Bioethanol is a renewable, alternative fuel traditionally produced by the yeast fermentation of sugar. When used as an oxygenated additive in gasoline to yield a cleaner-burning fuel, blends typically range from E10 (10% ethanol and 90% gasoline) to E25 (25% ethanol and 75% gasoline). It is also used in ¢flex-fuel¢ vehicles that can operate with higher ethanol percentages. In the USA, an E85 blend (85% ethanol and 15% gasoline) is used. In Brazil, where ethanol is made from sugar-cane, over half of their cars can operate on E100. Typically, commercial production of fuel ethanol involves

  • The breakdown of a starch (from corn, sugarcane, etc.) into simple sugars.
  • The fermentation of the sugars into ethanol using yeast.
  • The recovery of the ethanol, termed bioethanol, which is retained for use as a fuel. The by-product mass is sold for use as animal feed.

Many areas of the process are important to ensure a quality end product. However, none are more critical than the ethanol-producing step of fermentation. Residual sugars left unfermented not only lower ethanol concentrations, they also damage automotive engine components. Consequently, fuel ethanol producers continually monitor their process for purity levels, as well as for the presence of several contaminants.

Sigma-Aldrich offers many high-quality products to assist bioethanol producers and testing facilities with several key areas in the process:

  • Determining Purity: Necessary to be able to gauge the overall quality of the process and final product.
  • Testing for Contamination: Before being used or blended, E100 bioethanol must be tested for trace levels of contaminants that may cause problems in automotive engines.


  • ASTM D4806, Standard Specification for Denatured Fuel Ethanol for Blending with Gasolines for Use as Automotive Spark-Ignition Engine Fuel.
  • ASTM D5501, Standard Test Method for Determination of Ethanol Content of Denatured Fuel Ethanol by Gas Chromatography.

Determination of ethanol content as a measure of purity

Specifications for fuel ethanol that bulk producers and blenders must meet are outlined in ASTM D4806. This standard also requires bulk ethanol producers to render the fuel ethanol unfit for human consumption by adding a denaturant, typically natural gasoline. ASTM D4806 requires the fuel ethanol to contain a minimum of 92.1% ethanol by volume, with the denaturant volume ranging from 1.96% to 4.76%. Producers and blenders must monitor and report the content of ethanol and the denaturant to show they are in compliance. The GC-FID analytical procedure for measuring ethanol content in fuel ethanol can be found in ASTM D5501.

Once peak identifications are established, the quantitation of ethanol in samples may begin. To perform this, a mixture containing known amounts of each alcohol in proportion to what is expected in the final blend is injected into the GC column, using n-heptane as a solvent. Retention times of the fuel ethanol sample are then compared to the analytical standard to verify identity.

ASTM D5501 specifies the use of a long poly(dimethylsiloxane) capillary column, such as our Petrocol DH 150. These non-polar columns have considerable theoretical plate numbers and are designed for detailed hydrocarbon analysis (DHA). They are characterized by high efficiency and great reproducibility.

Testing for saccharide contamination

Efficient fermentation (the conversion of sugars to ethanol) is critical to the success of bulk fuel ethanol producers. Optimized fermentation leads to increased ethanol yields and profitability. Residual sugars left unfermented not only lower ethanol concentration, they also damage automotive engine components. Consequently, bulk fuel ethanol producers continually monitor the fermentation broth for the presence of unfermented sugars, also known as saccharides.

HPLC columns under saccharide contamination

The HPLC analysis utilizes a crosslinked polystyrene/divinylbenzene resin ion exchange column. Traditional methods require a balancing of resolution and run time:

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

Our SUPELCOGEL C-610H column has proven to be an excellent choice for this analysis, yielding a short 14 minute run time with resolution of eight key analytes. These columns contain a sulfonated divinylbenzene ion exchange resin with the cation in the hydrogen form.

Testing for chloride and sulfate contamination

Despite passing through several purification steps following fermentation, bulk bioethanol may contain the dissolved salts chloride and sulfate. Because of their ability to damage modern engines, determining chloride and sulfate levels in ethanol-based fuels is an important quality criterion. A standard method for detection of these analytes is ion chromatography (IC) configured with a conductivity detector. However, it lacks the capability to definitively identify the compounds.

An alternative method involves LC-MS. This is accomplished by post-column reaction with a di-/tri-cationic solution. This reaction causes positively charged adducts to be formed between the cationic reagents and the anionic chloride and sulfate. These adducts are detectable by the MS in the highly sensitive positive ESI mode. Advantages over IC methods are:

  • Definitive identification of analytes by their mass and spectra.
  • Easier sample preparation for complex matrices like biofuels.

Chemical standards

Independent of analytical techniques, the quality of quantitative results strongly depends on the precision and accuracy of the analytical reference standards. That is, even with a robust, well-calibrated method, results will have a high uncertainty when the reference materials are not well defined. Our TraceCERT product line of anion standards ensures the highest accuracy for analytical measurements.

Cation reagents

Several dication and trication reagents are available, providing the flexibility for users to design and make a specific solution combination best suited for their unique biofuel samples.


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