CN110794052A - Method for measuring adsorption capacity of montmorillonite - Google Patents
Method for measuring adsorption capacity of montmorillonite Download PDFInfo
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- CN110794052A CN110794052A CN201911044805.5A CN201911044805A CN110794052A CN 110794052 A CN110794052 A CN 110794052A CN 201911044805 A CN201911044805 A CN 201911044805A CN 110794052 A CN110794052 A CN 110794052A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
The invention provides a method for measuring montmorillonite adsorption force by using berberine sulfate, which comprises the following steps: preparing a berberine sulfate standard solution, taking montmorillonite fine powder, quantitatively adding a phosphate buffer solution with the pH value of 6.8, quantitatively adding the berberine sulfate standard solution, controlling the ratio of the sampling amount of the montmorillonite to the adding amount of the berberine sulfate to be a certain proportion, fully adsorbing excessive berberine sulfate by the montmorillonite, measuring the residual berberine sulfate by using a high performance liquid chromatography, and calculating the adsorption capacity of the montmorillonite. The method for measuring the adsorption capacity specificity, linearity, accuracy, precision, durability and the like of the medicinal montmorillonite is good, and the method is safe, non-toxic, convenient to purchase and manage, low in price and low in cost.
Description
Technical Field
The invention relates to a method for measuring the adsorption capacity of medicinal montmorillonite, in particular to a method for measuring the adsorption capacity of the montmorillonite on berberine sulfate by using a high performance liquid chromatography, and belongs to the technical field of medicines.
Background
The mineral name of bentonite is montmorillonite (see, Jujiaying, Strin eds., the development and application of bentonite in engineering, 1 st edition of month 07/2003, page 1). Montmorillonite powder is used as an anti-diarrhea drug in the medical field, and is marketed in more than 100 countries and regions around the world. The pharmacological basis of the medicinal montmorillonite is the function of adsorbing pathogenic bacteria, viruses and bacterial toxins, and the magnitude of the adsorption force is a key index of the montmorillonite with low curative effect. Accurate detection of the adsorption force of montmorillonite is a method for controlling the quality of products and ensuring the curative effect.
The method for detecting the montmorillonite adsorption force in the existing national drug standard is to measure the amount of adsorbed strychnine sulfate. The method has the advantages that the adsorption force is measured accurately, but the strychnine sulfate belongs to A-grade organic highly toxic substances, the environment can be polluted when the strychnine sulfate is used, the experimenter can be injured by careless operation in the using process, and great potential safety hazards exist. Moreover, no enterprise produces strychnine sulfate which can meet the experimental requirements in China, and needs to purchase imported reagents, so that the purchase is difficult and the price is high, and the enterprise cost is increased virtually.
In other fields, the content of montmorillonite in the mineral is determined by detecting the methylene blue adsorption capacity of the montmorillonite, which is called blue adsorption amount for short, and the method is widely applied. The method for measuring the blue absorption amount is mainly a halo method in the reported article. The halo method is convenient to operate, simple in instrument and most commonly applied. However, because the liquid drops are taken out from the test paper for multiple times, and the eye is used for observing the green ring to determine the end point, the size of the green ring is greatly different due to human judgment, and the end point judgment is liquid taking observation, the result error is large, and the accuracy and precision of the method are limited.
Zhengxihua notes (see, Zhengxihua main edition, applied technology of bentonite, 1 st edition at 09 month 2001, pages 10 to 11): the adsorption force of bentonite can be divided into three types, namely physical adsorption, chemical adsorption and ion exchange adsorption.
(1) And (4) carrying out physical adsorption. Physical adsorption is produced by intermolecular attraction between the adsorbent and the adsorbate. Physical adsorption is a reversible adsorption process, and the adsorption speed and the desorption speed are in dynamic balance under certain conditions. The main reason for the physical adsorption is that the surface molecules of bentonite have surface energy. The bentonite is highly dispersed in water, so that the physical adsorption phenomenon is very obvious.
(2) And (4) carrying out chemical adsorption. Chemisorption is generated by the chemical bonding forces between the adsorbent and the adsorbate, and chemisorption is generally irreversible.
(3) And (4) ion exchange adsorption. Bentonite mineral crystals are generally negatively charged and adsorb an equivalent amount of oppositely charged cations, which can exchange with cations in solution, a process known as ion exchange adsorption. The ion exchange adsorption is characterized in that: the same numbers are separated from each other, and the isoelectric values are exchanged. The reaction of ion exchange adsorption is reversible, and the rates of adsorption and desorption are influenced by the ion concentration, which follows the law of mass action.
The factors influencing the bentonite mineral adsorption are:
(1) the effect of the bentonite type. The adsorption capacity of the sodium bentonite is obviously stronger than that of other types of bentonite such as calcium bentonite.
(2) The influence of the size of the crushed particle size of the bentonite particles. According to the theory of solid adsorption, the bentonite mineral adsorption capacity after being crushed is obviously improved, and the finer the crushing is, the stronger the adsorption effect is.
(3) Influence of the solution medium. According to the theory of electric double layer, the mineral crystals of bentonite are negatively charged, ion exchange is carried out when the electric double layer is formed, and if the ion concentration in the solution is too high, the electric double layer of bentonite particles is compressed, dispersion and diffusion of bentonite are inhibited, and even the bentonite is coagulated and agglomerated.
In summary, the adsorption force of the montmorillonite comprises three types of physical adsorption, chemical adsorption and ion exchange adsorption, wherein the ion exchange adsorption capacity is not influenced by a set condition and is constant, and the montmorillonite belongs to isoelectric adsorption or stoichiometric reaction; the physical adsorption and chemical adsorption capacity can be determined by measuring the concentration of montmorillonite in the liquid, the dispersion degree of montmorillonite in the liquid, the granularity, the concentration of adsorbed ions, the pH value of the liquid, the temperature of the liquid, the chemical bond force between montmorillonite and adsorbed ions and other factors, and the physical adsorption and chemical adsorption capacity can be changed. An agent is selected to measure the adsorption force of the montmorillonite, and a series of scientific experiments are carried out to determine proper measuring conditions and methods.
The invention patent (CN 102406657 a) discloses (see paragraph 0039 of the specification) that the adsorption force of montmorillonite is derived from its internal charge imbalance. … …, the excess negative charge is compensated by cations adsorbed on the surface of the crystal layer, which are easily exchanged by other cations in the presence of water, known as exchange adsorption. The exchange adsorption has the following characteristics: (1) the same number of ions are exchanged, … … is a stoichiometric reaction, and the mass action law is met; (2) equal electrical quantities (or equal equivalents) are exchanged with each other, … …; (3) the exchange and adsorption of cations is reversible. According to the ion exchange adsorption characteristics of montmorillonite, the adsorbed agent can be a compound capable of dissociating positive ions in water or ethanol solution. These compounds include ammonium acetate, ammonium chloride, methylene blue, quinine sulfate, berberine, betaine, barium chloride, etc., which can dissociate positive ions in water or ethanol solution.
Orefin, norm H, indicates (orefin, usa, lead theory of clay colloid chemistry, 12 months, 1 st edition, 1982, page 169): the adsorption of organic cations is generally not limited by the cation exchange capacity equivalent of the clay. For example, it has been observed that the adsorption of montmorillonite clay to quaternary amine compounds having long chains and three methyl groups is approximately twice and half the cation exchange capacity of the clay.
The invention patent (CN 102406657A) only considers the ion exchange adsorption of montmorillonite, neglects the physical adsorption, the chemical adsorption and other factors influencing the adsorption, and has no general guiding significance. The specification also discloses (see paragraphs 0066 and 0067): experiment seven uses berberine to determine the adsorption force of montmorillonite used in the invention, uses high performance liquid chromatography to determine the adsorption force of montmorillonite to berberine sulfate, calculates the cation exchange capacity of montmorillonite to be 135mmol/100g, namely the adsorption force of montmorillonite used is 135mmol/100 g. The adsorption force of the montmorillonite comprises physical adsorption, chemical adsorption and ion exchange adsorption, and the physical adsorption and the chemical adsorption are influenced by various factors such as the concentration of the montmorillonite in the liquid, the dispersion degree granularity of the montmorillonite in the liquid, the concentration of adsorbed ions, the pH value of the liquid, the temperature of the liquid, the chemical bond force between the montmorillonite and the adsorbed ions and the like when the physical adsorption and the chemical adsorption are measured. The patent only discloses that the adsorption force of montmorillonite can be measured by berberine, and no specific measurement method is disclosed.
Disclosure of Invention
The invention aims to overcome the defects of the existing method for measuring the adsorption capacity of the medicinal montmorillonite and provide a novel method for measuring the adsorption capacity of the medicinal montmorillonite. The method for measuring the adsorption capacity of the medicinal montmorillonite is good in specificity, linearity, accuracy, precision, reproducibility, durability and the like, safe, nontoxic, convenient to purchase and manage, low in price and low in cost.
The purpose of the invention is realized by the following technical scheme.
A method for measuring the adsorption capacity of montmorillonite is characterized by comprising the following steps: the method for measuring the amount of berberine sulfate adsorbed by montmorillonite by high performance liquid chromatography comprises the following steps: preparing a berberine sulfate standard solution with the concentration of 1.5% -2.5%, taking 0.15-0.25 g of montmorillonite fine powder with the granularity of less than 45 mu m, quantitatively adding a phosphate buffer solution with the pH value of 6.8, quantitatively adding the berberine sulfate standard solution, controlling the ratio of the sampling amount of montmorillonite to the adding amount of berberine sulfate to be 0.8-1.2, fully adsorbing excessive berberine sulfate by montmorillonite, measuring the residual berberine sulfate by using a high performance liquid chromatography, and then calculating the adsorption force of the montmorillonite;
the particle size and the measuring method of the montmorillonite fine powder are as follows: taking 10g of montmorillonite, adding 500ml of water, stirring for 15 minutes at 5000 r/min, wetting a 45-micron pore size drug sieve which is dried to constant weight at 105 ℃ with water, pouring the stirred content into the drug sieve, washing the drug sieve with water until no suspension spots exist, drying the drug sieve for 3 hours at 105 ℃, and weighing. The weight of the unscreened granules should not exceed 1%.
The invention relates to a method for measuring the adsorption capacity of montmorillonite, which is characterized by comprising the following steps: the ratio of montmorillonite sampling amount to berberine sulfate addition amount is 1.
The invention relates to a method for measuring the adsorption capacity of montmorillonite, which is characterized by comprising the following steps: the high performance liquid chromatography for measuring berberine sulfate has the following chromatographic conditions and system applicability: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.02 to 0.05mol/l potassium dihydrogen phosphate solution =20 to 40: 80 to 60 adjusted to a pH value of 3.0 to 4.0 with phosphoric acid as a mobile phase, or acetonitrile-0.1 to 0.2g sodium dodecylsulfonate/100 ml 0.1 to 0.2% phosphoric acid solution =40 to 60: 60 to 40 as a mobile phase, or acetonitrile-phosphate buffer solution =40 to 60: 60 to 40 as a mobile phase, wherein the phosphate buffer solution is prepared by 0.05mol/l potassium dihydrogen phosphate: 0.05mol/l sodium heptanesulfonate =1:1, 0.2% triethylamine is contained, the pH value is adjusted to 3.0-4.0 by phosphoric acid, or acetonitrile-glacial acetic acid-triethylamine-water =34:1: 64 is used as a mobile phase, the detection wavelength is 230-420 nm, and the number of theoretical plates is not less than 3000 according to the berberine peak.
The invention relates to a method for measuring the adsorption capacity of montmorillonite, which is characterized by comprising the following steps:
1) preparing a standard berberine sulfate solution with the concentration of 2.0 percent: precisely weighing about 2.0g of berberine sulfate, placing into a 100ml measuring flask, adding appropriate amount of water, heating in water bath to dissolve, cooling, diluting with water to scale, and shaking to obtain standard berberine sulfate solution with concentration of 2.0%;
2) and (3) measuring the adsorption force:
according to high performance liquid chromatography 'Chinese pharmacopoeia' 2015 edition four-part general rules 0512 test
Octadecylsilane chemically bonded silica is used as a filler for chromatographic conditions and system applicability; acetonitrile-0.05 mol/l potassium dihydrogen phosphate solution = 25:75 adjusted to pH 3.0 with phosphoric acid as a mobile phase, or acetonitrile-0.1% sodium dodecylsulfate solution = 50: 50 per 100ml, or acetonitrile-phosphate buffer solution =40:60 as a mobile phase, wherein the phosphate buffer solution is prepared by 0.05mol/l potassium dihydrogen phosphate: 0.05mol/l sodium heptanesulfonate =1:1, containing 0.2% triethylamine, adjusted to pH 3.0 by phosphoric acid, or acetonitrile-glacial acetic acid-triethylamine water =34:1: 64 as a mobile phase, and the detection wavelength is 265nm or 350nm, and the theoretical plate number is not less than 3000 according to berberine peak;
preparation of control solutions: taking a berberine sulfate standard substance, precisely weighing, and preparing into a reference substance solution containing 20 μ g/ml berberine sulfate per 1ml by using methanol or flowing phase;
preparing a test solution: taking 0.2g of montmorillonite fine powder, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of phosphate buffer solution with the pH value of 6.8, shaking up, precisely adding 10ml of berberine sulfate standard solution with the concentration of 2.0%, placing in a water bath at 37 ℃, shaking for 1 hour, filtering, precisely measuring 10ml of subsequent filtrate, placing in a measuring flask with 250ml, diluting to scale with a mobile phase, shaking up, precisely measuring 5ml, placing in a measuring flask with 25ml, diluting to scale with the mobile phase, and shaking up to serve as a sample solution;
the determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
The advantageous effects of the present invention are further illustrated by the following experimental examples and methodological studies. Experimental examples and methodological studies are intended to further illustrate the beneficial effects of the present invention and are not intended to limit the invention.
Test example: the determination methodology of the adsorption force of the medicinal montmorillonite is verified
1. Test article and reagent
And (3) testing the sample: montmorillonite (batch number: 1607008, Shandong HongJitang pharmaceutical group GmbH)
Berberine sulfate: (batch No. 36XZM, Taixi Ai-Shanghai Kangcheng Industrial development Co., Ltd.)
2. Test methods and methodological validation
Referring to the downward adsorption force of montmorillonite in the second part of the year 2015 of Chinese pharmacopoeia, the method is tentatively determined as follows:
1) preparing a standard berberine sulfate solution with the concentration of 2.0 percent:
precisely weighing about 2.0g of berberine sulfate, placing into a 100ml measuring flask, adding appropriate amount of water, heating in water bath to dissolve, cooling, diluting with water to scale, and shaking to obtain standard berberine sulfate solution with concentration of 2.0%.
2) And (3) measuring the adsorption force:
performing high performance liquid chromatography (0512 in the four ministry of communications in the 2015 edition of Chinese pharmacopoeia)
Octadecylsilane chemically bonded silica is used as a filler for chromatographic conditions and system applicability; acetonitrile-0.02 mol/L potassium dihydrogen phosphate (pH value adjusted to 3.0 by phosphoric acid) (25: 75) is used as mobile phase, the detection wavelength is 265nm, and the number of theoretical plates is not less than 3000 according to berberine peak.
Preparation of reference substance solution taking berberine sulfate standard substance, precisely weighing, and preparing reference substance solution containing berberine sulfate 20 μ g/ml per 1ml with methanol.
Preparing a test solution, namely preparing 0.2g of montmorillonite, precisely weighing, placing the montmorillonite into a conical flask with a plug, precisely adding 10ml of phosphate buffer solution (pH 6.8), shaking up, precisely adding 10ml of 2.0% berberine sulfate standard solution, placing the solution into a water bath at 37 ℃, shaking up for 1 hour, filtering, precisely weighing 10ml of subsequent filtrate, placing the subsequent filtrate into a measuring flask with 250ml, diluting the subsequent filtrate to the scale with methanol, shaking up, precisely weighing 5ml, placing the subsequent filtrate into a measuring flask with 25ml, diluting the subsequent filtrate to the scale with methanol, and shaking up to obtain the test solution;
the determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.
Optimization of parameters in preparation process of test sample
2.1.1 adsorption reaction time Curve
Taking 7 parts of sample, testing according to the above method, setting reaction time at 1min, 5min, 10min, 30min, 60min, 90min and 120min, and measuring adsorption time curve, wherein the results are as follows (the adsorption force curve of different reaction times is shown in figure 1):
TABLE 1 results of adsorption force at different reaction times
| Reaction time (min) | 0 | 1 | 5 | 10 | 30 | 60 | 90 | 120 |
| Adsorption capacity (g/g) | 0 | 0.363 | 0.411 | 0.419 | 0.425 | 0.432 | 0.433 | 0.431 |
The results showed that the montmorillonite adsorbed berberine sulfate, rapidly reaching about 93% of the total adsorption within 5min, after which the adsorption rate slowed, slowly increased and finally reached equilibrium, so the tentative reaction time was 60 minutes.
2.1.2 relationship between berberine sulfate concentration and adsorption force
Taking 7 parts of sample, precisely adding 19ml, 18ml, 16ml, 12ml, 10ml, 8ml and 5ml of phosphate buffer solution (pH6.8) and 1ml, 2ml, 4ml, 8ml, 10ml, 12ml and 15ml of berberine sulfate solution (20 mg/ml) according to the test of the measuring method of the adsorption force, and measuring the relation between the adsorption force and the reaction concentration, wherein the results are as follows (the relation curve between the concentration of the berberine sulfate solution and the adsorption force is shown in figure 2):
TABLE 2 relationship between initial reaction concentration and adsorption force
| Initial concentration (mg/ml) | 1 | 2 | 4 | 8 | 10 | 12 | 15 |
| Adsorption force g/g | 0.401 | 0.405 | 0.412 | 0.430 | 0.433 | 0.435 | 0.437 |
The test result shows that the adsorption force result is increased along with the increase of the added amount of the berberine sulfate, and the adsorption force result is balanced when the added amount is 10ml, so the added amount of the tentative berberine sulfate standard solution is 10 ml.
2.1.3 method for measuring adsorption force
From the above examination, the provisional adsorption force measurement method is as follows:
1) preparing a standard berberine sulfate solution with the concentration of 2.0 percent:
precisely weighing about 2.0g of berberine sulfate, placing into a 100ml measuring flask, adding appropriate amount of water, heating in water bath to dissolve, cooling, diluting with water to scale, and shaking to obtain standard berberine sulfate solution with concentration of 2.0%.
2) And (3) measuring the adsorption force:
performing high performance liquid chromatography (0512 in the four ministry of communications in the 2015 edition of Chinese pharmacopoeia)
Octadecylsilane chemically bonded silica is used as a filler for chromatographic conditions and system applicability; acetonitrile-0.02 mol/L potassium dihydrogen phosphate (pH value adjusted to 3.0 by phosphoric acid) (25: 75) is used as mobile phase, the detection wavelength is 265nm, and the number of theoretical plates is not less than 3000 according to berberine peak.
Preparation of reference substance solution taking berberine sulfate standard substance, precisely weighing, and preparing reference substance solution containing berberine sulfate 20 μ g/ml per 1ml with methanol.
Preparing a test solution, namely preparing 0.2g of montmorillonite, precisely weighing, placing the montmorillonite into a conical flask with a plug, precisely adding 10ml of phosphate buffer solution (pH 6.8), shaking up, precisely adding 10ml of 2.0% berberine sulfate standard solution, placing the solution into a water bath at 37 ℃, shaking up for 1 hour, filtering, precisely weighing 10ml of subsequent filtrate, placing the subsequent filtrate into a measuring flask with 250ml, diluting the subsequent filtrate to the scale with methanol, shaking up, precisely weighing 5ml, placing the subsequent filtrate into a measuring flask with 25ml, diluting the subsequent filtrate to the scale with methanol, and shaking up to obtain the test solution;
the determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.
Methodology validation
The method is verified by referring to appendix 9101 of the four parts of the year 2015 of Chinese pharmacopoeia.
2.2.1 specificity
Preparing a test solution: a test solution was prepared from 0.20g of montmorillonite by the above-mentioned method for measuring adsorption force.
Preparation of negative solution: taking 0.20g of montmorillonite, precisely weighing, placing in a conical flask with a stopper, precisely adding 10ml of phosphate buffer solution (pH 6.8), precisely adding 10ml of water, and preparing a negative solution by the same method from 'placing in a water bath at 37 ℃ and shaking for 1 hour' according to the preparation method of the test solution.
The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
As a result: the specificity test result shows that the corresponding chromatographic peak of the test solution is detected within the retention time of the chromatographic peak of the reference solution, the negative solution has no chromatographic peak within the corresponding retention time, and other chromatographic peaks in the test solution are well separated from each target peak. The result shows that the negative has no interference to the determination, and the method has strong specificity in determination.
2.2.2 Linear relationship investigation test
Taking berberine sulfate reference solution, preparing into linear solutions containing 4, 10, 20, 40, 100 μ g/1 ml with methanol respectively, precisely sucking 10 μ l of linear solutions respectively, injecting into liquid chromatograph, and measuring.
TABLE 3 results of the Linear relationship test
| Concentration (μ g/ml) | 4.044 | 10.11 | 20.22 | 40.44 | 101.1 |
| Peak area | 190703 | 482544 | 1005696 | 1945438 | 4970938 |
Taking the concentration as an abscissa and the peak area as an ordinate, a linear regression equation y = 49204 x-12073 is obtained, and a correlation coefficient r =0.9999 (see fig. 3 for a linear relation graph).
The result shows that the berberine sulfate concentration is in a good linear relation with the peak area within the range of 4-100 mug/ml.
2.2.3 precision
2.2.3.1 repeatability
6 parts of the sample are taken, and a repeatability test is carried out according to an adsorption force measuring method, and the result is shown in a table 4.
TABLE 4 results of the repeatability tests
| Test No | 1 | 2 | 3 | 4 | 5 | 6 | Average | RSD% |
| Adsorption capacity (g/g) | 0.436 | 0.427 | 0.431 | 0.425 | 0.432 | 0.435 | 0.431 | 1.01% |
The result RSD% is less than 2.0%, which shows that the method has good repeatability.
2.2.3.2 different analysts
The test samples were taken and tested by different testers, and the results are shown in Table 5.
TABLE 5 precision of different experimenters
The result RSD% is less than 2.0%, which shows that the precision of different testers is good.
2.2.3.3 different analytical instruments
The test samples were taken and measured by different instruments, and the results are shown in Table 6.
TABLE 6 precision of different analytical instruments
The result RSD% is less than 2.0%, which shows that the precision of different analytical instruments is good.
2.2.3.4 different dates of analysis
The test samples were taken and measured on different days, and the results are shown in Table 7.
TABLE 7 results of different dates of analysis
| Date | Date 1 | | Date 3 | Average | RSD% |
| Adsorption capacity (g/g) | 0.432 | 0.434 | 0.429 | 0.431 | 0.53% |
The result RSD% is less than 2.0%, indicating that the precision is good at different analysis dates.
2.2.4 accuracy
Preparation of 1% berberine sulfate standard solution: taking 1.0g of berberine sulfate with known content, precisely weighing, and dissolving with water to 100ml to obtain standard berberine sulfate solution with concentration of 1%.
Taking 6 parts of conical flask with plug, respectively adding 10ml of phosphate buffer solution (pH 6.8) precisely, then adding 10ml of the berberine sulfate standard solution with the concentration of 1% precisely, adding 0.1g of berberine sulfate reference substance precisely, shaking up to dissolve, taking 0.20g of montmorillonite with known adsorption capacity of 0.431g/g, precisely weighing 6 parts, placing in the conical flask, and measuring according to the adsorption capacity measuring method from the beginning of 'placing in water bath at 37 ℃ and shaking for 1 hour', the results are as follows:
TABLE 8 recovery results
The result shows that the method has good accuracy.
2.2.5 durability
2.2.5.1 solution stability
Taking the test solution, standing at room temperature for 8 hours, precisely sucking 10 μ l of the test solution every 2 hours, injecting into a liquid chromatograph, and recording the chromatogram. The results are as follows.
TABLE 9 stability test results of solutions
| Time of | 0 | 2 | 4 | 6 | 8 hours | Average | RSD% |
| Peak area | 874561 | 892211 | 886376 | 887106 | 881673 | 884385.4 | 0.75 |
The results show that the solution is stable within 8 hours
2.2.5.2 different flow phase ratio
The adsorption force was measured by taking a sample and using acetonitrile-0.02 mol/L potassium dihydrogen phosphate (pH 3.0 adjusted with phosphoric acid) (22: 78), acetonitrile-0.02 mol/L potassium dihydrogen phosphate (pH 3.0 adjusted with phosphoric acid) (25: 75) and acetonitrile-0.02 mol/L potassium dihydrogen phosphate (pH 3.0 adjusted with phosphoric acid) (28: 72) as mobile phases, and the results were as follows:
TABLE 10 durability results for different mobile phase ratios
| Flow phase ratio | 22:78 | 25:75 | 28:72 | Average | RSD% |
| Adsorption capacity (g/g) | 0.435 | 0.432 | 0.438 | 0.435 | 0.69 |
The result shows that the method is used for measuring the potassium dihydrogen phosphate (the pH value is adjusted to be 3.0) with acetonitrile-0.02 mol/L (22-28: 78-72) and the result is accurate and reliable.
2.2.6 range
The amount of the test sample in the range of 80-120% of the test amount of the product is taken, and the test is carried out according to the method for measuring the adsorption force, and the result is shown in the table:
TABLE 11 Range of test results
The result shows that the sampling is carried out within the range of +/-20% of the testing amount, and the determination result is reliable.
The result of the above methodology verification shows that the method for determining the adsorption capacity of the medicinal montmorillonite is good in specificity, linearity, accuracy, precision, durability and the like, safe, nontoxic, convenient to purchase and manage, low in price and low in cost.
The method for measuring the adsorption capacity of the medicinal montmorillonite can be used for montmorillonite raw materials and montmorillonite preparations. The montmorillonite preparation comprises clinically acceptable dosage forms such as powder, tablets, capsules, granules, suspension, gels, dispersible tablets and the like.
Drawings
Figure 1, adsorption force versus time curve. It is shown that the montmorillonite rapidly adsorbed berberine sulfate to about 93% of the total adsorption within 5min, after which the adsorption rate slowed, slowly increased and finally reached equilibrium, so the tentative reaction time was 60 min.
FIG. 2 is a graph showing the relationship between the concentration of berberine sulfate solution and the adsorption force. As shown in the figure, the adsorption capacity is increased with the increase of the added berberine sulfate, and when the amount of the berberine sulfate solution added with 2.0 percent is more than 10ml, the adsorption capacity still continues to increase, but the increase of the adsorption capacity is slowed down, so that the added amount of the berberine sulfate standard solution is tentatively 10 ml.
Fig. 3, a linear range inspection diagram. The figure shows that the berberine sulfate concentration is in a good linear relation with the peak area within the range of 4-100 mu g/ml.
The invention is further illustrated by the following specific examples.
Detailed Description
Example 1: determination of adsorption force of medicinal montmorillonite
And (3) testing the sample: montmorillonite (batch number: 1611015, Shandong HongJitang pharmaceutical group GmbH)
Berberine sulfate: (batch No. 36XZM, Taixi Ai-Shanghai Kangcheng Industrial development Co., Ltd.)
Measurement method and results:
1) and (3) calibrating berberine sulfate content:
performing high performance liquid chromatography (0512 in the four ministry of communications in the 2015 edition of Chinese pharmacopoeia)
Octadecylsilane chemically bonded silica is used as a filler for chromatographic conditions and system applicability; acetonitrile-0.1% phosphoric acid solution (0.1 g sodium dodecyl sulfate per 100 ml) = 50: 50 is the mobile phase and the inspection wavelength is 350 nm. The number of theoretical plates is not less than 3000 calculated according to berberine peak.
Preparation of reference substance solution taking berberine sulfate standard substance, precisely weighing, and preparing reference substance solution containing berberine sulfate 20 μ g/ml per 1ml with methanol.
Preparing a test solution, precisely weighing about 20mg of the product, dissolving with methanol, and metering to 100ml, precisely weighing 5ml from the product, and metering to 50ml with methanol to obtain the test solution.
The determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.
2) Preparing a standard berberine sulfate solution with the concentration of 2.0 percent:
precisely weighing about 2.0g of berberine sulfate, placing into a 100ml measuring flask, adding appropriate amount of water, heating in water bath to dissolve, cooling, diluting with water to scale, and shaking to obtain standard berberine sulfate solution with concentration of 2.0%;
3) and (3) measuring the adsorption force:
performing high performance liquid chromatography (0512 in the four ministry of communications in the 2015 edition of Chinese pharmacopoeia)
Octadecylsilane chemically bonded silica is used as a filler for chromatographic conditions and system applicability; acetonitrile-0.1% phosphoric acid solution (0.1 g sodium dodecyl sulfate per 100 ml) = 50: 50 is the mobile phase and the inspection wavelength is 350 nm. The number of theoretical plates is not less than 3000 calculated according to berberine peak.
Preparation of reference substance solution taking berberine sulfate standard substance, precisely weighing, and preparing reference substance solution containing berberine sulfate 20 μ g/ml per 1ml with methanol.
Preparing a test solution, namely preparing 0.2g of montmorillonite, precisely weighing, placing the montmorillonite into a conical flask with a plug, precisely adding 10ml of phosphate buffer solution (pH 6.8), shaking up, precisely adding 10ml of 2.0% berberine sulfate standard solution, placing the solution into a water bath at 37 ℃, shaking up for 1 hour, filtering, precisely weighing 10ml of subsequent filtrate, placing the subsequent filtrate into a measuring flask with 250ml, diluting the subsequent filtrate to the scale with methanol, shaking up, precisely weighing 5ml, placing the subsequent filtrate into a measuring flask with 25ml, diluting the subsequent filtrate to the scale with methanol, and shaking up to obtain the test solution;
the determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.
As a result: the montmorillonite of the batch adsorbs berberine sulfate 0.428 g/g.
Example 2 method for measuring adsorption force of montmorillonite
Prescription: each bag contains 3g of montmorillonite, 0.749g of glucose, 0.007g of saccharin sodium and 0.004g of vanillin.
Montmorillonite powder: (batch number: 1608004)
Negative preparation: according to the proportion of the prescription, taking 7.49 g of glucose, 0.07 g of saccharin sodium and 0.04g of vanillin as auxiliary materials, and uniformly mixing to obtain the negative preparation.
Method for measuring adsorption force and results:
performing high performance liquid chromatography (0512 in the four ministry of communications in the 2015 edition of Chinese pharmacopoeia)
Octadecylsilane chemically bonded silica is used as a filler under chromatographic conditions and system applicability; the examination wavelength was 265nm using acetonitrile-phosphate buffer (0.05 mol/l potassium dihydrogen phosphate: 0.05mol/l sodium heptanesulfonate =1:1, containing 0.2% triethylamine, pH adjusted to 3.0 with phosphoric acid) =40:60 as mobile phase. The number of theoretical plates is not less than 3000 calculated according to berberine peaks;
preparing a reference substance solution, namely precisely weighing a berberine sulfate standard substance, and forming the reference substance solution with 20 mu g/ml of berberine sulfate per 1ml by using a mobile phase;
preparing a test solution, namely preparing 0.20g of montmorillonite powder, precisely weighing, placing the montmorillonite powder into a conical flask with a plug, precisely adding 10ml of phosphate buffer solution (pH 6.8), shaking up, precisely adding 10ml of 2.0% berberine sulfate standard solution, placing the solution into a water bath at 37 ℃, shaking up for 1 hour, filtering, precisely weighing 10ml of subsequent filtrate, placing the subsequent filtrate into a 250ml measuring flask, diluting the subsequent filtrate to a scale by using a mobile phase, shaking up, precisely weighing 5ml, placing the subsequent filtrate into a 25ml measuring flask, diluting the subsequent filtrate to a scale by using the mobile phase, and shaking up to obtain the test solution;
the determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.
Test of influence of auxiliary materials on adsorption force measurement: 0.05g of negative preparation mixed with auxiliary materials according to the proportion of the prescription is taken and tested according to the method.
As a result: the montmorillonite powder has berberine sulfate adsorption rate of 0.422g/g, and adjuvants do not adsorb berberine sulfate, and has no influence on measurement.
Example 3 method for measuring adsorption force of montmorillonite dispersible tablet
Prescription: each 1000 tablets contains 1000g of montmorillonite, 7g of glucose, 3g of saccharin sodium, 4g of vanillin, 100g of microcrystalline cellulose and 30g of sodium carboxymethyl starch.
Montmorillonite dispersible tablets: (batch number: 1611001)
Negative preparation: according to the proportion of the prescription, taking 7g of glucose, 3g of saccharin sodium, 4g of vanillin, 100g of micro-fine cellulose and 30g of sodium carboxymethyl starch as auxiliary materials, and uniformly mixing to obtain the negative preparation.
Method for measuring adsorption force and results:
performing high performance liquid chromatography (0512 in the four ministry of communications in the 2015 edition of Chinese pharmacopoeia)
Octadecylsilane chemically bonded silica is used as a filler under chromatographic conditions and system applicability; acetonitrile-glacial acetic acid-triethylamine-water =34:1:1:64 as mobile phase, and the checking wavelength is 350 nm. The number of theoretical plates is not less than 3000 calculated according to berberine peaks;
preparing a reference substance solution, namely precisely weighing a berberine sulfate standard substance, and preparing the reference substance solution with 20 microgram/ml of berberine sulfate per 1ml by using a mobile phase;
preparing a test solution, namely grinding montmorillonite dispersible tablets, weighing 0.3g, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of phosphate buffer solution (pH 6.8), shaking uniformly, precisely adding 10ml of 2.0% berberine sulfate standard solution, placing in a water bath at 37 ℃, shaking for 1 hour, filtering, precisely measuring 10ml of subsequent filtrate, placing in a 250ml measuring flask, diluting to a scale with a mobile phase, shaking uniformly, precisely measuring 5ml, placing in a 25ml measuring flask, diluting to a scale with a mobile phase, shaking uniformly, and taking as a test solution;
the determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.
Test of influence of auxiliary materials on adsorption force measurement: 0.03g of negative preparation mixed with auxiliary materials according to the proportion of the prescription is taken and tested according to the method.
As a result: the montmorillonite dispersible tablets have berberine sulfate adsorption rate of 0.429g/g, and the adjuvants do not adsorb berberine sulfate, and have no influence on the determination.
Example 4 method for measuring adsorption force of montmorillonite suspension
Prescription: each 1000ml of the product contains 150g of montmorillonite and 1g of ethylparaben.
Montmorillonite suspension: (batch number: 1702004)
Negative preparation: according to the proportion of the prescription, 1g of ethylparaben is taken, 1000ml of water is added, and the negative preparation is obtained after uniform mixing.
Method for measuring adsorption force and results:
performing high performance liquid chromatography (0512 in the four ministry of communications in the 2015 edition of Chinese pharmacopoeia)
Octadecylsilane chemically bonded silica is used as a filler under chromatographic conditions and system applicability; acetonitrile-0.1% phosphoric acid solution (0.1 g of sodium dodecylsulfonate per 100 ml) = 50: 50 is the mobile phase and the inspection wavelength is 250 nm. The number of theoretical plates is not less than 3000 calculated according to berberine peaks;
preparing a reference substance solution, namely precisely weighing a berberine sulfate standard substance, and preparing the reference substance solution with 20 mu g/ml of berberine sulfate per 1ml of methanol;
preparing a test solution, namely taking 10ml of montmorillonite suspension, evaporating to dryness in a water bath, grinding, weighing 0.20g, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of phosphate buffer solution (pH 6.8), shaking uniformly, precisely adding 10ml of berberine sulfate standard solution with the concentration of 1.8%, placing in a water bath at 37 ℃, shaking for 1 hour, filtering, precisely weighing 10ml of subsequent filtrate, placing in a 250ml measuring flask, diluting with methanol to scale, shaking uniformly, precisely weighing 5ml, placing in a 25ml measuring flask, diluting with methanol to scale, shaking uniformly to serve as the test solution;
the determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.
As a result: the adsorption capacity of the montmorillonite suspension in the batch is 0.414 g/g.
Claims (5)
1. A method for measuring the adsorption capacity of montmorillonite is characterized by comprising the following steps: the method for measuring the amount of berberine sulfate adsorbed by montmorillonite by high performance liquid chromatography comprises the following steps: preparing a berberine sulfate standard solution with the concentration of 1.5% -2.5%, taking 0.15-0.25 g of montmorillonite fine powder with the granularity of less than 45 mu m, quantitatively adding a phosphate buffer solution with the pH value of 6.8, quantitatively adding the berberine sulfate standard solution, controlling the ratio of the sampling amount of montmorillonite to the adding amount of berberine sulfate to be 0.8-1.2, fully adsorbing excessive berberine sulfate by the montmorillonite, measuring the residual berberine sulfate by using a high performance liquid chromatography, and calculating the adsorption capacity of the montmorillonite.
2. The method for measuring the adsorption ability of montmorillonite according to claim 1, characterized in that: the adsorption capacity of montmorillonite was determined using berberine sulphate.
3. The method for measuring the adsorption ability of montmorillonite according to claim 1, characterized in that: the ratio of montmorillonite sampling amount to berberine sulfate addition amount is 1.
4. The method for measuring the adsorption ability of montmorillonite according to claim 1, characterized in that: the high performance liquid chromatography for measuring berberine sulfate has the following chromatographic conditions and system applicability: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.02 to 0.05mol/l potassium dihydrogen phosphate solution =20 to 40: 80 to 60 adjusted to a pH value of 3.0 to 4.0 with phosphoric acid as a mobile phase, or acetonitrile-0.1 to 0.2g sodium dodecylsulfonate/100 ml 0.1 to 0.2% phosphoric acid solution =40 to 60: 60 to 40 as a mobile phase, or acetonitrile-phosphate buffer solution =40 to 60: 60 to 40 as a mobile phase, wherein the phosphate buffer solution is prepared by 0.05mol/l potassium dihydrogen phosphate: 0.05mol/l sodium heptanesulfonate =1:1, 0.2% triethylamine is contained, the pH value is adjusted to 3.0-4.0 by phosphoric acid, or acetonitrile-glacial acetic acid-triethylamine-water =34:1: 64 is used as a mobile phase, the detection wavelength is 230-420 nm, and the number of theoretical plates is not less than 3000 according to the berberine peak.
5. The method for measuring the adsorption ability of montmorillonite according to claim 4, characterized in that:
1) preparing a standard berberine sulfate solution with the concentration of 2.0 percent: precisely weighing about 2.0g of berberine sulfate, placing into a 100ml measuring flask, adding appropriate amount of water, heating in water bath to dissolve, cooling, diluting with water to scale, and shaking to obtain standard berberine sulfate solution with concentration of 2.0%;
2) and (3) measuring the adsorption force:
according to high performance liquid chromatography 'Chinese pharmacopoeia' 2015 edition four-part general rules 0512 test
Octadecylsilane chemically bonded silica is used as a filler for chromatographic conditions and system applicability; acetonitrile-0.05 mol/l potassium dihydrogen phosphate solution = 25:75 adjusted to pH 3.0 with phosphoric acid as a mobile phase, or acetonitrile-0.1% sodium dodecylsulfate solution = 50: 50 per 100ml, or acetonitrile-phosphate buffer solution =40:60 as a mobile phase, wherein the phosphate buffer solution is prepared by 0.05mol/l potassium dihydrogen phosphate: 0.05mol/l sodium heptanesulfonate =1:1, containing 0.2% triethylamine, adjusted to pH 3.0 by phosphoric acid, or acetonitrile-glacial acetic acid-triethylamine water =34:1: 64 as a mobile phase, and the detection wavelength is 265nm or 350nm, and the theoretical plate number is not less than 3000 according to berberine peak;
preparation of control solutions: taking a berberine sulfate standard substance, precisely weighing, and preparing into a reference substance solution containing 20 μ g/ml berberine sulfate per 1ml by using methanol or flowing phase;
preparing a test solution: taking 0.2g of montmorillonite fine powder with the particle size of less than 45 mu m, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of phosphate buffer solution with the pH value of 6.8, shaking up, precisely adding 10ml of berberine sulfate standard solution with the concentration of 2.0%, placing in a water bath at 37 ℃, shaking up for 1 hour, filtering, precisely measuring 10ml of subsequent filtrate, placing in a 250ml measuring flask, diluting to scale with a mobile phase, shaking up, precisely measuring 5ml, placing in a 25ml measuring flask, diluting to scale with the mobile phase, shaking up to scale, and taking as a sample solution;
the determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
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