Some standard content:
This standard is revised to adopt ISO5562-2:1999 "Test Methods for Orchidaceae" (English version). NY/T713--2003
This standard is redrafted based on ISO5562-2:1999. Appendix A lists the comparison of the chapter and article numbers of this standard with those of ISO standard 5562-2:1999.
Taking into account my country's national conditions, this standard has made some modifications when adopting ISO5562-2:1999. The relevant technical differences have been incorporated into the text and marked with a vertical single line in the margins of the clauses they involve. A list of these technical differences and their reasons is given in Appendix B for reference.
Appendix A and Appendix B of this standard are both informative appendices. This standard was proposed by the Agricultural Reclamation Bureau of the Ministry of Agriculture.
The drafting unit of this standard: Tropical Agricultural Products Quality Supervision, Inspection and Testing Center of the Ministry of Agriculture. The main drafters of this standard are: Peng Lixu, Fang Jia, Wu Liyu, He Limin, Feng Xinping, Wang Mingyue, Liu Hongsheng. 1 Scope
Determination of vanillin in vanilla pods
NY/T 713-—2003
This standard specifies the determination method of vanillin in vanilla fragrans belonging to the species Vanilla fragrans (Salisbury) Ames and syn. Vanilla planifolia Andrews.
This standard is applicable to the determination of vanillin in vanilla pods, cuts and powders. This standard is not applicable to the determination of vanillin in vanilla pod extracts.
This standard describes three analytical methods for the analysis of vanillin: a) high performance liquid chromatography;
b) ultraviolet spectrophotometry;
c) gas chromatography.
2 Normative references
The clauses in the following documents become the clauses of this standard through reference in this standard. For all dated references, all subsequent amendments (excluding errata) or revisions are not applicable to this standard. However, parties reaching an agreement based on this standard are encouraged to study whether the latest versions of these documents can be used. For all undated references, the latest versions are applicable to this standard. GB/T12806 Laboratory glassware single-line volumetric flask 3 High performance liquid chromatography (arbitration method)
3.1 Principle
The sample is extracted and diluted, internal standard is added, separated by high performance liquid chromatography (HPLC), and then the content of vanillin is determined by ultraviolet detector.
3.2 Reagents
Analytical reagents, distilled water, deionized water or water of equal purity. 3.2.1 Ethanol, 95% (volume fraction). 3.2.2 Methanol.
3.2.3 Phosphoric acid solution: c(H.PO4)=0.01mol/L. 3.2.4 Mobile phase: Mix 25 parts of methanol (3.2.2) with 75 parts of dilute phosphoric acid (3.2.3). Filter and degas with a membrane (3.3.3). 3.2.5 Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the reference standard, with a purity greater than 99%. 3.2.6 Internal standard: acetylsalicylic acid, with a purity greater than 99%. 3.2.7 Vanillin stock solution: Weigh a certain amount of vanillin (3.2.5) to an accuracy of 0.001g. Use the mobile phase to prepare a standard stock solution with a concentration of 1g/L.
3.2.8 Working solution
Dilute the stock solution (3.2.7) with the mobile phase (3.2.4) to obtain a working solution so that the concentration of the working solution meets the requirements in 3.2.8.1. The working concentration of vanillin solution is 0.1 g/L and can be stored in a dark place at 4°C for no more than 10 days. 3.2.9 Internal standard solution
Dissolve acetylsalicylic acid (3.2.6) in the mobile phase (3.2.4) to a concentration of 0.6 g/L and weigh to an accuracy of 0.001 g. Note: The internal standard concentration should be basically consistent with the vanillin concentration in the sample solution. 3.3 Instruments
3.3.1 Airtight grinder.
NY/T 713-2003
3.3.2 Single-label volumetric flasks with a capacity of 100 mL and 200 mL, in accordance with the requirements of GB/T12806. 3.3.3 Filter membrane with a pore size of 0.45 μm. 3.3.4 Continuous Soxhlet extractor, consisting of the following components (see Figure 1). Figure 1 Continuous Soxhlet Extractor
-125mL Soxhlet Extractor.
Condenser.
-250mL ground-mouth flask.
Heat source: electric heating mantle or oil bath with sliding rheostat or other heat control method. 3.3.5 High Performance Liquid Chromatography:
UV detector;
Injection device;
Chromatographic column: Cig silica gel column, 5μm～10μm particle size. 3.3.6 Syringe: for high performance liquid chromatography 3.3.7 Analytical balance, accuracy of 0.001g. 3.4 Preparation of Samples
Grind the sample (vanilla pods, cut segments or vanilla powder) and mix it thoroughly. 3.5 Weighing Samples
Weigh 20g of the prepared sample, accurate to 0.001g. 3.6 Extraction
Extract the sample (3.5) with about 200 mL of ethanol (3.2.1) in the extraction apparatus (3.3.4) for 16 h, i.e., the solvent is circulated 25 to 30 times.
Extract each 100 g of pods with 1 L of solvent.
Transfer the extract to a 200 mL volumetric flask (3.3.2). Rinse the flask in the extraction apparatus several times with a small amount of ethanol, add the washing liquid to the volumetric flask, make up to volume with ethanol and mix well. Dilute the solution 10 times with the mobile phase. Www.bzxZ.net
3.7 Analysis steps
3.7.1 Chromatographic operating conditions
The flow rate of the mobile phase (3.2.4) is about 1 mL/min, and the analysis is carried out at room temperature under the conditions specified in 3.3.5. 3.7.2 Calibration
NY/T 713-—2003
3.7.2.1 Inject the reference standard and internal standard separately, then mix and inject the two together. Adjust the operating conditions so that the resolution of the internal standard and sample peaks is greater than 1.
3.7.2.2 Filter the sample prepared in 3.6 through a membrane (3.3.3) and measure it on the instrument. 3.7.2.3 Mix 10 mL of the internal standard with 10 mL of the standard solution obtained in 3.2.7. Filter the sample through the membrane and inject it on the instrument to measure the peak height or peak area.
The correction factor (k) is given by equation (1).
Wherein:
k. Response coefficient of component i in the components to be determined; mass of component i in the solution on the machine, in milligrams (mg); m,
mass of the internal standard in the solution on the machine, in milligrams (mg); peak height of component i, in centimeters (cm); h. —peak height of the internal standard, in centimeters (cm). Note: The peak height in the formula can be replaced by its peak area. 3.7.3 Determination
3.7.3.1 Mix 10 mL of internal standard with 10 mL of sample solution prepared according to 3.6. Filter with a membrane. 3.7.3.2 Inject the solution obtained in 3.7.3.1 under the conditions specified in 3.7.2.3. The mass of the substance to be determined is calculated using formula (2): k; Xh; Xms
Wherein, the meanings of each symbol are the same as in 3.7.2.3. Note: The peak height in the formula can be replaced by its peak area. 4 Ultraviolet spectrophotometry
4.1 Principle
The vanillin contained in the sample is extracted with ethanol, and the vanillin content in the ethanol solution is determined by ultraviolet spectrophotometry. 4.2 Reagents
Analytical reagents, distilled water, deionized water or water of equal purity. 4.2.1 Ethanol, 96% (volume fraction). Sodium hydroxide solution, c(NaOH)=1 mol/I. 4.2.2 Extraction
4.2.3 Vanillin (4-hydroxy-3-methoxybenzaldehyde). 4.3 Apparatus
Airtight grinder.
100mL and 250mL single-label volumetric flasks, in accordance with the requirements of GB/T12806. 4.3.2
10mL, 20mL and 25mL pipettes.
Dryer.
Soxhlet extractor (3.3.4).
Spectrophotometer.
NY/T713-2003
4.3.7 Sample cell, optical path length 1 cm. 4.3.8 Weighing bottle, capacity 25 mL, with airtight stopper. 4.3.9 Analytical balance, accuracy 0.001 g. 4.4 Procedure
4.4.1 Determination of specific absorbance of vanillin
4.4.1.1 Preparation of standard solution
Weigh about 30 mg of vanillin (4.2.3) dried in the desiccator (4.3.4) in a weighing bottle (4.3.8) to the nearest 0.001 g. Dissolve it in about 20 mL of ethanol (4.2.1), transfer to a 250mL volumetric flask. Rinse the weighing bottle several times with ethanol and pour it into the volumetric flask, make up to volume with ethanol and shake well (solution A1). Use a pipette to transfer 25mL of solution A1 to a 100mL volumetric flask (4.3.2). Make up to volume with ethanol and shake well (solution B1). Use a pipette to transfer 10ml of solution B1 to a 100mL volumetric flask. Add 60mL of ethanol and 2mL of sodium hydroxide solution (4.2.2) and mix well. Make up to volume with ethanol and shake well (solution C1). 4.4.1.2 Preparation of reference solution
Pipette 2mL of sodium hydroxide solution (4.2.2) into a 100mL volumetric flask, make up to volume with ethanol and shake well to prepare a reference solution.
4.4.1.3 Determination
Use a spectrophotometer (4.3.6) and a sample cell (4.3.7) to measure and record the spectrum of solution C1 relative to the reference solution (4.4.1.2) in the wavelength range of 250nm to 420nm. 4.4.1.4 Calculation
The maximum absorption occurs at 350nm±3nm, and its absorbance should be between 0.2 and 0.8. Draw a baseline from approximately 270nm to 380nm.
Record the maximum absorbance (Amax) and the baseline absorbance (Abase) at the same wavelength, and calculate the specific absorbance (Ei%) of vanillin according to formula (3): Elg = 100(Amx - Abae)
Where:
mThe mass of vanillin used to prepare the solution, in milligrams (mg). 4.4.2 Preparation of the sample
Grind the sample and mix it evenly.
4.4.3 Weighing the sample
Weigh 20 g of the prepared sample (4.4.2) to the nearest 0.001 g. 4.4.4 Extraction
....( 3)
Extract the sample in the extraction apparatus (4.3.5) with about 200 mL of ethanol (4.2.1) for 16 h, i.e., the solvent is circulated 25 to 30 times. Transfer it to a 250 mL volumetric flask (4.3.2). Rinse the flask in the extraction apparatus several times with a small amount of ethanol, and then pour the washings into the volumetric flask (4.3.2). Make up to volume with ethanol and mix well (solution A2). 4.4.5 Preparation of reference solution
Use a pipette to add 2 mL of sodium hydroxide solution (4.2.2) to a 100 mL volumetric flask (4.3.2). Dilute to the mark with ethanol and mix well to prepare the reference solution.
4.4.6 Determination
Use a pipette to add 25 mL of A2 solution to a 100 mL volumetric flask, dilute to volume with ethanol and mix well (solution B2). Use a pipette to add 20 mL of IB2 solution to a 100 mL volumetric flask, dilute to volume with ethanol and mix well (solution C2). Use a pipette to add 10 mL of C2 solution to a 100 mL volumetric flask. Add about 60 mL of ethanol and 2 mL of sodium hydroxide solution (4.2.2). Dilute to volume with ethanol and mix well (solution D2). Use a spectrophotometer (4.3.6) and a sample cell (4.3.7) to measure and record the spectrum of D2 solution relative to the 4
reference solution (4.4.5) in the wavelength range of 250nm to 420nm.
4.4.7 Calculation of results
The vanillin content W, expressed as a percentage of the sample mass, is calculated according to formula (4): W, = 50 000(Amx -Aba)
Elo ×m
Where:
Amax maximum absorbance;
Abase—absorbance at the baseline at the same wavelength; E%——the specific absorbance of vanillin (4.4.1.4); m
The mass of the sample used for extraction, in grams (g). If the result is to be expressed as a content relative to dry matter, the moisture content of the product can be included in the calculation. 5 Gas chromatography
5.1 Principle
NY/T 713—2003
Extract the sample with anhydrous ethanol, add an internal standard and separate it by gas chromatography, and then determine the content of vanillin with FID detector. 5.2 Reagents
Analytically pure.
5.2.1 Anhydrous ethanol.
5.2.2 Anhydrous sodium sulfate.
5.2.3 Internal standard: eugenol (98%).
5.2.4 Vanillin standard: purity greater than 99%. 5.2.5 Preparation of standard solution: Accurately weigh the vanillin standard, dissolve it in anhydrous ethanol, and prepare a 1.00 mg/mL vanillin standard solution. Pipette 1.00 mL, 2.00 mL, 5.00 mL, and 10.00 mL of the standard solution into a 10 mL colorimetric tube, dilute to 10.00 mL with anhydrous ethanol, add 2.00 μL eugenol and a small amount of anhydrous sodium sulfate, shake well, and then measure on the machine. 5.3 Instruments
5.3.1 Gas chromatograph with FID detector. 5.3.2 Soxhlet extractor (3.3.4).
5.4 Sample preparation
Grind the sample thoroughly and make it uniform.
5.5 Analysis steps
5.5.1 Extraction
Weigh about 2.5 g of sample (5.4) to an accuracy of 0.001 g. Extract the sample with anhydrous ethanol for 16 hours, that is, the solvent is circulated 25 to 30 times. Combine the extracts, adjust the volume to 100mL, take 5.00mL of the extract and add 2.00uL of eugenol internal standard, shake well and put it on the machine. 5.5.2 Determination
5.5.2.1 Gas chromatography reference conditions
5.5.2.1.1 Chromatographic column: glass column, 1.1m×3.2mm, filled with OV-17ChromsorbW (AW-DMCS), or a packed column or capillary column with equivalent performance.
5.5.2.1.2 Temperature: column temperature 180℃, vaporization chamber and detection chamber temperature 240℃. 5.5.2.2 Chromatographic analysis
Pipette 1μL of sample and inject it into the gas chromatograph, record the retention time and peak height of the chromatographic peak. Pipette 1μL of mixed standard solution and inject it, record the retention time and peak height of the chromatographic peak. Qualitative analysis is performed by comparing the peak time of the component on the chromatogram with that of the standard component; quantitative analysis is performed by comparing the component with the standard using the internal standard method.
NY/T713—-2003
5.6 Result calculation
5.6.1 The correction factor (k:) is given by formula (5). Where:
The response coefficient of component 1 in the component to be determined; The mass of component i in the solution;
The mass of the internal standard in the solution;
The peak height of component i measured in the chromatogram;
The peak height of the internal standard measured in the chromatogram. Note: The peak height in the formula can be replaced by its peak area. The mass of the substance to be determined is calculated according to formula (6): 5.6.2
k: Xh; Xms
Where: The meaning of each symbol is the same as in 5.6.1. Note: The peak height in the formula can be replaced by its peak area. 5.6.3 The mass percentage of vanillin in vanilla pods is calculated according to formula (7): m% =
where:
m: - the mass of vanillin in the solution;
- the mass of the sample in the solution on the machine.
（5）
·（6)
Appendix A
（Informative Appendix）
Comparison table of chapter and article numbers of this standard and those of ISO5562-2A.1 Comparison table of chapter and article numbers of this standard and those of ISO 5562-2Chapter and article numbers of this standard
Appendix B
（Informative Appendix）
Chapter and article numbers of ISO5562-2
Technical differences between this standard and ISO5562-2 and their causesTable B.1 Technical differences between this standard and ISO5562-2 and their causesChapter and article numbers of this standard
Technical differences
Deleted ISO 5562-2 a); b) was changed to a), and the determination of "vanillic acid, 4-hydroxybenzaldehyde and 4-hydroxybenzoic acid" was deleted: c) was changed to b); c) was added to determine vanillin in vanilla pods by gas chromatography.
ISO 5562-2 "ISO 1042" was changed to GB/T 12806.
3\Terms and definitions" and 4 Determination of moisture content of vanilla beans and vanilla powder in ISO5562-2 were deleted. The content of this chapter is consistent with 4.2 in ISO 5562-2. The corresponding sections 3.2.5, 3.2.7 and 3.2.8 for the determination of "vanillic acid, 4-hydroxybenzaldehyde and 4-hydroxybenzoic acid" were deleted.
The content of this chapter is consistent with 4.2 in ISO5562-2.3. Only the serial number has been changed accordingly. \ISO1042\ in ISO5562-2 is changed to GB/T12806. This chapter is newly added.
NY/T 713—2003
a) in ISO 5562-2 is for the determination of moisture content of vanilla beans and vanilla powder, which is not within the scope of this standard. The added gas chromatography method for the determination of vanillin in vanilla pods is already a mature and common analytical method, so it is adopted in this standard.
In order to be consistent with domestic standards.
In order to be consistent with domestic standards.
In order to be consistent with the standard serial number. In order to be consistent with domestic standards.
The addition of gas chromatography determination makes the implementation of this standard more universal and the standard more operable.
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.