Separation and quantitation of galacturonic acid oligomers from 3 to over 25 residues in length by anion-exchange high-performance liquid chromatography.

A method for separation and quantitation of galacturonic acid oligomers from 3 to over 25 residues in length is described. Oligomers were labeled at the reducing end with 2-aminopyridine and then analyzed by anion-exchange high-performance liquid chromatography using a sodium acetate gradient. The amount of each oligogalacturonide present was determined by comparison to the response of an internal reference oligogalacturonide over a range from 0.5 to 20 nmol per oligomer. At least 5 h of incubation in the 2-aminopyridine reagent was required to obtain maximum and oligomer length-independent derivatization. To be analyzed using this technique, oligogalacturonides must possess a reducing terminus, they should be deesterified prior to derivatization if identification of the actual galacturonide chain length is desired, and they should fall within the range of 3 to over 25 galacturonide residues per oligomer. The wide range of oligogalacturonides separable, sensitivity of detection, ease of quantitation of chromatographic data, and ability to hydrolyze the 2-aminopyridinyl group from sugars makes this technique of potential use for numerous applications ranging from simple characterization of oligogalacturonide mixtures to purification of oligomers for use in bioassays.