2,5-Dihydroxybenzoic acid

HPLC Analysis of Dihydroxybenzoic Acids on Amaze TR Mixed-Mode Column
Application description

Dihydroxybenzoic acids are isomers with different positions of substitution in a benzene ring. When compounds have a similar structure, it is very hard to separate them using only one interaction whether it is reversed-phase, HILIC or ion-exchange. Since isomers have very similar properties, they often co-elute in RP or HILIC mode. In case of mixed-mode chromatography, the small difference between hydrophobic and ionic properties is explored to achieve good separation and the peak shape for dihydroxybenzoic acids. Isomers of dihydroxybenzoic acids are separated on a Amaze TR mixed-mod, reversed-phase anion- and cation-exchange column with good selectivity and peak shape. This method can be used for separation of other hydrophilic acidic compounds and acidic isomers. The method is reproducible and robust. It is compatible with major detection techniques including mass spectrometry (MS), Evaporative Light Scattering Detection(ELSD), ultraviolet detection and refractive index detection.

Conditions of Experiment
Column: Amaze TR
Separation Modes: reversed-phase, anion-exchange
Column Dimenstions: 4.6x50 mm, 3 um, 100A
Mobile Phase: 20% ACN with 15 mM AmFm pH 3
Detection: UV 255 nm
Sample: 0.3 mg/ml
Injection: 3 ul
Flow rate: 1 ml/min
Class of compounds: Aromatic acid, Isomer, Organic acid, Organic compound
Nature of compounds: Acidic, Hydrophilic, Neutral, Polar
Compounds: 3,4-Dihydroxybenzoic acid, 3,5-Dihydroxybenzoic acid, 2,3-Dihydroxybenzoic acid, 2,5-Dihydroxybenzoic acid, 2,4-Dihydroxybenzoic acid