. To learn more about SHARC 1 columns click here. To order this column click here. To see more chromatographic separations check our web site.

Application Compounds: 2,6-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid

Detection Technique: UV, LC/MS

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).