Uronic Acid and Wood Sugar Analysis for Biofuel Research

Monosaccharides are members of the most plentiful set of biomolecules in nature. They have a critical part to play in metabolism, structural biology, and energy storage. As such, studying this specific variety of carbohydrates is a priority, not only for the food industry, but also for a wide range of material and life sciences.

Hydroxyl groups being present allows for a particular and extremely sensitive type of analysis, which uses pulsed amperometric detection (PAD) with the DECADE Elite electrochemical detector as part of the purpose-built AZURA® High Performance Anion Exchange Chromatography (HPAEC) system.

Introduction

There can be variation in the sources for the diverse types of monosaccharides, from food samples, like honey [1] or fruits, to scientific applications, such as glycopeptides. They can even originate from fermentation processes, as is the case with the wood monosaccharides studied here.

The combination of the seven hemicellulosic sugars; fucose, rhamnose, arabinose, galactose, glucose, xylose and mannose, blended with the two uronic acids; galacturonic acid and glucuronic acid, extracted from wood by heat or chemical pre-treatment, are considered particularly interesting to those involved with the hunt for new biofuels.

Since they are considered to be comparatively more sustainable, it is anticipated that they will become a viable commercial alternative to fuels produced with corn and other food sources [2].

As carbohydrates are weak acids, with pKa values from 12 to 14, it is possible to partially or totally ionize them under basic conditions with a pH level greater than 12. Since these conditions are somewhat severe, monosaccharide analysis can only be successfully carried out with polymeric anion exchange columns.

The retention time with AZURA HPAEC has a relationship of inverse correlation with pKa value and demonstrates a notable increase with molecular weight of the monosaccharide.

Pyranose structure of the seven wood monosaccharides and the two uronic acids

Pyranose structure of the seven wood monosaccharides and the two uronic acids

Results

With the use of an analyte concentration of 0.1 mg/mL for the standard blend of the nine wood monosaccharides and acids, each component could be baseline-separated (Rs > 1.5) (Fig. 1). The separation of the analyte peaks rose with decreasing sample concentration.

The two monosaccharides xylose (6) and mannose (7) could not be baseline-separated when in concentrations stronger than 0.1 mg/mL. The signal to noise (S/N) ratio for each analyte was drawn from empiric data (Tab. 1).

Noise values were calculated for this concentration from two different baseline areas. For the monosaccharide sugars 1-7, the averaged noise was determined with 0.001 µA, while for the uronic acids 8-9 a value of 0.1 µA was determined. Figure 2 depicts concentration curves of all analytes from 0.0125 to 0.25 mg/mL.

Chromatogram of a standard mixture containing 0.1 mg/Ml fucose (1), rhamnose (2), arabinose (3), galactose (4), glucose (5), xylose (6), mannose (7), galacturonic acid (8) and  glucuronic acid (9). And a zoom into the peaks for the uronic acids

Figure 1. Chromatogram of a standard mixture containing 0.1 mg/Ml fucose (1), rhamnose (2), arabinose (3), galactose (4), glucose (5), xylose (6), mannose (7), galacturonic acid (8) and  glucuronic acid (9). And a zoom into the peaks for the uronic acids

Concentration curves of the described sugars and uronic acids in a concentration range between 0.0125 mg/mL to 0.25 mg/mL

Figure 2. Concentration curves of the described sugars and uronic acids in a concentration range between 0.0125 mg/mL to 0.25 mg/mL

Table 1. Empiric determined S/N ratios for a 10 μL injection

Analyte S/N
L-fucose 10000
L-rhamnose 3000
L-arabinose 4800
D-galactose 3800
D-glucose 2400
D-xylose 1600
D-mannose 1600
D-galacturonic acid 338
D-glucuronic acid 574

 

Materials and Methods

To carry out the analysis, researchers used the AZURA glass- and metal-free High Performance Anion Exchange Chromatography (HPAEC) system, which was made up of an AZURA P 6.1L LPG pump, an AZURA AS 6.1L autosampler and a DECADE Elite electrochemical detector, the latter of which had a secondary use in column tempering.

The analysis was based on a step-gradient with varied concentrations of NaOH solution (Tab. A2 & A3, additional material).

Carbonate ions, which are present in the mobile phase, can bind to the column material and lessen separation efficiency when low concentrations of NaOH are used. For this reason, it is advisable to employ a column regeneration with higher concentrations of NaOH for each run.

In addition, when preparing eluent, the contamination with carbonate ions should be minimized using carbonate-free 50% w/w NaOH solution, which is available commercially, and an ultrasonic degassing step before bringing the eluent into the system.

Fresh eluents should be used daily. Bearing in mind the high sensitivity of the DECADE Elite detector and the etching property of the NaOH, it is important to use only plastic eluent bottles, plastic eluent filters and metal-free system compartments, to avoid the unwelcome detection of unanticipated ions, silicates or borates.

To enable detection, an Antec electrochemical SenCell with Au working electrode, HyREF (Pd/H2) reference electrode and stainless steel auxiliary electrode was employed with a 4-step potential waveform (Fig. A1, additional material).

Conclusion

High Performance Anion Exchange Chromatography (HPAEC) with pulsed amperometric detection (PAD), using the AZURA HPAEC-PAD dedicated system and applying the developed method is an extremely sensitive system for the study of sugar monosaccharides and other carbohydrates.

The blend of seven monosaccharides and two uronic acids was able to be baseline-separated with very high S/N ratios. A quick and replicable analysis is possible, even for low concentrations, with a simple method employing varied concentrations of NaOH.

Aside from their use in research for biofuels, the sugars analyzed are components in many processes in nature and food applications. Therefore, the current application is appropriate for several issues in which there is a need for carbohydrates to be specifically separated and analyzed.

References

  1. H. Schlicke, K. Monks, KNAUER AppNote VFD0161, 2017.
  2. M. J. González-Muñoz, R. Alvarez, V. Santos, J. C. Parajó, Wood Science and Technology, 2012, 46, 1–3, 271–285

This information has been sourced, reviewed and adapted from materials provided by KNAUER Wissenschaftliche Geräte GmbH.

For more information on this source, please visit KNAUER Wissenschaftliche Geräte GmbH.

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