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High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection

HPAE-PAD is commonly used to determine anionic analytes without derivatization, with high selectivity and sensitivity in various samples matrices. It is best known for the analysis of carbohydrates, including mono-, di-, tri-, oligo- and polysaccharides, sugar alcohols, and amino sugars.

What is HPAE?

High-Performance Anion-Exchange Chromatography is used to separate anionic analytes which are either anions in their common form (e.g. amino acids) or analytes that can be ionized at high pH values (e.g., carbohydrates at >pH 12). Therefore, HPAE uses hydroxide-based eluents at high pH to produce anions from analytes that would not be anionic at neutral pH. Chromatography with these eluents is possible due the development of metal-free, polymer based chromatographic systems and pH stable (0 to 14) nonporous polymeric resins, in which small anion-exchange MicroBeads* carrying the anion-exchange functional groups are permanently attached electrostatically to a larger cation-exchange resin particle. The nonporous nature of the resin minimizes band-broadening and imparts highly effective separation of a wide variety of carbohydrates, including branched oligosaccharides. A range of different columns for carbohydrate separations from mono, di-, tri- to oligo- and polysaccharides is available under the Thermo Scientific Dionex CarboPac columns brand.

What is PAD?

The detection of underivatized analytes is performed using pulsed amperometric detection. PAD is the application of various potentials to a working electrode over a specific time period. The potential variations are known as a waveform, and result in oxidizing and reducing conditions on the electrode surface, resulting in oxidation of analytes bound to the working electrode surface. For example, hydroxyl groups in carbohydrates are oxidized on a working electrode surface and the resulting current is measured. The oxidation of a carbohydrate is performed at a specific potential and results in the loss of a proton, which results in a current flow which can then be measured at that  potential, ensuring selective and sensitive detection. After oxidation, a reduction and reoxidation step is applied to remove the bound analyte and renew the electrode surface. The variation of the potentials is performed as a pulse sequence and typically takes less than 1 second, thus allowing chromatographic data points to be recorded at least every second. Pico- and femtomol sensitivity can be achieved with PAD, making it one of the most sensitive detection techniques. Only a small portion of the sample is oxidized, so the rest can be collected if desired, for further analysis.

Gold electrodes are available in two different sizes, 1 and 3 mm. All electrode materials can be ordered either in conventional format (metal wire embedded in a polymeric block) or as disposable electrodes.

Superior Technology that Provides Excellent Results

HPAE can be performed on the Thermo Scientific Dionex ICS-5000 product line with standard bore (4 mm i.d.) microbore (2 mm i.d.) or capillary (0.4 mm i.d.) column formats. The Dionex ICS-5000 system’s dual capabilities allow for operation of two analyses at the same time (e.g., carbohydrate and amino acid analysis) or two-dimensional separations. To identify unknown analytes, the Dionex ICS-5000 configured for HPAE-PAD can also be coupled with a mass spectrometer using a carbohydrate membrane desalter (CMD) inline prior to the MS interface or to spot fractions onto MALDI targets for offline MALSI/MS analysis. Another option for identification of unknown analytes is offered by 3D Amperometry.

Maximize Convenience

The analysis of mono-, di-, and trisaccharides can be performed using Reagent-Free* IC with Eluent Generation, thus only requiring deionized water to electrolytically generate the eluent. This technology provides consistent results and the highest reproducibility, day-to-day, user-to-user, and lab-to-lab.

Carbohydrate Analysis

Typical examples of HPAE for carbohydrate analysis include:

  • Monosaccharides, disaccharides, trisaccharides, alditols, and glycols
  • Quantitative determination of glycoprotein monosaccharides
  • Single-unit resolution of glycoprotein oligosaccharides
  • Sialic acids
  • Carbohydrates and glycols in fermentation broths and other cultures