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(Roselle Red Pigment Determination by High Performance Liquid Chromatography)
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GH/T 1293-2020
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Basic data | Standard ID | GH/T 1293-2020 (GH/T1293-2020) | | Description (Translated English) | (Roselle Red Pigment Determination by High Performance Liquid Chromatography) | | Sector / Industry | Supply and Marketing Cooperatives Industry Standard (Recommended) | | Classification of Chinese Standard | B36 | | Classification of International Standard | 67.05 | | Date of Issue | 2020-06-04 | | Date of Implementation | 2020-09-01 | | Regulation (derived from) | Industry Standard Information Service Platform (2020.07.22) | | Issuing agency(ies) | All-China Federation of Supply and Marketing Cooperatives | GHT1293-2020: (Roselle Red Pigment Determination by High Performance Liquid Chromatography)---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
Determination of Roselle red pigment content-High-performance liquid chromatography
ICS 67.050
B 36
GH
Industry Standards for Supply and Marketing Cooperation of the People's Republic of China
Roselle red pigment content determination high performance liquid chromatography
2020- 06-04 release
2020-09-01 Implementation
Released by China National Supply and Marketing Cooperatives
Foreword
This standard was drafted in accordance with the rules given in GB/T 1.1-2009.
This standard was proposed by the All-China Federation of Supply and Marketing Cooperatives.
This standard is under the jurisdiction of the National Spice Standardization Technical Committee (SAC/TC 408).
Drafting organizations of this standard. China National Supply and Marketing Cooperatives Nanjing Institute of Comprehensive Utilization of Wild Plants, Jiangsu Physical and Chemical Testing Center, Jiangxi
Sujiaxin Testing Technology Co., Ltd., Nanjing Gaochun District Comprehensive Inspection and Testing Center, Anhui Huace Testing Technology Co., Ltd., Pony Test Set
Mission Jiangsu Co., Ltd.
The main drafters of this standard. Gao Hong, Huang Xiaode, Zhao Botao, Fu Lin, Luo Liang, Shan Chunsheng, Qian Hua, Jin Zhi, Chen Shirong, Zhang Weiming,
Zhang Fenglun, Chen Bin, Shi Zuhao, Yin Huili, Jin Ze, Guo Fangfang.
Roselle red pigment content determination high performance liquid chromatography
1 Scope
This standard specifies the high performance liquid chromatography method for the determination of the total content of main coloring substances in roselle red pigment.
This standard applies to the analysis and inspection of roselle calyx and roselle red products.
2 Normative references
The following documents are indispensable for the application of this document. For dated reference documents, only the dated version applies to this document.
For undated references, the latest version (including all amendments) applies to this document.
GB/T 6682 Analytical laboratory water specifications and test methods
GB/T 12729.2 Sampling method for spices and condiments
3 Terms and definitions
3.1
Roselle red pigment
The main coloring substances such as delphinium glucoside and cyanidin glucoside contained in the calyx of roselle.
3.2
Roselle red
A red pigment product extracted from the calyx of roselle.
4 Principle
After the main coloring substances in the sample are extracted by strong acidic solution of water, methanol or ethanol, they are hydrolyzed into aglycone in a boiling water bath, such as
Coloring substances such as cyanidin and delphinium are separated by a high performance liquid chromatography column and then detected by a visible light detector at 530 nm.
Qualitatively by the retention time of the standard, and quantitatively by the external standard method.
5 Reagents and equipment
5.1 Reagents
5.1.1 Water. Grade 1 water specified in GB/T 6682.
5.1.2 Methanol. chromatographically pure.
5.1.3 Anhydrous ethanol. chromatographically pure.
5.1.4 Formic acid. chromatographically pure.
5.1.5 Hydrochloric acid. analytically pure.
5.1.6 10% hydrochloric acid methanol solution (V/V). Take 10 mL of concentrated hydrochloric acid and 90 mL of methanol and mix well.
5.1.7 Extraction solvent. mix absolute ethanol, water, and concentrated hydrochloric acid in a ratio of 2.1.1 by volume.
5.1.8 Delphinium pigment. CAS number 528-53-0, purity ≥96%.
5.1.9 Centaurin. CAS No. 528-58-5, purity ≥98%.
5.2 Apparatus and materials
5.2.1 High performance liquid chromatograph equipped with visible light detector.
5.2.2 Analytical balance, with a sensitivity of 0.001 g and 0.0001 g.
5.2.3 Centrifuge, rotating speed ≥4000 r/min.
5.2.4 High-speed crusher.
5.2.5 Microporous filter membrane with a pore size of 0.22 μm.
5.2.6 Volumetric flask.
5.2.7 Micro injector.
6 Sample processing
6.1 Roselle Red
After vacuum drying at 50°C, it should be stored in a sealed airtight at room temperature and protected from light.
6.2 Calyx of Roselle
After vacuum drying at 50°C, it is crushed through a 20-mesh sieve by a high-speed pulverizer.
7 Preparation of test solution
7.1 Sample solution
Weigh the roselle red 0.2 g (accurate to 0.0001 g) and the roselle calyx 5 g (accurate to 0.001 g) to weigh the sample.
Add 50 mL extraction solvent to the flask, shake well, and ultrasonically extract for 30 min at an extraction temperature of 40 ℃, cool to room temperature, and use the extraction solvent to determine
The volume is 500 mL, and 10 mL is hydrolyzed in a boiling water bath for 1 h, taken out and cooled, and the volume is fixed to 100 mL, and filtered with a 0.22 μm microporous membrane for testing.
7.2 Mixed standard working solution
Accurately weigh 5 mg of delphinium pigment and cyanidin standard products (accurate to 0.0001 g) and use 10 mL of 10% hydrochloric acid methanol
After the solution is dissolved, mix and dilute to 100 mL, which is a 50 μg/mL mixed standard solution mother liquor. Accurately transfer roselle red pigment mixed label
The quasi-solution mother liquor is diluted with 10% hydrochloric acid methanol solution to mass concentrations of 1.25 μg/mL, 2.5 μg/mL, 5.0 μg/mL, 10.0 μg/mL,
20.0 μg/mL series of standard working solutions,
8 Determination
8.1 HPLC conditions
--Chromatographic column. C18 column, 250 mm×4.6 mm×5 μm or equivalent;
--Mobile phase. 0.5% formic acid aqueous solution, 0.5% formic acid acetonitrile solution;
--Detection wavelength. 530 nm;
--Column temperature. 30 ℃;
--Flow rate. 0.6 mL/min;
--Sampling volume. 20 μL;
--See Table 1 for gradient elution conditions.
8.2 Standard curve drawing
According to 8.1 chromatographic conditions, a series of standard working solutions were injected into the liquid chromatograph for determination. Take the measured peak area as the ordinate,
The mass concentration of the corresponding standard solution is the abscissa, and the standard curve is drawn.
8.3 Chromatographic analysis
According to 8.1 chromatographic conditions, the sample solution (7.1) is injected into the liquid chromatograph for determination, qualitatively based on the retention time, and the peak area of the sample solution
Bring it into the standard curve for quantification. The mass concentration of roselle red pigment in the sample should be within the mass concentration range of the standard working curve, beyond the linear range
The test solution should be tested after dilution.
9 Result calculation
The total content of roselle red pigment in the sample is the sum of delphinium and cyanidin. The content is calculated by mass fraction X, and the unit is microgram per
Gram (μg/g), calculated according to formula (1).
X-The sum of the content of delphinium and cyanidin in the sample, in micrograms per gram (μg/g);
V-constant volume volume, the unit is (mL);
m-the mass of the sample, in grams (g);
Ci--the mass concentration corresponding to the peak area of component i in the sample to be tested, in micrograms per milliliter (μg/mL).
K--The dilution factor of the sample to be tested.
Take the arithmetic mean of the two measured values whose absolute difference does not exceed 10% of the arithmetic mean as the measurement result, and keep decimals in the calculation result
The last 2 digits of the point.
AA
Appendix A
(Informative appendix)
Typical liquid chromatogram
A.1 The typical chromatogram of the reference substance is shown in Figure A.1.
Note. 1 delphinium pigment 2 cyanidin
Figure A.1 Standard liquid chromatogram
A.2 A typical liquid chromatogram of Roselle Red is shown in Figure A.2.
Note. 1 Delphinium pigment 2 cyanidin
Figure A.2 Roselle red liquid chromatogram
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