Evaluation method of multi-peak granularity particle counting standard substance

Abstract

The invention discloses an evaluation method of a multi-peak particle size particle counting standard substance, and belongs to the technical field of particle size analysis. According to the method, the quantity value of the mixed standard substance can be accurately evaluated, and the uncertainty can be reliably calculated. The method comprises the following steps: pre-treating narrow-distribution particle counting standard substances with various particle sizes, namely sub-standard substances; independently evaluating the magnitude of each sub-standard substance; obtaining a proportion result of the plurality of sub-standard substances, and mixing the sub-standard substances to obtain a multi-peak granularity particle counting standard substance, namely a mixed standard substance; and calculating the quantity value of the mixed standard substance according to the fixed value result and the proportion result of the sub-standard substance. In addition, according to the method, the uncertainty of the quantity value of the mixed standard substance can be calculated by evaluating the uncertainty introduced in the pre-treatment process of the standard substance, the valuing process of the standard substance before mixing and the sampling and mixing process. The method is simple and convenient to operate, high in accuracy and suitable for calibrating the particle counting instrument, and ensures the accuracy of a detection result.

Description

Technical Field

[0001] The present invention belongs to the technical field of particle size analysis, and in particular relates to a particle counting particle standard material, and more particularly to an evaluation method for a multi-peak particle size particle counting standard material. Background Art

[0002] Instruments that can be used for particle counting, such as particle analyzers, Coulter particle counters, and particle size analyzers, all need to be calibrated regularly with particle counting particle standard materials. Therefore, the accurate determination of particle counting particle standard materials becomes the key to the accuracy of the particle counting instrument test results.

[0003] Traditional particle counting particle standard materials are mainly monodisperse particle size particles. In recent years, with the rapid development of science and technology, scientific research and production in related fields have had more and more demands for mixed particle standard materials with multiple particle size counts. The mixed standard material can have a multi-peak distribution of particle size. In the use of standard materials, not only can the performance of the instrument be judged according to the measured particle number concentration, but also the resolution of the particle size analyzer can be quickly and intuitively judged according to the separation between the peaks of the particle number concentration distribution curve. There is no standard material in my country that can identify the particle size resolution of particle counting instruments. In 2017, the International Committee for Standardization has set the method of separately determining the values ​​of samples of different particle sizes and then weighing and mixing to prepare standard materials as a recommended standard (ISO / TS 14411-1:2017). The present invention aims to provide a method for evaluating the value of multi-peak particle counting standard materials to meet the needs of high-precision particle counting. Summary of the invention

[0004] The object of the present invention is to provide a method for evaluating a multi-modal particle size particle counting standard material, which method can accurately evaluate the value of a mixed standard material and reliably calculate the uncertainty.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The evaluation methods include:

[0007] (1) Evaluate the value of the mixed standard;

[0008] (2) Calculate the uncertainty of the value of the mixed standard;

[0009] The step (1) of evaluating the amount of the mixed standard comprises the following steps:

[0010] 1) Pre-treat the narrow distribution particle counting standard materials of various particle sizes, i.e., the separated standards respectively;

[0011] 2) Evaluate the values ​​of the standard components before mixing one by one to obtain the fixed value results of the standard components;

[0012] 3) obtaining the ratio results between the plurality of the sub-standard materials, and mixing the sub-standard materials to obtain a multi-modal particle size counting standard material, i.e., a mixed standard material;

[0013] 4) The value of the mixed standard is calculated based on the set value results and proportion results of the separate standard.

[0014] Furthermore, the method for pre-treating the separated standard substances in step 1) includes:

[0015] When the particle concentration of the standard substance is ≤2000 / mL, ultrasonic treatment is used;

[0016] When the particle concentration of the standard substance is >2000 / mL, dilute it to a particle concentration of ≤2000 / mL, and then use ultrasonic treatment to disperse the particles.

[0017] Furthermore, the determination result of the sub-standard substance in step 2) is obtained by one of the methods in patent CN108387503A and the counting method of directly observing the blood cell counting plate under a microscope.

[0018] Furthermore, in step 3), the standard substance is transferred by a single-standard pipette, and mixed and fixed to volume by a volumetric flask to obtain a mixed standard substance.

[0019] Furthermore, the uncertainty of the value of the mixed standard in step (2) includes:

[0020] Uncertainty is introduced in the process of pretreatment of the standard substances, uncertainty is introduced in the process of determining the value of the standard substances before mixing, and uncertainty is introduced in the process of sampling and mixing.

[0021] Furthermore, the uncertainty introduced in the pretreatment process of the standard substance is the uncertainty of the dilution process, which specifically includes:

[0022] The uncertainty is introduced by the pipette volume calibration, the uncertainty is introduced by the repeatability of the pipette liquid collection operation, the uncertainty is introduced by the volume calibration of the volumetric flask, the uncertainty is introduced by the repeatability of the volumetric flask volume setting operation and the uncertainty is introduced by the inhomogeneity of the solution in the volumetric flask.

[0023] Furthermore, the uncertainty introduced by the pipette volume correction and the uncertainty introduced by the volumetric flask volume correction need to be provided by the manufacturer respectively, the uncertainty introduced by the repeatability of the pipette liquid collection operation and the uncertainty introduced by the repeatability of the volumetric flask volume setting operation need to be measured no less than 3 times to calculate the relative standard deviation, and the uncertainty introduced by the non-uniformity of the solution in the volumetric flask needs to be sampled no less than 10 times at different heights of the volumetric flask after sufficient stirring to calculate the relative standard deviation.

[0024] Furthermore, the uncertainty introduced in the process of determining the value of the standard substance before mixing is calculated according to the method specified in JJF1343-2012 "General principles and statistical principles for the determination of the value of reference materials".

[0025] Furthermore, the sampling and mixing process introduces uncertainties including:

[0026] Uncertainty is introduced by volume calibration of single-label pipettes, uncertainty is introduced by the temperature of single-label pipettes and solutions being different from the calibration temperature, uncertainty is introduced by the repeatability of liquid collection operation of single-label pipettes, uncertainty is introduced by volume calibration of volumetric flasks, uncertainty is introduced by the temperature of volumetric flasks and solutions being different from the calibration temperature, and uncertainty is introduced by the repeatability of volumetric flask volume adjustment operation.

[0027] Beneficial effects of the present invention:

[0028] (1) The evaluation method of the present invention can accurately evaluate the value of a mixed standard; the method pre-treats narrow distribution particle counting standard materials of various particle sizes, i.e., fractional standards; evaluates the value of each fractional standard separately; obtains the ratio results between the plurality of fractional standards, and mixes the fractional standards to obtain a multi-peak particle counting standard material, i.e., a mixed standard; calculates the value of the mixed standard based on the determination results and the ratio results of the fractional standards; the determination method is accurate, reliable, and practical.

[0029] (2) The evaluation method of the present invention can also reliably calculate the uncertainty of the value of the mixed standard; by evaluating the uncertainty introduced by the pretreatment process of the separate standard, the determination process of the separate standard before mixing, and the sampling and mixing process, the uncertainty of the value of the mixed standard is calculated. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described below in conjunction with the accompanying drawings.

[0031] Figure 1 It is a flow chart of a method for evaluating the value of a multi-modal particle counting standard material according to an embodiment of the present invention;

[0032] Figure 2 It is a flow chart of a solution for calculating the uncertainty of the value of a multi-modal particle counting standard material according to an embodiment of the present invention;

[0033] Figure 3 This is a microscope photograph of the multimodal particle size counting standard material of Example 1 of the present invention;

[0034] Figure 4 This is a microscope photograph of the multimodal particle size counting standard material of Example 3 of the present invention. DETAILED DESCRIPTION

[0035] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Example 1

[0037] This embodiment provides an evaluation method for a multimodal particle size particle counting standard material, the evaluation method comprising:

[0038] (1) Evaluate the value of the mixed standard. The flow chart of the value evaluation method is as follows: Figure 1 As shown;

[0039] (2) Calculate the uncertainty of the value of the mixed standard. The flowchart of the uncertainty calculation scheme is as follows: Figure 2 As shown;

[0040] The specific steps include:

[0041] The solutions of three narrow distribution particle counting standard substances (i.e., standard substances) of particle size are:

[0042] The nominal value is 4.5×10 5 The particle counting particle standard material is a monodisperse polystyrene latex particle with an average particle size of 0.8 μm (1-1 standard);

[0043] The nominal value is 5.0×10 5 The particle counting particle standard material is a monodisperse polystyrene latex particle with an average particle size of 2 μm (1-2 point standard);

[0044] The nominal value is 2.5×10 5 The particle counting particle standard material is a monodisperse polystyrene latex particle with an average particle size of 5 μm (1-3 points standard).

[0045] (1) Evaluating the value of the mixed standard obtained by mixing the above three sub-standards, comprising the following steps:

[0046] 1) Pre-treat the above three kinds of standard substances separately:

[0047] Dilute each standard substance 1000 times: use an accurately calibrated 1mL pipette to transfer 1mL to a 1000mL volumetric flask and dilute to the mark with ultrapure water; use ultrasonic treatment before measurement to disperse the particles in the diluted solution to be tested;

[0048] 2) Evaluate the values ​​of the standard components before mixing one by one to obtain the fixed value results of the standard components;

[0049] This embodiment uses the method in patent CN108387503A to obtain the fixed value result of the sub-standard object, and the specific steps are as follows:

[0050] Pre-treating the mesh filter membrane: soaking the mesh filter membrane with glycerol; the mesh filter membrane in this embodiment is a mixed cellulose ester mesh filter membrane produced by Beijing Beihua Liming Membrane Separation Technology Co., Ltd., with a diameter of 25 mm, and 55 small squares on each mesh filter membrane. The pore size of the filter membrane used for different fractions is different. For the 1-1 fraction, the pore size of the filter membrane is 0.45 μm, for the 1-2 fraction, the pore size of the filter membrane is 1 μm, and for the 1-3 fraction, the pore size of the filter membrane is 5 μm;

[0051] Prepare a sample by intercepting a certain volume of particles in the pretreated test solution using a pretreated mesh filter membrane: place the pretreated mesh filter membrane on a suction filter; use an accurately calibrated 1 mL pipette to draw 1 mL of the pretreated test solution and drop it on the pretreated mesh filter membrane; remove the diluent: place the mesh filter membrane that intercepts the particles in the pretreated test solution on a glass slide;

[0052] Microscope counting of samples: Figure 3 The figure shows a microscope photograph of the multi-peak particle size particle counting standard material of this embodiment; a BX53 microscope (OLYMPUS, Japan) is used, and a micrometer ruler verified by the National Institute of Metrology is used to calibrate the eyepiece scale (at a certain magnification), or to calibrate the "pixel length measurement standard" for automatic sampling; the magnification of the eyepiece is ×10, the magnification of the objective lens is ×100, and the number of particles trapped on the grid filter is counted grid by grid, and the total number of particles trapped on the grid filter is accumulated;

[0053] The particle concentration of the solution to be tested is calculated based on the total number of particles retained on the mesh filter membrane; the particle concentration of the solution to be tested is calculated according to the following calculation formula:

[0054] Particle concentration of the test solution = total number of particles trapped on the mesh filter membrane ÷ volume of the test solution after pretreatment through the mesh filter membrane × dilution multiple of the test solution;

[0055] After multiple measurements, the average particle concentration was taken as the fixed value result, and the fixed value result of the 1-1 standard was 4.53×10 5 / mL; the result of the 1-2 standard is 4.95×10 5 / mL; the result of the standard substance of 1-3 points is 2.36×10 5 Pieces / mL;

[0056] 3) Mix the three standard samples to obtain a mixed standard:

[0057] Take 10 mL of the standard substance for 1-1, 10 mL of the standard substance for 1-2, and 10 mL of the standard substance for 1-3;

[0058] 4) Calculate the value of the mixed standard substance based on the above-mentioned determination results and proportion results:

[0059] That is, the number of monodisperse polystyrene latex particles with an average particle size of 0.8 μm is 1.51×10 5 The counting result of monodisperse polystyrene latex particles with an average particle size of 2 μm was 1.65×10 5 The counting result of monodisperse polystyrene latex particles with an average particle size of 5 μm was 0.79×10 5 Pieces / mL.

[0060] (2) Calculate the uncertainty of the value of the mixed standard substance. The uncertainty of the value of the mixed standard substance includes:

[0061] 1) Uncertainty is introduced in the pretreatment process of the standard substance, including the following parts:

[0062] The uncertainty introduced by the pipette volume correction and the uncertainty introduced by the volumetric flask volume correction are provided by the manufacturer respectively. The uncertainty introduced by the repeatability of the pipette liquid taking operation and the repeatability of the volumetric flask volume setting operation are measured three times to calculate the relative standard deviation. The uncertainty introduced by the inhomogeneity of the solution in the volumetric flask is calculated by sampling 10 times at different heights of the volumetric flask after sufficient stirring.

[0063] The calculation shows that the uncertainty introduced by the pretreatment process of the 1-1 standard substance is 1.04%; the uncertainty introduced by the pretreatment process of the 1-2 standard substance is 1.02%; the uncertainty introduced by the pretreatment process of the 1-3 standard substance is 1.04%;

[0064] 2) Uncertainty is introduced in the process of determining the value of the standard substance before mixing, and is calculated according to the method specified in JJF1343-2012 "General Principles and Statistical Principles for Determining the Value of Standard Materials":

[0065] The result of determination of the monodisperse polystyrene latex particles with an average particle size of 0.8 μm is 4.53×10 5 The relative standard deviation of the three measurements was 1.14%, and the uncertainty introduced in the determination process of the 1-1 standard was calculated to be 3.3%. The particle concentration of the particle counting standard material was tested using a calibrated insoluble particle analyzer (GWF-5JA, Tianhe, Tianjin), and the result was 4.48×10 5 Pieces / mL;

[0066] The result of determination of the monodisperse polystyrene latex particles with an average particle size of 2 μm is 4.95×10 5 The relative standard deviation of the three measurements was 1.49%, and the uncertainty introduced in the determination process of the 1-2 point standard was calculated to be 4.3%. The particle concentration of the particle counting particle standard material was tested using a calibrated insoluble particle analyzer (GWF-5JA, Tianhe, Tianjin), and the result was 4.98×10 5 Pieces / mL;

[0067] The result of determination of the monodisperse polystyrene latex particles with an average particle size of 5 μm is 2.36×10 5 The relative standard deviation of the three measurements was 1.27%, and the uncertainty introduced in the determination process of the 1-3 points standard was calculated to be 5.0%. The particle concentration of the particle counting particle standard material was tested using a calibrated insoluble particle analyzer (GWF-5JA, Tianhe, Tianjin), and the result was 2.38×10 6 Pieces / mL;

[0068] The test results are close to the set value results, indicating that the set value method is accurate, reliable and feasible;

[0069] 3) The sampling and mixing process introduces uncertainty, including the following:

[0070] The combined relative standard uncertainty of the uncertainty introduced by the single-standard pipette volume calibration, the uncertainty introduced by the temperature difference between the single-standard pipette and the solution and the calibration temperature, and the uncertainty introduced by the repeatability of the single-standard pipette liquid extraction operation is 0.0983%, and the combined relative standard uncertainty of the uncertainty introduced by the volume calibration of the volumetric flask, the uncertainty introduced by the temperature difference between the volumetric flask and the solution and the calibration temperature, and the repeatability of the volumetric flask volume determination operation is 0.0377%;

[0071] According to the method specified in formula (71) and formula (72) in Chapter 8 of JJF1343-2012 "General Principles and Statistical Principles for the Determination of Reference Materials", the expanded uncertainty is obtained by adding the square root of each uncertainty and multiplying it by the expansion factor k. The expanded uncertainty is the combination of each uncertainty. The calculation formula for the expanded uncertainty (U) is as follows:

[0072]

[0073] The expanded uncertainty of the 1-1 standard substance was calculated to be 6.8%;

[0074] Similarly, the expanded uncertainty of the 1-2 point standard is 8.6%; the expanded uncertainty of the 1-3 point standard is 10.0%.

[0075] Example 2

[0076] This embodiment provides an evaluation method for a multimodal particle size particle counting standard material, the evaluation method comprising:

[0077] (1) Evaluate the value of the mixed standard. The flow chart of the value evaluation method is as follows: Figure 1 As shown;

[0078] (2) Calculate the uncertainty of the value of the mixed standard. The flowchart of the uncertainty calculation scheme is as follows: Figure 2 As shown;

[0079] The specific steps include:

[0080] The solutions of two narrow distribution particle counting standard substances (i.e., standard substances) of particle size are:

[0081] The nominal value is 3.6×10 5 The particle counting particle standard material is a monodisperse polystyrene latex particle with an average particle size of 5 μm (2-1 standard);

[0082] The nominal value is 2.4×10 5 The particle counting particle standard material is a monodisperse polystyrene latex particle with an average particle size of 10 μm (2-2 point standard).

[0083] (1) Evaluating the value of the mixed standard obtained by mixing the two separate standard substances, comprising the following steps:

[0084] 1) Pre-treat the above two kinds of standard substances separately:

[0085] Dilute each standard substance 1000 times: use an accurately calibrated 1mL pipette to transfer 1mL to a 1000mL volumetric flask and dilute to the mark with ultrapure water; use ultrasonic treatment before measurement to disperse the particles in the diluted solution to be tested;

[0086] 2) Evaluate the values ​​of the standard components before mixing one by one to obtain the fixed value results of the standard components;

[0087] This embodiment uses the method in patent CN108387503A to obtain the fixed value result of the sub-standard object, and the specific steps are as follows:

[0088] The uncertainty of the dilution process includes the uncertainty introduced by the pipette volume calibration, the uncertainty introduced by the repeatability of the pipette liquid collection operation, the uncertainty introduced by the volume calibration of the volumetric flask, the uncertainty introduced by the repeatability of the volumetric flask volume setting operation, and the uncertainty introduced by the inhomogeneity of the solution in the volumetric flask.

[0089] Pre-treating the mesh filter membrane: soaking the mesh filter membrane with glycerol; the mesh filter membrane in this embodiment is a mixed cellulose ester mesh filter membrane produced by Beijing Beihua Liming Membrane Separation Technology Co., Ltd., with a diameter of 25 mm, and 55 small squares on each mesh filter membrane. The pore size of the filter membrane used for different separation targets is different. For the 2-1 separation target, the pore size of the filter membrane is 1 μm, and for the 2-2 separation target, the pore size of the filter membrane is 5 μm;

[0090] Prepare a sample by intercepting a certain volume of particles in the pretreated test solution using a pretreated mesh filter membrane: place the pretreated mesh filter membrane on a suction filter; use an accurately calibrated 1 mL pipette to draw 1 mL of the pretreated test solution and drop it on the pretreated mesh filter membrane; remove the diluent: place the mesh filter membrane that intercepts the particles in the pretreated test solution on a glass slide;

[0091] Microscope counting of samples: use a BX53 microscope (OLYMPUS, Japan), and use a micrometer ruler verified by the National Institute of Metrology to calibrate the eyepiece scale (at a certain magnification), or calibrate the "pixel length measurement standard" for automatic sampling; the magnification of the eyepiece is ×10, the magnification of the objective lens is ×40, and the number of particles trapped on the grid filter is counted grid by grid, and the total number of particles trapped on the grid filter is accumulated;

[0092] Calculate the particle concentration of the solution to be tested based on the total number of particles trapped on the mesh filter. Calculate the particle concentration of the solution to be tested according to the following calculation formula:

[0093] The particle concentration of the test solution = the total number of particles retained on the mesh filter membrane ÷ the volume of the pretreated test solution after passing through the mesh filter membrane × the dilution multiple of the test solution.

[0094] After multiple measurements, the average particle concentration was taken as the fixed value result, and the fixed value result of the 2-1 standard was 3.53×10 5 / mL; the result of the 2-2 standard is 2.55×10 5 Pieces / mL;

[0095] 3) Mix the two separate standard samples to obtain a mixed standard:

[0096] Take 5 mL of the 2-1 fraction and 15 mL of the 2-2 fraction;

[0097] 4) Calculate the value of the mixed standard substance based on the above-mentioned determination results and proportion results:

[0098] That is, the number of monodisperse polystyrene latex particles with an average particle size of 5 μm is 0.88×10 5 The counting result of monodisperse polystyrene latex particles with an average particle size of 10 μm was 1.91×10 5Pieces / mL.

[0099] (2) Calculate the uncertainty of the value of the mixed standard substance. The uncertainty of the value of the mixed standard substance includes:

[0100] 1) Uncertainty is introduced in the pretreatment process of the standard substance, including the following parts:

[0101] The uncertainty introduced by the pipette volume correction and the uncertainty introduced by the volumetric flask volume correction are provided by the manufacturer respectively. The uncertainty introduced by the repeatability of the pipette liquid taking operation and the repeatability of the volumetric flask volume setting operation are measured three times to calculate the relative standard deviation. The uncertainty introduced by the inhomogeneity of the solution in the volumetric flask is calculated by sampling 10 times at different heights of the volumetric flask after sufficient stirring.

[0102] The calculation shows that the uncertainty introduced by the pretreatment process of the 2-1 standard substance is 1.06%; the uncertainty introduced by the pretreatment process of the 2-2 standard substance is 1.01%;

[0103] 2) Uncertainty is introduced in the process of determining the value of the standard substance before mixing, and is calculated according to the method specified in JJF1343-2012 "General Principles and Statistical Principles for Determining the Value of Standard Materials":

[0104] The result of determination of the monodisperse polystyrene latex particles with an average particle size of 5 μm is 3.53×10 5 The relative standard deviation of the three measurements was 1.26%, and the uncertainty introduced in the determination process of the 2-1 standard was calculated to be 3.5%. The particle concentration of the particle counting standard material was tested using a calibrated insoluble particle analyzer (GWF-5JA, Tianhe, Tianjin), and the result was 3.48×10 5 Pieces / mL;

[0105] The result of determination of the monodisperse polystyrene latex particles with an average particle size of 10 μm is 2.55×10 5 The relative standard deviation of the three measurements was 1.49%, and the uncertainty introduced in the determination process of the 2-2 standard was calculated to be 4.8%. The particle concentration of the particle counting particle standard material was tested using a calibrated insoluble particle analyzer (GWF-5JA, Tianhe, Tianjin), and the result was 2.48×10 5 Pieces / mL;

[0106] The test results are close to the set value results, indicating that the set value method is accurate, reliable and feasible;

[0107] 3) The sampling and mixing process introduces uncertainty, including the following:

[0108] The combined relative standard uncertainty of the uncertainty introduced by the single-standard pipette volume calibration, the uncertainty introduced by the temperature difference between the single-standard pipette and the solution and the calibration temperature, and the uncertainty introduced by the repeatability of the single-standard pipette liquid extraction operation is 0.0966%, and the combined relative standard uncertainty of the uncertainty introduced by the volume calibration of the volumetric flask, the uncertainty introduced by the temperature difference between the volumetric flask and the solution and the calibration temperature, and the repeatability of the volumetric flask volume determination operation is 0.0353%;

[0109] According to the method specified in formula (71) and formula (72) in Chapter 8 of JJF1343-2012 "General Principles and Statistical Principles for the Determination of Reference Materials", the expanded uncertainty is obtained by adding the square root of each uncertainty and multiplying it by the expansion factor k. The expanded uncertainty is the combination of each uncertainty. The calculation formula for the expanded uncertainty (U) is as follows:

[0110]

[0111] The expanded uncertainty of the 2-1 standard substance was calculated to be 7.1%;

[0112] Similarly, the expanded uncertainty of the 2-2 standard is 9.7%.

[0113] Example 3

[0114] This embodiment provides an evaluation method for a multimodal particle size particle counting standard material, the evaluation method comprising:

[0115] (1) Evaluate the value of the mixed standard. The flow chart of the value evaluation method is as follows: Figure 1 As shown;

[0116] (2) Calculate the uncertainty of the value of the mixed standard. The flowchart of the uncertainty calculation scheme is as follows: Figure 2 As shown;

[0117] The specific steps include:

[0118] The solutions of two narrow distribution particle counting standard substances (i.e., standard substances) of particle size are:

[0119] The nominal value is 3.0×10 6 The particle counting particle standard material is a monodisperse polystyrene latex particle with an average particle size of 1 μm (3-1 standard);

[0120] The nominal value is 1.0×10 6 The particle counting particle standard material is a monodisperse polystyrene latex particle with an average particle size of 5 μm (3-2 point standard).

[0121] (1) Evaluating the value of the mixed standard obtained by mixing the two separate standard substances, comprising the following steps:

[0122] 1) Pre-treat the above two kinds of standard substances separately:

[0123] Dilute each standard substance 10000 times: use an accurately calibrated 1mL pipette to transfer 1mL to a 1000mL volumetric flask, dilute to the mark with ultrapure water, then use an accurately calibrated 1mL pipette to transfer 1mL to a 10mL volumetric flask, dilute to the mark with ultrapure water; use ultrasonic treatment before measurement to disperse the particles in the diluted solution to be tested;

[0124] 2) Evaluate the values ​​of the standard components before mixing one by one to obtain the fixed value results of the standard components;

[0125] This embodiment uses a microscope to directly observe the counting method of the blood cell counting plate to obtain the fixed value result of the sub-standard object. The specific steps are as follows:

[0126] Microscope counting of samples: Figure 4 This is a microscope photo of the multi-peak particle counting standard material of the embodiment of the present invention; a BX53 microscope (Japan OLYMPUS) is used, and the eyepiece scale is calibrated with a micrometer ruler verified by the China Institute of Metrology (under a certain magnification), or the automatic sampling "pixel length measurement standard" is calibrated; the number of particles is counted using a microscope. Since the particle size of latex particles has a certain distribution width, in order to accurately and consistently count the number of particles and to avoid the interference of "background" tiny particles in the dispersant, when counting particles in a standard suspension with a nominal particle size of 1 um, the average particle size is used. The size at 50% of the particle size is the upper and lower limits of the number of particles to be counted; the number average distribution curve of the particles is actually observed, and there are very few particles outside this limit, which will not cause obvious interference; in order to eliminate the influence of small sampling volume and poor representativeness of microscope measurement, the number of particles falling into the microscope field of view should be greater than 100; samples are taken from different parts of the sample bottle, and more than 10 slides are prepared to increase the number of parallel measurements and the cumulative number of measured particles (about 1500-2000 particles); the accuracy and reliability of the measurement are improved, and the average value of the counts of each sample measured in parallel is used as the standard value of the particle count;

[0127] Use a micrometer ruler to accurately measure the side length of the grid on the blood cell counting plate, and calculate the grid area to be 1.003mm 2 ; The depth of the pool under the grid was accurately measured and calculated by the China Institute of Metrology, which was 95.5um. The volume of the selected XB-K-25 blood cell counting plate sampling pool was calculated to be 0.0958mm and the depth was 95.5um. The volume of the selected blood cell counting plate sampling pool was calculated;

[0128] In order to overcome the influence of poor representativeness of the suspension, for each sample that needs to be fixed, the suspension is removed from different parts, and the sampling is carried out in parallel for more than 10 times, so that the total count accumulates to 1000-2000 particles; the uncertainty caused by statistical calculation is about 4%; the nominal value of the sampling volume of the blood cell counting plate is 0.1mm; due to the original design accuracy and production quality of the plate, the pool depth is uneven, the two supporting surfaces of the cover glass are not at the same height, and there are large differences between the plates, so the sampling volume of each blood cell counting plate used needs to be calibrated, and the actual calibration value is used for the particle concentration calculation; in this study, the selected XB-K-25 blood cell counting plate was calibrated, and a calibrated plate was repeatedly used in actual measurement to eliminate the systematic error between plates. The calibration value of the center grid area of ​​the plate is 1.003mm 2 , the relative error of calibration is 0.1%; the pool depth is determined by the multi-point measurement (6-point) method at the length of the National Institute of Metrology of China, and the average pool depth is 95.5μm, the detection limit is 0.1um, the relative measurement error is 0.9%, and the sampling volume is 95.8μL calculated by multiplying the pool area by the pool depth. The relative error of calibration is 0.1%; the particle count in the above grid divided by the volume size is the particle count concentration value;

[0129] After multiple measurements, the average particle concentration was taken as the fixed value result, and the fixed value result of the 3-1 standard was 3.13×10 6 / mL; the result of the 3-2 standard is 1.05×10 6 Pieces / mL;

[0130] 3) Mix the two separate standard samples to obtain a mixed standard:

[0131] Take 1 mL of the 3-1 fraction and 4 mL of the 3-2 fraction;

[0132] 4) Calculate the value of the mixed standard substance based on the above-mentioned determination results and proportion results:

[0133] That is, the number of monodisperse polystyrene latex particles with an average particle size of 1 μm is 0.63×10 6 The counting result of monodisperse polystyrene latex particles with an average particle size of 5 μm was 0.84×10 6 Pieces / mL.

[0134] (2) Calculate the uncertainty of the value of the mixed standard substance. The uncertainty of the value of the mixed standard substance includes:

[0135] 1) Uncertainty is introduced in the pretreatment process of the standard substance, including the following parts:

[0136] The uncertainty introduced by the pipette volume correction and the uncertainty introduced by the volumetric flask volume correction are provided by the manufacturer respectively. The uncertainty introduced by the repeatability of the pipette liquid taking operation and the repeatability of the volumetric flask volume setting operation are measured three times to calculate the relative standard deviation. The uncertainty introduced by the inhomogeneity of the solution in the volumetric flask is calculated by sampling 10 times at different heights of the volumetric flask after sufficient stirring.

[0137] The uncertainty introduced by the pretreatment process of the 3-1 standard substance is calculated to be 0.89%; the uncertainty introduced by the pretreatment process of the 3-2 standard substance is calculated to be 0.98%;

[0138] 2) The process of determining the value of the standard substance before mixing introduces uncertainty.

[0139] The uncertainty of the direct value of the microscope is synthesized by the uncertainty of the microscope measurement and the uncertainty introduced by the pipette; it is calculated according to the method specified in JJF1343-2012 "General Principles and Statistical Principles for the Determination of Reference Materials":

[0140] The result of determination of the monodisperse polystyrene latex particles with an average particle size of 1 μm is 3.13×10 6 The relative standard deviation of 10 measurements was 2.26%, and the uncertainty introduced in the determination process of the 3-1 standard was calculated to be 4.5%. The particle concentration of the particle counting standard material was tested using a calibrated insoluble particle analyzer (GWF-5JA, Tianhe, Tianjin), and the result was 3.18×10 6 Pieces / mL;

[0141] The result of determination of the monodisperse polystyrene latex particles with an average particle size of 5 μm is 1.05×10 6 The relative standard deviation of 10 measurements was 2.49%, and the uncertainty introduced in the determination process of the 3-2 standard substance was calculated to be 5.8%. The particle concentration of the particle counting particle standard substance was tested using a calibrated insoluble particle analyzer (GWF-5JA, Tianhe, Tianjin), and the result was 2.56×10 6 Pieces / mL;

[0142] The test results are close to the set value results, indicating that the set value method is accurate, reliable and feasible;

[0143] 3) Uncertainties introduced by the sampling and mixing process include:

[0144] The combined relative standard uncertainty of the uncertainty introduced by the single-standard pipette volume calibration, the uncertainty introduced by the temperature difference between the single-standard pipette and the solution and the calibration temperature, and the uncertainty introduced by the repeatability of the single-standard pipette liquid extraction operation is 0.1001%, and the combined relative standard uncertainty of the uncertainty introduced by the volume calibration of the volumetric flask, the uncertainty introduced by the temperature difference between the volumetric flask and the solution and the calibration temperature, and the repeatability of the volumetric flask volume determination operation is 0.0227%;

[0145] According to the method specified in formula (71) and formula (72) in Chapter 8 of JJF1343-2012 "General Principles and Statistical Principles for the Determination of Reference Materials", the expanded uncertainty is obtained by adding the square root of each uncertainty and multiplying it by the expansion factor k. The expanded uncertainty is the combination of each uncertainty. The calculation formula for the expanded uncertainty (U) is as follows:

[0146]

[0147] The expanded uncertainty of the 3-1 standard substance is calculated to be 9.1%;

[0148] Similarly, the expanded uncertainty of the 3-2 point standard is 11.7%.

[0149] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0150] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for evaluating a multimodal particle size counting reference material, characterized in that: The evaluation methods include: (1) Evaluate the value of the mixed standard; (2) Calculate the uncertainty of the value of the mixed standard; The step (1) of evaluating the amount of the mixed standard comprises the following steps: 1) Pre-treat the narrow distribution particle counting standard materials of various particle sizes, i.e., the separated standards respectively; 2) Evaluate the values ​​of the standard components before mixing one by one to obtain the fixed value results of the standard components; 3) obtaining the ratio results between the plurality of the sub-standard materials, and mixing the sub-standard materials to obtain a multi-modal particle size counting standard material, i.e., a mixed standard material; 4) The value of the mixed standard is calculated based on the set value results and proportion results of the separate standard.

2. The method for evaluating a multimodal particle counting reference material according to claim 1, characterized in that: The method for pre-treating the separated standard substances in step 1) includes: When the particle concentration of the standard substance is ≤2000 / mL, ultrasonic treatment is used; When the particle concentration of the standard substance is >2000 / mL, dilute it to a particle concentration of ≤2000 / mL, and then use ultrasonic treatment to disperse the particles.

3. The method for evaluating a multimodal particle counting reference material according to claim 1, characterized in that: The determination result of the standard substance in step 2) is obtained by one of the methods in patent CN108387503A and the counting method of directly observing the blood cell counting plate under a microscope.

4. The method for evaluating a multimodal particle counting reference material according to claim 1, characterized in that: In step 3), the standard substance is transferred by a single-standard pipette, and mixed and fixed to volume by a volumetric flask to obtain a mixed standard substance.

5. The method for evaluating a multimodal particle counting reference material according to claim 1, characterized in that: The uncertainty of the value of the mixed standard in step (2) includes: Uncertainty is introduced in the process of pretreatment of the standard substances, uncertainty is introduced in the process of determining the value of the standard substances before mixing, and uncertainty is introduced in the process of sampling and mixing.

6. The method for evaluating a multimodal particle counting reference material according to claim 5, characterized in that: The uncertainty introduced in the pretreatment process of the standard substance includes the uncertainty of the dilution process, and the uncertainty of the dilution process specifically includes: The uncertainty is introduced by the pipette volume calibration, the uncertainty is introduced by the repeatability of the pipette liquid collection operation, the uncertainty is introduced by the volume calibration of the volumetric flask, the uncertainty is introduced by the repeatability of the volumetric flask volume setting operation and the uncertainty is introduced by the inhomogeneity of the solution in the volumetric flask.

7. The method for evaluating a multimodal particle counting reference material according to claim 5, characterized in that: The uncertainty introduced in the process of determining the value of the standard substance before mixing is calculated according to the method specified in JJF1343-2012 "General principles and statistical principles for the determination of the value of reference materials".

8. The method for evaluating a multimodal particle counting reference material according to claim 5, characterized in that: The sampling and mixing process introduces uncertainties including: Uncertainty is introduced by volume calibration of single-label pipettes, uncertainty is introduced by the temperature of single-label pipettes and solutions being different from the calibration temperature, uncertainty is introduced by the repeatability of liquid collection operation of single-label pipettes, uncertainty is introduced by volume calibration of volumetric flasks, uncertainty is introduced by the temperature of volumetric flasks and solutions being different from the calibration temperature, and uncertainty is introduced by the repeatability of volumetric flask volume adjustment operation.

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