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HPAEC with PAD Thoughtful Designs: Innovations in Carbohydrate Analysis

Author / Editor: Detlef Jensen* / Dr. Ilka Ottleben

Carbohydrates are the most abundant class of biomolecules. But — their analysis is not always trivial. This article describes how electrolytic and highly automated inline eluent generation can be successfully applied to carbohydrate separations using HPAEC in combination with PAD.

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Fig 1 Schematic setup of electrolytic Eluent Generation (RFIC); the KOH eluent is produced electrolytically in the eluent generator cartridge (EGC).
Fig 1 Schematic setup of electrolytic Eluent Generation (RFIC); the KOH eluent is produced electrolytically in the eluent generator cartridge (EGC).
(Sources: Thermo Fisher Scientific)

Neutral carbohydrates are poly­alcohols with an acid dissociation constant (pKa) above twelve that can be separated at high pH using high performance anion exchange chromatography (HPAEC). Under these conditions, their detection predominantly is carried out by pulsed amperometry (PAD) [1-3]. Modern column chemistry and detection capabilities allow for a comprehensive analysis of alditols and monosaccharides as well as oligo- and polysaccharides [4,5].

Driven by the application of continuously and dynamically regenerated desalting devices, known as electrolytically regenerated suppressors in ion chromatography (IC), the combination of HPAEC with mass spectrometry has become a key tool in modern glycan analysis [6]. High pH eluents in HPAEC are based on aqueous solutions of sodium or potassium hydroxide (KOH).

Additional Information

Carbohydrates play vital roles in a variety of biological functions, including cellular communication, gene expression, immunology, organism defense mechanisms, and growth and development. They are difficult to analyze as they are very polar compounds, exhibit similar structural characteristics, and do not have a suitable chromophore. An improved chromatographic technique known as high-performance anion exchange chromatography (HPAEC) takes advantage of the weakly acidic nature of carbohydrates to give highly selective separations at high pH using a strong anion-exchange stationary phase. Coupled with pulsed amperometric detection (PAD), it permits direct quantification of non-derivatized carbohydrates at high femtomolar concentration levels with minimal sample preparation and cleanup. Quote: Monika Verma, Thermo Fisher ­Scientific, Sunnyvale, CA, USA

However, slight deviations in manual preparation of eluents can interfere with chromatographic measurements. Consequently, the manual preparation is one of the major contributors to uncertainty in analytical results. Precautions can be introduced to avoid contamination by the absorption of carbon dioxide from the surrounding atmosphere [7], but these steps need to be carefully executed by all laboratory team members in the same way. Introducing automated eluent generation can improve confidence in analytical results.

Results and Discussion

Originally developed for its application in IC, Reagent-free ion chromatography (RFIC) is capable of generating high purity hydroxide eluents based on an electrolytic process. It allows for in situ eluent preparation, avoiding the manual handling of external chemicals [8,9]. Figure 1 shows the general setup of an RFIC instrument applied to HPAEC-PAD. The electrolytic process, combining hydroxide ions from the feed water with the counter ions from the eluent generator cartridge (Elugen EGC), happens in the high-pressure part of the IC instrument. By preventing carbon dioxide-contamination, this approach ensures that high-purity eluents can be generated automatically and user-independently. A Chromatography Data and Management System (Thermo Scientific Chromeleon 7 CDS) controls the defined hydroxide concentration and chosen elution program (isocratic, gradient) by automatically adjusting the electrolytic process. In essence, the user only has to ­deploy demineralized water of ASTM Type I quality or better.

Gallery with 6 images

This instrument setup can be used for the determination of mono-, di- or oligosaccharides and aminoglycosides. Figure 2 shows the fast separation of biofuel sugars from lignocellulosic-biomass-derived samples. Patil and Rohrer used a Thermo Scientific Dionex Carbopac SA10-4µm to not only reduce the analytical cycle time (< 10 minutes) but also improve the chromatographic resolution [10]. A small injection volume was applied, combined with a thicker gasket, to manage the high sugar concentrations present in the hydrolysates, avoid a massive dilution of the sample and shift the linearity of the calibration functions towards higher concentrations [11]. The low eluent concentration and the high carbohydrate concentrations in this example are noteworthy.

The same Eluent Generation approach can be used to assay the tobramycin content and quantify the related impurities of tobramycin-based antibiotics. Aminoglycosides were separated at 0.5 ml/min on the Dionex Carbopac PA1 column using a 2 mmol/L KOH eluent and detected by PAD. Figure 3 shows the separation of tobramycin with kanamycin impurities [12]. To ensure that the oxygen dip (a dip in the baseline that is the result of having less dissolved oxygen in the sample than in the eluent) from the previous injection did not appear near a peak of interest, the cycle time was adjusted to 16 minutes. The analytical conditions support the trace determination of impurities in tobramycin and other aminoglycosides like neomycin or paromomycin [13,14]. The elution of streptomycin, kanamycin, and amikacin requires elevated hydroxide concentrations [15,16]. In addition to the HPAEC approaches, the use of a new column, Thermo Scientifc Dionex Ionpac AmG C18, facilitates conventional ion-pair separations of aminoglycosides, followed by PAD or charged aerosol detection (CAD) [17].