Analysis of FAMEs in Biodiesel Fuel: Pro EZGC Modeling Software Ensures Proper Column Selection
Abstract
Polar columns were evaluated for the analysis of fatty acids methyl esters (FAMEs) in finished B100 biodiesel according to method EN 14103 (2011). Using Restek’s Pro EZGC chromatogram modeler, a high cyano phase Rt-2330 column and a polyethylene glycol phase FAMEWAX column were compared. The modeling software predicted an unacceptable coelution between the internal standard (C19:0 FAME) and FAME C18:2 when using the Rt-2330 column. However, the modeler also predicted that the FAMEWAX column would separate all the compounds of interest, which was demonstrated empirically. In addition, the results on the FAMEWAX column showed excellent repeatability for both total FAMEs and the linolenic acid methyl ester component.
Introduction
Biodiesel is a diesel fuel made from plant or animal fat feedstocks. These biologically sourced fats, predominantly triglycerides (1), are converted into fatty acid methyl esters (FAMEs) via a transesterification reaction that occurs in the presence of methanol and a basic or acidic catalyst. This reaction produces biodiesel fuel and also generates glycerol as a byproduct. The biodiesel FAME profile is determined by the type of fat that is used in the reaction and, therefore, the specific composition of biodiesel can vary from saturated to unsaturated FAMEs. The ester composition of biodiesel is used to determine product quality and to calculate its cetane number. According to the method EN 14214, which regulates biodiesel quality, ester content in 100% biodiesel (B100 product) has to be greater than 90% total fatty acid methyl esters by mass. In addition, the linolenic acid methyl ester (methyl linolenate) content must be between 1% and 15% by mass (2).
European standard method EN 14103 (2011) is widely used for the analysis of FAMEs in biodiesel. It is specifically used to determine both the FAME composition and, simultaneously, the linolenic acid methyl ester concentration. Linolenic acid methyl ester is a methyl ester of a polyunsaturated fatty acid where both trans and cis isomers can be present. A high concentration of linolenic acid methyl ester is undesirable because its poor oxidation stability can change fuel properties and form undesirable species (3).
According to method EN 14103 (2011), polar FAMEs in biodiesel can be resolved and quantitated using gas chromatography and highly polar capillary column (2). Polar columns, such as high cyano (Rt-2330) or polyethylene glycol columns (FAMEWAX) offer excellent retention and selectivity for polar FAME compounds. This application note uses the Pro EZGC chromatogram modeling software to assess the performance of two polar analytical columns for EN 14103 (2011) biodiesel analysis and then compares the model output to empirical data.
Experimental
Rt-2330 and FAMEWAX columns were selected for this experiment because they are highly polar phases that have been shown to generally perform well for FAMEs analysis. Both columns were initially evaluated using two criteria: selectivity using Pro EZGC chromatogram modeling software and overall method suitability. The Pro EZGC modeler conditions were customized to match EN 14103 (2011) operating conditions and the most prevalent FAMEs were chosen and modeled along with C19:0 as an internal standard.
The results from the most promising modeled chromatogram were confirmed in the laboratory by analyzing a Restek food industry FAME standard (30 mg/mL in methylene chloride, cat.# 35077) and a single compound FAME C19:0 internal standard (10 mg/mL in toluene, cat.# 35055) on a FAMEWAX 30 m x 0.25 mm x 0.25 µm column (cat.# 12497). Commercially obtained canola and soy biodiesel B100 samples were also analyzed following method EN 14103 (2011).
In order to assess repeatability, total ester content and linolenic acid methyl ester content over multiple analyses were calculated as described in the method.
Results and Discussion
The modeled Pro EZGC chromatogram for the Rt-2330 column (Figure 1) clearly illustrates a coelution between the internal standard FAME C19:0 and trans C18:2 FAME, which can be present in biodiesel. This means that the Rt-2330 column is not suitable for the analysis of FAMEs in biodiesel under the method conditions because the internal standard was not completely resolved. Predicting this problem using the Pro EZGC chromatogram modeler took only minutes on the computer, providing a substantial savings of time and money compared to determining this experimentally in the lab.
Figure 1: Pro EZGC modeling software predicts the coelution of internal standard FAME C19:0 with trans C18:2 FAME on an Rt-2330 column. This allows analysts to remove the column from consideration without the time and expense of testing its performance in the lab.
The Pro EZGC chromatogram modeler is not only useful in determining that a column will not work, it also can be used to predict what column phase will work best for the analysis of FAMEs in biodiesel according to method EN 14103 (2011). As shown in Figure 2, the modeled FAMEWAX chromatogram predicts that the critical separation of linolenic acid methyl ester will be achieved as well as complete resolution of the internal standard. While another coelution is predicted later in the chromatogram, the internal standard is completely resolved and the other coelution is not critical because the FAME peak areas will be summed according to the method. Based on the promising nature of the modeled output, chromatographic results were confirmed in the lab and the actual FAMEWAX column analysis sufficiently matched the predicted results. As shown in Figure 3, the selectivity of the FAMEWAX column separated all critical components.
Figure 2: The Pro EZGC chromatogram modeler predicts good separation of the internal standard and linolenic acid methyl ester from FAMEs that are commonly present in biodiesel using a FAMEWAX capillary column.
Figure 3: Chromatogram overlay of a FAME standard and a C19:0 internal standard analyzed on a FAMEWAX column closely match modeled results.
| Peaks | tR (min) | Conc. (wt.%) | Structural Nomenclature | |
|---|---|---|---|---|
| 1. | Methyl capronate | 3.629 | 1.2 | C6:0 |
| 2. | Methyl caprylate | 6.237 | 1.2 | C8:0 |
| 3. | Methyl caprate | 8.787 | 1.2 | C10:0 |
| 4. | Methyl undecanoate | 9.971 | 0.6 | C11:0 |
| 5. | Methyl laurate | 11.105 | 1.2 | C12:0 |
| 6. | Methyl tridecanoate | 12.179 | 0.6 | C13:0 |
| 7. | Methyl myristate | 13.215 | 1.2 | C14:0 |
| 8. | Methyl myristoleate | 13.549 | 0.6 | C14:1 (cis-9) |
| 9. | Methyl pentadecanoate | 14.196 | 0.6 | C15:0 |
| 10. | Methyl pentadecenoate | 14.524 | 0.6 | C15:1 (cis-10) |
| 11. | Methyl palmitate | 15.152 | 1.8 | C16:0 |
| 12. | Methyl palmitoleate | 15.355 | 0.6 | C16:1 (cis-9) |
| 13. | Methyl margarate | 16.052 | 0.6 | C17:0 |
| 14. | Methyl heptadecenoate | 16.261 | 0.6 | C17:1 (cis-10) |
| 15. | Methyl stearate | 16.995 | 1.2 | C18:0 |
| 16. | Methyl oleate | 17.156 | 1.2 | C18:1 (cis-9) |
| 17. | Methyl elaidate | 17.168 | 1.2 | C18:1 (trans-9) |
| 18. | Methyl linoleate | 17.583 | 0.6 | C18:2 (all-cis-9,12) |
| Peaks | tR (min) | Conc. (wt.%) | Structural Nomenclature | |
|---|---|---|---|---|
| 19. | Methyl linolelaidate | 17.641 | 0.6 | C18:2 (all-trans-9,12) |
| 20. | Methyl γ-linolenate | 17.874 | 0.6 | C18:3 (all-cis-6,9,12) |
| 21. | Methyl nonadecanoate | 18.052 | 2.0 | C19:0 |
| 22. | Methyl α-linolenate | 18.223 | 0.6 | C18:3 (all-cis-9,12,15) |
| 23. | Methyl arachidate | 19.075 | 1.2 | C20:0 |
| 24. | Methyl (Z)-11-eicosenoate | 19.255 | 0.6 | C20:1 (cis-11) |
| 25. | Methyl 11,14-eicosadienoate | 19.761 | 0.6 | C20:2 (all-cis-11,14) |
| 26. | Methyl eicosa-8,11,14-trienoate | 20.046 | 0.6 | C20:3 (all-cis-8,11,14) |
| 27. | Methyl heneicosanoate | 20.197 | 0.6 | C21:0 |
| 28. | Methyl arachidonate | 20.290 | 0.6 | C20:4 (all-cis-5,8,11,14) |
| 29. | Methyl 11,14,17-eicosatrienoate | 20.488 | 0.6 | C20:3 (all-cis-11,14,17) |
| 30. | Methyl 5,8,11,14,17-eicosapentanoate | 21.036 | 0.6 | C20:5 (all-cis-5,8,11,14,17) |
| 31. | Methyl behenate | 21.39 | 1.2 | C22:0 |
| 32. | Methyl erucate | 21.595 | 0.6 | C22:1 (cis-13) |
| 33. | Methyl docosadienoate | 22.150 | 0.6 | C22:2 (all-cis-13,16) |
| 34. | Methyl tricosanoate | 22.584 | 0.6 | C23:0 |
| 35. | Methyl lignocerate | 23.826 | 1.2 | C24:0 |
| 36. | Methyl docosahexaenoate | 23.863 | 0.6 | C22:6 (all-cis-4,7,10,13,16,19) |
| 37. | Methyl nervonate | 24.055 | 0.6 | C24:1 (cis-15) |
| Column | FAMEWAX, 30 m, 0.25 mm ID, 0.25 µm (cat.# 12497) |
|---|---|
| Standard/Sample | |
| Food industry FAME mix (cat.# 35077) | |
| Methyl nonadecanoate | |
| Diluent: | Methyl nonadecanoate was dissolved in toluene. |
| Injection | |
| Inj. Vol.: | 1 µL split (split ratio 100:1) |
| Liner: | Topaz 4.0 mm ID Precision inlet liner w/wool (cat.# 23305) |
| Inj. Temp.: | 240 °C |
| Oven | |
| Oven Temp.: | 60 °C (hold 2 min) to 200 °C at 10 °C/min to 240 °C at 5 °C/min (hold 7 min) |
| Carrier Gas | H2, constant flow |
| Flow Rate: | 1.7 mL/min |
| Detector | FID @ 250 °C |
|---|---|
| Instrument | Agilent 7890B GC |
| Notes | This chromatogram is an overlay of two injections: food industry FAME standard (black) and C19:0 methyl ester in toluene (red). An excellent separation of C19:0 (used in EN 14103 as an internal standard) and the most prevalent FAMEs found in biodiesel blends was achieved. Note that C4:0 from the food industry FAME standard elutes in the solvent front. Reference standard cat.# 35055 was used to produce this chromatogram, but has since been discontinued. For assistance choosing a replacement for this application, contact Restek Technical Service or your local Restek representative. |
After empirically demonstrating that good separation of FAMEs in the reference standard was achieved using a FAMEWAX column, commercially obtained biodiesel samples were also analyzed according to method EN14103. Good chromatographic results were obtained as shown in Figures 4 and 5. In addition, repeatability was assessed in order to evaluate the potential for carryover or poor sample transfer onto the column. Calculations of total ester content and the linolenic acid methyl ester content (all isomers combined) were highly repeatable, indicating consistent chromatographic performance and no observable issues with carryover or sample transfer for the analysis of FAMEs in biodiesel (Table I).
Figure 4: Chromatographic analysis of canola biodiesel according to method EN 14103 (2011). The red overlay is a linolenic acid methyl ester isomer mix standard (Sigma-Aldrich, L6031-25 mg) showing that all the isomers are separated from the C19:0 internal standard.
| Peaks | tR (min) | Structural Nomenclature | |
|---|---|---|---|
| 1. | Methyl palmitate | 15.149 | C16:0 |
| 2. | Methyl stearate | 16.995 | C18:0 |
| 3. | Methyl oleate | 17.167 | C18:1 (cis-9) |
| 4. | Methyl linoleate | 17.619 | C18:2 (cis-9,12) |
| 5. | Methyl nonadecanoate | 18.044 | C19:0 |
| 6. | Methyl linolenate | 18.233 | C18:3 (cis-9,12,15) |
| 7. | Methyl erucate | 21.359 | C22:1 (cis-13) |
| Column | FAMEWAX, 30 m, 0.25 mm ID, 0.25 µm (cat.# 12497) |
|---|---|
| Standard/Sample | |
| Canola B100 biodiesel | |
| Methyl nonadecanoate | |
| Diluent: | Toluene |
| Conc.: | 10 mg/mL, EN 14103 (2011) method preparation |
| Injection | |
| Inj. Vol.: | 1 µL split (split ratio 100:1) |
| Liner: | Topaz 4.0 mm ID Precision inlet liner w/ wool (cat.# 23305) |
| Inj. Temp.: | 240 °C |
| Oven | |
| Oven Temp.: | 60 °C (hold 2 min) to 200 °C at 10 °C/min to 240 °C at 5 °C/min (hold 7 min) |
| Carrier Gas | H2, constant flow |
| Flow Rate: | 1.7 mL/min |
| Detector | FID @ 250 °C |
|---|---|
| Instrument | Agilent 7890B GC |
| Notes | The chromatogram in black is canola biodiesel analyzed according to method EN 14103 (2011). The overlaid red chromatogram is a linolenic acid isomer standard (Supelco L6031). Linolenic acid can be present in different cis/trans conformations. All the linolenic acid isomers should be included in the calculation. Reference standard cat.# 35055 was used to produce this chromatogram, but has since been discontinued. For assistance choosing a replacement for this application, contact Restek Technical Service or your local Restek representative. |
Figure 5: Chromatographic analysis of soy biodiesel according to method EN 14103 (2011).
| Peaks | tR (min) | Structural Nomenclature | |
|---|---|---|---|
| 1. | Methyl palmitate | 15.149 | C16:0 |
| 2. | Methyl stearate | 16.997 | C18:0 |
| 3. | Methyl oleate | 17.171 | C18:1 (cis-9) |
| 4. | Methyl linoleate | 17.621 | C18:2 (cis-9,12) |
| 5. | Methyl nonadecanoate | 18.052 | C19:0 |
| 6. | Methyl linolenate | 18.235 | C18:3 (cis-9,12,15) |
| Column | FAMEWAX, 30 m, 0.25 mm ID, 0.25 µm (cat.# 12497) |
|---|---|
| Standard/Sample | |
| Soy B100 biodiesel | |
| Methyl nonadecanoate | |
| Diluent: | Toluene |
| Conc.: | 10 mg/mL, EN 14103 (2011) method preparation |
| Injection | |
| Inj. Vol.: | 1 µL split (split ratio 100:1) |
| Liner: | Topaz 4.0 mm ID Precision inlet liner w/ wool (cat.# 23305) |
| Inj. Temp.: | 240 °C |
| Oven | |
| Oven Temp.: | 60 °C (hold 2 min) to 200 °C at 10 °C/min to 240 °C at 5 °C/min (hold 7 min) |
| Carrier Gas | H2, constant flow |
| Flow Rate: | 1.7 mL/min |
| Detector | FID @ 250 °C |
|---|---|
| Instrument | Agilent 7890B GC |
| Notes | Reference standard cat.# 35055 was used to produce this chromatogram, but has since been discontinued. For assistance choosing a replacement for this application, contact Restek Technical Service or your local Restek representative. |
Table I: Total FAME and linolenic methyl ester weight percents with analysis of precision.
|
Soy B100 |
Canola B100 |
|||
|
Total FAME (wt%) |
C18:3 (wt%) |
Total FAME (wt%) |
C18:3 (wt%) |
|
|
Run 1 |
90.69 |
7.32 |
92.70 |
7.39 |
|
Run 2 |
90.70 |
7.32 |
92.76 |
7.39 |
|
Run 3 |
90.64 |
7.32 |
92.68 |
7.39 |
|
Run 4 |
90.65 |
7.32 |
92.74 |
7.39 |
|
Run 5 |
90.66 |
7.32 |
92.78 |
7.40 |
|
Run 6 |
90.55 |
7.31 |
92.61 |
7.39 |
|
Run 7 |
90.64 |
7.31 |
92.62 |
7.39 |
|
%RSD |
0.05 |
0.05 |
0.07 |
0.05 |
Conclusion
Using the Pro EZGC chromatogram modeler was a quick and easy way of identifying a column that would successfully analyze FAMEs in biodiesel, without spending any money or any time in the lab. While this work used the analytical conditions described in method EN 14103, the Pro EZGC modeler also can be used to further optimize these conditions and achieve a faster analysis while still maintaining resolution between all the targeted compounds. Empirical analysis of FAMEs in biodiesel samples confirmed that the selectivity of the FAMEWAX column allowed all critical compounds to be separated. Excellent peak shape and low bleed provided accurate, precise, and repeatable quantification of analytes.
References
- F. Gunstone, The chemistry of oils and fats: sources, composition, properties and uses, Wiley-Blackwell, 2009. https://www.wiley.com/The+Chemistry+of+Oils+and+Fats%3A+Sources%2C+Composition%2C+Properties+and+Uses-p-9781405150026
- DIN EN 14103, Fat and oil derivatives - Fatty Acid Methyl Esters (FAME) - Determination of ester and linolenic acid methyl ester contents, 2011. https://www.din.de/en/getting-involved/standards-committees/nmp/wdc-beuth:din21:232191873
- J. Pullen, K. Saeed, An overview of biodiesel oxidation stability, Renewable and Sustainable Energy Reviews 16 (2012) 5924-5950. https://doi.org/10.1016/j.rser.2012.06.024