Fuel Ethanol: Fermentation Analysis by HPLC

By: Steve Cecil, Reporter US Volume 26.3

One Source Solution of Column and Quantitative Calibration Standard


Fuel ethanol continues to be the mainstay in the biofuel arena, with increasing production yield and higher conversion percentages of corn-to-ethanol driving discussion of both economic and environmental viability of the product.

Ethanol is traditionally produced by the fermentation of sugar by yeast. Typically, commercial production of fuel ethanol involves breakdown of the starch into simple sugars, yeast fermentation of these sugars, and finally recovery of the main ethanol product and byproducts (e.g., animal feed).

Many areas of the process are important to ensure a quality end product, such as the breakdown of the corn substrate to fermentable sugars and distillation. However, none are more critical than the ethanol-producing step of fermentation.

Optimized fermentation leads to increased ethanol yield and profitability of the biofuel facility. Residual sugars left unfermented lower ethanol concentrations, increase plant water usage and often require additional fermentation equipment cleaning and maintenance. Consequently, fuel ethanol producers continually look for more efficient processing techniques.

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Importance of HPLC Analysis of Residual Saccharides

A key measurement is the residual sugar and ethanol concentrations in the fermentation broth. Fuel ethanol facilities use High-Performance Liquid Chromatography (HPLC) as the technique of choice to monitor the ethanol fermentation process. HPLC permits detailed monitoring of the complete cycle, including conversion of the sugars to ethanol and ethanol breakdown to acetic acid.

The HPLC analysis utilizes a crosslinked polystyrene / divinylbenzene resin ion exchange column. Traditional methods suffer from a) poor resolution or b) long run times.

  1. Methods with fast run times (< 12 minutes) sacrifice resolution of the early eluting saccharides. These fast methods often show co-elution of the dextrin, maltotriose and maltose peaks.
  2. Methods focused on improved resolution of the simple sugars suffer from long run times. To achieve improved resolution for the early eluting saccharides, these methods have run times exceeding 22 minutes.

Neither of these is an acceptable compromise for your lab. The SUPELCOGEL™ C-610H column has proven to be an excellent choice for this analysis, yielding a shorter run time as well as resolution of all eight key components. Figure 1 illustrates the improved separation of components in the Supelco Fuel Ethanol Residual Saccharides Mix.

Figure 1 Fuel Ethanol Residual Saccharides Mix Run on the SUPELCOGEL C-610H HPLC Column (59320-U)

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Importance of Fuel Ethanol Residual Saccharides Mix

It is critical to ensure the analysis is calibrated through a commercially available quantitative calibration standard. The Supelco Fuel Ethanol Residual Saccharides Mix contains key components used to monitor the fermentation process. These components include dextrin (DP4+), maltotriose (DP3), maltose (DP2), D-glucose, and ethanol.

In addition to the saccharides and ethanol components, acetic acid, lactic acid and glycerol are included in the quantitative standard. Lactic acid and acetic acid are breakdown products produced during fermentation. Glycerol is also added to measure the stress being placed on the yeast during fermentation. Figure 1 illustrates the Fuel Ethanol Residual Saccharides Mix run on the SUPELCOGEL C-610H HPLC column.

Utilizing Supelco’s Fuel Ethanol Residual Saccharides Mix in conjunction with the SUPELCOGEL C-610H column can lead to improved fermentation and higher ethanol yields. Both of these products are backed by strong technical support from the people you trust at Sigma-Aldrich.

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