Sigma-Aldrich Introduces New Standards Kit for ASTM D5501: Determination of Ethanol Content of Denatured Fuel Ethanol

By: Vicki Yearick, Steve Cecil, Reporter US Volume 27.2

Current public focus on reduction of the use of fossil fuels and replacement of these fuels with cleaner-burning renewable fuels has played a key role in the growth of the fuel ethanol industry. Large demand for blending ethanol into gasoline began with its use as an oxygenated gasoline additive. Currently the US EPA has set the denatured ethanol-to-gasoline blend rate at 10 percent, but discussions are being held to increase this level to 15 percent or higher. Automotive companies are also producing flex-fuel vehicles; able to operate on ethanol-to-gasoline blends of up to 85 percent denatured ethanol, called E85.

With all this increased attention to fuel ethanol, producers and blenders have increased awareness of mandated fuel ethanol specifications, outlined in ASTM D4806 - Specification for Denatured Fuel Ethanol for Blending with Gasoline for Use as Automotive Spark-Ignition Engine Fuel.

Ethanol producers are required by law 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 with state and federal laws. Monitoring is accomplished by following the analytical method ASTM D5501 - Determination of Ethanol Content of Denatured Fuel Ethanol by Gas Chromatography.

The GC method ASTM D5501 specifies the use of temperature program and a flame ionization detector to analyze the sample on a long polydimethylsiloxane capillary column, such as the Supelco Petrocol DH 150 (Cat. No. 24155). The method cites establishing peak identification, followed by the quantitation of ethanol.

The identification of ethanol and methanol is performed by injecting into the GC column a mixture containing known amounts of each alcohol in proportion to what is expected in the final blend, using n-heptane as a solvent. Retention times of the fuel ethanol sample are then compared to the analytical standard to verify identity.

Quantitation requires preparation of six multi-component calibration solutions, each containing ethanol, methanol and n-heptane in varying concentrations, to establish a linearity curve for the GC system. Because D4806 specifies a minimum ethanol content of 92.1% for denatured fuel ethanol, the ethanol content found in the six solutions range from 92 to 97%. N-heptane is included in the solutions in-place of the denaturant.

The D5501 calibration solutions and the denatured fuel ethanol samples are individually analyzed using the column conditions and temperature program shown in Figure 1. The mass relative response factors for the fuel ethanol sample are calculated to the nearest 0.01 mass percent, and then compared to the values obtained for each of the six calibration solutions to determine the ethanol content in the denatured fuel ethanol sample for reporting purposes.

Figure 1. E85 Denatured Fuel Ethanol

Preparation of these calibration standards is time consuming and requires maintaining an inventory of high purity raw materials. Calibration standards preparation is made easier by using Sigma-Aldrich’s ASTM D5501 Denatured Fuel Ethanol Standards Kit. This kit contains pre-made Supelco brand multi-component, quantitative solutions covering the required range for accurate calibration, per the ASTM D5501. A certificate of analyses is provided for each calibration solution. To learn more about this kit and suggested analytical columns, please visit

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