GC/FID system with GERSTEL Dual Rail MPS used to automate ASTM Method D6584-07: “Standard Test Method for the Determination of Free and Total Glycerin in B-100 Biodiesel Methyl Esters by Gas Chromatography”.
Samples are prepared during GC analysis of the preceding sample. Whenever the GC becomes ready, the next sample is ready to be injected ensuring best possible system utilization. The screenshot (MAESTRO Sequence Scheduler) illustrates the high efficiency of the biodiesel analysis using MPS GC/FID..
Automated process steps with the MPS
 1. Addition of 100 μL butanetriol solution as internal standard 1
 2. Addition of 100 μL tricaprine solution as internal standard 2
 3. Addition of 100 μL derivatization reagent
 4. Mix (1 min)
 5. Wait (15 min)
 6. Dilution with 8 mL heptane
 7. Mix (1 min)
 8. Sample injection 1 μL on-column
Analysis conditions
CIS: On-column, 60 °C (0.05 min)
0,2 °C/s 230 °C (2 min)
0,5 °C/s 380 °C (10 min)
Column: 10 m Rtx-Biodiesel TG (Restek), ID = 0.32 mm, df = 0.1 µm
Carrier gas: Helium, 3 mL/min (constant flow)
GC Oven: 50 °C (1 min) 15 °C/min
180 °C 7 °C/min
230 °C 30 °C/min
380 °C (10 min)
FID: 380 °C
Chromatogram of a biodiesel standard.
Chromatogram of a real biodiesel sample analyzed using the MPS-GC/FID system.
Diolein calibration curve

Automated determination of glycerin in biodiesel

Sustainable fuel

When Biodiesel is produced, glycerin is generated as a by-product, and it must be removed since it can cause damage to diesel engines. EU- and U.S. guidelines specify the maximum allowable concentrations of free and total glycerin in Biodiesel. A standard method based on GC/FID is available, but it is relatively labor intensive. If the right autosampler and sample preparation robot is used, the entire process can be automated.

Biodiesel is in many ways comparable to mineral oil based diesel fuel. However, unlike conventional diesel fuel, biodiesel is not obtained from crude oil but from renewable raw materials: In the U.S. mainly from soybean oil, in Europe often from rapeseed oil. When the books are balanced as to how environmentally friendly we are, Biodiesel is counted as “renewable energy” and is regarded as a sustainable fuel if certain criteria are met.
Chemically speaking, Biodiesel consists of fatty acid methyl esters (FAMEs). Depending on which raw material was used to produce the fuel, the FAMEs are classified as either soy methyl esters (SMEs) or rape methyl esters (RMEs). Regardless of type and origin of the basic biogenic raw material, FAMEs are produced by trans-esterification of fats and oils (triglycerides). In the process of the alkaline or acidic catalyzed reaction, the trivalent alcohol glycerin is substituted by methanol in order to ensure adequate viscosity of the resulting fuel at a wide temperature range. In the U.S., Biodiesel for use in Diesel engines must conform to ASTM D6751 and the amount of Glycerin is determined using ASTM Method D6584-07 “Standard Test Method for the Determination of Free and Total Glycerin in B-100 Biodiesel Methyl Esters by Gas Chromatography”.

Glycerin, an undesirable travel companion

Apart from FAMEs or SMEs and RMEs, substandard glycerin (SSG) is generated during the Biodiesel production process and a residue is formed consisting of glycerin, water, catalyst, excess methanol and free fatty acids. The SSG by-product is toxic and flammable, but unsuitable as a fuel and generally undesirable, since it forms solid sediment, which can block the fuel filter. When separated from biodiesel, SSG can be purified and re-introduced into the production stream. Additionally, SSG is an important raw material in the production of pharmaceutical and industrial glycerin.

Improved analytical efficiency with automation

Pure Biodiesel is referred to as B 100 diesel. Some engines can operate on B 100 diesel, but in most cases a mixture of the biogenic fuel and mineral oil based diesel is used. In Germany, the Biofuel Quota Act was enacted in 2007, making it mandatory to add as much as five percent biodiesel to conventional diesel (B 5). If you are not sure whether your vehicle should be operated on pure biodiesel or on a mixture such as B 5, you should contact the vehicle manufacturer for advice.
Determining whether biodiesel is free from glycerin requires a suitable analysis method. For the determination of the amount of free and total glycerin and of mono-, di- and triglycerides, European standard EN 14105 and its American counterpart ASTM Method D6584 prescribe the use of gas chromatography (GC) with flame ionization detection (FID). “The analytes must first be transferred into a form that is suitable for GC and this is done through derivatization, which is normally a tedious, labor intensive and time-consuming task”, Dr. John R. Stuff explains. Dr. Stuff and Jacqueline A. Whitecavage, experienced application chemists from GERSTEL, Inc. in Baltimore, MD set out to automate the method from A to Z, reducing the workload while maximizing sample throughput. The sample preparation steps were transferred to the autosampler and fully synchronized with the GC run to ensure that the GC never has to wait for the next sample to be ready.

Dual syringe system for liquid handling

For the analysis, Stuff and Whitecavage used a GC 6890 from Agilent® Technologies with a GERSTEL Cooled Injection System (CIS 4) and FID. Automated sample preparation and sample introduction to the GC was performed using the Dual Rail version of the GERSTEL MultiPurpose Sampler (MPS). The MPS was equipped with two different syringe sizes, a 10 μL on-column syringe and an 80 μL sideport syringe with dilutor module. Biodiesel standards containing glycerin, monoolein, diolein, triolein, butanetriol, and tricaprin, all in pyridine, as specified in ASTM D6584-07 were purchased. Biodiesel B-100 was purchased locally. The glycerin, mono-, di- and triolein standards as well as butanetriol, tricaprin and MSTFA were placed in separate vials in the MPS for further processing. Biodiesel B-100 samples were weighed directly into 10 mL screw cap vials and placed on the MPS tray. The samples and standards were prepared by the MPS based on a GERSTEL MAESTRO Prep-Sequence. ASTM D6584-7 specifies preparation of a five point calibration curve for glycerin, mono-, di-, and triolein from stock standards. Heptane was used for rinsing and dilution. Derivatization was performed using N-methyl-N-trimethylsilyl trifluoracetamide (MSTFA). To reduce the required number of manual steps, the GERSTEL, Inc. scientists set up the MultiPurpose Sampler (MPS) for automated sample preparation. The necessary instructions: Add, Move, Mix, Dilute, Wait, and Inject are selected via mouse click in the menu of the MAESTRO control software PrepBuilder and added to the individual prep method. MAESTRO operates fully integrated into the ChemStation® and GC MassHunter® software (Agilent Technologies). “The manual effort is reduced to the weighing of 100 mg of the sample into 10 mL headspace vials and placing them in the MPS sample tray”, explains Jacqueline Whitecavage. Standards were prepared in empty 10 mL vials placed on the autosampler. All further steps are fully automated and synchronized for best possible throughput: Standard preparation, adding internal standards and derivatization reagent, mixing, incubating, rinsing, and introducing the sample into the CIS. The automated steps of the procedure are performed by the MPS. Processing a sample in the MPS requires approximately 27 minutes. The GC run takes a total of 38 minutes including a seven-minute cool-down phase. “We optimized the method to ensure maximum throughput”, said John R. Stuff. In other words, the MPS prepares the calibration standards at the required concentration levels, after which the first sample is derivatized and injected into the CIS. The next sample is always prepared “just-in-time” for when the GC is ready for the next run. The scientists are satisfied with their results: “The results show good linearity for the standards and a good reproducibility (RSDs range from 2.1 to 2.5 %) for the biodiesel sample.” Automating the sample preparation process means that valuable resources can be used more efficiently and laboratory staff is less exposed to potentially toxic solvents and reagents. Furthermore, system productivity can be maintained overnight and throughout the weekend meaning that valuable instrumentation is used much more efficiently.