HPLC separation of Dihydroxybenzoic acid in Hydrogen-Bonding mode on SHARC 1 HPLC column



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


Column Sharc 1, 3.2×100 mm, 5 µm, 100A
Mobile Phase MeCN/MeOH
Buffer AmFm, Formic acid
Flow Rate 1.0 ml/min
Detection UV, 270 nm



Class of Compounds
Drug, Acid, Hydrophilic, Ionizable, Vitamin, Supplements
Analyzing Compounds 2,6-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid


Application Column

Primesep D

The Primesep family of mixed-mode columns offers a wide variety of stationary phases, boasting unprecedented selectivity in the separation of a broad array of chemical compounds across multiple applications. Corresponding Primesep guard columns, available with all stationary phases, do not require holders. SIELC provides a method development service available to all customers. Inquire about our specially-tailored custom LC-phases for specific separations.

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The SHARC™ family of innovative columns represents the first commercially available columns primarily utilizing separation based on hydrogen bonding. SHARC stands for Specific Hydrogen-bond Adsorption Resolution Column. Hydrogen bonding involves an interaction or attraction between a bound hydrogen atom and molecules containing electronegative atoms, such as oxygen, nitrogen, and fluorine.

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Application Analytes:
2,4-Dihydroxybenzoic Acid
2,5-Dihydroxybenzoic Acid
3,5-Dihydroxybenzoic Acid

Application Detection:
UV Detection
SIELC Technologies usually develops more than one method for each compound. Therefore, this particular method may not be the best available method from our portfolio for your specific application. Before you decide to implement this method in your research, please send us an email to research@sielc.com so we can ensure you get optimal results for your compound/s of interest.