WO2016004779A1 - 一种缓冲体系及采用该缓冲体系以连续流动法测定烟草或烟草制品中总植物碱的方法 - Google Patents

一种缓冲体系及采用该缓冲体系以连续流动法测定烟草或烟草制品中总植物碱的方法 Download PDF

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WO2016004779A1
WO2016004779A1 PCT/CN2015/076250 CN2015076250W WO2016004779A1 WO 2016004779 A1 WO2016004779 A1 WO 2016004779A1 CN 2015076250 W CN2015076250 W CN 2015076250W WO 2016004779 A1 WO2016004779 A1 WO 2016004779A1
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water
solution
volumetric flask
beaker
tobacco
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PCT/CN2015/076250
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English (en)
French (fr)
Inventor
张威
胡清源
刘楠
罗安娜
何声宝
王英元
冯晓民
马雁军
邢军
侯宏卫
刘洋
王颖
辛宝珺
张洪非
姜兴益
杨进
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国家烟草质量监督检验中心
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Priority claimed from CN201410322455.5A external-priority patent/CN104132895B/zh
Priority claimed from CN201410324273.1A external-priority patent/CN104132937B/zh
Application filed by 国家烟草质量监督检验中心 filed Critical 国家烟草质量监督检验中心
Priority to EP15819267.4A priority Critical patent/EP3168605B1/en
Publication of WO2016004779A1 publication Critical patent/WO2016004779A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
    • G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/10—Chemical features of tobacco products or tobacco substitutes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4055—Concentrating samples by solubility techniques
    • G01N2001/4061—Solvent extraction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G01N2001/4088—Concentrating samples by other techniques involving separation of suspended solids filtration

Definitions

  • the invention belongs to the technical field of tobacco chemical component detection, and particularly relates to a buffer system and a method for determining total plant alkali in tobacco or tobacco products by the continuous flow method using the buffer system.
  • Nicotine is an important component of tobacco, and smokeless alkali is not called tobacco.
  • information on the content of nicotine is usually obtained by measuring the content of total plant alkaloids in tobacco.
  • the tobacco industry currently publishes standards for the determination of total alkaloids in tobacco: GB/T23225-2008 "Determination of Total Alkaloids in Tobacco and Tobacco Products", YC/T160-2002 “Determination of Total Alkaloids in Tobacco and Tobacco Products Continuous Flow Method" and YC/T 468-2013 "Determination of Total Alkaloids in Tobacco and Tobacco Products (Continuous Flow (KC) Method".
  • the continuous flow method belongs to the instrument analysis method and is suitable for large-volume injection, and is currently widely used.
  • YC/T160-2002 Determination of Total Alkaloids in Tobacco and Tobacco Products by Continuous Flow Method” is based on the principle that potassium cyanide and chloramine T are used to form cyanogen chloride on the line, so that the pyridine ring of nicotine is broken, and then the amino group The benzenesulfonic acid reacted and the reaction product was measured at a wavelength of 460 nm using a colorimeter.
  • potassium cyanide is relatively stable, it is a highly toxic substance and is at the forefront of toxic chemicals. There are many scruples about the use of potassium cyanide, and its sales channels are very strict.
  • the method controls the acid-base environment of the reaction by buffering the solution A and the buffer solution B.
  • the buffering ability of the buffer solution B is not strong, such as the pH of some chemical analysis laboratory water is between 5.0 and 6.0, the buffering capacity of the buffer solution B does not meet the test requirements.
  • the pH requirement of the actual use of water in this method is higher than the requirements of the first grade water in GB6682/T-2008 "Analytical laboratory water specifications and test methods".
  • the chemical analysis laboratory needs to be the first grade water. Performing reprocessing has certain limitations in the practical use of the method.
  • YC/T 468-2013 Determination of Total Alkaloids in Tobacco and Tobacco Products by Continuous Flow (Potassium Cyanate) Method
  • the principle of the determination of cyanogen chloride by using potassium thiocyanate and sodium dichloroisocyanurate The pyridine ring of the base was cleaved and further reacted with p-aminobenzenesulfonic acid, and the reaction product was measured with a colorimeter at a wavelength of 460 nm.
  • YC/T 468-2013 does not use potassium cyanide, which improves the operational safety of total plant alkali determination.
  • the present invention provides a novel buffer system and a method for determining total plant alkaloids in tobacco or tobacco products using the buffer system in a continuous flow method.
  • the buffer system is suitable for the determination of total alkaloids in tobacco and tobacco products using a continuous flow (potassium thiocyanate) method.
  • the buffer system of the invention can control the acid-base environment of the reaction while reducing the amount of the chemical used in the preparation, ensuring that the cyanide ring of the plant base is broken by the cyanogen chloride, and then reacting with the p-aminobenzenesulfonic acid to make the reaction Under the required acid-base environment conditions, the buffer system is used to reduce the amount of use while ensuring consistency with the YC/T 468-2013 method test results.
  • the present invention is achieved by the following technical solutions.
  • the present invention provides a buffer system suitable for continuous flow method for determining total plant alkali in tobacco and tobacco products, the buffer system comprising buffer solution A and buffer solution B;
  • the buffer solution A is prepared by weighing disodium hydrogen phosphate 60.5-83.0 g, preferably 71.6 g; trisodium phosphate 10.1-20.2 g, preferably 15.2 g, into a beaker, dissolving in water, and then transferring to a 1 L volumetric flask. Medium to constant volume with water;
  • Buffer solution B is prepared by weighing 5.5-8.0 g of p-aminobenzenesulfonic acid, preferably 7.0 g; disodium hydrogen phosphate 60.5-83.0 g, preferably 71.6 g; sodium dihydrogen phosphate 5.0-7.0 g, preferably 6.2 g and sodium citrate 10.0-12.1 g, preferably 11.8 g into a beaker, dissolved in water, then transferred to a 1 L volumetric flask and brought to volume with water.
  • the above disodium hydrogen phosphate, trisodium phosphate, sodium dihydrogen phosphate, sodium citrate may be in a conventionally present form, for example, disodium hydrogen phosphate is present in the form of 12 crystal waters, and trisodium phosphate is in the form of 12 crystal waters.
  • disodium hydrogen phosphate is present in the form of 12 crystal waters
  • trisodium phosphate is in the form of 12 crystal waters.
  • sodium dihydrogen phosphate exists in the form of two crystal waters
  • sodium citrate exists in the form of two crystal waters.
  • the ratio between the buffer solution A and the buffer solution B is from 1:2 to 1:1 (volume ratio), preferably 3:4 (volume ratio).
  • the total plant alkali (in terms of nicotine) in the tobacco product is reacted, the pyridine ring of nicotine is cleaved, and then reacted with p-aminobenzenesulfonic acid, and the reaction product is determined by a colorimeter at 460 nm, the method comprising:
  • the buffer solution A and the buffer solution B are used to control the pH of the reaction system between 6.0 and 7.5;
  • the buffer solution A is prepared by weighing disodium hydrogen phosphate 60.5-83.0 g, preferably 71.6 g; trisodium phosphate 10.1-20.2 g, preferably 15.2 g, into a beaker, dissolving in water, and then transferring to a 1 L volumetric flask. Medium to constant volume with water;
  • Buffer solution B is prepared by weighing 5.5-8.0 g of p-aminobenzenesulfonic acid, preferably 7.0 g; disodium hydrogen phosphate 60.5-83.0 g, preferably 71.6 g; sodium dihydrogen phosphate 5.0-7.0 g, preferably 6.2 g and sodium citrate 10.0-12.1 g, preferably 11.8 g into a beaker, dissolved in water, then transferred to a 1 L volumetric flask and brought to volume with water.
  • the above disodium hydrogen phosphate, trisodium phosphate, sodium dihydrogen phosphate, sodium citrate may be in a conventionally present form, for example, disodium hydrogen phosphate is present in the form of 12 crystal water, and trisodium phosphate is present in the form of 12 crystal water, phosphoric acid.
  • Sodium dihydrogen is present in the form of two crystal waters, and sodium citrate is present in the form of two crystal waters.
  • the ratio of the buffer solution A to the buffer solution B is 1:2 to 1:1 (volume ratio), preferably 3:4 (volume ratio);
  • the above method further comprises:
  • the sample was prepared by preparing a tobacco sample according to YC/T31 and measuring its moisture content, and then extracting the tobacco sample with 5% acetic acid and filtering to obtain a filtrate.
  • the above method further comprises:
  • the standard solution is prepared as follows:
  • the nicotine or nicotine salt is dissolved in 5% acetic acid to prepare at least 5 working standards, and the concentration of nicotine in the standard solution is 0.5-7%;
  • the above method further comprises:
  • Detoxification solution A is prepared by weighing 1g of citric acid and 10g of ferrous sulfate into a beaker, dissolving it in water, then transferring it to a 1L volumetric flask, and diluting it to the mark with water; the main function of the detoxification solution A is to reduce the non-reactive reaction.
  • the toxicity of cyanogen chloride A is the same as the detoxification solution A in YC/T160-2002 (potassium cyanide method).
  • Detoxification solution B is prepared by weighing 10 g of sodium carbonate into a beaker, dissolving it in water, then transferring it to a 1 L volumetric flask, and diluting it to the mark with water; the main function of the detoxification solution B is to reduce the cyanogen chloride which is not involved in the reaction. Toxic, the detoxification solution B is the same as YC/T160-2002 (potassium cyanide method) detoxification solution B.
  • Potassium thiocyanate solution is prepared by weighing potassium thiocyanate into a beaker, dissolving it in water, then transferring it to a 250 mL volumetric flask, and diluting it with water.
  • concentration of the solution ranges from 0.09 to 0.21 mol/L, preferably 0.12 mol. /L.
  • the sodium dichloroisocyanurate solution is prepared by weighing sodium dichloroisocyanurate into a beaker, dissolving it in water, and then transferring it into a 250 mL volumetric flask, and diluting it with water.
  • the concentration of the solution ranges from 0.02 to 0.06 mol/L. It is preferably 0.04 mol/L.
  • the concentration ratio of the potassium thiocyanate solution and the sodium dichloroisocyanurate solution is from 2.5:1 to 3.5:1, preferably 3:1.
  • the above method further comprises:
  • X is the instrumental observation of the sample in milligrams per milliliter (mg/mL);
  • V volume of the extract added, in milliliters (mL);
  • W moisture content of the sample, % (mass fraction);
  • the total plant alkali calculated by the above formula is the total plant alkali content on a dry basis.
  • the operating parameters of the continuous flow analyzer are: injection cleaning ratio of 1.5, injection frequency of 48 cups/hour, gain of 18-60, and light intensity of 2.85 volts.
  • the above method comprises:
  • the nicotine or nicotine salt is dissolved in 5% acetic acid to prepare at least 5 working standards, and the concentration of nicotine in the standard solution is 0.5-7%;
  • Potassium thiocyanate solution is prepared by weighing potassium thiocyanate into a beaker, dissolving it in water, then transferring it to a 250 mL volumetric flask, and diluting it with water.
  • concentration of the solution ranges from 0.09 to 0.21 mol/L, preferably 0.12 mol. /L.
  • the sodium dichloroisocyanurate solution is prepared by weighing sodium dichloroisocyanurate into a beaker, dissolving it in water, and then transferring it into a 250 mL volumetric flask, and diluting it with water.
  • the concentration of the solution ranges from 0.02 to 0.06 mol/L. It is preferably 0.04 mol/L.
  • buffer solution A Weigh 60.5-83.0 g of disodium hydrogen phosphate, preferably 71.6 g, trisodium phosphate 10.10.2 g, preferably 15.2 g into a beaker, dissolve in water, and then transfer to a 1 L volumetric flask, with water Make up to the scale;
  • buffer solution B 5.5-8.0 g of p-aminobenzenesulfonic acid, preferably 7.0 g; disodium hydrogen phosphate 60.5-83.0 g, preferably 71.6 g; sodium dihydrogen phosphate 5.0-7.0 g, preferably 6.2 g And sodium citrate 10.0-12.1g, preferably 11.8g into a beaker, dissolved in water, then transferred to a 1L volumetric flask, and dilute to the mark with water;
  • the above disodium hydrogen phosphate, trisodium phosphate, sodium dihydrogen phosphate, sodium citrate may be in a conventionally present form, for example, disodium hydrogen phosphate is present in the form of 12 crystal water, and trisodium phosphate is present in the form of 12 crystal water, phosphoric acid.
  • Sodium dihydrogen is present in the form of two crystal waters, and sodium citrate is present in the form of two crystal waters.
  • Detoxification solution A is prepared by weighing 1g of citric acid and 10g of ferrous sulfate into a beaker, dissolving it in water, then transferring it to a 1L volumetric flask, and diluting it to the mark with water; the main function of the detoxification solution A is to reduce the non-reactive reaction.
  • the toxicity of cyanogen chloride A is the same as the detoxification solution A in YC/T160-2002 (potassium cyanide method).
  • Detoxification solution B is prepared by weighing 10 g of sodium carbonate into a beaker, dissolving it in water, then transferring it to a 1 L volumetric flask, and diluting it to the mark with water; the main function of the detoxification solution B is to reduce cyanogen chloride which is not involved in the reaction. Toxic, the detoxification solution B is the same as YC/T160-2002 (potassium cyanide method) detoxification solution B.
  • X is the instrumental observation of the sample in milligrams per milliliter (mg/mL);
  • V volume of the extract added, in milliliters (mL);
  • W moisture content of the sample, % (mass fraction);
  • the ratio of the buffer solution A to the buffer solution B is 1:2 to 1:1 (volume ratio), preferably 3:4 (volume ratio), and the ratio is such that the pH of the reaction system is controlled. Between 6.0 and 7.5;
  • the concentration ratio of the potassium thiocyanate solution and the sodium dichloroisocyanurate solution is from 2.5:1 to 3.5:1, preferably 3:1.
  • the operating parameters of the continuous flow analyzer are: injection cleaning ratio of 1.5, injection frequency of 48 cups/hour, gain of 18-60, and light intensity of 2.85 volts.
  • the preparation of the sample may vary.
  • the pump tube on the total alkaloid module is inserted into the peristaltic pump as shown in Fig. 1 (the inner diameters of different pump tubes on the module are different, the flow rate of the corresponding reagent is different, and the flow rate of the reaction reagent is controlled to control the participating reaction reagents.
  • the ratio The purpose of the ratio; the air pump tube is added to make the solution in the pipeline mix evenly), the pump cover is covered, the reagent tube is placed in the water; the power is turned on, the detector is turned on, the peristaltic pump, the autosampler, the computer; Turn the switch on the peristaltic pump to Run; double-click the icon of the continuous flow analysis workstation connected to the computer; click the charting on the workstation, after the connection is successful, the peristaltic pump runs, the water is pumped into the pipeline for cleaning, the cleaning time is 20 ⁇ 30min; After the baseline is stable (the baseline fluctuation is less than ⁇ 1%), put the different pipelines into the corresponding reagents, continue to run, and adjust the baseline to about 5% in the charting window popped up by the workstation; In the drop-down menu, Analyze the prompt to compile the running program.
  • click Run in the workstation After the baseline is stable, click Run in the workstation. It can be run; when the program is finished, put the different pipelines back into the water and wash for 20 ⁇ 30min; after the cleaning is completed, turn off the autosampler, peristaltic pump, detector, remove the pump cover and turn off the power.
  • the above step (1) is specifically: weighing the smoke sample (the smoke sample is prepared according to YC/T 31) into a 50 mL stoppered flask, adding 5% acetic acid for 30 minutes, and filtering using a qualitative filter paper.
  • step (2) is specifically as follows: according to FIG. 1, the pump tube is mounted on the peristaltic pump, and the pump cover is added to wash the pipeline with water.
  • the buffer solution A is prepared by weighing 71.6 g of disodium hydrogen phosphate and 15.2 g of trisodium phosphate into a beaker, dissolving in water, and then transferring to a 1 L volumetric flask, and diluting to a volume with water.
  • disodium hydrogen phosphate and trisodium phosphate may be in a conventionally existing form, for example, disodium hydrogen phosphate is present in the form of 12 water of crystallization, and trisodium phosphate is present in the form of 12 crystal water.
  • the buffer solution B is prepared by weighing 7 g of p-aminobenzenesulfonic acid, 71.6 g of disodium hydrogen phosphate, 6.2 g of sodium dihydrogen phosphate, and 11.8 g of sodium citrate into a beaker, dissolving in water, and then Transfer to a 1L volumetric flask and bring up to volume with water.
  • disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate may be in a conventional form, for example, disodium hydrogen phosphate is present in the form of 12 water of crystallization, sodium dihydrogen phosphate is present in the form of 2 crystal water, sodium citrate is Two crystal water forms exist.
  • the detoxification solution A is prepared by weighing 1 g of citric acid and 10 g of ferrous sulfate into a beaker, dissolving in water, and then transferring to a 1 L volumetric flask, and diluting to a volume with water.
  • the main role of detoxification solution A is to reduce the toxicity of cyanogen chloride that is not involved in the reaction.
  • the detoxification solution A is the same as the YC/T160-2002 (potassium cyanide method) detoxification solution A.
  • the detoxification liquid B is prepared by weighing 10 g of sodium carbonate into a beaker, dissolving it in water, then transferring it into a 1 L volumetric flask, and diluting it to the mark with water.
  • the main role of detoxification solution B is to reduce the toxicity of cyanogen chloride that is not involved in the reaction.
  • the detoxifying solution B is the same as the YC/T160-2002 (potassium cyanide method) detoxifying solution B.
  • the potassium thiocyanate solution is prepared by weighing an appropriate amount of potassium thiocyanate into a beaker, dissolving it in water, and then transferring it into a 250 mL volumetric flask and diluting it with water.
  • the solution concentration ranges from 0.09 to 0.21 mol/L.
  • the sodium dichloroisocyanurate solution is prepared by weighing an appropriate amount of sodium dichloroisocyanurate into a beaker, dissolving it in water, and then transferring it into a 250 mL volumetric flask, and diluting it with water.
  • the solution concentration ranges from 0.02 to 0.06 mol/L.
  • the concentration ratio of the potassium thiocyanate solution and the sodium dichloroisocyanurate solution is about 3: 1.
  • the standard solution is prepared by weighing an appropriate amount of nicotine in a 250 mL volumetric flask, dissolving with 5% acetic acid, and diluting to a volume.
  • This solution is a nicotine stock solution with a nicotine content of about 1.6 mg/mL.
  • the operating parameters of the continuous flow analyzer are: injection cleaning ratio of 1.5, injection frequency of 48 cups/hour, gain of 18-60, and light intensity of 2.85 volts.
  • the invention adopts a new buffer solution A to control the acid-base environment of the reaction before the dialysis machine, and ensures that the pH value of the solution before the dialysis device is between 6.0 and 11.0, and does not affect the acid-base environment controlled by the buffer solution B;
  • the invention adopts a new buffer solution B to control the acid-base environment of the reaction, adjust the pH value of the solution between 6.0 and 7.5, ensure that the cyanide ring of the plant base breaks the pyridine ring, and further reacts with p-aminobenzenesulfonic acid. , carried out under the acid-base environment conditions required for the reaction;
  • the invention establishes a new buffer system suitable for continuous flow (potassium thiocyanate) determination of total alkaloids in tobacco and tobacco products;
  • the continuous flow method using the buffer system of the present invention reduces the amount of chemicals used in the preparation of buffer solution A and buffer solution B as compared with YC/T 468-2013, while ensuring that the data is consistent with YC/T468-2013.
  • Figure 1 is a piping diagram for the determination of total plant alkaloids in tobacco and tobacco products using the continuous flow method of the buffer system of the present invention.
  • Example 1 Determination of total plant alkaloids in flue-cured tobacco
  • the flue-cured tobacco was prepared as a sample according to YC/T31, and its moisture content was determined. Then, 0.25 g of the sample was weighed into a 50 mL stoppered flask, 25 mL of 5% acetic acid was added, and the stopper was placed to oscillate on the shaker (rotation speed). >150 rpm) extraction for 30 min. The extract was filtered with a fast qualitative filter paper, and the first 2 mL to 3 mL of the filtrate was discarded, and the subsequent filtrate was collected for analysis.
  • Buffer solution A is prepared by weighing 60.5 g of disodium hydrogen phosphate (12 crystal water) and 12.2 g of trisodium phosphate (12 crystal water) into a beaker, dissolving in water, and then transferring to a 1 L volumetric flask. Tolerance to the scale.
  • the buffer solution B was prepared by weighing 5.5 g of p-aminobenzenesulfonic acid, disodium hydrogen phosphate (12 crystal water), 60.5 g, sodium dihydrogen phosphate (2 crystal water), 5.0 g, and sodium citrate (2 crystals). Water) 10.0 g into a beaker, dissolved in water, then transferred to a 1 L volumetric flask and dilute to volume with water.
  • the ratio of the buffer solution A to the buffer solution B was 3:4 (volume ratio).
  • the preparation of detoxification solution A is:
  • the preparation of detoxification solution B is:
  • the analytical instrument used in the present invention is an AA3 continuous flow analyzer manufactured by Brown Rupee Company of Germany.
  • the pipeline for the determination of total alkaloids in tobacco and tobacco products by continuous flow is shown in Figure 1.
  • the pump tube on the total plant alkali module is inserted into the peristaltic pump as shown in Fig. 1 (the inner diameters of different pump tubes on the module are different, the flow rate of the corresponding reagent is different, and the flow rate of the reaction reagent is controlled to achieve the purpose of controlling the proportion of the reagents involved in the reaction.
  • the air pump tube is added to make the solution in the pipeline mix evenly.
  • the peak height of the total plant base peak was subjected to regression analysis for its corresponding concentration to obtain a standard curve. Then, by measuring the peak height of the sample, the corresponding concentration of the sample, that is, the instrument observation value, is obtained from the standard curve.
  • the total plant alkali content on a dry basis is given by:
  • X is the instrumental observation of the sample in milligrams per milliliter (mg/mL);
  • V volume of the extract added, in milliliters (where the volume of the extract is 25mL);
  • W moisture content of the sample, % (mass fraction);
  • the total plant alkali content in the flue-cured tobacco was calculated from the above formula to be 0.5 to 3%.
  • Example 2 Determination of total plant alkaloids in Burley tobacco
  • the burley tobacco was prepared as a sample according to YC/T31, and its moisture content was determined. Then, 0.25 g of burley tobacco sample was weighed into a 50 mL stoppered flask, 25 mL of 5% acetic acid was added, and the stopper was placed to oscillate. The mixture was shaken (rotation > 150 rpm) for 30 min. The extract was filtered with a fast qualitative filter paper, and the first 2 mL to 3 mL of the filtrate was discarded, and the subsequent filtrate was collected for analysis.
  • Buffer solution A was prepared by weighing 83.0 g of disodium hydrogen phosphate (12 crystal water) and 18.4 g of trisodium phosphate (12 crystal water) into a beaker, dissolving in water, and then transferring to a 1 L volumetric flask. Tolerance to the scale.
  • Buffer solution B was prepared by weighing 8.0 g of p-aminobenzenesulfonic acid, disodium hydrogen phosphate (12 crystal water), 83.0 g, sodium dihydrogen phosphate (2 crystal water), 7.0 g, and sodium citrate (2 crystals). Water) 12.1 grams into the beaker, dissolved in water, then transferred to a 1L volumetric flask, and dilute to the mark with water.
  • the ratio of the amount of the buffer solution A to the buffer solution B is 3:4 (volume ratio).
  • the preparation of detoxification solution A is:
  • the preparation of detoxification solution B is:
  • the analytical instrument used in the present invention is an AA3 continuous flow analyzer manufactured by Brown Rupee Company of Germany.
  • the pipeline for the determination of total alkaloids in tobacco and tobacco products by continuous flow is shown in Figure 1.
  • the pump tube on the total plant alkali module is inserted into the peristaltic pump as shown in Fig. 1 (the inner diameters of different pump tubes on the module are different, the flow rate of the corresponding reagent is different, and the flow rate of the reaction reagent is controlled to achieve the purpose of controlling the proportion of the reagents involved in the reaction.
  • the air pump tube is added to make the solution in the pipeline mix evenly.
  • the peak height of the total plant base peak was subjected to regression analysis for its corresponding concentration to obtain a standard curve. Then, by measuring the peak height of the sample, the corresponding concentration of the sample, that is, the instrument observation value, is obtained from the standard curve.
  • the total plant alkali content on a dry basis is given by:
  • X is the instrumental observation of the sample in milligrams per milliliter (mg/mL);
  • V volume of the extract to be added, in milliliters (where the volume of the extract is 25 mL);
  • W moisture content of the sample, % (mass fraction);
  • the total plant alkali content in burley tobacco was calculated from the above formula to be 1 to 6%.
  • Example 3 Determination of total plant alkaloids in flue-cured tobacco
  • the flue-cured tobacco was prepared as a sample according to YC/T31, and its moisture content was determined. Then, 0.25 g of the sample was weighed into a 50 mL stoppered flask, 25 mL of 5% acetic acid was added, and the stopper was placed to oscillate on the shaker (rotation speed). >150 rpm) extraction for 30 min. The extract was filtered with a fast qualitative filter paper, and the first 2 mL to 3 mL of the filtrate was discarded, and the subsequent filtrate was collected for analysis.
  • Buffer solution A was prepared by weighing 71.6 g of disodium hydrogen phosphate (12 crystal water) and 15.2 g of trisodium phosphate (12 crystal water) into a beaker, dissolving in water, and then transferring to a 1 L volumetric flask. Tolerance to the scale.
  • the buffer solution B was prepared by weighing 7 g of p-aminobenzenesulfonic acid, disodium hydrogen phosphate (12 crystal water), 71.6 g, sodium dihydrogen phosphate (2 crystal water), 6.2 g, and sodium citrate (2 crystal water). ) 11.8 grams into the beaker, dissolved in water, then transferred to a 1L volumetric flask, and dilute to the mark with water.
  • the ratio of the buffer solution A to the buffer solution B was 3:4 (volume ratio).
  • the preparation of detoxification solution A is:
  • the preparation of detoxification solution B is:
  • the analytical instrument used in the present invention is an AA3 continuous flow analyzer manufactured by Brown Rupee Company of Germany.
  • the pipeline for the determination of total alkaloids in tobacco and tobacco products by continuous flow is shown in Figure 1.
  • the pump tube on the total plant alkali module is inserted into the peristaltic pump as shown in Fig. 1 (the inner diameters of different pump tubes on the module are different, the flow rate of the corresponding reagent is different, and the flow rate of the reaction reagent is controlled to achieve the purpose of controlling the proportion of the reagents involved in the reaction.
  • the air pump tube is added to make the solution in the pipeline mix evenly.
  • the peak height of the total plant base peak was subjected to regression analysis for its corresponding concentration to obtain a standard curve. Then, by measuring the peak height of the sample, the corresponding concentration of the sample, that is, the instrument observation value, is obtained from the standard curve.
  • the total plant alkali content on a dry basis is given by:
  • X is the instrumental observation of the sample in milligrams per milliliter (mg/mL);
  • V volume of the extract to be added, in milliliters (where the volume of the extract is 25 mL);
  • W moisture content of the sample, % (mass fraction);
  • the total plant alkali content in the flue-cured tobacco was calculated from the above formula to be 0.5 to 3%.
  • the burley tobacco was prepared as a sample according to YC/T31, and its moisture content was determined. Then, 0.25 g of burley tobacco sample was weighed into a 50 mL stoppered flask, 25 mL of 5% acetic acid was added, and the stopper was placed to oscillate. The mixture was shaken (rotation > 150 rpm) for 30 min. The extract was filtered with a fast qualitative filter paper, and the first 2 mL to 3 mL of the filtrate was discarded, and the subsequent filtrate was collected for analysis.
  • Buffer solution A was prepared by weighing 71.6 g of disodium hydrogen phosphate (12 crystal water) and 15.2 g of trisodium phosphate (12 crystal water) into a beaker, dissolving in water, and then transferring to a 1 L volumetric flask. Tolerance to the scale.
  • the buffer solution B was prepared by weighing 7 g of p-aminobenzenesulfonic acid, disodium hydrogen phosphate (12 crystal water), 71.6 g, sodium dihydrogen phosphate (2 crystal water), 6.2 g, and sodium citrate (2 crystal water). ) 11.8 grams into the beaker, dissolved in water, then transferred to a 1L volumetric flask, and dilute to the mark with water.
  • the ratio of the amount of the buffer solution A to the buffer solution B is 3:4 (volume ratio).
  • the preparation of detoxification solution A is:
  • the preparation of detoxification solution B is:
  • the analytical instrument used in the present invention is an AA3 continuous flow analyzer manufactured by Brown Rupee Company of Germany.
  • the pipeline for the determination of total alkaloids in tobacco and tobacco products by continuous flow is shown in Figure 1.
  • the pump tube on the total plant alkali module is inserted into the peristaltic pump as shown in Fig. 1 (the inner diameters of different pump tubes on the module are different, the flow rate of the corresponding reagent is different, and the flow rate of the reaction reagent is controlled to achieve the purpose of controlling the proportion of the reagents involved in the reaction.
  • the air pump tube is added to make the solution in the pipeline mix evenly.
  • the peak height of the total plant base peak was subjected to regression analysis for its corresponding concentration to obtain a standard curve. Then, by measuring the peak height of the sample, the corresponding concentration of the sample, that is, the instrument observation value, is obtained from the standard curve.
  • the total plant alkali content on a dry basis is given by:
  • X is the instrumental observation of the sample in milligrams per milliliter (mg/mL);
  • V volume of the extract to be added, in milliliters (where the volume of the extract is 25 mL);
  • W moisture content of the sample, % (mass fraction);
  • the total plant alkali content in burley tobacco was calculated from the above formula to be 1 to 5%.
  • Example 5 Determination of total plant alkaloids in cigarettes
  • the cigarette cut tobacco was prepared into a sample according to YC/T31, and its moisture content was determined. Then, 0.25 g of the cigarette sample was weighed into a 50 mL stoppered flask, 25 mL of 5% acetic acid was added, and the stopper was placed to oscillate on the shaker (rotation speed). >150 rpm) extraction for 30 min. The extract was filtered with a fast qualitative filter paper, and the first 2 mL to 3 mL of the filtrate was discarded, and the subsequent filtrate was collected for analysis.
  • Buffer solution A was prepared by weighing 71.6 g of disodium hydrogen phosphate (12 crystal water) and 15.2 g of trisodium phosphate (12 crystal water) into a beaker, dissolving in water, and then transferring to a 1 L volumetric flask. Tolerance to the scale.
  • the buffer solution B was prepared by weighing 7 g of p-aminobenzenesulfonic acid, disodium hydrogen phosphate (12 crystal water), 71.6 g, sodium dihydrogen phosphate (2 crystal water), 6.2 g, and sodium citrate (2 crystal water). ) 11.8 grams into the beaker, dissolved in water, then transferred to a 1L volumetric flask, and dilute to the mark with water.
  • the ratio of the buffer solution A to the buffer solution B is 3:4 (volume ratio)
  • the preparation of detoxification solution A is:
  • the preparation of detoxification solution B is:
  • the analytical instrument used in the present invention is an AA3 continuous flow analyzer manufactured by Brown Rupee Company of Germany.
  • the pipeline for the determination of total alkaloids in tobacco and tobacco products by continuous flow is shown in Figure 1.
  • the pump tube on the total plant alkali module is inserted into the peristaltic pump as shown in Fig. 1 (the inner diameters of different pump tubes on the module are different, the flow rate of the corresponding reagent is different, and the flow rate of the reaction reagent is controlled to achieve the purpose of controlling the proportion of the reagents involved in the reaction.
  • the air pump tube is added to make the solution in the pipeline mix evenly.
  • the peak height of the total plant base peak was subjected to regression analysis for its corresponding concentration to obtain a standard curve. Then, by measuring the peak height of the sample, the corresponding concentration of the sample, that is, the instrument observation value, is obtained from the standard curve.
  • the total plant alkali content on a dry basis is given by:
  • X is the instrumental observation of the sample in milligrams per milliliter (mg/mL);
  • V volume of the extract to be added, in milliliters (where the volume of the extract is 25 mL);
  • W moisture content of the sample, % (mass fraction);
  • the total plant alkali content in the cigarette was calculated from the above formula to be 0.5 to 4%.
  • Example 6 Comparative study of continuous flow method using the buffer system of the present invention and continuous flow method using other buffer systems
  • Table 1 compares the results of the continuous flow method of the buffer system of the present invention (used in Example 1) with the YC/T 468-2013 method.
  • Table 2 compares the results of the continuous flow method of the buffer system of the present invention (used in Example 2) with the YC/T 468-2013 method.
  • Table 3 compares the results of the continuous flow method of the buffer system of the present invention (used in Examples 3-5) with the YC/T 468-2013 method.
  • the inventors have also employed other buffer systems (other than the buffer system differs from the present invention, the remaining reagents are the same as in Examples 3 to 5 of the present invention) for flue-cured tobacco, burley tobacco, oriental tobacco, flue-cured cigarettes, and mixed cigarettes. Five samples were measured, and the measurement results are shown in Tables 4 to 9. It can be seen from Tables 4 to 9 that the absolute difference between the measurement results using other buffer systems and the YC/T 468-2013 method is large.
  • Table 10 shows the use of the buffer system of the present invention for the same sample (the buffer solution A and the buffer solution B used in the embodiment 3-5 of the present invention, the buffer solution A and the buffer solution B are used in an amount ratio and in Example 3

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Abstract

本发明提供一种缓冲体系及采用该缓冲体系以连续流动法测定烟草或烟草制品中总植物碱的方法。该缓冲体系包含缓冲溶液A和缓冲溶液B；其中，缓冲溶液A的配制为：称取磷酸氢二钠60.5-83.0g，优选为71.6g、磷酸三钠10.1-20.2g，优选为15.2g至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度；缓冲溶液B的配制为：称取对氨基苯磺酸5.5-8.0g，优选为7.0g、磷酸氢二钠60.5-83.0g，优选为71.6g、磷酸二氢钠5.0-7.0g，优选为6.2g和柠檬酸钠10.0-12.1g，优选为11.8g至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。

Description

一种缓冲体系及采用该缓冲体系以连续流动法测定烟草或烟草制品中总植物碱的方法 技术领域
本发明属于烟草化学成分检测技术领域，具体涉及一种缓冲体系及采用该缓冲体系以连续流动法测定烟草或烟草制品中总植物碱的方法。
背景技术
烟碱是烟草的重要成分，无烟碱也就称不上烟草。在烟草常规分析中，通常通过测定烟草中总植物碱的含量来获知烟碱的含量信息。烟草行业目前发布的关于烟草中总植物碱测定的标准有：GB/T23225-2008“烟草及烟草制品总植物碱的测定光度法”、YC/T160-2002“烟草及烟草制品总植物碱的测定连续流动法”以及YC/T 468-2013“烟草及烟草制品总植物碱的测定连续流动(硫氰酸钾)法”。
连续流动法属于仪器分析方法，适合大批量进样，目前应用较为广泛。YC/T160-2002“烟草及烟草制品总植物碱的测定连续流动法”的测定原理是采用氰化钾和氯胺T在线生成氯化氰，使烟碱的吡啶环断开，进而与对氨基苯磺酸发生反应，反应产物在460nm波长处用比色计进行测定。氰化钾虽然较为稳定，但它是一种剧毒的物质，位于有毒化学品前列。对于氰化钾的使用，存在诸多顾忌，其销售渠道把关很严，很多实验室购买周期很长甚至无法买到，使用者还需要在公安机关备案等等。由于该方法对反应适宜的酸碱环境有要求，因此该方法是通过缓冲溶液A和缓冲溶液B来控制反应的酸碱环境。在该方法中由于缓冲溶液B的缓冲能力不强，如一些化学分析实验室用水的pH在5.0～6.0之间，该缓冲溶液B的缓冲能力达不到测试需要。该方法实际使用水的pH要求要高于GB6682/T-2008“分析实验室用水规格和试验方法”中一级水的要求，为了达到该方法的用水要求，化学分析实验室需要对一级水进行再处理，使该方法实际使用具有一定的局限性。
YC/T 468-2013“烟草及烟草制品总植物碱的测定连续流动(硫氰酸钾)法”的测定原理是采用硫氰酸钾和二氯异氰尿酸钠在线生成氯化氰，使烟碱的吡啶环断开，进而与对氨基苯磺酸发生反应，反应产物在460nm波长处用比色计进行测定。相较于YC/T160-2002，YC/T 468-2013不使用氰化钾，提高了总植物碱测定的操作安全性。在YC/T 468-2013中对反应适宜的酸碱环境也有要求，同样是通过缓冲溶液A和缓冲溶液B来控制反应的酸碱环境，虽然能够有效控制反应的酸碱环境，但是该方法缓冲溶液A和缓冲溶液B配制时化学品用量较大，同样对该方法的使用带来不便。
发明内容
为了克服现有技术的缺陷，本发明提供一种新的缓冲体系以及采用该缓冲体系以连续流动法测定烟草或烟草制品中总植物碱的方法。该缓冲体系适用于采用连续流动(硫氰酸钾)法测定烟草及烟草制品中的总植物碱。采用本发明的缓冲体系在减少了配制时化学品的使用量的同时能控制反应的酸碱环境，保证氯化氰使植物碱的吡啶环断裂，进而与对氨基苯磺酸的反应，使反应在需要的酸碱环境条件下进行，采用该缓冲体系在减少使用量的同时保证了同YC/T 468-2013方法测试结果的一致性。
本发明是采用如下技术方案来实现的。
一方面，本发明提供一种适用于连续流动法测定烟草及烟草制品中总植物碱的缓冲体系，该缓冲体系包含缓冲溶液A和缓冲溶液B；
其中，缓冲溶液A的配制为：称取磷酸氢二钠60.5-83.0g，优选为71.6g；磷酸三钠10.1-20.2g，优选为15.2g至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度；
缓冲溶液B的配制为：称取对氨基苯磺酸5.5-8.0g，优选为7.0g；磷酸氢二钠60.5-83.0g，优选为71.6g；磷酸二氢钠5.0-7.0g，优选为6.2g和柠檬酸钠10.0-12.1g，优选为11.8g至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。
上述的磷酸氢二钠、磷酸三钠、磷酸二氢钠、柠檬酸钠可以为常规存在形式，例如磷酸氢二钠以12个结晶水形式存在，磷酸三钠以12个结晶水形 式存在，磷酸二氢钠以2个结晶水形式存在，柠檬酸钠以2个结晶水形式存在。
优选地，在上述缓冲体系中，缓冲溶液A与缓冲溶液B之间的用量比为1：2至1：1(体积比)，优选为3：4(体积比)。
另一方面，一种用连续流动法测定烟草或烟草制品中总植物碱的方法，该方法采用硫氰酸钾和二氯异氰尿酸钠在线生成氯化氰，氯化氰与所述烟草或烟草制品中的总植物碱(以烟碱计)反应，使烟碱的吡啶环断开，进而与对氨基苯磺酸发生反应，反应产物用比色计在460nm处测定，该方法包括：
采用缓冲溶液A和缓冲溶液B将反应体系的pH值控制在6.0～7.5之间；
其中，缓冲溶液A的配制为：称取磷酸氢二钠60.5-83.0g，优选为71.6g；磷酸三钠10.1-20.2g，优选为15.2g至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度；
缓冲溶液B的配制为：称取对氨基苯磺酸5.5-8.0g，优选为7.0g；磷酸氢二钠60.5-83.0g，优选为71.6g；磷酸二氢钠5.0-7.0g，优选为6.2g和柠檬酸钠10.0-12.1g，优选为11.8g至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。
上述的磷酸氢二钠、磷酸三钠、磷酸二氢钠、柠檬酸钠可以为常规存在形式，例如磷酸氢二钠以12个结晶水形式存在，磷酸三钠以12个结晶水形式存在，磷酸二氢钠以2个结晶水形式存在，柠檬酸钠以2个结晶水形式存在。
优选地，上述方法中，缓冲溶液A与缓冲溶液B的用量比为1：2至1：1(体积比)，优选为3：4(体积比)；
优选地，上述方法还包括：
样品的制备为：按YC/T31制备烟草试样，并测定其水分含量，然后使用5％醋酸对烟草试样进行萃取，过滤，得滤液。
优选地，上述方法还包括：
标准溶液的配制为：
称取烟碱或烟碱盐以5％的醋酸溶解，配制成至少5个工作标准液，所配制的标准溶液中烟碱含量浓度范围为0.5～7％；
优选地，上述方法还包括：
解毒液A的配制为：称取1g柠檬酸、10g硫酸亚铁至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度；该解毒液A的主要作用是降低未参与反应的氯化氰的毒性，该解毒液A与YC/T160-2002(氰化钾法)中的解毒液A相同。
解毒液B的配制为：称取10g碳酸钠至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度；该解毒液B的主要作用是降低未参与反应的氯化氰的毒性，该解毒液B与YC/T160-2002(氰化钾法)解毒液B相同。
硫氰酸钾溶液的配制为：称取硫氰酸钾至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容，该溶液浓度范围为0.09～0.21mol/L，优选为0.12mol/L。
二氯异氰尿酸钠溶液的配制为：称取二氯异氰尿酸钠至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容，该溶液浓度范围为0.02～0.06mol/L，优选为0.04mol/L。
优选地，上述方法中，硫氰酸钾溶液和二氯异氰尿酸钠溶液浓度比为2.5：1至3.5：1，优选为3：1。
优选地，上述方法还包括：
使用连续流动分析仪进行检测，然后根据下式计算样品中的总植物碱：
Figure PCTCN2015076250-appb-000001
式中，X：样品的仪器观测值，单位毫克每毫升(mg/mL)；
V：加入的萃取液体积，单位为毫升(mL)；
m：样品质量，单位为克(g)；
W：试样的水分百分含量，％(质量分数)；
上式计算的总植物碱为以干基计的总植物碱的含量。
优选地，上述方法中，连续流动分析仪的运行参数为：进样清洗比为1.5，进样频率48杯/小时，增益18-60，光强2.85伏。
优选地，上述方法包括：
(1)样品的制备：按YC/T31制备烟草试样，并测定其水分含量，然后使用5％醋酸对烟草试样进行萃取，过滤，得滤液备用；(2)标准溶液的配制：
称取烟碱或烟碱盐以5％的醋酸溶解，配制成至少5个工作标准液，所配制的标准溶液中烟碱含量浓度范围为0.5～7％；
(3)反应试剂的配制：
硫氰酸钾溶液的配制为：称取硫氰酸钾至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容，该溶液浓度范围为0.09～0.21mol/L，优选为0.12mol/L。
二氯异氰尿酸钠溶液的配制为：称取二氯异氰尿酸钠至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容，该溶液浓度范围为0.02～0.06mol/L，优选为0.04mol/L。
缓冲溶液A的配制：称取磷酸氢二钠60.5-83.0g，优选为71.6g、磷酸三钠10.1-20.2g，优选为15.2g至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度；
缓冲溶液B的配制：称取对氨基苯磺酸5.5-8.0g，优选为7.0g；磷酸氢二钠60.5-83.0g，优选为71.6g；磷酸二氢钠5.0-7.0g，优选为6.2g和柠檬酸钠10.0-12.1g，优选为11.8g至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度；
上述的磷酸氢二钠、磷酸三钠、磷酸二氢钠、柠檬酸钠可以为常规存在形式，例如磷酸氢二钠以12个结晶水形式存在，磷酸三钠以12个结晶水形式存在，磷酸二氢钠以2个结晶水形式存在，柠檬酸钠以2个结晶水形式存在。
解毒液A的配制为：称取1g柠檬酸、10g硫酸亚铁至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度；该解毒液A的主要作用是降低未参与反应的氯化氰的毒性，该解毒液A与YC/T160-2002(氰化钾法)中的解毒液A相同。
解毒液B的配制为：称取10g碳酸钠至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度；该解毒液B的主要作用是降低未参与反应的氯化氰的毒性，该解毒液B与YC/T160-2002(氰化钾法)解毒液B相同。
(4)使用连续流动分析仪在步骤(3)的反应体系下检测步骤(1)的滤液和步骤(2)的工作标准液；
(5)根据下式计算样品中的总植物碱：
Figure PCTCN2015076250-appb-000002
式中，X：样品的仪器观测值，单位毫克每毫升(mg/mL)；
V：加入的萃取液体积，单位为毫升(mL)；
m：样品质量，单位为克(g)；
W：试样的水分百分含量，％(质量分数)；
优选地，上述方法中，缓冲溶液A与缓冲溶液B的用量比为1：2至1：1(体积比)，优选为3：4(体积比)，该用量比使得反应体系的pH值控制在6.0～7.5之间；
优选地，上述方法中，硫氰酸钾溶液和二氯异氰尿酸钠溶液浓度比为2.5：1至3.5：1，优选为3：1。
优选地，上述方法中，连续流动分析仪的运行参数为：进样清洗比为1.5，进样频率48杯/小时，增益18-60，光强2.85伏。
在上述方法中，样品的制备、标准溶液的配制、反应试剂的配制顺序可以变化。
在一个具体实施方案中，本发明的方法具体步骤如下：
(1)使用5％醋酸萃取烟末试样(烟末试样按YC/T 31来制备)中的总植物碱；
(2)按照图1所示连接管路：
(3)配置一定浓度的缓冲溶液A和B、解毒液A、解毒液B、硫氰酸钾溶液和二氯异氰尿酸钠溶液；
(4)使用5％醋酸配制总植物碱标准溶液；
(5)将总植物碱模块上的泵管按照图1所示卡入蠕动泵(模块上不同泵管的内径不同，对应的试剂的流速不同，通过控制反应试剂的流速来达到控制参与反应试剂比例的目的；加入空气泵管是为了使管路中的溶液混合均匀)，将泵盖盖上，将试剂管放入水中；打开电源，打开检测器，蠕动泵，自动进样器，计算机；将蠕动泵上的开关拨至Run；双击计算机中连接连续流动分析工作站图标；点击工作站上的charting，连接成功后，蠕动泵运行，水就被泵入管路中清洗，清洗时间为20～30min；待基线稳定(此时基线波动小于±1％)后，将不同的管路放入所对应的试剂中，继续运行，期间在工作站所弹出的charting窗口中调节基线至5％左右；按照工作站的下拉菜单中Analysis的提示编制运行程序，待基线稳定，点击工作站中的Run即 可运行；待程序运行结束，将不同的管路重新放回水中，清洗20～30min；清洗完成后，依次关闭自动进样器，蠕动泵，检测器，将泵盖取下，关闭电源。
上述步骤(1)具体为：称取烟末试样(烟末试样按YC/T 31来制备)至50mL具塞三角瓶中，加入5％醋酸振荡30min，使用定性滤纸过滤。
上述步骤(2)具体为：按照图1所示，将泵管安装在蠕动泵上，加上泵盖，用水清洗管路。
上述步骤(3)中，缓冲溶液A的配制为：称取71.6克的磷酸氢二钠、15.2克的磷酸三钠至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。其中，磷酸氢二钠、磷酸三钠可以为常规存在形式，例如磷酸氢二钠以12个结晶水形式存在，磷酸三钠以12个结晶水形式存在。
上述步骤(3)中，缓冲溶液B的配制为：称取7g对氨基苯磺酸、71.6克磷酸氢二钠、6.2克磷酸二氢钠和11.8克柠檬酸钠至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。其中，磷酸氢二钠、磷酸二氢钠、柠檬酸钠可以为常规存在形式，例如磷酸氢二钠以12个结晶水形式存在，磷酸二氢钠以2个结晶水形式存在，柠檬酸钠以2个结晶水形式存在。
上述步骤(3)中，解毒液A的配制为：称取1g柠檬酸、10g硫酸亚铁至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。解毒液A的主要作用是降低未参与反应氯化氰的毒性。步骤(3)中解毒液A与YC/T160-2002(氰化钾法)解毒液A相同。
上述步骤(3)中解毒液B的配制为：称取10g碳酸钠至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。解毒液B的主要作用是降低未参与反应氯化氰的毒性。步骤(3)中解毒液B与YC/T160-2002(氰化钾法)解毒液B相同。
上述步骤(3)中，硫氰酸钾溶液的配制为：称取适量硫氰酸钾至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容。该溶液浓度范围为0.09～0.21mol/L。
上述步骤(3)中，二氯异氰尿酸钠溶液的配制为：称取适量二氯异氰尿酸钠至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容。该溶液浓度范围为0.02～0.06mol/L。
上述步骤(3)中，硫氰酸钾溶液和二氯异氰尿酸钠溶液浓度配比约为3： 1。
上述步骤(4)中，标准溶液配制为：称取适量烟碱于250mL容量瓶中，用5％醋酸溶解，定容至刻度。此溶液为烟碱储备液，烟碱含量约1.6mg/mL左右。由烟碱储备液制备至少5个工作标准液，其浓度范围应覆盖预计检测到的样品含量。
上述步骤(5)中，连续流动分析仪的运行参数为：进样清洗比为1.5，进样频率48杯/小时，增益18-60，光强2.85伏。
上述连续流动法测定烟草及烟草制品中总植物碱中，样品前处理与YC/T 468-2013“烟草及烟草制品总植物碱的测定连续流动(硫氰酸钾)法”一致，改变了缓冲溶液A和缓冲溶液B，建立了一种新的缓冲体系，减少配制缓冲溶液A和缓冲溶液B时化学品的使用量，同时保证了同YC/T468-2013方法测试结果的一致性。
本发明至少存在以下有益效果：
本发明采用一种新的缓冲溶液A能控制渗析器前反应的酸碱环境，保证渗析器前溶液pH值在6.0～11.0之间并不会对缓冲溶液B控制的酸碱环境造成影响；
本发明采用一种新的缓冲溶液B能控制反应的酸碱环境，调节溶液pH值在6.0～7.5之间，保证氯化氰使植物碱的吡啶环断裂，进而与对氨基苯磺酸的反应，在反应需要的酸碱环境条件下进行；
本发明建立了一种新的适用于连续流动(硫氰酸钾)法测定烟草及烟草制品中总植物碱的缓冲体系；
采用本发明的缓冲体系的连续流动法与YC/T 468-2013相比，减少了配制缓冲溶液A和缓冲溶液B时化学品的使用量，同时保证数据与YC/T468-2013一致。
附图说明
图1为采用本发明的缓冲体系的连续流动法测定烟草及烟草制品中总植物碱的管路图。
具体实施方式
下面结合实施例对本发明做进一步的说明。这些实施例旨在帮助阐述发 明的内容而不是限制本发明的范围。
实施例1：烤烟中总植物碱的测定
1)样品前处理
将烤烟烟末按照YC/T31制备成试样，并测定其水分含量，然后称取0.25g试样于50mL具塞三角瓶中，加入25mL5％醋酸，盖上塞子，在振荡器上振荡(转速>150rpm)萃取30min。用快速定性滤纸过滤萃取液，弃去前2mL～3mL滤液，收集后续滤液作分析之用。
2)反应试剂的配制
缓冲溶液A的配制为：称取磷酸氢二钠(12个结晶水)60.5克、磷酸三钠(12个结晶水)12.2克至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。
缓冲溶液B的配制为：称取5.5g对氨基苯磺酸、磷酸氢二钠(12个结晶水)60.5克、磷酸二氢钠(2个结晶水)5.0克和柠檬酸钠(2个结晶水)10.0克至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。
在以下测定时，缓冲溶液A和缓冲溶液B的用量比为3：4(体积比)。
解毒液A、解毒液B、硫氰酸钾溶液和二氯异氰尿酸钠溶液的配制与YC/T 468-2013“烟草及烟草制品总植物碱的测定连续流动(硫氰酸钾)法”中对应试剂相同。
具体如下：
解毒溶液A的配制为：
称取1g柠檬酸、10g硫酸亚铁至烧杯中，用水溶解，然后转入1000mL容量瓶中，用水定容至刻度。
解毒溶液B的配制为：
称取10g碳酸钠至烧杯中，用水溶解，然后转入1000mL容量瓶中，用水定容至刻度。
硫氰酸钾溶液的配制为：
称取2.88g硫氰酸钾至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容至刻度。
二氯异氰尿酸钠溶液的配制为：
称取2.20g二氯异氰尿酸钠至烧杯中，用水溶解，然后转入250mL容 量瓶中，用水定容至刻度。该溶液应现配现用。
3)标准溶液的配制
称取适量总植物碱标液(国家烟草质量监督检验中心，有证标物)于250mL容量瓶中，精确至0.0001g，用水溶解，定容至刻度，此溶液为烟碱标准储备液，烟碱含量应在1.6mg/mL左右。分别移取12.5mL、10mL、5mL、2.5mL、0.5mL烟碱标准储备液至100mL容量瓶中，用5％的醋酸定容，得到工作标液的浓度分别为1.2105mg/mL、0.9683mg/mL、0.4842mg/mL、0.2421mg/mL、0.0484mg/mL。
4)本发明中所使用的分析仪器为德国布朗卢比公司生产的AA3型连续流动分析仪。采用连续流动法测定烟草及烟草制品中总植物碱的管路如图1所示。将总植物碱模块上的泵管按照图1所示卡入蠕动泵(模块上不同泵管的内径不同，对应的试剂的流速不同，通过控制反应试剂的流速来达到控制参与反应试剂比例的目的；加入空气泵管是为了使管路中的溶液混合均匀)，将泵盖盖上，将试剂管放入水中；打开电源，打开检测器，蠕动泵，自动进样器，计算机；将蠕动泵上的开关拨至Run，双击连接连续流动分析工作站；点击工作站上的charting，连接成功后，蠕动泵运行，水就被泵入管路中清洗，清洗时间为20～30min；待基线稳定(此时基线波动小于±1％)后，将不同的管路放入所对应的试剂中，继续运行，期间在工作站所弹出的charting窗口中调节基线至5％左右；按照工作站的下拉菜单中Analysis的提示编制运行程序，待基线稳定，点击工作站中的Run即可运行；待程序运行结束，将不同的管路重新放回水中，清洗20～30min；清洗完成后，依次关闭自动进样器，蠕动泵，检测器，将泵盖取下，关闭电源。
5)标准曲线采用一次曲线；
6)数据计算
总植物碱峰的峰高对其相应浓度进行回归分析，得到标准曲线。然后通过检测样品的峰高，由标准曲线得到样品对应的浓度，即仪器观测值。
以干基计的总植物碱含量，由下式得出：
Figure PCTCN2015076250-appb-000003
式中，X：样品的仪器观测值，单位毫克每毫升(mg/mL)；
V：加入的萃取液体积，单位为毫升(此处萃取液体积为 25mL)；
m：样品质量，单位为克(g)；
W：试样的水分百分含量，％(质量分数)；
由以上公式算出烤烟中总植物碱含量为0.5～3％。
实施例2：白肋烟中总植物碱的测定
1)样品前处理
将白肋烟烟末按照YC/T31制备成试样，并测定其水分含量，然后称取0.25g白肋烟试样于50mL具塞三角瓶中，加入25mL5％醋酸，盖上塞子，在振荡器上振荡(转速>150rpm)萃取30min。用快速定性滤纸过滤萃取液，弃去前2mL～3mL滤液，收集后续滤液作分析之用。
2)反应试剂的配制
缓冲溶液A的配制为：称取磷酸氢二钠(12个结晶水)83.0克、磷酸三钠(12个结晶水)18.4克至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。
缓冲溶液B的配制为：称取8.0g对氨基苯磺酸、磷酸氢二钠(12个结晶水)83.0克、磷酸二氢钠(2个结晶水)7.0克和柠檬酸钠(2个结晶水)12.1克至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。
在以下测定时，缓冲溶液A和缓冲溶液B的用量比为请补充3：4(体积比)。
解毒液A、解毒液B、硫氰酸钾溶液和二氯异氰尿酸钠溶液的配制与YC/T 468-2013“烟草及烟草制品总植物碱的测定连续流动(硫氰酸钾)法”中对应试剂相同。
具体如下：
解毒溶液A的配制为：
称取1g柠檬酸、10g硫酸亚铁至烧杯中，用水溶解，然后转入1000mL容量瓶中，用水定容至刻度。
解毒溶液B的配制为：
称取10g碳酸钠至烧杯中，用水溶解，然后转入1000mL容量瓶中，用水定容至刻度。
硫氰酸钾溶液的配制为：
称取2.88g硫氰酸钾至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容至刻度。
二氯异氰尿酸钠溶液的配制为：
称取2.20g二氯异氰尿酸钠至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容至刻度。该溶液应现配现用。
3)标准溶液的配制
称取适量总植物碱标液(国家烟草质量监督检验中心，有证标物)于250mL容量瓶中，精确至0.0001g，用水溶解，定容至刻度，此溶液为烟碱标准储备液，烟碱含量应在1.6mg/mL左右。分别移取12.5mL、10mL、5mL、2.5mL、0.5mL烟碱标准储备液至100mL容量瓶中，用5％的醋酸定容，得到工作标液的浓度分别为1.2105mg/mL、0.9683mg/mL、0.4842mg/mL、0.2421mg/mL、0.0484mg/mL。
4)本发明中所使用的分析仪器为德国布朗卢比公司生产的AA3型连续流动分析仪。采用连续流动法测定烟草及烟草制品中总植物碱的管路如图1所示。将总植物碱模块上的泵管按照图1所示卡入蠕动泵(模块上不同泵管的内径不同，对应的试剂的流速不同，通过控制反应试剂的流速来达到控制参与反应试剂比例的目的；加入空气泵管是为了使管路中的溶液混合均匀)，将泵盖盖上，将试剂管放入水中；打开电源，打开检测器，蠕动泵，自动进样器，计算机；将蠕动泵上的开关拨至Run，双击连接连续流动分析工作站；点击工作站上的charting，连接成功后，蠕动泵运行，水就被泵入管路中清洗，清洗时间为20～30min；待基线稳定(此时基线波动小于±1％)后，将不同的管路放入所对应的试剂中，继续运行，期间在工作站所弹出的charting窗口中调节基线至5％左右；按照工作站的下拉菜单中Analysis的提示编制运行程序，待基线稳定，点击工作站中的Run即可运行；待程序运行结束，将不同的管路重新放回水中，清洗20～30min；清洗完成后，依次关闭自动进样器，蠕动泵，检测器，将泵盖取下，关闭电源。
5)标准曲线采用一次曲线；
6)数据计算
总植物碱峰的峰高对其相应浓度进行回归分析，得到标准曲线。然后通过检测样品的峰高，由标准曲线得到样品对应的浓度，即仪器观测值。
以干基计的总植物碱含量，由下式得出：
Figure PCTCN2015076250-appb-000004
式中，X：样品的仪器观测值，单位毫克每毫升(mg/mL)；
V：加入的萃取液体积，单位为毫升(此处萃取液体积为25mL)；
m：样品质量，单位为克(g)；
W：试样的水分百分含量，％(质量分数)；
由以上公式算出白肋烟中总植物碱含量为1～6％。
实施例3：烤烟中总植物碱的测定
1)样品前处理
将烤烟烟末按照YC/T31制备成试样，并测定其水分含量，然后称取0.25g试样于50mL具塞三角瓶中，加入25mL5％醋酸，盖上塞子，在振荡器上振荡(转速>150rpm)萃取30min。用快速定性滤纸过滤萃取液，弃去前2mL～3mL滤液，收集后续滤液作分析之用。
2)反应试剂的配制
缓冲溶液A的配制为：称取磷酸氢二钠(12个结晶水)71.6克、磷酸三钠(12个结晶水)15.2克至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。
缓冲溶液B的配制为：称取7g对氨基苯磺酸、磷酸氢二钠(12个结晶水)71.6克、磷酸二氢钠(2个结晶水)6.2克和柠檬酸钠(2个结晶水)11.8克至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容用水定容至刻度。
在以下测定时，缓冲溶液A和缓冲溶液B的用量比为3：4(体积比)。
解毒液A、解毒液B、硫氰酸钾溶液和二氯异氰尿酸钠溶液的配制与YC/T 468-2013“烟草及烟草制品总植物碱的测定连续流动(硫氰酸钾)法”中对应试剂相同。
具体如下：
解毒溶液A的配制为：
称取1g柠檬酸、10g硫酸亚铁至烧杯中，用水溶解，然后转入1000mL容量瓶中，用水定容至刻度。
解毒溶液B的配制为：
称取10g碳酸钠至烧杯中，用水溶解，然后转入1000mL容量瓶中，用水定容至刻度。
硫氰酸钾溶液的配制为：
称取2.88g硫氰酸钾至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容至刻度。
二氯异氰尿酸钠溶液的配制为：
称取2.20g二氯异氰尿酸钠至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容至刻度。该溶液应现配现用。
3)标准溶液的配制
称取适量总植物碱标液(国家烟草质量监督检验中心，有证标物)于250mL容量瓶中，精确至0.0001g，用水溶解，定容至刻度，此溶液为烟碱标准储备液，烟碱含量应在1.6mg/mL左右。分别移取12.5mL、10mL、5mL、2.5mL、0.5mL烟碱标准储备液至100mL容量瓶中，用5％的醋酸定容，得到工作标液的浓度分别为1.2105mg/mL、0.9683mg/mL、0.4842mg/mL、0.2421mg/mL、0.0484mg/mL。
4)本发明中所使用的分析仪器为德国布朗卢比公司生产的AA3型连续流动分析仪。采用连续流动法测定烟草及烟草制品中总植物碱的管路如图1所示。将总植物碱模块上的泵管按照图1所示卡入蠕动泵(模块上不同泵管的内径不同，对应的试剂的流速不同，通过控制反应试剂的流速来达到控制参与反应试剂比例的目的；加入空气泵管是为了使管路中的溶液混合均匀)，将泵盖盖上，将试剂管放入水中；打开电源，打开检测器，蠕动泵，自动进样器，计算机；将蠕动泵上的开关拨至Run，双击连接连续流动分析工作站；点击工作站上的charting，连接成功后，蠕动泵运行，水就被泵入管路中清洗，清洗时间为20～30min；待基线稳定(此时基线波动小于±1％)后，将不同的管路放入所对应的试剂中，继续运行，期间在工作站所弹出的charting窗口中调节基线至5％左右；按照工作站的下拉菜单中Analysis的提示编制运行程序，待基线稳定，点击工作站中的Run即可运行；待程序运行结束，将不同的管路重新放回水中，清洗20～30min；清洗完成后，依次关闭自动进样器，蠕动泵，检测器，将泵盖取下，关闭电源。
5)标准曲线采用一次曲线；
6)数据计算
总植物碱峰的峰高对其相应浓度进行回归分析，得到标准曲线。然后通过检测样品的峰高，由标准曲线得到样品对应的浓度，即仪器观测值。
以干基计的总植物碱含量，由下式得出：
Figure PCTCN2015076250-appb-000005
式中，X：样品的仪器观测值，单位毫克每毫升(mg/mL)；
V：加入的萃取液体积，单位为毫升(此处萃取液体积为25mL)；
m：样品质量，单位为克(g)；
W：试样的水分百分含量，％(质量分数)；
由以上公式算出烤烟中总植物碱含量为0.5～3％。
实施例4：白肋烟中总植物碱的测定
1)样品前处理
将白肋烟烟末按照YC/T31制备成试样，并测定其水分含量，然后称取0.25g白肋烟试样于50mL具塞三角瓶中，加入25mL5％醋酸，盖上塞子，在振荡器上振荡(转速>150rpm)萃取30min。用快速定性滤纸过滤萃取液，弃去前2mL～3mL滤液，收集后续滤液作分析之用。
2)反应试剂的配制
缓冲溶液A的配制为：称取磷酸氢二钠(12个结晶水)71.6克、磷酸三钠(12个结晶水)15.2克至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。
缓冲溶液B的配制为：称取7g对氨基苯磺酸、磷酸氢二钠(12个结晶水)71.6克、磷酸二氢钠(2个结晶水)6.2克和柠檬酸钠(2个结晶水)11.8克至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容用水定容至刻度。
在以下测定时，缓冲溶液A和缓冲溶液B的用量比为请补充3：4(体积比)。
解毒液A、解毒液B、硫氰酸钾溶液和二氯异氰尿酸钠溶液的配制与YC/T 468-2013“烟草及烟草制品总植物碱的测定连续流动(硫氰酸钾)法”中对应试剂相同。
具体如下：
解毒溶液A的配制为：
称取1g柠檬酸、10g硫酸亚铁至烧杯中，用水溶解，然后转入1000mL容量瓶中，用水定容至刻度。
解毒溶液B的配制为：
称取10g碳酸钠至烧杯中，用水溶解，然后转入1000mL容量瓶中，用水定容至刻度。
硫氰酸钾溶液的配制为：
称取2.88g硫氰酸钾至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容至刻度。
二氯异氰尿酸钠溶液的配制为：
称取2.20g二氯异氰尿酸钠至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容至刻度。该溶液应现配现用。
3)标准溶液的配制
称取适量总植物碱标液(国家烟草质量监督检验中心，有证标物)于250mL容量瓶中，精确至0.0001g，用水溶解，定容至刻度，此溶液为烟碱标准储备液，烟碱含量应在1.6mg/mL左右。分别移取12.5mL、10mL、5mL、2.5mL、0.5mL烟碱标准储备液至100mL容量瓶中，用5％的醋酸定容，得到工作标液的浓度分别为1.2105mg/mL、0.9683mg/mL、0.4842mg/mL、0.2421mg/mL、0.0484mg/mL。
4)本发明中所使用的分析仪器为德国布朗卢比公司生产的AA3型连续流动分析仪。采用连续流动法测定烟草及烟草制品中总植物碱的管路如图1所示。将总植物碱模块上的泵管按照图1所示卡入蠕动泵(模块上不同泵管的内径不同，对应的试剂的流速不同，通过控制反应试剂的流速来达到控制参与反应试剂比例的目的；加入空气泵管是为了使管路中的溶液混合均匀)，将泵盖盖上，将试剂管放入水中；打开电源，打开检测器，蠕动泵，自动进样器，计算机；将蠕动泵上的开关拨至Run，双击连接连续流动分析工作站；点击工作站上的charting，连接成功后，蠕动泵运行，水就被泵入管路中清洗，清洗时间为20～30min；待基线稳定(此时基线波动小于±1％)后，将不同的管路放入所对应的试剂中，继续运行，期间在工作站所弹出的charting窗口中调节基线至5％左右；按照工作站的下拉菜单中Analysis的提示编制运行程序，待基线稳定，点击工作站中的Run即可运行；待程序运行结束， 将不同的管路重新放回水中，清洗20～30min；清洗完成后，依次关闭自动进样器，蠕动泵，检测器，将泵盖取下，关闭电源。
5)标准曲线采用一次曲线；
6)数据计算
总植物碱峰的峰高对其相应浓度进行回归分析，得到标准曲线。然后通过检测样品的峰高，由标准曲线得到样品对应的浓度，即仪器观测值。
以干基计的总植物碱含量，由下式得出：
Figure PCTCN2015076250-appb-000006
式中，X：样品的仪器观测值，单位毫克每毫升(mg/mL)；
V：加入的萃取液体积，单位为毫升(此处萃取液体积为25mL)；
m：样品质量，单位为克(g)；
W：试样的水分百分含量，％(质量分数)；
由以上公式算出白肋烟中总植物碱含量为1～5％。
实施例5：卷烟中总植物碱的测定
1)样品前处理
将卷烟烟丝按照YC/T31制备成试样，并测定其水分含量，然后称取0.25g卷烟试样于50mL具塞三角瓶中，加入25mL5％醋酸，盖上塞子,在振荡器上振荡(转速>150rpm)萃取30min。用快速定性滤纸过滤萃取液，弃去前2mL～3mL滤液，收集后续滤液作分析之用。
2)反应试剂的配制
缓冲溶液A的配制为：称取磷酸氢二钠(12个结晶水)71.6克、磷酸三钠(12个结晶水)15.2克至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。
缓冲溶液B的配制为：称取7g对氨基苯磺酸、磷酸氢二钠(12个结晶水)71.6克、磷酸二氢钠(2个结晶水)6.2克和柠檬酸钠(2个结晶水)11.8克至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。
在以下测定时，缓冲溶液A和缓冲溶液B的用量比为3：4(体积比)
解毒液A、解毒液B、硫氰酸钾溶液和二氯异氰尿酸钠溶液的配制与 YC/T 468-2013“烟草及烟草制品总植物碱的测定连续流动(硫氰酸钾)法”中对应试剂相同。
具体如下：
解毒溶液A的配制为：
称取1g柠檬酸、10g硫酸亚铁至烧杯中，用水溶解，然后转入1000mL容量瓶中，用水定容至刻度。
解毒溶液B的配制为：
称取10g碳酸钠至烧杯中，用水溶解，然后转入1000mL容量瓶中，用水定容至刻度。
硫氰酸钾溶液的配制为：
称取2.88g硫氰酸钾至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容至刻度。
二氯异氰尿酸钠溶液的配制为：
称取2.20g二氯异氰尿酸钠至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容至刻度。该溶液应现配现用。
3)标准溶液的配制
称取适量总植物碱标液(国家烟草质量监督检验中心，有证标物)于250mL容量瓶中，精确至0.0001g，用水溶解，定容至刻度，此溶液为烟碱标准储备液，烟碱含量应在1.6mg/mL左右。分别移取12.5mL、10mL、5mL、2.5mL、0.5mL烟碱标准储备液至100mL容量瓶中，用5％的醋酸定容，得到工作标液的浓度分别为1.2105mg/mL、0.9683mg/mL、0.4842mg/mL、0.2421mg/mL、0.0484mg/mL。
4)本发明中所使用的分析仪器为德国布朗卢比公司生产的AA3型连续流动分析仪。采用连续流动法测定烟草及烟草制品中总植物碱的管路如图1所示。将总植物碱模块上的泵管按照图1所示卡入蠕动泵(模块上不同泵管的内径不同，对应的试剂的流速不同，通过控制反应试剂的流速来达到控制参与反应试剂比例的目的；加入空气泵管是为了使管路中的溶液混合均匀)，将泵盖盖上，将试剂管放入水中；打开电源，打开检测器，蠕动泵，自动进样器，计算机；将蠕动泵上的开关拨至Run，双击连接连续流动分析工作站；点击工作站上的charting，连接成功后，蠕动泵运行，水就被泵入管路中清洗，清洗时间为20～30min；待基线稳定(此时基线波动小于±1％)后，将 不同的管路放入所对应的试剂中，继续运行，期间在工作站所弹出的charting窗口中调节基线至5％左右；按照工作站的下拉菜单中Analysis的提示编制运行程序，待基线稳定，点击工作站中的Run即可运行；待程序运行结束，将不同的管路重新放回水中，清洗20～30min；清洗完成后，依次关闭自动进样器，蠕动泵，检测器，将泵盖取下，关闭电源。
5)标准曲线采用一次曲线；
6)数据计算
总植物碱峰的峰高对其相应浓度进行回归分析，得到标准曲线。然后通过检测样品的峰高，由标准曲线得到样品对应的浓度，即仪器观测值。
以干基计的总植物碱含量，由下式得出：
Figure PCTCN2015076250-appb-000007
式中，X：样品的仪器观测值，单位毫克每毫升(mg/mL)；
V：加入的萃取液体积，单位为毫升(此处萃取液体积为25mL)；
m：样品质量，单位为克(g)；
W：试样的水分百分含量，％(质量分数)；
由以上公式算出卷烟中总植物碱含量为0.5～4％。
实施例6：采用本发明缓冲体系的连续流动法与采用其他缓冲体系的连续流动法检测结果的对比研究
如无特殊说明，本实施例使用的试剂及方法均与YC/T 468-2013相一致。
分别采用本发明实施例1、实施例2和实施例3-5使用的缓冲体系对烤烟，白肋烟，香料烟、烤烟型卷烟和混合型卷烟五种样品进行测定，其测定结果见表1～表3。从表1～表3可知：同一样品采用本发明(实施例1、实施例2和实施例3-5使用)缓冲体系的连续流动法测定结果和YC/T 468-2013方法的测定结果进行配对t检验(α＝0.05)，结果，采用本发明缓冲体系的连续流动法结果为P实施例1＝0.85>0.05、P实施例2＝0.92>0.05、P实施例 3-5＝0.35>0.05，说明两种分析方法没有显著性差异，表明采用本发明缓冲体系的连续流动法与YC/T 468-2013标准方法结果一致；在本发明实施例1、实施例2和实施例3-5使用的缓冲体系中，实施例3-5使用的缓冲体系与 YC/T 468-2013标准方法结果差值最小，为优选的条件。
表1采用本发明缓冲体系(实施例1中使用)的连续流动法与YC/T 468-2013方法测定结果比对
Figure PCTCN2015076250-appb-000008
表2采用本发明缓冲体系(实施例2中使用)的连续流动法与YC/T 468-2013方法测定结果比对
Figure PCTCN2015076250-appb-000009
表3采用本发明缓冲体系(实施例3-5中使用)的连续流动法与YC/T 468-2013方法测定结果比对
Figure PCTCN2015076250-appb-000010
Figure PCTCN2015076250-appb-000011
此外，发明人还采用其他缓冲体系(除缓冲体系与本发明不同之外，其余试剂与本发明的实施例3～5相同)对烤烟，白肋烟，香料烟、烤烟型卷烟和混合型卷烟五种样品进行测定，其测定结果见表4～表9。从表4～表9可知：采用其他缓冲体系测定结果相对于YC/T 468-2013方法的绝对差值均较大。在表10中列出了对同一样品分别采用本发明缓冲体系(本发明的实施例3-5中使用的缓冲溶液A和缓冲溶液B，缓冲溶液A和缓冲溶液B的用量比与实施例3-5相同)的连续流动法、其他缓冲体系的连续流动法和YC/T 468-2013方法的测定结果进行配对t检验(α＝0.05)，结果，采用本发明缓冲体系的连续流动法结果为P实施例3-5＝0.35>0.05，说明两种分析方法没有显著性差异，表明采用本发明缓冲体系的连续流动法与YC/T 468-2013标准方法结果一致；采用其他缓冲体系的检测方法P<0.05，说明这些分析方法存在显著性差异，表明采用其他缓冲体系的方法与YC/T 468-2013标准方法测试结果不一致。
表4磷酸氢二钠、硼砂体系与YC/T 468-2013方法测定结果比对
Figure PCTCN2015076250-appb-000012
Figure PCTCN2015076250-appb-000013
表5磷酸二氢钠、氢氧化钠体系与YC/T 468-2013方法测定结果比对
Figure PCTCN2015076250-appb-000014
表6磷酸钠、柠檬酸体系与YC/T 468-2013方法测定结果比对
Figure PCTCN2015076250-appb-000015
Figure PCTCN2015076250-appb-000016
表7柠檬酸钠、柠檬酸体系与YC/T 468-2013方法测定结果比对
Figure PCTCN2015076250-appb-000017
表8巴比妥纳、盐酸体系与YC/T 468-2013方法测定结果比对
Figure PCTCN2015076250-appb-000018
Figure PCTCN2015076250-appb-000019
表9三羟甲基氨基甲烷、盐酸体系与YC/T 468方法测定结果比对
Figure PCTCN2015076250-appb-000020
表10不同缓冲体系与YC/T 468-2013方法测定结果配对t检验
Figure PCTCN2015076250-appb-000021

Claims (12)

  1. 一种适用于连续流动法测定烟草及烟草制品中总植物碱的缓冲溶液，该缓冲溶液包含缓冲溶液A和缓冲溶液B；
    其中，缓冲溶液A的配制为：称取磷酸氢二钠60.5-83.0g，优选为71.6g；磷酸三钠10.1-20.2g，优选为15.2g至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度；
    缓冲溶液B的配制为：称取对氨基苯磺酸5.5-8.0g，优选为7.0g；磷酸氢二钠60.5-83.0g，优选为71.6g；磷酸二氢钠5.0-7.0g，优选为6.2g和柠檬酸钠10.0-12.1g，优选为11.8g至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。
  2. 根据权利要求1所述的缓冲溶液，其特征在于，缓冲溶液A与缓冲溶液B之间的用量比为1：2至1：1，优选为3：4。
  3. 一种用连续流动法测定烟草或烟草制品中总植物碱的方法，该方法采用硫氰酸钾和二氯异氰尿酸钠在线生成氯化氰，氯化氰与所述烟草或烟草制品中的总植物碱(以烟碱计)反应，使烟碱的吡啶环断开，进而与对氨基苯磺酸发生反应，反应产物用比色计在460nm处测定，该方法包括：
    采用缓冲溶液A和/或缓冲溶液B将反应体系的pH值控制在6.0～7.5之间；
    其中，缓冲溶液A的配制为：称取磷酸氢二钠60.5-83.0g，优选为71.6g；磷酸三钠10.1-20.2g，优选为15.2g至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度；
    缓冲溶液B的配制为：称取对氨基苯磺酸5.5-8.0g，优选为7.0g；磷酸氢二钠60.5-83.0g，优选为71.6g；磷酸二氢钠5.0-7.0g，优选为6.2g和柠檬酸钠10.0-12.1g，优选为11.8g至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度。
  4. 根据权利要求3所述的方法，其特征在于，缓冲溶液A与缓冲溶液B的用量比为1：2至1：1，优选为3：4。
  5. 根据权利要求3或4所述的方法，其特征在于，所述方法还包括：
    样品的制备为：按YC/T31制备烟草试样，并测定其水分含量，然后使用5％醋酸对烟草试样进行萃取，过滤，得滤液。
  6. 根据权利要求3至5中任一项所述的方法，其特征在于，所述方法还包括：
    标准溶液的配制为：称取烟碱或烟碱盐以5％的醋酸溶解，配制成至少5个工作标准液，所配制的标准溶液中烟碱含量浓度范围为0.5～7％；
  7. 根据权利要求3至6中任一项所述的方法，其特征在于，所述方法还包括：
    解毒液A的配制为：称取1g柠檬酸、10g硫酸亚铁至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度；
    解毒液B的配制为：称取10g碳酸钠至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度；
    硫氰酸钾溶液的配制为：称取硫氰酸钾至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容，该溶液浓度范围为0.09～0.21mol/L，优选为0.12mol/L；
    二氯异氰尿酸钠溶液的配制为：称取二氯异氰尿酸钠至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容，该溶液浓度范围为0.02～0.06mol/L，优选为0.04mol/L；
    优选地，上述方法中，硫氰酸钾溶液和二氯异氰尿酸钠溶液浓度比为2.5：1至3.5：1，优选为3：1。
  8. 根据权利要求3至7中任一项所述的方法，其特征在于，所述方法还包括：
    使用连续流动分析仪进行检测，然后根据下式计算样品中的总植物碱：
    Figure PCTCN2015076250-appb-100001
    式中，X：样品的仪器观测值，单位毫克每毫升(mg/mL)；
    V：加入的萃取液体积，单位为毫升(mL)；
    m：样品质量，单位为克(g)；
    W：试样的水分百分含量，％(质量分数)；
    上式计算的总植物碱为以干基计的总植物碱的含量。
  9. 根据权利要求3至8中任一项所述的方法，其特征在于，所述方法中连续流动分析仪的运行参数为：进样清洗比为1.5，进样频率48杯/小时，增益18-60，光强2.85伏。
  10. 根据权利要求3所述的方法，其特征在于，所述方法包括：
    (1)样品的制备：按YC/T31制备烟草试样，并测定其水分含量，然后使用5％醋酸对烟草试样进行萃取，过滤，得滤液备用；
    (2)标准溶液的配制：
    称取烟碱或烟碱盐以5％的醋酸溶解，配制成至少5个工作标准液，所配制的标准溶液中烟碱含量浓度范围为0.5～7％；
    (3)反应试剂的配制：
    硫氰酸钾溶液的配制为：称取硫氰酸钾至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容，该溶液浓度范围为0.09～0.21mol/L，优选为0.12mol/L；
    二氯异氰尿酸钠溶液的配制为：称取二氯异氰尿酸钠至烧杯中，用水溶解，然后转入250mL容量瓶中，用水定容，该溶液浓度范围为0.02～0.06mol/L，优选为0.04mol/L；
    缓冲溶液A的配制：称取磷酸氢二钠60.5-83.0g，优选为71.6g、磷酸三钠10.1-20.2g，优选为15.2g至烧杯中，用水溶解，然后转入1L容量瓶 中，用水定容至刻度；
    缓冲溶液B的配制：称取对氨基苯磺酸5.5-8.0g，优选为7.0g、磷酸氢二钠60.5-83.0g，优选为71.6g、磷酸二氢钠5.0-7.0g，优选为6.2g和柠檬酸钠10.0-12.1g，优选为11.8g至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度；
    解毒液A的配制为：称取1g柠檬酸、10g硫酸亚铁至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度；
    解毒液B的配制为：称取10g碳酸钠至烧杯中，用水溶解，然后转入1L容量瓶中，用水定容至刻度；
    (4)使用连续流动分析仪在步骤(3)的反应体系下检测步骤(1)的滤液和步骤(2)的工作标准液；
    (5)根据下式计算样品中的总植物碱：
    Figure PCTCN2015076250-appb-100002
    式中，X：样品的仪器观测值，单位毫克每毫升(mg/mL)；
    V：加入的萃取液体积，单位为毫升(mL)；
    m：样品质量，单位为克(g)；
    W：试样的水分百分含量，％(质量分数)；
    上式计算的总植物碱为以干基计的总植物碱的含量。
  11. 根据权利要求10所述的方法，其特征在于，缓冲溶液A与缓冲溶液B的用量比为1：2至1：1，优选为3：4，该用量比使得反应体系的pH值控制在6.0～7.5之间；优选地，硫氰酸钾溶液和二氯异氰尿酸钠溶液浓度比为2.5：1至3.5：1，优选为3：1。
  12. 根据权利要求10或11所述的方法，其特征在于，连续流动分析仪的运行参数为：进样清洗比为1.5，进样频率48杯/小时，增益18-60，光强2.85伏。
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