HPLC analysis of carbohydrates on POLYSPHER®CH OH columns using pulsed amperometric detection (PAD) with sodium hydroxide as post column detection reagent

Abstract

Carbohydrates have been separated on POLYSPHER(R)CH OH columns using pulsed amperometric detection (PAD) and UV detection (lambda =196 nm) in series and pure water as mobile phase. Nearly baseline separations have been obtained for the glycoprotein carbohydrates of sialic acid ( N-acetylneuraminic acid, NANA), N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). As carbohydrates dissolved and eluted with pure water are present in the neutral form they are not detectable with PAD in contrast to carbohydrate anions formed at high pH values. Therefore an additional NaOH post column reagent has been continuously pumped through a mixing chamber into the mobile phase to form carbohydrate anions resulting in improved detection limits. Monosaccharides as well as glycoprotein carbohydrates could be detected in the microg/ml-range. This method has been applied successfully to the analysis of sugars in fruit juice. With only 2 microl of juice per 50 ml water, the determination of the main constituents, sucrose, glucose and fructose, was possible in a few minutes without sample preparation.

Simultaneous Determination of Monosaccharides in Glycoproteins by Capillary Electrophoresis

“…Another disadvantage is that sialic acids cannot be determined in the same analysis with neutral and amino sugars. While high-performance anion-exchange chromatography with pulsed-amperometry detection (HPAEC-PAD) 2 (7,8) is easy, sensitive, and useful for the analysis of underivatized neutral and amino sugars liberated from glycoproteins, hydrolyzed amino acids and peptides cause significant interference (7).…”

Speciation of iron coordinated by phytosiderophores by use of HPLC with pulsed amperometric detection and AAS

“…With the exception of some minor peaks in the retention range below 6 min the phytosiderophores are the only analytes detectable by pulsed amperometric detection (PAD). This high selectivity and sensitivity of PAD for compounds containing aliphatic hydroxy groups is well-known and has been used mainly for detection of carbohydrates [15,16,17,18,19,20,21], although amino acids [22,23] and even metal species [24,25] are also detectable by PAD. The above mentioned peaks in the retention range below 6 min are amino acids, which are electroactive under the experimental conditions used.…”

Rapid screening of airway secretions for fucose by parallel ligand-exchange chromatography with post-column derivatization and fluorescence detection

“…Because this process is difficult to automate, it was performed manually. We chose sulfuric acid as hydrolyzing agent, because of the high recovery expected [12] and its compatibility with chromatographic separation on the Aminex column. Trifluoroacetic acid (TFA) is retained on the Aminex column, and is, therefore, less suitable than sulfuric acid.…”