Analyzing Carbohydrates by HPAE-PAD Ion Chromatography

Carbohydrates are carbon sources essential for cell growth and product synthesis and are commonly analyzed in the food and beverage and biopharmaceutical/pharmaceutical industries. Fast and accurate determination of carbohydrates is vital for food and beverage quality control, nutritional labeling, and identification of possible adulteration. In the biopharmaceutical industry, characterization of glycoproteins routinely involves carbohydrate analysis, and changes in protein glycosylation are often studied in cancer research to identify potential biomarkers. There are over 30 approved glycoprotein-based biodrugs on the market, and the number is increasing rapidly.

The US FDA and the European Medicines Agency have increased pressure on biopharmaceutical manufacturers to demonstrate satisfactory programs for understanding, measuring, and controlling glycosylation in glycoprotein-based drugs. Carbohydrates are difficult to analyze due to their highly polar nature, similar structural characteristics, and lack of a suitable chromophore. By using HPAE-PAD, manufacturers can easily and effectively separate and quantify underivatized carbohydrates.

HPAE-PAD – High-Performance Anion Exchange chromatography with Pulsed Amperometric Detection – is a technique that was developed specifically for the separation and detection of carbohydrates. Unlike other carbohydrate analysis techniques, HPAE-PAD allows for highly selective separation and quantification of non-derivatized carbohydrates.

High-Performance Anion Exchange (HPAE) chromatography is used to separate analytes that are ionized under high pH conditions, such as carbohydrates (pKas are usually between 12-13). This is accomplished through hydroxide-based eluents and a strong anion-exchange stationary phase. The columns’ nonporous resins have small anion-exchange microbeads carrying the anion-exchange functional groups. These small anion-exchange microbeads are permanently attached electrostatically to a larger cation-exchange resin particle. The nonporous nature of the resin minimizes band-broadening and allows for highly effective separation of a wide variety of carbohydrates, including branched oligosaccharides.