Free and Total Glycerin in Biodiesel by Gas Chromatography

Mar 02, 2007

Rudolph Diesel originally designed his 1892 prototype of the diesel engine to run on a renewable fuel made from peanut oil and other vegetable oils. Today, as oil prices continue to climb, the world's interest in alternative fuels is stronger than ever. One of the most popular fuel alternatives is biodiesel. Biodiesel is a renewable fuel from natural oils like soybean oil, rapeseed oil or animal fats and can be used as a substitute for diesel fuel. Biodiesel is often mixed with petroleum based diesel; the common nomenclature for blended fuel is noted as the percentage of biodiesel in the blend. B100 is 100% biodiesel. ASTM method D6751 specifications serve as the benchmark, which identifies the standards that pure biodiesel (B100) must meet before being used as fuel or being blended with petrodiesel.


The gas chromatograph used in this application note is a PerkinElmer Clarus GC with a programmable on-column (POC) injector and flame ionization detector (FID). The capillary column used was an Elite-5HT (15 m × 0.32 mm × 0.10 μm), with a guard column of (254 mm × 0.53 mm i.d.). The carrier gas, helium, is programmed with a constant flow of 3 mL/min. The GC oven-temperature program is as follows: initial temperature 50 °C, hold for 1 min, ramp at 15 °C/min to 180 °C, no hold, ramp at 7 °C/min to 230 °C, no hold, ramp at 30 °C/min to 380 °C, hold for 10 min. A cold on-column injection of 1 μL introduced the sample; the POC injector is programmed to track the GC oven temperature. The FID conditions are as follows: temperature: 380 °C, range: 1, attenuation: 4, air flow: 450 mL/min, hydrogen flow: 45 mL/min.

Results and Discussion

Figure 1
A typical chromatogram for biodiesel prepared from soybean oil is shown in Figure 1. Total monoglycerides are calculated with a summed peak group including all five monoglyceride peaks, but not including C24 carboxylic acid methyl ester peak, eluting between the monoglycerides. The total diglycerides are calculated as a summed time group, including all peaks eluting with diolein ±0.35 min. Total triglycerides are calculated as a summed time group, including all peaks eluting with triolein ±1 min. Free glycerin is calculated by the area of the glycerin peak. Total glycerin is calculated by the sum of free glycerin plus a percentage of total mono-, di- and triglycerides, as formulated in ASTM D6584.


Alternative fuel interest continues to increase with higher prices at the pump. With this increased interest, both established fuel suppliers and new entrepreneurs are doing their best to respond to this interest by producing quality, alternative fuel products. The ASTM method discussed in this paper uses gas chromatography (GC) to measure the quality of biodiesel. The GC method presented here is simple, sensitive and reliable and requires only a small amount of sample preparation.

Timothy Ruppel and Gerald Hall, PerkinElmer Life and Analytical Sciences, Shelton, Connecticut, USA.


1. ASTM Method D6751 03a, Standard Specification for Biodiesel Fuel (B100) Blend Stock for Distillate Fuels.

2. ASTM Method D6584, Test Method for Determination of Free and Total Glycerin in B100 Biodiesel Methyl Esters by Gas Chromatography.

3. US Energy Pollution Act of 1992.

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