January 2023

Biofuels, Alternative/Renewable Fuels

Fast ion chromatography analysis for determining biofuel sugars

All industrial processes require reliable, accurate and timely methods for analyzing process chemistry.

Shevlin, C., Thermo Fisher Scientific

All industrial processes require reliable, accurate and timely methods for analyzing process chemistry. The biofuel industry faces unique challenges in its production processes, which must generate pure products from complex raw feedstock materials that are often highly variable in their original composition. This necessitates the use of advanced analytical tools to ensure quality control along the entire production workflow, as well as highlight issues with process variations long before yield is adversely affected.

Below are three key stages of process analysis within the biofuel production workflow (FIG. 1):

  1. In biofeedstock characterization, accurate compositional analysis of biomass raw material (algae, crops or cellulosic plants) is essential for determining production yield, which, in turn, influences the ability to produce robust cost estimates for biofuel consumption.
  2. For bioalcohol production, it is crucial to determine the most favorable conditions for converting complex carbohydrates into fermentable sugars (via fermentation monitoring) to maximize final product yield. For producing biodiesel from algae, all cell products and carbohydrate breakdown products should be analyzed to determine which sugars are best absorbed by the algae for nutrient recycling.
  3. The final step is quality control analysis, which is critical for identifying trace contaminants and ensuring product purity in both bioalcohol and biodiesel manufacturing.
FIG. 1. Biofuel production analysis workflow for bioalcohol and biodiesel.

The challenges of carbohydrate analysis

Lignocellulosic biomass is the most abundant biomass in the world and an important renewable source for alternative energy generation. With the continued exploration of new types of cellulosic biomass as raw materials, it is important for biofuel producers to find new approaches that will maximize energy production from these starting materials.

An essential aspect of determining the efficiency of biomass-to-biofuel conversion is monitoring the release of 5- and 6-carbon sugars, as this is directly related to yield and process economics. However, robust quantification of the diverse mix of sugars in hemicellulose is challenging, and the methods that are currently used suffer from several drawbacks, including:

  • Low throughput: Sample preparation for current carbohydrate analysis methods is time-consuming. This can reduce the lab’s throughput and influence how quickly results are obtained. It also drives up the cost per sample because of the materials and reagents required for sample preparation. Longer run times are also needed to sufficiently resolve complex matrices.
  • Poor analyte resolution: Biomass often contains a complex mixture of 5- and 6-carbon sugars, and some contain structurally similar sugars that are challenging to separate. This is the case for the closely related sugars known as galactosamine and glucosamine, which are commonly found in algal biomass hydrolysates. Sugars tend to not separate well when using high-performance liquid chromatography (HPLC), which can result in poor data quality. Moreover, because similar mono- and di-saccharide sugars have the same mass-to-charge ratio, they might not be distinguishable when using a single quadrupole mass spectrometer (MS). A more advanced MS could be utilized, but this requires considerable investment.
  • Lack of broad applicability: The increasing range of biomass source materials being explored requires analysis methods that are applicable to highly variable biomass feedstock materials. Conventional carbohydrate analysis methods cannot handle the divergent ratios of sugars in these samples.

Faster accurate analysis of sugars in complex matrices

HPLC and MS approaches have several limitations. An alternative is to use ion exchange chromatography. This technology provides the capability to develop a detailed understanding of the chemical composition and trace contaminant analysis across every step of biofuel production. It is also typically more robust for sugar analysis than spectroscopy-based techniques.

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) has been shown to deliver fast determination of carbohydrates in biomass hydrolysate samples. This method has been successfully used to resolve and detect the eight common fermentable sugars derived from lignocellulosic biomass—xylose, sucrose, arabinose, galactose, glucose, mannose, fructose and cellobiose—in 6 min.

This advancement on previous chromatography methods was achieved through a combination of smaller resin particle size and shorter column length and provides significant time savings. The shorter run time allows for faster sample turnarounds and reduced eluent consumption, improving overall process economics.

Accurate analysis of diverse samples

When tested for its accuracy and robustness, the improved HPAE-PAD method was comparable with longer HPAE-PAD analysis protocols. Using HPAE-PAD for lignocellulosic biomass analysis, peak area calibration curves of more than two orders of magnitude wide were generated for all eight sugars. This shows that the calibration range can handle a wide concentration divergence without the need for sample dilution, thus avoiding potentialdilution errors.

Moreover, the accuracy of the HPAE-PAD method was demonstrated with a recovery study of 10 biomass hydrolysate samples spiked with 50%–150% of the original sugar amounts found in each sample.

Adaptable for algal biomass

The HPAE-PAD method used for lignocellulosic biomass analysis is not optimal for algal biomass hydrolysates because it is unable to resolve the key amino sugars (galactosamine and glucosamine) that occur frequently in algae. Instead, two adapted HPAE-PAD methods were developed that can achieve similar resolution and accuracy to the protocol used for lignocellulose biomass.

The first method separates 12 common carbohydrate sugars in less than 15 min but is unable to resolve uronic acids. These key components are present at low concentrations and strongly bind to anion-exchange columns. A second, longer method using acetate as a pusher does extend the run time, but successfully separates the uronic acids and the 12 other sugars.

For a quick quantification of sugars present in an algal biomass sample, the shorter method can be used—whereas, for more detailed sugar composition calculations, the longer method would be required. Both methods can be run simultaneously on a dual-pump ion chromatography (IC) system.

These methods are convenient, precise and robust for algal biomass sugar analysis, and will improve the reliability of biomass-to-biofuel efficiency calculations. Moreover, using HPAE-PAD provides the required separation and specificity for mono- and di-saccharide sugars at a very economical price point vs. other methods, such as advanced MS. 

Integrated analysis for improved process monitoring

Online IC provides fast, reliable and accurate methodologies for the in-process analysis and characterization of biofuel production. This technology is also applicable in a range of other fields where early detection of process variations is critical.  

The process analyzers described here enable the measurement of critical analytes in a range of process operations, from detecting corrosive ions and additives in power plant coolants, to identifying trace contaminants in ultra-pure water and characterizing environmental contaminants in surface and groundwater.

Online IC or liquid chromatography (LC) systems can be easily configured for process analysis at any scale: from helping laboratories fine-tune process chemistry in the research and development (R&D) phase, to pilot plant and large-scale manufacturing with continuous monitoring.

A key advantage of the HPAE-PAD system described here is its modular design, which enables integration with time-saving technologies, such as online sample preparation tools and corresponding detectors. This allows exploration of the benefits of online IC analysis on a small-scale basis, so that customized monitoring systems can be established for the lab and for the plant, and modules can be added as needed to meet the scale required at different stages of R&D and production. For further exploration or monitoring, online analysis can also be converted to conventional IC/HPLC for grab sample analysis.


All industrial processes require robust, accurate and timely process analysis and quality control. Online IC and HPLC systems offer fast, accurate and reliable analyses of challenging biological samples. Using these methods for continuous process monitoring enables earlier detection of variations, long before yield is impacted or costly process shutdowns become necessary. Today’s modular IC and HPLC systems enable easy configuration and integration with other analyzers and detectors, as well as the ability to scale up from the lab to large-scale manufacturing—this provides confidence in process chemistry at every stage, from raw materials to the final product. HP

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