GB/T 28769-2012 Determination of free glycerol content in fatty acid methyl esters — Gas chromatography

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Card type: Test method

1. Method number / full title / English title / replacement relationship

• Standard number: GB/T 28769-2012
• Chinese full title: Determination of free glycerol content in fatty acid methyl esters — Gas chromatography
• English title: Test method for determination of free glycerol content in Fatty Acid Methyl Esters (FAME) by gas chromatography
• Replacement relationship: not provided in the available source (no replacement of any standard is indicated)
• Adoption relationship: modified adoption by the redrafting method of European Standard EN 14106:2003 "Animal and vegetable fats and oils derivatives — Fatty acid methyl esters — Determination of free glycerol content"
• Main differences from EN 14106:2003: in 9.1, a chromatographic condition using splitless injection was added to improve the sensitivity of the determination
• Publication/implementation: published on 2012-11-05, implemented on 2013-03-01
• Responsible committee: National Technical Committee for Standardization of Petroleum Products and Lubricants, Subcommittee on Petroleum Fuels and Lubricants (SAC/TC 280/SC 1)
• Drafting organization: Sinopec Research Institute of Petroleum Processing
• Main drafter: Li Changxiu

2. Scope of application and non-applicability

• 1.1 This standard specifies a method for the determination of free glycerol content in fatty acid methyl esters (FAME) in the mass fraction range of 0.005% to 0.07% by gas chromatography.
• 1.2 This standard is applicable to evaluating the quality of fatty acid methyl esters of pure biodiesel. Glycerol, as a by-product of the transesterification reaction, affects fuel performance at certain concentrations.
• Non-applicability: not provided in the available source (apart from 11.1 indicating that the precision values do not apply to samples beyond the sample types and concentration ranges listed in Annex A, no other non-applicable situations are listed).

3. Method principle

• 4 Method summary: Ethanol, water, n-hexane and a certain amount of internal standard are added to a known amount of sample, forming a two-phase mixture. Free glycerol is quantitatively transferred to the lower layer solution. The lower layer solution is analyzed by gas chromatography to quantitatively determine the free glycerol content.

4. Reagents and materials (purity / preparation)

• General: Unless otherwise specified, all reagents are of analytical grade.
• 5.1 n-Hexane.
• 5.2 1,4- Butanediol: purity not less than 99%.
• 5.3 Ethanol: purity not less than 95%.
• 5.4 Formic acid: purity not less than 99%.
• 5.5 Glycerol: purity not less than 99%.
• 5.6 Carrier gas: helium or hydrogen, meeting the requirements for gas chromatography carrier gas, generally with purity not less than 99.99%.
• 5.7 Auxiliary gases:
  • Hydrogen: purity not less than 99.99%, free of water and organic compounds;
  • Air: dry air, free of organic compounds.
• 5.8 Internal standard solution: accurately weigh about 80 mg (to the nearest 0.1 mg) of 1,4- butanediol into a 100 mL volumetric flask, dissolve with a small amount of distilled water, add 1 mL formic acid, and dilute to the mark with distilled water. The solution prepared by this procedure is stable at room temperature for 24 h.

5. Apparatus and equipment

• 6.1 Gas chromatograph with the following configuration:
  • 6.1.1 Column oven: temperature control precision of setting ±1 ℃ (original source-text extraction is "[unreadable characters]1°C", requires confirmation against the source standard (source-text extraction)).
  • 6.1.2 Injection port: split/splitless injection port or packed column injection port.
  • 6.1.3 Flame ionization detector (FID) and signal conversion/amplification system.
  • 6.1.4 Recorder/integrator, capable of being connected to the signal conversion/amplification system, with a maximum response time of less than 1 s (original source-text extraction is "[unreadable characters] s", value requires confirmation against the source standard (source-text extraction)), and adjustable chart speed.
  • 6.1.5 Capillary column: type PoraPLOT Q, length 10 m, internal diameter 0.32 mm, film thickness 10 μm (original source-text extraction is "10km", requires confirmation against the source standard (source-text extraction)); or a packed column with stationary phase CHROMOSORB 101 (original source-text extraction is "HROMOSORBIO1", requires confirmation against the source standard (source-text extraction)), internal diameter 4 mm, length 1 m. The column used shall completely separate the chromatographic peaks of glycerol and the internal standard (the baseline between the two peaks approaches zero), with an analysis time of less than 15 min, and the correction factor for glycerol determined according to 9.3 shall not exceed 2.5.
  • Note: It has been noted that capillary columns from different manufacturers differ in performance, and some may cause problems in use. Capillary columns using a stationary phase for free fatty acid analysis (FFAP) or a polyethylene glycol stationary phase have been suggested. Regardless of the type of column used, its separation performance must meet the specified requirements.
  • 6.1.6 Flow controller, for controlling carrier gas and auxiliary gas flow rates.
• 6.2 Microsyringe for gas chromatography: 5 μL or 10 μL (original source-text extraction is ".5[unreadable character]l or 10/L", requires confirmation against the source standard (source-text extraction)).
• 6.3 Volumetric flasks: 50 mL and 100 mL.
• 6.4 Pipette: 1 μL (original source-text extraction is "1 LL", requires confirmation against the source standard (source-text extraction)).
• 6.5 Graduated pipette: 5 mL (original source-text extraction is "5m", requires confirmation against the source standard (source-text extraction)).
• 6.6 Analytical balance: readability 1 mg.
• 6.7 Glass conical centrifuge tube: 10 mL.
• 6.8 Centrifuge: speed up to 2000 r/min (original source-text extraction is "2000 t/min", requires confirmation against the source standard (source-text extraction)).

6. Recommended operating conditions

• 9.1.1 The following conditions may be used for a capillary column:
  • Splitless mode:
    • a) Column oven temperature: initial temperature 150 ℃, hold for 1 min, heating rate 40 ℃/min, final temperature 210 ℃, hold for 12 min;
    • b) Inlet temperature: 250 ℃;
    • c) Detector temperature: 250 ℃;
    • d) After injection of 0.5 min, open the split valve, split ratio about 20:1 (original source-text extraction is "approximately 20:", requires confirmation against the source standard (source-text extraction));
    • e) Carrier gas flow rate: 3 mL/min.
  • Split mode:
    • a) Column oven temperature: 210 ℃;
    • b) Inlet temperature: 230 ℃;
    • c) Detector temperature: 250 ℃;
    • d) Split ratio: about 50:1 (original source-text extraction is "approximately 501", requires confirmation against the source standard (source-text extraction));
    • e) Carrier gas flow rate: 1 mL/min to 2 mL/min.
• 9.1.2 The following conditions may be used for a packed column:
  • Column oven temperature: 200 ℃;
  • Inlet temperature: 230 ℃;
  • Detector temperature: 250 ℃;
  • Carrier gas flow rate: 20 mL/min to 30 mL/min.
• Note: It is recommended to use the splitless injection mode chromatographic conditions for a capillary column to ensure the sensitivity of the determination.

7. Sampling and sample preparation

• 7.1 Sample according to the method of GB/T 4756.
• 7.2 During sampling, consideration should be given to the strong affinity of free glycerol in fatty acid methyl esters for glass containers; therefore, glass containers should be avoided for collecting and storing samples, and suitable plastic containers may be used.
• 8 Sample preparation: Prepare the sample according to the requirements of GB/T 15687. The sample must not be heated or filtered.

8. Analytical procedure

• 9.2 Qualitative identification of chromatographic peaks: Based on the retention times of glycerol and the internal standard under the same conditions, the chromatographic peaks of glycerol and the internal standard in the sample can be identified. Figure 1 is a typical chromatogram (Figure 1 legend: 1——1,4- butanediol; 2—— glycerol).
• 9.3 Determination of glycerol correction factor: Accurately weigh about 100 mg (to the nearest 0.1 mg) of glycerol and 100 mg (to the nearest 0.1 mg) of 1,4- butanediol into a 100 mL volumetric flask, add 50 mL ethanol (see 5.3) to dissolve, and dilute to the mark with distilled water. During preparation, pay attention to the exothermic effect and volume decrease when water/alcohol are mixed. Inject 1 μL of this mixed solution into the chromatographic system and analyze according to the aforementioned chromatographic conditions, at least three parallel determinations, and calculate the correction factor for glycerol. This solution is stable at room temperature for several weeks. Calculate the correction factor F for glycerol from the chromatogram results and equation (1). The glycerol correction factor F calculated according to equation (2) shall not exceed 2.5 (see the note in 6.1.5).
• 9.4 Quantitative analysis:
  • 9.4.1 Sample preparation: Weigh approximately 3.5 g (accurate to 0.1 mg) of sample (approximately 4 mL) into a 10 mL centrifuge tube, add 1 mL ethanol (see 5.3), and shake gently to mix uniformly. Accurately add 1 mL internal standard solution (see 5.8) and 4 mL n-hexane, tightly close the tube stopper and shake vigorously for 5 min. Centrifuge for 15 min. Take the lower layer solution for chromatographic analysis.
  • 9.4.2 Chromatographic analysis: Use a microsyringe to take approximately 1 μL of the lower layer solution, then draw in a certain amount of air so that no solution remains in the needle ("hot needle" technique), carefully wipe the syringe and needle clean with a tissue, insert the syringe needle through the septum into the injection port, wait for 5 s, quickly inject the sample into the vaporization chamber, and withdraw the injection needle after 5 s. Mark the injection time on the chromatogram, and continue for a few minutes after the glycerol chromatographic peak elutes.

9. Qualitative and Quantitative Determination and Calculation Formulas

• Qualitative: see 9.2, determine the chromatographic peaks based on the retention times of glycerol and the internal standard under the same conditions.

• Calculation of glycerol correction factor F (Equation 1):

  F = (A2/M2) / (A1/M1) (the original text source-text extraction is typeset as "A2/M2 • (1)", the fraction structure requires confirmation against the source standard (source-text extraction))

  Where:

  • A1——1,4- butanediol peak area;
  • A2—— glycerol peak area;
  • M1—— mass of 1,4- butanediol in the solution, in milligrams (mg);
  • M2—— mass of glycerol in the solution, in milligrams (mg).

• Calculation of free glycerol mass fraction (Equation 2):

  w = (A2/A1) × F × (m1/m) × 100%

  Where:

  • A1, A2, F—— have the same meanings as in Equation (1);
  • m1—— mass of 1,4- butanediol added to the sample, in milligrams (mg);
  • m—— mass of the sample, in milligrams (mg).

• Expression of results: expressed as mass fraction (%), accurate to the 2th decimal place.

10. Precision

• 11.1 Detailed inter-laboratory precision determination data are given in Appendix A. The precision values derived therefrom are not applicable to testing samples outside the listed sample types and concentration ranges. Judge the reliability of test results according to the following provisions (95% confidence level).
• 11.2 Repeatability (r): In the same laboratory, by the same operator, using the same instrument and equipment, under the same experimental conditions, the difference between two replicate determination results on the same sample shall not exceed the repeatability (r) value in Table 1.
• 11.3 Reproducibility (R): In different laboratories, by different operators, using different instruments, the absolute difference between two single and independent determination results on the same sample shall not exceed the reproducibility (R) value in Table 1.
• Table 1 Determination repeatability and reproducibility (r and R):
  • Repeatability: r = 0.466 4w − 0.0012
  • Reproducibility: R = 0.781 2w + 0.0032
  • Note: w is the average of two determination results, mass fraction (%).
• Appendix A (informative appendix) Inter-laboratory test results:
  • The laboratory collaborative test involved the analysis results of 5 samples by 7 laboratories in 5 European countries.
  • Sample 1: fatty acid methyl esters prepared from sunflower seed oil.
  • Sample 2: fatty acid methyl esters prepared from rapeseed oil 1.
  • Sample 3: fatty acid methyl esters prepared from a mixture of rapeseed oil and sunflower seed oil (75:25).
  • Sample 4: fatty acid methyl esters prepared from rapeseed oil 2.
  • Sample 5: fatty acid methyl esters prepared from a mixture of rapeseed oil and sunflower seed oil (25:75).
  • This test project was organized by the European Committee for Standardization CEN/TC 307/WG1 working group in 1999, and the test results obtained were statistically analyzed in accordance with the requirements of ISO 4259.
  • Table A.1 Precision data (the original text source-text extraction table values are severely misaligned, each value requires confirmation against the source standard (source-text extraction)):
    • Number of participating laboratories/N: 7 (samples 1～4), the value corresponding to sample 5 in the original text requires confirmation against the source standard (source-text extraction)
    • Number of laboratories after removing outlier laboratories/N: 6 (listed in the original text), the corresponding sample requires confirmation against the source standard (source-text extraction)
    • Average determination value (mass fraction)/%: 0.018, 0.014, 0.032, 0.001, 0.048 (listed in the original text, the corresponding sample requires confirmation against the source standard (source-text extraction))
    • Standard deviation of repeatability determination (mass fraction)/%: 0.001, 0.001, 0.005, 0.000, 0.006 (listed in the original text, the corresponding sample requires confirmation against the source standard (source-text extraction))
    • Standard deviation of reproducibility determination (mass fraction)/%: 0.003, 0.004, 0.007, 0.002, 0.012 (listed in the original text, the corresponding sample requires confirmation against the source standard (source-text extraction))
    • Repeatability allowable difference (mass fraction) r/%: 0.004, 0.004, 0.018, 0.001, 0.020 (listed in the original text, the corresponding sample requires confirmation against the source standard (source-text extraction))
    • Reproducibility allowable difference (mass fraction) R/%: 0.011, 0.015, 0.025, 0.006, 0.042 (listed in the original text, the corresponding sample requires confirmation against the source standard (source-text extraction))

11. Report

• 12 The test report shall state:
  • Necessary information to identify the sample;
  • Sampling method used;
  • Analysis method and standard number;
  • Operational details not listed in this standard, and other factors that may affect the analysis results;
  • Determination results (see Chapter 10).

12. Normative References

• GB/T 4756 Petroleum liquids — Manual sampling (GB/T 4756—1998, eqv ISO 3170:1988)
• GB/T 15687 Animal and vegetable fats and oils — Preparation of test samples (GB/T 15687—2008, ISO 661:2003, IDT)
• Reference: EN 14106: 2003 (modified adoption); ISO 4259 (Appendix A statistical analysis reference); GB/T 1.1—2009 (foreword, drafting rules)

13. Source and verification

This page is an AI translation of the available Chinese method card. Human verification is pending. It is not an official translation of the standard. Check the source standard and product certificate before use.

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