Biodiesel is a renewable, alternative diesel fuel produced from vegetable oils, animal fats, or recycled restaurant grease. This non-toxic, biodegradable liquid fuel consists of mono-alkyl esters of long chain fatty acids (also known as fatty acid methyl esters, or FAMEs) and may be used alone or blended with petroleum-based diesel fuels. The most common process for producing biodiesel involves two steps:

  • Through the transesterification reaction, triglycerides (i.e. oils or fats) are chemically reacted with an alcohol, usually methanol, in the presence of a catalyst, like sodium or potassium hydroxide, yielding fatty acid methyl esters (FAMEs) and glycerin by-product.
  • The FAMEs and glycerin by-product are then separated and purified. Biodiesel is the name given to the FAME fraction retained for use as fuel. The glycerin fraction is sold for use in soaps and other products.

The resulting biodiesel contains no sulfur or fossil fuel aromatics. Biodiesel is almost 10% oxygen, making it an oxygenated fuel, which aids combustion in fuel-rich circumstances. Biodiesel can be used pure (B100 biodiesel = 100% biodiesel) or blended (for example, B20 biodiesel = 20% biodiesel and 80% petroleum diesel).

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

  • Manufacturing: The efficient and complete separation of FAMEs from the glycerin by-product results in purer material and less re-processing.
  • 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, B100 biodiesel must be tested for trace levels of contaminants that may cause problems in diesel engines.


  • ASTM D6584, Standard Test Method for Determination of Total Monoglyceride, Total Diglyceride, Total Triglyceride, and Free and Total Glycerin in B-100 Biodiesel Methyl Esters by Gas Chromatography.
  • ASTM D6751, Standard Specification for Biodiesel Fuel Blend Stock (B100) for Middle Distillate Fuels.
  • DIN EN 14103, Fat and Oil Derivates - Fatty Acid Methylesters (FAME) - Determination of Ester and Linolenic Acid Methyl Ester Contents.
  • DIN EN 14105, Fat and Oil Derivatives - Fatty Acid Methyl Esters (FAME) - Determination of Free and Total Glycerol and Mono-, Di-, Tri-glyceride Content.
  • DIN EN 14110, Fat and Oil Derivatives - Fatty Acid Methyl Esters (FAME) - Determination of Methanol Content.
  • DIN EN 14214, Automotive Fuels - Fatty Acid Methyl Esters (FAME) for Diesel Engines - Requirements and Test Methods.
  • ISO 12937, Petroleum Products - Determination of Water - Coulometric Karl Fischer Titration Method.

Bulk separation of FAMEs and glycerin by-product

One of the later steps in the manufacture of biodiesel is the separation of FAMEs and glycerin by-product into two fractions. The FAME fraction is then further purified for use as fuel. Residual glycerin not only lowers the FAME concentration (biodiesel quality), it can also damage automotive engine components. DOWEX DR-G8 dried cation exchange resin is designed as processing aid to reduce and remove trace levels of glycerin, salts, soaps, and other organics from crude biodiesel streams.

Determination of FAME profile as a measure of purity

Biodiesel is produced around the world using a variety of biomass starting materials, including soy, canola oil, palm oil, distilled palm oil, and tallow. The biomass starting material used by a given manufacturing facility depends on how plentiful it is in their region of the world. It is important for bulk biodiesel producers to measure the FAME profile, as it is an indicator of the amount of useable fuel in the final B100 biodiesel product. DIN EN 14103 specifies a procedure for determining the FAME profile in B100 biodiesel samples.

Capillary GC Columns

Chemical standards

We offer two standards that contain the same list of C4 to C24 FAMEs, including two C18:3 linolenic acid methyl ester isomers. These standards are very useful in identifying the FAMEs present in B100 biodiesel regardless of the source of biomass starting material. Our 37-Component FAME Mix is made in methylene chloride, and is ready for GC analysis. Our C4-C24 F.A.M.E. Mix is a NEAT standard that requires the user to dilute in the injection solvent of their choice prior to GC analysis.

Characterized reference oils

These Characterized Reference Oils can be used as part of a quality control program, providing an excellent means of standardizing procedures and comparing results between facilities. They can also be used to identify the source of oils in unknown samples through fingerprinting techniques.

Testing for glycerin/glyceride contamination

ASTM D6751 outlines specifications that must be met for B100 biodiesel to be considered an acceptable fuel source, and include a maximum limit for glycerin content. Specifications are set for free glycerin and also for total glycerin (bound in mono-, di-, and tri-glycerides). The need to verify that the levels of these contaminants are below established guidelines is because of their potential to clog fuel systems. The source of each contaminant can be traced back to a specific manufacturing step:

  • Triglycerides remain if not converted to FAMEs during the transesterification reaction step in the manufacturing process.
  • Mono- and di-glycerides remain due to the incomplete conversion of triglycerides to FAMEs during the transesterification reaction step in the manufacturing process.
  • Glycerin remains due to incomplete separation of FAMEs and glycerin into two fractions during a later step in the manufacturing process.

The actual procedures for testing B100 biodiesel for glycerin/glycerides can be found in ASTM D6584 and DIN EN 14105. Both methods provide for the quantitative determination by silylating the sample with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) followed by high temperature gas chromatography using cool-on-column (COC) injection beginning at 50 °C. Use of a heated injection port can lead to sample discrimination and is not suggested as a replacement for COC. The syringe needle used must have a diameter small enough to fit inside the 0.53 mm I.D. guard column. Automated injection is highly recommended for consistency.

Capillary GC Columns

GC column accessories

To cut a metal capillary GC column, score hard/saw with a ceramic wafer, grab the end to be discarded with needle-nose pliers, and then snap off.

Chemical standards

Glycerin determination by ASTM D6584 and/or DIN EN 14105 requires the use of two internal standards and multi-component calibration solutions, each containing glycerin, monoolein, diolein, and triolein, in varying concentrations. Analysts following ASTM D6584 are required to prepare five different multi-component solutions, while those adhering to DIN EN 14105 must prepare only four different multi-component solutions. Although these chemicals are readily available, the preparation can be time consuming and requires working with pyridine.

Calibration standards preparation for both methods is made easier with pre-made, multi-component, varied concentration solutions. Use of these standards saves time and reduces exposure to pyridine. Step-by-step preparation instructions are included, and were purposefully developed to be similar to those used in preparing B100 biodiesel samples. This ensures that error, due to variation in the preparation of samples vs. standards, will be minimized.

ASTM D6584 and DIN EN 14105 both specify the use of two internal standards, butanetriol and tricaprin. Both of these internal standards are added to every B100 biodiesel sample prior to analysis. They are used to compensate for run-to-run injection variations through comparison to the internal standard retention times and levels in the calibration standards.

Preparation of ASTM D6584 calibration standards involves transferring a known amount of each of our five standard solutions into separate vials along with both internal standards (butanetriol and tricaprin) and MSTFA silylation reagent. After allowing the standard mixtures 20 minutes to fully derivatize, each is diluted with n-heptane. The standards are now ready for GC analysis.

We offer each ASTM D6584 analyte as an individual reference material for those who wish to prepare their own calibration standards.

Due to the possible overlap of FAME and derivatized monoglyceride peaks in the chromatographic analysis, DIN EN 14015 also recommends use of a monoglyceride mixture containing monopalmitin, monolein, and monostearin to aid in peak identification. We offer a monoglyceride stock solution that can be used for this purpose.


ASTM D6584 and DIN EN 14105 both require that the sample undergo silylation with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) to convert any residual mono- and di-glycerides into methyl esters prior to GC analysis. Because of the moisture-sensitive nature of derivatization-grade MSTFA, special precautions should be taken. It is strongly recommended to purchase and use the smallest reagent package practical for the work at hand. Storing the reagent in a tightly sealed container, such as a refrigerated desiccator, will provide the best shelf life. Carefully drying all glassware and syringes that will come into contact with the MSTFA reagent during sample preparation minimizes the opportunity for the reagent to become inactive.

It is important to use only high quality derivatization reagents, to ensure that no artifacts are introduced during the processing of samples. The storage conditions of all derivatization reagents should be strictly adhered to, as some are susceptible to degradation during long-term storage.

All of our high-purity GC solvents are manufactured and bottled under oxygen-free conditions and sealed with a PTFE-lined cap to prevent product contamination and degradation.

Micro Reaction Vessels and Caps

Block Heater

Testing for methanol contamination

Because methanol is commonly used as the catalyst for the transesterification reaction during manufacturing, it may remain at residual levels in the final B100 biodiesel product. Too much methanol in a fuel will cause engine stress. Therefore, its level must be below set specifications for a fuel to be acceptable. DIN EN 14110 describes the headspace analysis of biodiesel for methanol, and is used to verify the residual methanol level is below set guidelines. A capillary GC column with a non-polar phase is most suitable for this application, allowing the more volatile compounds (analyte methanol and internal standard 2-propanol) to elute prior to the biodiesel components (FAMEs). Our Equity-1 columns are made with a poly(dimethylsiloxane) phase, which allows separation of analytes primarily according to boiling point. Several dimensions of this column are available to suit the needs and desires of the analyst.

As an alternative to traditional headspace, headspace solid phase microextraction (SPME) is also able to perform this application, requiring considerably less equilibration time (5 min. vs. 45 min.) while exhibiting excellent sensitivity at the low end of the required calibration range.

Whether by traditional headspace, or headspace SPME, we have many high-quality products for the determination of methanol in B100 biodiesel.

Capillary GC Columns

Gas syringes and headspace vials

All syringes in the Series A-2 line have PTFE-tipped plungers, and easy-to-use push-button valves. Simply push the red side to close, and the green side to open. Additionally, an oversized flange at the top of the syringe barrel simplifies handling during operation.

Our headspace vial convenience kits provide vials, closures, and septa all in one package. This insures that each component fits together properly, and eliminates the guess-work in identifying the correct items. Helpful accessories are also offered.

SPME fibers and holders

Solid phase microextraction (SPME) is a solvent-less extraction technique. Two components are required to perform SPME:

  • A SPME fiber assembly.
  • The appropriate holder to operate the fiber assembly.

Fiber assemblies coated with a polyacrylate material are suitable for the small, volatile, polar analyte (methanol) and internal standard (2-propanol) required for this application.

For manual, hands-on sampling/analysis with manual fiber assemblies, use P/N 57330-U.
For automated sampling/analysis with autosampler fiber assemblies, use:

  • P/N 57347-U for CTC CombiPal, Gerstel MPS 2, and Thermo TriPlus autosamplers.
  • P/N 57331 for Varian autosamplers.

Testing for chloride and sulfate contamination

Despite passing through several purification steps following fermentation, bulk biodiesel may contain the dissolved salts chloride and sulfate. Because of their ability to damage modern engines, determining chloride and sulfate levels in biodiesel-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.


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