Sucrose purity determination method based on ion chromatography-pulsed amperometric detector

Abstract

The invention discloses a sucrose purity determination method, which innovatively utilizes an ion chromatography-pulsed ampere detector to determine trace organic impurities, combines a firing residue weighing method to determine inorganic impurities and a Karl Fischer Coulomb method to determine moisture, and calculates the content (purity) of main components according to a mass balance principle. The method has the advantages of simplicity and convenience in operation, reliable result, high sensitivity and the like, can be widely applied to the fields of sucrose quality control and quality evaluation, and has important significance in improving sucrose product quality and promoting development of related industries.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry and food testing technology, and in particular to a method for accurately determining the purity of sucrose by measuring the organic impurity content of sucrose based on an ion chromatography-pulsed amperometric detector and combining the inorganic impurity content and moisture content. Background Art

[0002] As the world's most important natural sweetener and a fundamental raw material in the food industry, sucrose is widely used in a wide range of industries, including food, beverages, pharmaceuticals, and chemicals. Its purity is not only a core indicator for measuring product quality, but also a key parameter that directly impacts downstream product performance, process efficiency, economic benefits, and even safety and compliance. Accurately measuring sucrose purity is of great significance on multiple levels.

[0003] Ensuring Product Quality and Safety: Impurities in sucrose, such as organic impurities (glucose, fructose), inorganic impurities, and moisture, can significantly affect the product's sensory qualities (color, flavor), physical properties (crystallinity, solubility, viscosity), and storage stability. Optimizing Production Processes and Controlling Costs: In the sugar industry, purity is the core criterion for evaluating raw material value and guiding production processes. Raw material purity directly affects refining yield, energy consumption, and auxiliary material consumption. Real-time, accurate monitoring of the purity of intermediate and final products is key to optimizing process parameters, improving production efficiency, and reducing production costs. Ensuring Fair Trade and Economic Value: Sucrose is a major internationally traded commodity, and its price is closely linked to its purity. The market value of sucrose of different purity grades varies significantly. Buyers and sellers rely on recognized and accurate purity determination methods as the basis for settlement. Inaccurate or inconsistent results can directly lead to trade disputes, economic losses, and undermine market trust. Therefore, establishing reliable and standardized purity determination methods is crucial to maintaining fair trade.

[0004] In the field of purity determination of high-purity organic matter, the mass balance method is generally used. The mass balance method first determines the content of organic impurities, inorganic impurities and water, and then deducts the content of the above impurities from 100% based on the mass balance principle to calculate the content of the main component (i.e., purity). Among them, the organic impurity content is usually determined by liquid chromatography or gas chromatography. For sucrose, gas chromatograph cannot be used, and liquid chromatography is usually used to measure the organic impurity content. However, since sucrose and its organic impurities have very weak absorption in the ultraviolet wavelength range, they cannot be detected by ultraviolet detectors or diode array detectors. Only evaporative light scattering detectors can be used for detection. The detection limit of the evaporative light scattering detector for measuring organic impurities (glucose and fructose, etc.) is about 1 mg / L. Considering that the concentration of the sucrose sample is about 1000 mg / L, the detection limit of the organic impurity content is about 0.1%. The organic impurity content of high-purity sucrose may be close to or lower than the above detection limit, resulting in the content cannot be accurately measured.

[0005] Inorganic impurity content is primarily determined by the ignition residue weighing method, with a detection limit of typically 0.01%. Moisture is primarily determined by the Karl Fischer coulometric method, with a detection limit of typically 0.01%. Therefore, the primary challenge in determining sucrose purity is the lack of sensitivity in methods for measuring organic impurities, leading to inaccurate or undetectable organic impurity levels, severely impacting quality control and process improvements for sucrose products. Therefore, there is an urgent need to develop a highly sensitive detection method for determining the value of organic impurities in high-purity sucrose. Summary of the Invention

[0006] The present invention aims to provide an innovative method for determining sucrose purity. This method uses high-performance anion exchange chromatography coupled with pulsed amperometric electrochemical detection (HPAEC-PAD) to determine the organic impurity content, combined with the ignition residue method to determine the inorganic impurity content and Karl Fischer coulometric determination of the water content, to accurately determine the sucrose purity. The method demonstrates very low uncertainty when determining sucrose purity.

[0007] To achieve the purpose of the above invention, the inventors of the present invention conducted a series of research and put in creative work, and obtained innovative results with important practical value.

[0008] The present invention provides a method for determining sucrose purity, which comprises the following steps:

[0009] (1) Determination of organic impurity content X by HPAEC-PAD Org ;

[0010] (2) Determination of inorganic impurity content X by ignition residue method Inorg ;

[0011] (3) Determination of water content X by Karl Fischer coulometric method W ;

[0012] (4) Based on the mass balance principle, the sucrose purity X is calculated by the following formula P :

[0013] X P =1-X Org -X W -X Inorg .

[0014] According to the present invention, each determination in steps (1) to (3) is repeated 6 times, and the following steps are further included:

[0015] (5) Determine the uncertainty u of the organic impurity content Org , uncertainty of inorganic impurity content u Inorg, uncertainty of moisture content u W , the uncertainty of sucrose purity U is calculated by the following formula:

[0016]

[0017] U=2u P

[0018] According to the present invention, in step (1),

[0019] A low-concentration sucrose sample solution to be tested is prepared, with a concentration of 5-20 mg / L, preferably 10 mg / L, for determining the chromatographic peak retention times of sucrose and various organic impurities.

[0020] Prepare a high-concentration sucrose test sample solution with a concentration of 500-5000 mg / L, and use it to measure the peak area data of each organic impurity. Repeat 6 times, substitute the peak area data into the standard curve formula of each organic impurity to obtain the concentration of each organic impurity, divide it by the concentration of the sucrose test sample solution, and then take the average value to calculate the content and standard deviation SD of each organic impurity in the sucrose test sample;

[0021] Preferably, the organic impurities are glucose and fructose, and the content of glucose in the sucrose sample to be tested is X Org,1 , the fructose content in the sucrose sample to be tested is X Org,2 , calculate the sucrose purity X P The formula is:

[0022] X P =1-X Org,1 -X Org,2 -X W -X Inorg

[0023] Preferably, during detection, the detector is turned off before the sucrose chromatographic peak retention time and turned back on after the sucrose chromatographic peak retention time. The timing of turning off and turning on the detector is selected to avoid contamination of the electrode by the high-concentration sucrose solution and to avoid missed detection of organic impurities.

[0024] Preferably, the standard curve of organic impurities is obtained by the following method: preparing a mixed solution containing each organic impurity, diluting it to obtain 5-7 standard solutions with concentration gradients, measuring the peak area data of each organic impurity in the standard solution of each concentration, repeating 6 times, taking the average value, and plotting the concentration of each organic impurity as the abscissa and the average peak area as the ordinate for linear fitting to obtain the standard curve formula of each organic impurity.

[0025] Those skilled in the art can select an instrument suitable for the present invention and set chromatographic conditions. In one embodiment, a 930 Compact IC Flex HPAEC-PAD instrument was used, along with a Metrosep Carb 2 column (250 mm length, 4 mm inner diameter), an Au electrode, a 100 μL loop volume, and a 200 mmol / L NaOH solution as the eluent. The eluent was delivered at a flow rate of 0.5 mL / min. The column temperature was controlled at a constant temperature of 30°C. The pulse integration amperometric potential was 0.05 V (0-300 ms), 0.55 V (300-350 ms), and -0.1 V (350-550 ms). The integration time was 200 ms. Under these instrument and chromatographic conditions, the retention times of sucrose, glucose, and fructose were 23.98 min, 9.94 min, and 11.56 min, respectively.

[0026] According to the present invention, in step (2),

[0027] The sucrose sample to be tested was burned in a crucible to fully decompose the organic components and measure the mass of inorganic impurities. This was repeated 6 times. The mass of inorganic impurities was divided by the mass of the sucrose sample to be tested and the average was taken to calculate the inorganic impurity content and standard deviation SD of the sucrose sample to be tested.

[0028] Preferably, the mass of the sucrose sample to be tested is between 200-400 mg;

[0029] Preferably, the calcination temperature and time are as follows: heating to 200° C. and holding the temperature for 1 hour, then heating to 300° C. and holding the temperature for 1 hour, then heating to 500° C. and holding the temperature for 5 hours.

[0030] According to the present invention, in step (3),

[0031] The sucrose sample to be tested is titrated in a Karl Fischer coulometric titrator to measure the electrolysis charge, which is repeated 6 times. The amount of iodine is calculated according to Faraday's law, which is equal to the amount of water. The amount of water is converted into the mass of water and then divided by the mass of the sucrose sample to be tested and the average is taken to calculate the moisture content and standard deviation SD of the sucrose sample to be tested;

[0032] Preferably, the mass of the sucrose sample to be tested is between 200-600 mg.

[0033] According to the present invention, in step (5),

[0034] Uncertainty of organic impurity content u Org The calculation formula is as follows:

[0035]

[0036] The standard deviation SD of each organic impurity content in the sucrose sample to be tested is divided by As the uncertainty of its Class A method assessment (u A );

[0037] Divide the detection limit of each organic impurity concentration by the concentration of the sucrose sample solution to obtain the detection limit of each organic impurity content in the sucrose sample, and then divide it by The uncertainty introduced by the detection limit is used as the uncertainty of the Class B method evaluation (u B );

[0038] Preferably, the uncertainty of the glucose content in the sucrose sample is u Org,1 , the uncertainty of fructose content in the sucrose sample is u Org,2 , the formula for calculating the uncertainty U of sucrose purity is:

[0039]

[0040] U=2u P

[0041] Preferably, the method for determining the detection limit of each organic impurity concentration is as follows: when drawing the organic impurity standard curve, the peak area of ​​each organic impurity in the standard solution of each concentration measured is substituted into the standard curve formula to obtain the concentration measurement value of each prepared concentration, and its standard deviation SD is calculated. The concentration preparation value is used as the horizontal coordinate and three times the standard deviation of the concentration measurement value, that is, SD×3, is used as the vertical coordinate. A linear fit is drawn to obtain a fitted straight line, and its intercept (in the present invention, the vertical intercept) is the concentration detection limit of each organic impurity.

[0042] Those skilled in the art can choose known methods to determine the uncertainty of the inorganic impurity content u Inorg , uncertainty of moisture content u W .For example:

[0043] In one embodiment, the uncertainty of the inorganic impurity content u Inorg The calculation formula is as follows:

[0044]

[0045] The standard deviation SD of the average value of inorganic impurities in the sucrose sample to be tested is divided by As the uncertainty of the evaluation of the type A method ( A );

[0046] Divide the detection limit of inorganic impurities by the average mass of the sucrose sample to obtain the detection limit of inorganic impurities in the sucrose sample, and then divide it by The uncertainty introduced by the detection limit is used as the uncertainty of the Class B method evaluation (uB );

[0047] Among them, the determination method of the mass detection limit of inorganic impurities is: using NaCl as the inorganic impurity standard substance to determine the detection limit of the inorganic impurity content determination method: prepare a NaCl aqueous solution with a concentration of 100 mg / L, take 50 μL, 100 μL, 200 μL, 350 μL and 500 μL respectively, of which the mass of sodium chloride is 5 μg, 10 μg, 20 μg, 35 μg and 50 μg respectively, and determine according to the inorganic impurity content determination method to obtain the measured value, repeat 6 times, use the prepared value of NaCl mass as the horizontal axis, and three times the standard deviation of its measured value, that is, SD×3, as the vertical axis, draw a graph for linear fitting, and obtain a fitting straight line, whose intercept is the mass detection limit of inorganic impurities.

[0048] In one embodiment, the uncertainty in moisture content u W The calculation formula is as follows:

[0049]

[0050] The standard deviation SD of the average water content in the sucrose sample to be tested is divided by As the uncertainty of the evaluation of the type A method ( A );

[0051] Divide the water content detection limit by the average mass of the sucrose sample to obtain the water content detection limit in the sucrose sample, and then divide it by The uncertainty introduced by the detection limit is used as the uncertainty of the Class B method evaluation (u B );

[0052] Among them, the method for determining the water mass detection limit is: use liquid water standard material (GBW 13513) to determine the detection limit of the water content determination method: take volumes of 10 μL, 20 μL, 50 μL, 100 μL and 200 μL of the standard material respectively, its water mass = volume × solution density (0.86 g / mL) × standard material water content (87 μg / g), and measure according to the water content determination method to obtain the measured value, repeat 6 times, use the theoretical value of the water mass as the horizontal axis, and three times the standard deviation of the measured value, that is, SD×3, as the vertical axis, draw a linear fit, and obtain a fitted straight line, the intercept of which is the detection limit of the water mass.

[0053] The method of the present invention has the advantages of simple operation, reliable results, high sensitivity, etc., and can be widely used in the field of sucrose quality control and quality assessment. It is of great significance for improving the quality of sucrose products and promoting the development of related industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 .Standard curves for glucose and sucrose

[0055] Figure 2 .Determination of detection limits of glucose and sucrose concentrations

[0056] Figure 3 .Determination of mass detection limit of inorganic impurities

[0057] Figure 4 .Determination of detection limit of water quality

[0058] Figure 5 HPLC method standard curve of glucose and sucrose

[0059] Figure 6 . HPLC method for determination of detection limit of glucose and sucrose concentration

[0060] Figure 7 Typical chromatogram of sucrose organic impurities determined by ion chromatography-amperometric detection

[0061] Figure 8 Typical chromatogram of sucrose determination using ion chromatography-amperometric detection DETAILED DESCRIPTION

[0062] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content of the present invention being recorded, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the limited scope of the present invention equally.

[0063] The method of the present invention was used to determine the purity of sucrose from three different brands of commercially available sugars. The specific process is as follows:

[0064] Example 1

[0065] Follow the steps below to measure the organic impurities, inorganic impurities, and moisture content in Sigma sucrose.

[0066] ①Organic impurity content (X Org ) determination:

[0067] Instruments and Materials: A 930 Compact IC Flex HPAEC-PAD instrument (Metrohm, Switzerland) was used, along with a Metrosep Carb 2 column (Metrohm, Switzerland) with a length of 250 mm and an inner diameter of 4 mm and an Au electrode. The quantitative loop volume was 100 μL, and the eluent was 200 mmol / L NaOH solution (Merck, Germany).

[0068] Chromatographic conditions: The eluent was delivered at a flow rate of 0.5 mL / min; the column temperature was controlled at 30 °C; the pulse amperometric potential was 0.05 v (0-300 ms), 0.55 v (300-350 ms), -0.1 v (350-550 ms); the integration time was 200 ms.

[0069] Preparation of sample solution: 500.0 mg of sucrose sample was weighed into a 100 mL volumetric flask, and deionized water was added to the mark to prepare a 5000.0 mg / L high-concentration sucrose sample solution. Then, 1.0 mg of sucrose sample was weighed into a 100 mL volumetric flask, and deionized water was added to the mark to prepare a 10.0 mg / L low-concentration sucrose sample solution.

[0070] Detection: The sucrose sample solution was filtered through a 0.45 μm organic phase filter. First, the 10.0 mg / L sucrose sample solution was measured, 100 μL of which was injected to determine the chromatographic peak retention time of the main component (i.e., sucrose) at 23.98 min, and the chromatographic peak retention times of the organic impurities at 9.94 min and 11.56 min, respectively. Then, the 5000.0 mg / L sucrose sample solution was measured, 100 μL of which was injected repeatedly 6 times. The detector was turned off before the retention time of the sucrose chromatographic peak (19 min) and turned on again at 35 min to avoid the contamination of the electrode by the high-concentration sucrose solution and the missed detection of the organic impurities. Figure 7 ) The chromatographic peak retention time and peak area data of each organic impurity were recorded automatically by the chromatographic workstation.

[0071] Qualitative identification of organic impurities: 1.0 mg of glucose and fructose was weighed into a 100 mL volumetric flask, respectively, and a small amount of deionized water was added to the volumetric flask to dissolve the glucose and fructose together. Then, deionized water was added to the mark to prepare a 10.0 mg / L glucose and fructose mixed solution. The solution was filtered through a 0.45 μm organic phase filter. 100 μL of the solution was injected to determine the chromatographic peak retention times of glucose and fructose at 9.94 min and 11.56 min, respectively, which were consistent with the chromatographic peak retention times of the organic impurities in the sucrose sample, thereby determining that the organic impurities in the sucrose sample were glucose and fructose.

[0072] Drawing of organic impurity standard curve: weigh 10.0 mg of glucose and fructose respectively, add them to a 100 mL volumetric flask, add a small amount of deionized water to the volumetric flask, dissolve the glucose and fructose together, and then continue to add deionized water to the scale line to prepare a glucose and fructose mixed solution with a concentration of 100.0 mg / L, and then dilute the solution to 1.0 mg / L, 0.8 mg / L, 0.6 mg / L, 0.4 mg / L, 0.2 mg / L, and 0.1 mg / L, respectively, to obtain a standard solution; filter through a 0.45 μm organic phase filter membrane; inject 100 μL of each, record the peak area data of glucose and fructose at each concentration, repeat 6 times, take the average value, use the glucose and fructose concentrations as the horizontal axis and the average value of their respective peak areas as the vertical axis, draw a graph for linear fitting, and obtain Figure 1 The standard curve is shown, and the formula is as follows:

[0073] Glucose standard curve formula: y = 7.3162x - 0.0767 (R 2 =0.9993)(1)

[0074] Fructose standard curve formula: y = 4.3085x - 0.0196 (R 2 =0.9986)(2)

[0075] In the above formula:

[0076] x——concentration of organic impurities, mg / L;

[0077] y——Chromatographic peak area of ​​organic impurities.

[0078] Substitute the glucose chromatographic peak area obtained from the above 6 tests into formula (1) to obtain the glucose concentration, then divide it by the sucrose concentration of 5000.0 mg / L, and then take the average value to calculate the glucose content (in mass percentage) in the sucrose sample to be tested. Org,1 The fructose concentration is 0.0013% and the standard deviation SD is 0.00013%. Similarly, the fructose chromatographic peak area obtained from the above 6 tests is substituted into formula (2) to obtain the fructose concentration, which is then divided by the sucrose concentration of 5000.0 mg / L. The average value is then taken to calculate the fructose content (in mass percentage) in the sucrose sample to be tested. Org,2 The value is 0.0014% and the standard deviation SD is 0.00021%.

[0079] ②Inorganic impurity content (X Inorg ) determination:

[0080] Instrument preparation: Use an electronic balance with an accuracy of 0.001 mg.

[0081] Crucible Pretreatment: Aluminum foil was cut into six 5 cm × 5 cm square sheets and folded into a square crucible. The crucible was placed in a 5E-MF6000 muffle furnace (Changsha Kaiyuan Instrument Co., Ltd., China) and calcined at 500°C for 24 h to completely remove impurities and residues. After the crucibles were cooled to room temperature in the muffle furnace, the masses of the six crucibles were accurately weighed using an electronic balance and recorded. The crucibles were then stored in a desiccator until further use.

[0082] Sample Weighing and Ignition: Use an electronic balance to accurately weigh six sucrose samples (masses of 277.445 mg, 300.893 mg, 284.658 mg, 337.312 mg, 285.260 mg, and 388.598 mg, respectively) for an average mass of 312.361 mg. These samples are then placed into six pre-treated and weighed crucibles. The crucibles are then slowly placed into a muffle furnace with the door closed. The temperature is raised from room temperature to 200°C, held constant for 1 hour, then to 300°C, held constant for 1 hour, and then to 500°C, held constant for 5 hours. This temperature profile fully decomposes the organic components in the sucrose samples, leaving inorganic impurities in the crucibles.

[0083] Cooling and weighing: After the burning is completed, turn off the power of the muffle furnace and let the crucible cool naturally to room temperature in the furnace. Then take out the crucible and put it in a desiccator for testing. Use an electronic balance to accurately weigh the total mass of the crucible and inorganic residue and record it. Subtract the mass of the crucible from the total mass of the crucible and inorganic residue to get the mass of the inorganic impurities. Then divide it by the mass of the sucrose sample to be tested and take the average value to calculate the inorganic impurity content (in mass percentage) in the sucrose sample to be tested X Inorg The value is 0.0059% and the standard deviation SD is 0.0012%.

[0084] ③ Moisture content (X W ) determination:

[0085] Instrument preparation: A DL39 Karl Fischer coulometric titrator (Mettler-Toledo, USA) was placed in a water-free glove box. An appropriate amount of Coulomat AK reagent (Fluka, Germany) was added to the titration cell. The instrument was turned on and automatically pre-titrated.

[0086] Sample weighing and injection: Use an electronic balance to accurately weigh 6 portions of sucrose samples (masses of 246.3 mg, 436.3 mg, 381.9 mg, 441.3 mg, 346.7 mg, and 538.5 mg, respectively), with an average mass of 398.5 mg. Quickly transfer the samples to the glove box and then quickly add them to the titration cell of the Karl Fischer coulometric moisture analyzer; immediately start the instrument measurement program, and the instrument automatically electrolyzes iodine molecules to react with water. After the titration is completed, the instrument calculates the amount of iodine substance based on the electrolysis quantity and Faraday's law, which is equal to the amount of water substance. After converting it into the mass of water, it is divided by the mass of the sucrose sample to be tested and the average is taken to calculate the water content (in mass percentage) in the sucrose sample to be tested X W The value is 0.0011% and the standard deviation SD is 0.000085%.

[0087] ④Purity (X P ) Calculation: Based on the mass balance principle, the purity Xp of the sucrose sample to be tested is calculated to be 99.9903% by the following formula:

[0088] X P =1-X Org,1 -X Org,2 -X W -X Inorg

[0089] ⑤Uncertainty assessment

[0090] Uncertainty evaluation of organic impurity content:

[0091] The standard deviation SD of the mean value of glucose and fructose content in the sucrose sample to be tested is divided by As the uncertainty of the evaluation of the respective Class A method ( A ), namely glucose u A 0.000053%, fructose u A It is 0.000085%.

[0092] Substitute the peak areas measured 6 times at each preparation concentration into the standard curve formula when drawing the standard curve to obtain the concentration measurement value at each preparation concentration, calculate its standard deviation SD, use the preparation value of the concentration as the horizontal axis and three times the standard deviation of the measurement value, i.e. SD×3, as the vertical axis, and draw a linear fit to obtain Figure 2Fit the straight line, and its intercept is the concentration detection limit. Divide the concentration detection limit by the concentration of the sucrose sample solution, 5000.0 mg / L, to get the detection limit of the organic impurities in the sucrose sample. The detection limit of glucose concentration is 0.0652 mg / L, and the detection limit of glucose content in the sucrose sample is 0.0013%; the detection limit of fructose concentration is 0.0824 mg / L, and the detection limit of fructose content in the sucrose sample is 0.0016%. Divide the detection limit of organic impurities in the sucrose sample by The uncertainty introduced by the detection limit is used as the uncertainty of the Class B method evaluation (u B ), glucose B 0.00075%, fructose u B It is 0.00095%.

[0093] The uncertainty of the organic impurity content in the sucrose sample is calculated according to the following formula:

[0094]

[0095] Finally, the uncertainty of glucose content in the sucrose sample is u Org,1 The uncertainty of fructose content in the sucrose sample is 0.00075%. Org,2 It is 0.00096%.

[0096] Uncertainty assessment of inorganic impurity content:

[0097] Divide the standard deviation SD of the mean value of inorganic impurities in the sucrose sample by As the uncertainty of the evaluation of the type A method ( A ), that is, inorganic impurities u A It is 0.00047%.

[0098] The detection limit of the inorganic impurity content determination method was determined using NaCl as the inorganic impurity standard substance. Weigh 100 mg of NaCl, add it to a 1000 mL volumetric flask, add deionized water to the scale line, and prepare a NaCl aqueous solution with a concentration of 100 mg / L. Take 50 μL, 100 μL, 200 μL, 350 μL and 500 μL respectively, of which the mass of sodium chloride is 5 μg, 10 μg, 20 μg, 35 μg and 50 μg respectively. Determine according to the inorganic impurity content determination method to obtain the measured value. Repeat 6 times, with the prepared value of NaCl mass as the horizontal axis and three times the standard deviation of its measured value, i.e. SD×3, as the vertical axis. Draw a graph for linear fitting and obtain Figure 3The intercept of the fitted straight line is the detection limit of inorganic impurities, which is 5.7575μg. The detection limit of inorganic impurities is divided by the average mass of the sucrose sample to be tested, 312.361mg, to obtain the detection limit of inorganic impurities in the sucrose sample to be tested, which is 0.0018%. The uncertainty introduced by the detection limit is used as the uncertainty of the Class B method evaluation (u B ), which is 0.0011%.

[0099] Uncertainty u of the inorganic impurity content in the sucrose sample to be tested Inorg Calculate according to the following formula:

[0100]

[0101] Finally, the uncertainty of the inorganic impurity content in the sucrose sample is u Inorg It is 0.0012%.

[0102] Uncertainty assessment of moisture content:

[0103] Divide the standard deviation SD of the mean water content in the sucrose sample by As the uncertainty of the evaluation of the type A method ( A ), that is, the moisture content u A It is 0.000035%.

[0104] The detection limit of the water content determination method was determined using a liquid water standard substance (GBW 13513). Take 10 μL, 20 μL, 50 μL, 100 μL, and 200 μL of the standard substance, respectively. The water mass = volume × solution density (0.86 g / mL) × water content of the standard substance (87 μg / g), which are 0.7482 μg, 1.4964 μg, 3.7410 μg, 7.4820 μg, and 14.9640 μg, respectively. Determine according to the water content determination method and obtain the measured values. Repeat 6 times, with the theoretical value of the water mass as the horizontal axis and three times the standard deviation of the measured value, i.e., SD×3, as the vertical axis. A linear fit is plotted to obtain the value. Figure 4 The intercept of the fitted straight line is the detection limit of water content, which is 1.4312μg. The detection limit of water content is divided by the average mass of sucrose sample to be tested, 398.5mg, to obtain the detection limit of water content in sucrose sample to be tested, which is 0.00036%. The uncertainty introduced by the detection limit is used as the uncertainty of the Class B method evaluation (u B ), which is 0.00021%.

[0105] Uncertainty u of the water content in the sucrose sample to be tested W Calculate according to the following formula:

[0106]

[0107] Finally, the uncertainty of the water content in the sucrose sample is u W It is 0.00021%.

[0108] Evaluation of purity uncertainty: The purity uncertainty U of the sucrose sample to be tested is calculated according to the following formula:

[0109]

[0110] U=2u P

[0111] Finally, u P The purity uncertainty U of the sucrose sample to be tested is 0.0017% and 0.0034%.

[0112] Therefore, the results are as follows:

[0113] X Org,1 is 0.0013%, SD is 0.00013%, u Org,1 0.00075%; X Org,2 is 0.0014%, SD is 0.00021%, u Org,2 It is 0.00096%.

[0114] X Inorg is 0.0059%, SD is 0.0012%, u Inorg It is 0.0012%.

[0115] X W is 0.0011%, SD is 0.000085%, u W It is 0.00021%.

[0116] X p It is 99.9903%, and U is 0.0034% (k=2).

[0117] ⑥Comparative experiment:

[0118] The organic impurities were determined using a 1290 Infinity II liquid chromatography-evaporative light scattering detector (HPLC-ELSD) (Agilent, USA) equipped with an Asahipak NH2-50 column according to the above detection step ①. The results showed that no organic impurities were detected. A and fructose A Both are 0.

[0119] When drawing the standard curve, the concentrations of glucose and fructose in the glucose and fructose standard solutions were 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L and 25 mg / L, respectively.

[0120] Draw the standard curve Figure 5 , the standard curve formula is as follows:

[0121] Glucose standard curve formula: y = 0.6171x - 1.1684 (R 2 =0.9962)

[0122] Fructose standard curve formula: y = 0.7034x - 1.923 (R 2 =0.9982)

[0123] In the above formula:

[0124] x——concentration of organic impurities, mg / L;

[0125] y——Chromatographic peak area of ​​organic impurities.

[0126] Substitute the peak areas measured 6 times at each preparation concentration into the above standard curve formula when drawing the standard curve to obtain the concentration measurement value at each preparation concentration, calculate its standard deviation SD, use the preparation value of the concentration as the horizontal axis and three times the standard deviation of the measured value, i.e. SD×3, as the vertical axis, and draw a linear fit to obtain Figure 6 Fit the straight line, and its intercept is the concentration detection limit. Divide the concentration detection limit by the concentration of the sucrose sample solution, 5000.0 mg / L, to get the detection limit of the organic impurity content in the sucrose sample. The detection limit of glucose concentration is 1.4085 mg / L, the detection limit of fructose concentration is 1.1781 mg / L, the detection limit of glucose content in the sucrose sample is 0.028%, and the detection limit of fructose content in the sucrose sample is 0.024%. Divide the detection limit of organic impurity content in the sucrose sample by The uncertainty introduced by the detection limit is used as the uncertainty of the Class B method evaluation (u B ), glucose B 0.016%, fructose u B Finally, the uncertainty of the glucose content in the sucrose sample is u Org,1-HPLC The uncertainty of fructose content in the sucrose sample is 0.016%. Org,2-HPLC The inorganic impurity content and uncertainty, moisture content and uncertainty are calculated using the above data to obtain X p The purity uncertainty U of this method is much larger than the U obtained based on HPAEC-PAD (0.0034%).

[0127] Example 2

[0128] The organic impurities, inorganic impurities and moisture content in TCI sucrose were measured according to the procedure of Example 1. For the determination of organic impurities, the TCI sample solution was prepared at 500.0 mg / L and the eluent concentration was 100 mmol / L.

[0129] X Org,1 was 0.0064%, SD was 0.00065%, u Org,1 was 0.0062%; X Org,2 was 0.0053%, SD was 0.00073%, u Org,2 was 0.0074%.

[0130] X Inorg was 0.0059%, SD was 0.0013%, u Inorg was 0.0011%.

[0131] X W was 0.0033%, SD was 0.00018%, u W was 0.00042%.

[0132] X p was 99.9791%, U was 0.019% (k = 2).

[0133] Example 3

[0134] The organic impurities, inorganic impurities and moisture content in innochem sucrose were measured according to the procedure of Example 1. For the determination of organic impurities, the innochem sample solution was prepared at 1000.0 mg / L and the eluent concentration was 50 mmol / L.

[0135] X Org,1 was 0.0014%, SD was 0.000085%, u Org,1 was 0.0019%; X Org,2 was 0.0015%, SD was 0.00018%, u Org,2 was 0.0024%.

[0136] X Inorg was 0.0097%, SD was 0.0017%, u Inorg was 0.0015%.

[0137] X W was 0.0017%, SD was 0.00024%, u W was 0.00044%.

[0138] X p It is 99.9857% and U is 0.0069% (k=2).

[0139] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A method for determining sucrose purity, wherein: The steps include: (1) Determination of organic impurity content X by HPAEC-PAD Org ; (2) Determination of inorganic impurity content X by ignition residue method Inorg ; (3) Determination of water content X by Karl Fischer coulometric method W ; (4) Based on the mass balance principle, the sucrose purity X is calculated by the following formula: P : 。 2. The assay method according to claim 1, wherein Each determination in steps (1) to (3) was repeated 6 times, and further included the following steps: (5) Determine the uncertainty u of the organic impurity content Org , uncertainty of inorganic impurity content u Inorg , uncertainty of moisture content u W , the uncertainty U of sucrose purity is calculated by the following formula: ， 。 3. The measuring method according to any one of claims 1 or 2, wherein In step (1), Prepare a low-concentration sucrose sample solution to be tested, the concentration of which is 5-20 mg / L, preferably 10 mg / L, for determining the chromatographic peak retention time of sucrose and various organic impurities; Prepare a high-concentration sucrose test sample solution with a concentration of 500-5000 mg / L, and use it to determine the peak area of ​​each organic impurity. Repeat 6 times, substitute the peak area into the standard curve formula of each organic impurity to obtain the concentration of each organic impurity, divide it by the concentration of the sucrose test sample solution, and then take the average value to calculate the content of each organic impurity in the sucrose test sample and the standard deviation SD; Preferably, the organic impurities are glucose and fructose, and the glucose content in the sucrose sample to be tested is X Org,1 , the fructose content in the sucrose sample to be tested is X Org,2 , calculate the sucrose purity X P The formula is: ； Preferably, during detection, the detector is turned off before the retention time of the sucrose chromatographic peak, and is then turned back on after the retention time of the sucrose chromatographic peak.

4. The assay method according to claim 3, wherein The standard curve of organic impurities is obtained by the following method: preparing a mixed solution containing each organic impurity, diluting it to obtain 5-7 standard solutions with concentration gradients, measuring the peak area data of each organic impurity in the standard solution of each concentration, repeating 6 times, taking the average value, and plotting the concentration of each organic impurity as the abscissa and the average peak area as the ordinate for linear fitting to obtain the standard curve formula of each organic impurity.

5. The measuring method according to any one of claims 1 or 2, wherein In step (2), The sucrose sample to be tested was burned in a crucible to fully decompose the organic components and measure the mass of inorganic impurities. This was repeated 6 times. The mass of inorganic impurities was divided by the mass of the sucrose sample to be tested and the average was taken to calculate the inorganic impurity content and standard deviation SD of the sucrose sample to be tested. Preferably, the mass of the sucrose sample to be tested is between 200-400 mg; Preferably, the temperature and time of the burning are: heating to 200° C. and holding the temperature for 1 hour, then heating to 300° C. and holding the temperature for 1 hour, then heating to 500° C. and holding the temperature for 5 hours.

6. The measuring method according to any one of claims 1 or 2, wherein In step (3), The sucrose sample to be tested is titrated in a Karl Fischer coulometric titrator to measure the electrolysis charge, which is repeated 6 times. The amount of iodine is calculated according to Faraday's law, which is equal to the amount of water. The amount of water is converted into the mass of water and then divided by the mass of the sucrose sample to be tested and the average is taken to calculate the moisture content and standard deviation SD of the sucrose sample to be tested; Preferably, the mass of the sucrose sample to be tested is between 200-600 mg.

7. The measuring method according to any one of claims 1 or 2, wherein In step (5), Uncertainty of organic impurity content u Org The calculation formula is as follows: ， The standard deviation SD of each organic impurity content in the sucrose sample to be tested is divided by As the uncertainty of its Class A method assessment ( A ); Divide the detection limit of each organic impurity concentration by the concentration of the sucrose sample solution to obtain the detection limit of each organic impurity content in the sucrose sample, and then divide it by , the uncertainty introduced by the detection limit is obtained, which is used as the uncertainty of the Class B method assessment (u B ); Preferably, the uncertainty of the glucose content in the sucrose sample is u Org,1 , the uncertainty of fructose content in the sucrose sample is u Org,2 , the formula for calculating the uncertainty U of sucrose purity is: ， ； Preferably, the method for determining the detection limit of each organic impurity concentration is as follows: when drawing the organic impurity standard curve, the peak area of ​​each organic impurity in the standard solution of each concentration measured is substituted into the standard curve formula to obtain the concentration measurement value of each prepared concentration, and its standard deviation SD is calculated. The concentration preparation value is used as the horizontal coordinate and three times the standard deviation of the concentration measurement value, that is, SD×3, is used as the vertical coordinate. A linear fit is drawn to obtain a fitted straight line, and its intercept is the concentration detection limit of each organic impurity.

8. The assay method according to claim 7, wherein In step (5), Uncertainty of inorganic impurity content u Inorg The calculation formula is as follows: ， The standard deviation SD of the inorganic impurities content in the sucrose sample to be tested is divided by As the uncertainty of the evaluation of the type A method ( A ); Divide the detection limit of inorganic impurities by the average mass of the sucrose sample to obtain the detection limit of inorganic impurities in the sucrose sample, and then divide it by , the uncertainty introduced by the detection limit is obtained, which is used as the uncertainty of the Class B method assessment (u B ); Among them, the determination method of the mass detection limit of inorganic impurities is: using NaCl as the inorganic impurity standard substance to determine the detection limit of the inorganic impurity content determination method: prepare a NaCl aqueous solution with a concentration of 100 mg / L, take 50 μL, 100 μL, 200 μL, 350 μL and 500 μL respectively, of which the mass of sodium chloride is 5 μg, 10 μg, 20 μg, 35 μg and 50 μg respectively, and determine according to the inorganic impurity content determination method to obtain the measured value, repeat 6 times, use the prepared value of NaCl mass as the horizontal axis, and three times the standard deviation of its measured value, that is, SD×3, as the vertical axis, draw a graph for linear fitting, and obtain a fitting straight line, whose intercept is the mass detection limit of inorganic impurities.

9. The assay method according to claim 7, wherein In step (5), Uncertainty of moisture content u W The calculation formula is as follows: ， The standard deviation SD of the average water content in the sucrose sample to be tested is divided by As the uncertainty of the evaluation of the type A method ( A ); Divide the water content detection limit by the average mass of the sucrose sample to obtain the water content detection limit in the sucrose sample, and then divide it by , the uncertainty introduced by the detection limit is obtained, which is used as the uncertainty of the Class B method assessment (u B ); Among them, the method for determining the detection limit of water mass is as follows: use liquid water standard material (GBW 13513) to determine the detection limit of the water content determination method: take volumes of 10 μL, 20 μL, 50 μL, 100 μL and 200 μL of standard material respectively, and its water mass = volume × solution density (0.86 g / mL) × water content of standard material (87 μg / g), and measure according to the water content determination method to obtain the measured value. Repeat 6 times, with the theoretical value of water mass as the horizontal axis and three times the standard deviation of its measured value, that is, SD×3, as the vertical axis, draw a linear fit to obtain a fitting line, and its intercept is the detection limit of water mass.

Images