SH/T 0796-2007 B-100 Determination of free glycerol and total glycerol content in biodiesel fatty acid methyl esters (gas chromatography)

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Card type: Test method

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

• Standard number: SH/T 0796-2007 (the file name is marked as SHT-796-2007, the text page header is SH/T 0796-2007, requires confirmation against the source standard (source-text extraction))
• Chinese name: B-100 Determination of free glycerol and total glycerol content in biodiesel fatty acid methyl esters (gas chromatography)
• English name: not provided in the available source
• Replacement relationship: not provided in the available source

2. Scope of application and non-applicability

• Applicable to the determination of free glycerol and total glycerol content in B-100 biodiesel fatty acid methyl esters by gas chromatography (1.1).
• Free glycerol detection range: 0.005% (mass fraction) to 0.05% (mass fraction) (1.1).
• Total glycerol detection range: 0.05% (mass fraction) to 0.5% (mass fraction) (1.1).
• Not applicable to the determination of free glycerol and total glycerol content in biodiesel synthesized from vegetable oils containing relatively more glyceryl dodecanoate (such as coconut oil and palm kernel oil, the source text writes "palm phase kernel oil", requires confirmation against the source standard (source-text extraction)) (1.1).
• Uses International System of Units [SI] units (1.2).
• The standard involves certain safety issues, but does not provide recommendations for all safety issues; users are responsible for establishing appropriate safety and health measures and determining appropriate regulations before use (1.3).

3. Method principle

After the sample is silylated with N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA), it is analyzed by gas chromatography. The quantification process uses dual internal standards and four reference substances to correct the results. For the determination of mono-fatty acid glycerides, di-fatty acid glycerides and tri-fatty acid glycerides, glyceryl monooleate, glyceryl dioleate and glyceryl trioleate are used as standard reference substances, respectively. The average conversion factor is used to calculate the content of bound glycerol in the test sample (Chapter 4).

4. Reagents and materials (purity / preparation)

• Reagent purity: all reagents used are analytical grade; other grades of reagents may also be used, provided that they do not reduce analytical precision (7.1).
• n-Heptane: analytical grade (7.2).
• N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA): analytical grade (7.3).
• Pyridine: analytical grade (7.4).
• Carrier gas: use high-purity hydrogen, helium or nitrogen (the source text writes "hydrogen, hydrogen or nitrogen", requires confirmation against the source standard (source-text extraction)), purity greater than 99.99%; purify the carrier gas using molecular sieves or other adsorbents to remove moisture, oxygen and hydrocarbon impurities, and select the carrier gas pressure to ensure the carrier gas flow rate (7.5).
• Microliter syringe or pipette: capacity 100 μL and 250 μL (7.6).
• Sample bottles with caps: with polytetrafluoroethylene gaskets, capacity 2 mL and 10 mL (7.7).
• Volumetric flasks: 10 mL, 25 mL, 50 mL (7.8).

Preparation of calibration standards (9.1): prepare the standards as listed in Table 2, weigh the pure components in a volumetric flask to an accuracy of 0.1 mg, dilute to the mark with pyridine, and store the prepared standard solutions in a refrigerator for later use.

Table 2 Stock solutions of each component (the source table header and part of the content have severe source-text extraction noise; the following is the identifiable content, requires confirmation against the source standard (source-text extraction)):

Preparation of mixed standards (9.2): according to the amounts listed in Table 3, use a microsyringe or pipette to transfer the specified volumes of calibration standards into 10 mL capped sample bottles to prepare five mixed standard solutions. To each of the five standard solutions, respectively add 100 μL MSTFA, cap the bottles and shake, allow to stand at room temperature for 15 min~20 min, add about 8 mL n-heptane, shake well and set aside.

Table 3 Standard solutions (volume unit: μL, source-text extraction severe noise, requires confirmation against the source standard (source-text extraction)):

5. Instruments and equipment

• Gas chromatograph system (6.1): refer to GB/T 4946 for detailed names and definitions.
• Gas chromatograph: the instrument configuration must meet the requirements of the chromatographic analysis conditions specified in Table 1 (6.1.1).
• Chromatographic column: an open tubular column with 5% phenyl dimethyl polysiloxane cross-linked bonded stationary phase or 100% dimethyl polysiloxane stationary phase, with a maximum column operating temperature of up to 400℃ (the source text writes "400T", requires confirmation against the source standard (source-text extraction)). A column length of 10 m or 15 m, an inner diameter of 0.32 mm, and a film thickness of 0.1 μm (the source text writes "0.1wm", requires confirmation against the source standard (source-text extraction)) can achieve good separation. Other columns with the same or better column efficiency and selectivity may also be used. It is recommended to use a high-temperature column of 2 m~5 m length and inner diameter 0.53 mm connected before the analytical column and to the injection port, in order to use an autosampler and extend column service life (6.1.2).
• Electronic data acquisition system (6.2):
  • Integrator or computer: capable of real-time plotting and chromatographic data conversion, automatically integrating and measuring the area and retention time of chromatographic peaks (6.2.1).
  • The data processing system can implement a multi-level internal standard calibration calculation process, calculate the correlation coefficient of the calibration curve, and process the data using the internal standard method (6.2.2).

6. Recommended operating conditions

Table 1 Chromatographic analysis conditions (source-text extraction severe noise, requires confirmation against the source standard (source-text extraction)):

7. Sampling and test sample preparation

• Directly weigh about 100 mg of test sample in a 10 mL sample bottle, accurate to 0.1 mg, then use a microsyringe or pipette to accurately transfer each 100 μL of internal standard and silylation reagent (MSTFA) into the sample bottle, shake well, allow to stand at room temperature for 15 min~20 min, add about 8 mL n-heptane to the sample bottle, shake well and set aside (10.1).
• If an autosampler is used for injection analysis, take a certain amount of the mixed solution into a 2 mL capped sample bottle for injection analysis (9.3).

8. Analysis procedure

• Set the instrument parameters with reference to condition 1 or condition 2 in Table 1 (10.1).
• Inject the silylation product of the 1 μL sample into the on-column injection port and run the instrument to obtain a chromatogram and area integration report (10.2).
• Peak identification: identify based on the retention time of the standard sample; the identification of other peaks refers to the relative retention time in Table 4 and the reference chromatogram shown in Figure 1. Mono-fatty acid glycerides, di-fatty acid glycerides, and tri-fatty acid glycerides are separated according to differences in carbon number (10.3).
• Mono-fatty acid glycerides consist of four overlapping peaks; their relative retention times with respect to the internal standard tridecanoin are 0.76 and 0.83–0.86; the relative retention time of the group of peaks corresponding to the fatty acid methyl ester of C24 is 0.80–0.82, and cannot be counted as mono-fatty acid glycerides (10.4).
• Di-fatty acid glycerides are likewise separated according to carbon number, but due to changes in the position of double bonds in the molecule, the chromatographic peaks are difficult to separate to baseline. The 3 to 4 peaks with relative retention times of 1.05–1.09 belong to the chromatographic peaks of di-fatty acid glycerides (carbon numbers 34, 36, and 38). Tri-fatty acid glycerides are also separated according to carbon number. Chromatographic peaks with relative retention times between 1.16–1.31 are calculated as tri-fatty acid glycerides (carbon numbers 52, 54, 56, and 58) (10.5).

Table 4 Relative retention times of components:

*When n-pentadecane is used as the internal standard, the relative retention time of glycerol is 0.71.

Figure 1 Typical chromatogram (peak assignments): 1—glycerol; 2—1,2,4-butanetriol (internal standard 1); 3—n-pentadecane (alternative internal standard 1); 4—C14fatty acid methyl ester; 5—C16fatty acid methyl ester; 6—C18fatty acid methyl ester; 7—C18fatty acid methyl ester (original text reads "Gz", requires confirmation against the source standard (source-text extraction)); 8—C18mono-fatty acid glycerides; 9—C18fatty acid methyl ester (original text reads "Gaa", requires confirmation against the source standard (source-text extraction)); 10—C18mono-fatty acid (a.stearic acid; b.oleic acid, linoleic acid, linolenic acid) glycerides; 11—tridecanoin; 12—di-fatty acid glycerides (carbon numbers: 34, 36, 38); 13—tri-fatty acid glycerides (carbon numbers: 52, 54, 56, 58).

9. Qualitative and Quantitative Calculation Formulas

• The calibration standard is analyzed using the same chromatographic conditions as the sample analysis. Inject the silylated product of the 1 μL mixed standard into a cold on-column injector and start acquisition to obtain the chromatogram and integration report. For each reference substance, calculate the response ratio (rspi) and mass ratio (amti) according to equations (1) and (2) (9.4.1):
  • rspi = (Ai/Ais) (1), where Ai— is the area of the reference substance, and Ais— is the area of the internal standard.
  • amti = (Wi/Wis) (2), where Wi— is the mass of the reference substance (mg), and Wis— is the mass of the internal standard (mg).
  • Plot the standard curve for each reference substance with rsp as the y axis and amt as the x axis.
• Calculate the correlation coefficient r² for each reference substance according to equation (3). The correlation coefficient r² should be no less than 0.99; if the calculated r² is below this value, check the instrument parameters and hardware configuration and rerun the calibration process (9.4.2). The original formula content of equations (3) (4) (5) source-text extraction is severely noisy, requires confirmation against the source standard (source-text extraction).
• Standard curve equation: The standard curve equation for each reference substance is expressed by equation (6) (9.4.3):
  • Wi/Wis = a × (Ai/Ais) + b (6)
  • Where: Wi— is the mass of the reference substance (mg); Wis— is the mass of the internal standard (mg); Ai— is the peak area of the reference substance; Ais— is the peak area of the internal standard; a— is the slope of the calibration curve equation; b— is the intercept of the calibration curve equation.
• The mass fraction of glycerol is calculated according to equation (7) (11.1.1):
  • G = (ag × Ag/Ais1 + bg) × Wis1 × 100 / Ws (7)
  • Where: G— is the content of glycerol in the sample, % (mass fraction); Ag— is the peak area of glycerol; Ais1— is the peak area of the internal standard 1; Wis1— is the mass of the internal standard 1 (mg); Ws— is the mass of the sample (mg); ag— is the slope of the calibration curve equation; bg— is the intercept of the calibration curve equation.
• The mass fraction of each component of mono-, di-, and tri-glycerides is calculated according to equation (8) (11.1.2):
  • CL = (aa × Aai/Ais2 + ba) × Wis2 × 100 / Ws (8)
  • Where: CL— is the content of each fatty acid glyceride in the sample, % (mass fraction); Aai— is the peak area of each fatty acid glyceride; Ais2— is the peak area of the internal standard 2; Wis2— is the mass of the internal standard 2 (mg); Ws— is the mass of the sample (mg); aa— is the slope of the calibration curve equation for mono-, di-, and tri-fatty acid glycerides; ba— is the intercept of the calibration curve equation for mono-, di-, and tri-fatty acid glycerides.
• The mass fraction of total glycerol is calculated according to equation (9) (11.1.3):
  • Total glycerol mass fraction = free glycerol mass fraction + bound glycerol mass fraction (9)
  • The free glycerol mass fraction is obtained from equation (7).
  • Bound glycerol mass fraction = Σ(GLm, GLd, GLt)
  • Where: GLm = 0.2591 × Σ mass fraction of mono-fatty acid glycerides (obtained from equation (8)); GLd = 0.1488 × Σ mass fraction of di-fatty acid glycerides (obtained from equation (8)); GLt = 0.1044 × Σ mass fraction of tri-fatty acid glycerides (obtained from equation (8)).
• Report the content of free glycerol and total glycerol, accurate to 0.001% (mass fraction) (11.2).

10. Precision

• Repeatability (12.1): The difference between two test results obtained by the same operator using the same instrument on the same sample shall not exceed the value listed in Table 5 (95% confidence level).

Table 5 Repeatability:

• Bias (12.2): Since there is no standard substance available for testing, the bias of the method has not been determined.

11. Report

• Report the content of free glycerol and total glycerol, accurate to 0.001% (mass fraction) (11.2).

12. Normative References

• GB/T 4946 Terminology for gas chromatography (Chapter 2)
• GB/T 9722 Chemical reagents—General rules for gas chromatography (original text reads "CB/T9722", requires confirmation against the source standard (source-text extraction)) (Chapter 2)

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.

14. Quick Reference Q&A