U.S. Reporter 29.5

Gas Chromatography, Supelco Reporter 29.5

EN 14110 (Determination of Methanol Impurity in B100 Biodiesel) Using SPME on the Equity-1
US EPA Method 8141 (Organophosphorous Pesticides) on the SLB-5ms and SPB-608

EN 14110 (Determination of Methanol Impurity in B100 Biodiesel) Using SPME on the Equity-1 back to top
Katherine K. Stenerson and Michael D. Buchanan
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:
  1. 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.
  2. 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% biomass-based diesel) or blended (for example, B20 biodiesel = 20% biomass-based diesel and 80% petroleum-based diesel).

Before being used or blended, B100 biodiesel must be tested for trace levels of contaminants that may cause problems in diesel engines. One of these contaminants is methanol. Because methanol is commonly used as the alcohol 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
European Standard Method DIN EN 14110 describes the headspace gas chromatography (GC) analysis of biodiesel for methanol, and is used to verify the residual methanol level is below set guidelines (1). A poly(dimethylsiloxane) phase is one of the column chemistries recommended in EN 14110 for the analysis methanol in biodiesel. This phase will elute analytes primarily according to boiling point, so under the proper analysis conditions, methanol and the internal standard 2-propanol should be resolved. Our Equity®-1 columns are made with poly(dimethylsiloxane) phase, and are available in a variety of dimensions to suit the needs of the application. For this analysis, a column with the dimensions described in the analytical conditions of the EN method was used.

Solid Phase Microextraction (SPME)
EN 14110 specifies the use of headspace analysis with a 45 minute sample equilibration time at 80 °C prior to GC analysis. In this study, solid phase microextraction (SPME) was evaluated as a possible alternative to headspace.

Linearity Evaluation
The three calibration standards described in EN 14110 were made and analyzed. Each standard consisted of a B100 biodiesel sample spiked with methanol plus 2-propanol (internal standard). Each standard was extracted using SPME and analyzed by GC on an Equity-1. An 85 ìm polyacrylate fused silica fiber assembly was selected, as this phase possesses good affinity for polar analytes. A five minute extraction time at 60 °C was found to yield sufficient response for the lowest level calibration standard. The resulting chromatogram for the mid-level calibration standard is shown in Figure 1.
Figure 1. Spiked B100 Biodiesel Sample
sample/matrix:   2 g of B100 biodiesel containing 0.01% methanol and 0.0785% 2-propanol in a 4 mL vial
SPME fiber:   85 µm polyacrylate (57304)
extraction:   headspace, 60 °C for 5 min.
desorption process:   250 °C for 0.75 min.
column:   Equity-1, 30 m x 0.32 mm I.D., 1.0 µm (28057-U)
oven:   50 °C
inj.:   250 °C
det.:   FID, 200 °C
carrier gas:   helium, 30 cm/sec
liner:   0.75 mm I.D., SPME type, straight design (unpacked)

Calibration factors were calculated per EN14110, and then the average, standard deviation, and %RSD of the calibration factors were determined. Per the method, the %RSD value must be <15 for the calibration to be acceptable. A 5 %RSD was obtained with the SPME calibration, showing that SPME is a viable extraction technique for this method. Complete calibration data is shown in Table 1.
Table 1. 3-Point Calibration Results
(wt/wt %)
Area Counts
Area Counts*
0.01 515 1947 0.4819
0.1 4718 1787 0.4826
0.5 28995 2410 0.5294

Whether headspace or SPME is used for the extraction of methanol from biodiesel, the Equity-1 column can be used in the subsequent GC analysis, as it provides sufficient resolution of the methanol and 2-propanol internal standard.

SPME required considerably less equilibration time than the headspace method (5 min. vs. 45 min.), and exhibited excellent sensitivity at the low end of the required calibration range. SPME also proved to be quantitative, as evidenced by the linearity evaluation done using the three standard levels described in EN 14110.

  1. DIN EN 14110, “Fat and Oil Derivatives - Fatty Acid Methyl Esters (FAME) - Determination of Methanol Content”

Featured Products
Description Cat. No.
SPME Fiber Assemblies, 85 µm polyacrylate fused silica, 24 ga for manual holder, 3 ea 57304
SPME Fiber Holder, for manual use, 1 ea 57330-U
Equity-1 Capillary GC Column, 30 m x 0.32 mm I.D., 1.0 µm, 1 ea 28057-U
Molded Thermogreen™ LB-2 Septa, 11 mm diameter, 50 ea 28336-U
Inlet Liner, 0.75 mm I.D., direct (SPME) type, straight design, for Agilent®, 5 ea 2637505

Related Products
Description Cat. No.
SPME Fiber Assemblies, 85 µm Polyacrylate Fused Silica, 3 ea
23 ga for Autosampler Holder and Merlin Microseal™ Systems 57294-U
24 ga for Autosampler Holder 57305
SPME Fiber Holders, 1 ea
For CTC CombiPAL, Gerstel ® MPS 2, and Thermo® TriPlus™ Autosamplers 57347-U
For Varian® Autosamplers 57331
Equity-1 Capillary GC Columns, 1 ea
30 m x 0.25 mm I.D., 0.25 µm 28046-U
60 m x 0.25 mm I.D., 0.25 µm 28047-U
60 m x 0.32 mm I.D., 1.00 µm 28058-U
Merlin Microseal Systems (required a nut and a SPME septum)
For Agilent (nut only) 22582
For Varian 1079 Injector (1 nut, 1 inlet adapter, 1 o-ring, and 1 non-SPME septum) 24817-U
For Varian CP-1177 Injector (1 nut, 1 inlet adapter, 1 o-ring, and 1 non-SPME septum) 22609-U
Replacement SPME septum (fits Agilent and Varian) 24818-U
Molded Thermogreen LB-2 GC Septa, 50 ea
9.5 mm diameter 28331-U
10 mm diameter 28333-U
11.5 mm diameter 29446-U
17 mm diameter 29452-U
Inlet Liners, 0.75 mm I.D., Direct (SPME) Type, Straight Design, 5 ea
For Finnigan (9001GCQ) 2637505
For PerkinElmer® (AutoSystem™) 2631205
For Shimadzu® (9A, 15A, and 16, with SPL-G9/15 Injector) 2632905
For Shimadzu (14, 15A, and 16, with SPL-14 Injector) 2633505
For Shimadzu (17A, with SPL-17 Injector) 2633905
For Thermo (ThermoQuest 8000 and TRACE®) 2876605-U
For Varian (1075 and 1077 Injector) 2635805
For Varian (1078 and 1079 Injector) 2637805
For Varian (1093-94 SPI Injector) 2636405
For Varian (CP-1177 Injector) 2637505
Related Information
Additional information about this, or other analytical methodologies used for biofuel (biodiesel or bioethanol) is available at our web node: sigma-aldrich.com/biofuels
NEW! Handbook of SPME by Janusz Pawliszyn
This new 400-page book contains comprehensive descriptions of the fundamental principles of solid phase microextraction (SPME), recent applications, SPME devices and procedures published to date. SPME protocols are presented in a step-by-step fashion, providing useful tips and potential pitfalls. The important steps in SPME method development and optimization including calibration are clearly discussed to assist new users of the technology. This handbook enables researchers at all stages of their careers to effectively apply this convenient and solvent-free sample preparation technique to solve their analytical challenges in an effective way. This up-to-date handbook contains 13 chapters with topics including: Theory of SPME, SPME devices and fiber coatings, commercial devices and coatings, automated SPME systems, calibration of extraction step, SPME method development, ligand-receptor binding, in-vivo SPME, review of different application areas including: environmental, food and fragrance, forensic and drug analysis, as well as SPME protocols.

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US EPA Method 8141 (Organophosphorous Pesticides) on the SLB-5ms and SPB-608 back to top
Katherine K. Stenerson and Michael D. Buchanan

Several methods and multiple literature references exist for the analysis of pesticides and herbicides, as these compounds are widely used against specific animal, insect, or plant life. The most commonly used pesticides and herbicides contain chlorine substitution. Therefore, the use of an electron capture detector (ECD), a specialized detector for detecting chlorinated analytes, is employed during analysis. Non-chlorinated pesticides and herbicides, those which contain phosphorous or nitrogen substitution, are also used to deter the spread of specific biologics. Due to the different chemistry of this group, different methodology must be employed for their analysis.

US EPA Method 8141 describes the analysis of a variety of organophosphorus pesticides and triazine herbicides (sometimes collectively referred to as OP-Pest) using gas chromatography (GC) following their extraction from solid waste or ground water samples (1). This method requires the use of either a nitrogen phosphorous detector (NPD) or a flame photometric detector (FPD), two specialized detectors that should be used in conjunction with stable, low bleed columns for maximum sensitivity.

This method also requires confirmatory analysis; the analysis of all standards, sample extracts, and QA/QC extracts on two columns, each with a different selectivity. If a peak is found within an analyte’s retention time window on the primary column, it must be “confirmed” on the confirmatory column in order to be considered positive. This technique reduces the occurrence of false positives when confirmation by mass spectrometry (MS) detection and spectral library matching is not feasible.

Chromatographic Results
Figure 1 shows the GC-NPD analysis of many of the target compounds listed in US EPA Method 8141 on a non-polar SLB™- 5ms. No coelutions were noted under these run conditions, making this column a viable choice as the primary column for this method. The same mix analyzed on an intermediate polar SPB™-608 column is shown in Figure 2. The alternative selectivity of this column results in a different elution order, making this column a suitable choice as a confirmatory column for this method. Identical run conditions were used to generate both chromatograms, allowing a direct comparison of elution orders and retention times. This also allows both columns to be used in the same GC oven.
Figure 1. OP-Pest Standard on the SLB-5ms
column:   SLB-5ms, 30 m x 0.25 mm I.D., 0.25 ìm (28471-U)
oven:   60 °C (1 min.), 5 °C/min. to 200 °C, 15 °C/min. to 300 °C (5 min.)
inj.:   250 °C
det.:   NPD, 320 °C
carrier gas:   helium, 1.5 mL/min.
injection:   2.0 µL, splitless (0.75 min.)
liner:   4 mm I.D., single taper
sample:   nitrogen-phosphorous pesticide standard, each analyte at 100 ppb in MTBE
1. Dichlorvos
2. Hexamethyl phosphoramide (HMPA)
3. 1-Bromo-2-nitrobenzene (I.S.)
4. Mevinphos isomers
5. Trichlorfon
6. Tetraethyl pyrophosphate (TEPP)
7. Thionazin (Zinophos)
8. Demeton-O
9. Ethoprop
10. Tributyl phosphate (surr.)
11. Naled
12. Dicrotophos
13. Sulfotepp
14. Monocrotophos
15. Phorate
16. Dimethoate
17. Simazine
18. Atrazine
19. Dioxathion
20. Terbufos
21. Fonophos
22. Diazinon
23. Disulfoton
24. Phosphamidon
25. Dichlorofenthion
26. Methyl chloropyrifos
27. Methyl parathion
28. Ronnel
29. Fenitrothion
30. Malathion
31. Aspon
32. Chloropyrifos
33. Fenthion
34. Ethyl parathion
35. Trichloronate
36. Chlorfenvinphos
37. Crotoxyphos
38. Stirophos
39. Tokuthion
40. DEF (from oxidation of
Merphos in the injection port)
41. Fensulfothion
42. Ethion
43. Bolstar
44. Famphur
45. Carbophenothion
46. Triphenyl phosphate (surr.)
47. Phosmet
48. EPN
49. Leptophos
50. Methyl azinphos
51. Tri-o-cresyl phosphate (TOCP)
52. Ethyl azinphos
53. Coumaphos
Figure 2. OP-Pest Standard on the SPB-608
column: SPB-608, 30 m x 0.25 mm I.D., 0.25 µm (24103-U)
Other conditions and peak IDs are the same as Figure 1

One Injection, Two Columns
Instead of separate injections on each column, it is possible to perform a single injection and split it to each column. This is only possible if the run conditions for each column are identical. Simply follow this procedure:
  1. Connect a short guard column/retention gap from the injection port to a “Y” connector (the deactivation of the fused silica should match the polarity of the injection solvent)
  2. Connect each column to the “Y” connector
  3. Connect each column to its detector
  4. Weekly, clip 4-6 inches off the front of the guard column/ retention gap
  5. When the guard column/retention gap becomes shorter than 9 inches in length:
    1. Replace the guard column/retention gap
    2. Replace the “Y” connector
    3. Clip 4-6 inches off the front of each analytical column
Applying a small drop of polyimide sealing resin on the outside of guard columns/retention gaps and analytical columns prior to inserting into “Y” GlasSeal™ connectors makes very durable permanent seals.

As shown here, an SLB-5ms/SPB-608 column set can be used to perform US EPA Method 8141, and similar methods, for the confirmatory analysis of organophosphorous pesticides. This is the same column combination recommended for the GC analysis of chlorinated pesticides (2).

  1. US EPA Method 8141B “Organophosphorous Compounds by Gas Chromatography” Revision 2, February 2007.
  2. K.K. Stenerson, “Dual Column Analysis of Organochlorine Pesticides Using SLB-5ms and SPB-608” Supelco Reporter 24.2 (2006), p 6.

Related Information
To view our full-line of GC columns for environmental applications, visit sigma-aldrich.com/gc-enviro

Featured Products
Description Cat. No.
SLB-5ms, 30 m x 0.25 mm I.D., 0.25 µm 28471-U
SPB-608, 30 m x 0.25 mm I.D., 0.25 µm 24103-U

Related Products
Description Cat. No.
Fused Silica Tubing, Intermediate Polar Deactivated
3 m x 0.25 mm I.D. 25727
5 m x 0.25 mm I.D. 25747
“Y” GlasSeal Column Connector, Fused Silica
1 ea 23631
3 ea 23632
Polyimide Sealing Resin
5 g bottle 23817