This standard specifies the principle, instruments and utensils, reagents and solutions, operation methods and result calculation methods for the determination of caffeine in tea by high performance liquid chromatography and ultraviolet spectrophotometry. This standard is applicable to the determination of caffeine in tea. GB/T 8312-2002 Determination of caffeine in tea GB/T8312-2002 standard download decompression password: www.bzxz.net

GB/T8312--2002
This standard is a revision of GB/T8312-1987 "Determination of caffeine in tea". This standard \ "First method High performance liquid chromatography method" is equivalent to ISO10727:1995 "Determination of caffeine content in tea and solid instant tea by high performance liquid chromatography". The main difference between this standard and ISO10727:1995 is that the equivalent ISO10727:1995 method is used as the second method of this standard.
The main difference between this standard and GB/T8312-1987 is that the first method High performance liquid chromatography method is added, and the "ultraviolet spectrophotometry method" in GB/T8312-1987 is replaced by " as the second method of this standard. The first and second methods of this standard can obtain the same results. This standard will replace GB/T8312-1987 from the date of implementation. This standard is proposed by the All-China Federation of Supply and Marketing Cooperatives. This standard is under the jurisdiction of the Hangzhou Tea Research Institute of the All-China Federation of Supply and Marketing Cooperatives. The drafting unit of this standard: Hangzhou Tea Research Institute of the All-China Federation of Supply and Marketing Cooperatives. The main drafters of this standard: Zhou Weilong, Sun Anhua, Zhong Luo. This standard is interpreted by the Hangzhou Tea Research Institute of the All-China Federation of Supply and Marketing Cooperatives. 144
GB/T8312—2002
ISO Foreword
ISO (International Organization for Standardization) is an international alliance of national standardization organizations (ISO member bodies). Usually, the work of formulating international standards is undertaken by ISO technical committees. Each member body has the right to be a representative of the project committee if it is interested. Official or non-official international standard organizations associated with ISO also participate in the work. ISO and the International Electrotechnical Commission (IEC) work closely on electrotechnical standard documents.
The draft international standards adopted by the technical committees are submitted to the member bodies for voting. The publication of international standards must obtain the approval of not less than 75% of the member bodies.
International Standard ISO10727 was formulated by ISO/TC34 Agricultural Food Technical Committee. Annexes A and B of International Standard IS010727 are for reference only. 145
1 Scope
National Standard of the People's Republic of China
Determination of Caffeine in Tea
TeaDetermination of caffeine contentGB/T 8312--2002
eqv ISo 10727:1995
Replaces GB/T83121987
This standard specifies the principle, instruments and utensils, reagents and solutions, operation methods and result calculation methods for the determination of caffeine in tea by high performance liquid chromatography and ultraviolet spectrophotometry. This standard is applicable to the determination of caffeine in tea. 2 Referenced standards
The provisions contained in the following standards constitute the provisions of this standard through reference in this standard. When this standard was published, the versions shown were All standards will be revised. Parties using this standard should explore the possibility of using the latest version of the following standards. GB/T8302-2002 Tea sampling
GB/T8303-2002 Preparation of ground tea samples and determination of dry matter content Method 1 High performance liquid chromatography
3 Principle
After the caffeine in tea leaves is extracted by mixing boiling water and magnesium oxide, it is detected by high performance liquid chromatography, C1s separation column, and ultraviolet detector, and compared with the standard series for quantitative analysis.
4 Instruments and appliances
Routine laboratory instruments and the following items:
4.1 High performance liquid chromatograph.
4.2 Ultraviolet detector: detection wavelength 280nm. 4.3 Analytical column: C18ODS column).
4.4 Analytical balance: sensitivity 0.0001g.
5 Reagents and solutions
Methanol is chromatographic grade, water is double distilled water. 5.1 Magnesium oxide: analytical grade.
5.2 HPLC mobile phase: Take 600mL methanol and pour it into 1400ml double distilled water, mix, and degas. 5.3 Caffeine standard solution: Weigh 125mg caffeine (purity not less than 99%) and add ethanol: water (1:4) to dissolve, and make up to 250mL. When using, after the standard solution reaches room temperature, take 2ml and add water to 100mL as working solution. 6 Operation method
6.1 Sampling
Approved by the General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China on June 17, 2002 146
Implemented on December 1, 2002
According to the provisions of GB/T8302.
6.2 Sample preparation
According to the provisions of GB/T8303.
6.3 Determination steps
6.3.1 Test solution preparationwwW.bzxz.Net
GB/T8312—2002
Weigh 1.0g (accurate to 0.0001g) of ground tea sample (6.2), place it in a 500mL flask, add 4.5g of magnesium oxide and 300ml of boiling water, heat in a boiling water bath, and extract for 20min (shake once every 5min). After the extraction is completed, filter under reduced pressure while hot, transfer the filtrate to a 500mL volumetric flask, cool it, dilute it to the mark with water, and mix it. Take a portion of the test solution, filter it through a 0.45μm filter membrane, and set it aside. 6.3.2 Chromatographic conditions
6.3.2.1 Detection wavelength: UV detector, wavelength 280nm. 6.3.2.2 Mobile phase: water: methanol volume fraction is 7:3. 6.3.2.3 Flow rate: 0.5 mL/min~1.5 mL/min. 6.3.2.4 Column temperature: 40℃.
6.3.2.5 Injection volume: 10μl~~20μl. 6.3.3 Determination
Accurately pipette 10uL~20μL of the preparation solution (6.3.1), inject it into the high performance liquid chromatograph, and use the caffeine standard solution (5.3) to make a standard curve, and perform chromatographic determination.
7 Calculation of results
7.1 Calculation method
Compare the peak areas of the sample and the standard sample and calculate according to formula (1): C×
Caffeine (%) M,×100×100
Wherein: Ci-
Caffeine content in the test solution, ug;
Total volume of sample, mL;
Injection volume, μL;
Mass of sample, g;
ml—substance content in sample, %.
If the requirements of repeatability (7.2) are met, take the arithmetic mean of the two determinations as the result, and keep one decimal place after the result. 7.2 Repeatability
The difference between the two determination values ​​of the same sample shall not exceed 0.2g for every 100g of sample. Method 2 Ultraviolet spectrophotometry
8 Principle
Caffeine in tea is easily soluble in water. After removing interfering substances, its content is determined using a specific wavelength. 9 Instruments and appliances
Conventional laboratory instruments and the following items:
9.1 Ultraviolet spectrophotometer.
9.2 Analytical balance: sensitivity 0.001g.
(1)
10 Reagents and solutions
GB/T 83122002
The reagents used should be analytical grade (AR), and the water should be distilled water. 10.1 Basic lead acetate solution: Weigh 50g basic lead acetate, add 100ml water, let stand overnight, pour out the supernatant and filter. 10.2 Hydrochloric acid: 0.01mol/L solution, take 0.9mL concentrated hydrochloric acid, dilute 1L with water, and shake well. 10.3 Sulfuric acid: 4.5mol/L solution, take 250mL of concentrated sulfuric acid, dilute to 11 with water, and shake well. 10.4 Caffeine standard solution: weigh 100mg of caffeine (purity not less than 99%) and dissolve it in 100ml of water as the mother solution, accurately take 5ml and add water to 100mL as the working solution (1mL contains 0.05mg of coffee). 11 Determination steps
11.1 Preparation of test solution
Weigh 3g (accurate to 0.001g) of ground sample (6.2) in a 500mL conical flask, add 450mL of boiling distilled water, immediately transfer to a boiling water bath, and extract for 45min (shake once every 10min). After the extraction is completed, filter under reduced pressure while hot. Transfer the filtrate to a 500mL volumetric flask, wash the residue with a small amount of hot distilled water 2 to 3 times, and filter the filtrate into the above volumetric flask, cool it and dilute it to the scale with steam water. 11.2 Use a pipette to accurately pipette 10 ml of the test solution (11.1) into a 100 ml volumetric flask, add 4 ml of 0.01 mol/l hydrochloric acid (10.2) and 1 ml of basic lead acetate solution (10.1), dilute to the mark with water, mix, let stand to clarify and filter, accurately pipette 25 ml of the filtrate into a 50 ml volumetric flask, add 0.1 ml of 4.5 mol/l sulfuric acid solution (10.3) Dilute to the mark with water, mix, let stand to clarify and filter. Use a 10mm colorimetric cup to measure the absorbance (A) at a wavelength of 274nm, using the reagent blank solution as a reference. 11.3 Preparation of the coffee standard curve
Pick 0, 1, 2, 3, 4, 5, and 6mL of caffeine working solution (10.4) into a set of 25ml volumetric flasks, add 1.0mL of hydrochloric acid (10.2) to each, dilute to the mark with water, mix, and use a 10mm quartz colorimetric cup to measure the absorbance (A) at a wavelength of 274nm, using the reagent blank solution as a reference. Draw a standard curve with the measured absorbance and the corresponding caffeine concentration. 12 Calculation of results
The caffeine content in tea is expressed as dry mass fraction and calculated according to formula (2): 2××
Caffeine (%)=
Mz × m2
25×100
Wherein: C. According to the absorbance (A) measured by the sample, the corresponding caffeine content found from the caffeine standard curve, mg/ml.LTotal amount of test solution, ml
MAmount of sample, g;
mDry matter content of sample, %.
If the repeatability (7.2) is met, take the arithmetic mean of the two measurements as the result and keep one decimal place. 148
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