CN111830144A - High performance liquid detection method for surfactant triton X-100 - Google Patents
High performance liquid detection method for surfactant triton X-100 Download PDFInfo
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- CN111830144A CN111830144A CN201911064164.XA CN201911064164A CN111830144A CN 111830144 A CN111830144 A CN 111830144A CN 201911064164 A CN201911064164 A CN 201911064164A CN 111830144 A CN111830144 A CN 111830144A
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- G—PHYSICS
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- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a high performance liquid detection method of triton X-100, which adopts a reversed phase C18 chromatographic column and an ELSD detector, wherein a mobile phase is a mixed solution of methanol and water, and isocratic elution is adopted. The method has the advantages that sample introduction is 5-20 ul, triton X-100 can be effectively detected, the lowest detection concentration is 0.5 mu g/ml, an HPLC spectrum base line is stable, and drifting is avoided. The method can be used for measuring the content of the triton X-100, has the advantages of high sensitivity, good linear relation, good reproducibility, high precision and high accuracy, and has important research value in the aspects of industrial analysis, product formula research and the like.
Description
Technical Field
The invention relates to the technical field of industrial analysis, in particular to a high-efficiency liquid phase detection method of triton X-100.
Background
Triton X-100 (English: Triton X-100) is a nonionic surfactant that can lyse cellular membranes in immunocytochemistry by solubilizing lipid components on the cellular membranes, thereby allowing antibodies to enter the cells and bind to antigens. With a hydrophilic polyethylene glycol chain (n is usually 9 or 10) and a lipophilic hydrocarbon radical (4- (1,1,3, 3-tetramethylbutyl) -phenyl radical). Triton X-100 is not dissociated in water, has high stability in solution, is not easily influenced by strong electrolyte inorganic salts, and can be combined with lipids such as phospholipid in a biological membrane to form a soluble compound; the hydrophobic end can also be combined with the hydrophobic region of the membrane protein to form a complex, and the complex is dissolved in the solution.
As a surfactant with high utilization rate, the detection of the triton X-100 can be carried out by liquid chromatography and then an ultraviolet detector, but the detection sensitivity of the method is not high, the operation is more complicated and the accuracy is general because the molecules of the triton X-100 absorb less in the ultraviolet wavelength range.
Disclosure of Invention
The invention aims to provide a surfactant triton X-100 high-efficiency liquid phase detection method which has the advantages of good separation effect, high precision and accuracy, safe and simple operation, convenient and quick treatment and suitability for high-throughput screening and accurate quantification, so as to solve the problems of low detection sensitivity, complex operation and general accuracy in the conventional detection method.
In order to solve the problems, the invention provides a high performance liquid detection method of surfactant triton X-100, which is characterized by comprising the following steps:
(1) preparation of control solutions:
precisely weighing a proper amount of Triton X-100 reference substances, dissolving by using a mobile phase and fixing the volume to obtain a plurality of reference substance solutions with a certain concentration gradient;
(2) preparation of a test solution:
precisely weighing a proper amount of a test sample, ultrasonically dissolving the test sample by using a mobile phase, and fixing the volume, and filtering the test sample by using a 0.22 mu m filter membrane if the solution is turbid to obtain a test sample solution;
(3) and (3) determination:
and performing liquid chromatography determination on the plurality of reference substance solutions and the test solution, recording a chromatogram, preparing a linear correlation working curve according to the spectrum data and the concentration data of the plurality of reference substance solutions, substituting the linear correlation working curve into the spectrum data of the test sample to calculate and obtain the concentration of the test solution, and completing the determination of the content of the triton X-100.
Preferably, in the step (1), the number of the control solutions is 5, and the concentrations of the control solutions are 20, 40, 60, 80 and 100 μ g/ml in sequence.
Preferably, in the step (3), the chromatographic conditions measured are as follows:
the chromatographic column adopts a reversed phase C18 chromatographic column;
the detector adopts an ELSD detector;
the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is methanol; mobile phase B is water; and the volume ratio of methanol to water is 90: 10.
preferably, the length of the chromatographic column is 250 mm.
Preferably, the temperature of the chromatographic column is controlled to be 30-40 ℃.
Preferably, the ELSD detector has an atomization temperature of 40-45 ℃, an evaporation temperature of 55-60 ℃ and a carrier gas pressure of 25-30 psi.
Preferably, the flow rate of the mobile phase is 1.0 ml/min.
Preferably, the sample injection amount is 5 to 20 μ l.
More preferably, in the step (3), the flow rate is 1.0ml/min, the column temperature is 35 ℃, the ELSD detector atomization temperature is 45 ℃, the ELSD detector evaporation temperature is 60 ℃, the detector carrier gas purge pressure is 26psi, and the sample injection amount is 20 μ L.
The technical scheme has the following beneficial technical effects:
1. the method disclosed by the invention has high sensitivity in measuring the triton X-100, and the lowest detection limit is 0.5 mu g/ml.
2. The method is used for measuring the content of the triton X-100, has good linear relation and high reproducibility, realizes the aim of rapid separation and detection, and has important research value in industrial product analysis and formula research.
3. The invention is improved and optimized aiming at the detection method in the prior art, and compared with the method in the prior art, the detection method has the advantages of high sensitivity, good separation effect, high precision and accuracy, safe and simple operation, convenient and quick treatment, and suitability for high-throughput screening and accurate quantification. The method has important research value for industrial product analysis and formula research.
Drawings
FIGS. 1-5 are HPLC profiles of Triton X-100 control using the method of the present invention;
FIG. 6 is a graph of the operation of Triton X-100 plotted using the method of the present invention.
Detailed description of the preferred embodiments
The invention is further described with reference to specific examples.
The following examples are not provided to limit the scope of the present invention, nor are the steps described to limit the order of execution. Modifications of the invention which are obvious to those skilled in the art in view of the prior art are also within the scope of the invention as claimed.
The inventor of the application obtains a high performance liquid detection method of the triton X-100 through extensive research and a large number of experiments, the method provides a chromatographic analysis method capable of effectively detecting the triton X-100, the sensitivity is high, and the minimum detection limit is 0.5 mug/ml.
The high performance liquid detection method of triton X-100 adopted by the inventor has the following chromatographic conditions:
the chromatographic column adopts a reversed phase C18 chromatographic column, such as a Waters Xbridge chromatographic column using octadecylsilane chemically bonded silica as a filler;
the detector adopts an evaporative light scattering ELSD detector;
the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is methanol; mobile phase B is water; the volume ratio of methanol to water is 90: 10;
in a preferred embodiment of the invention, the column length of the chromatography column is 250 mm.
In a preferred embodiment of the present invention, the flow rate is 0.8 to 1.2ml/min, more preferably 1.0 ml/min.
In a preferred embodiment of the present invention, the column temperature is controlled to be 30 to 40 ℃, and more preferably 35 ℃.
In a preferred embodiment of the present invention, the ELSD detector has an atomization temperature of 40-45 deg.C, more preferably 45 deg.C.
In a preferred embodiment of the present invention, the ELSD detector has an evaporation temperature of 55-60 deg.C, more preferably 60 deg.C.
In a preferred embodiment of the present invention, the ELSD detector nitrogen purge pressure is 25-30 psi, more preferably 26 psi.
In a preferred embodiment of the invention, the sample injection amount is 5-20 μ l, and more preferably 20 μ l.
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example one
The experimental conditions in the examples of the invention are as follows:
the instrument comprises the following steps: high performance liquid chromatography (Shimadzu LC-20 AT);
a chromatographic column: a Waters XBridge C18 chromatography column, column length 250 mm;
test solution: precisely weighing 10.0mg of Triton X-100 sample, placing in a 10ml volumetric flask, dissolving with mobile phase (methanol: water: 90: 10) and fixing volume to obtain sample solution;
flow rate: 1.0 ml/min;
ELSD detector atomization temperature: 45 ℃;
ELSD detector carrier gas pressure: 26 psi;
mobile phase: methanol/water 90/10 (v/v).
Based on the experimental conditions, the inventor also provides a high performance liquid phase method for measuring the content of triton X-100, which comprises the following steps:
(1) preparation of control solutions:
precisely weighing a proper amount of Triton X-100 reference substance, dissolving with a mobile phase and fixing the volume to obtain a stock solution of 1mg/ml, and diluting with the mobile phase for the second time to obtain a reference substance solution of 20, 40, 60, 80 and 100 mu g/ml;
(2) preparation of a test solution:
precisely weighing a proper amount of a test sample, ultrasonically dissolving the test sample by using a mobile phase, fixing the volume, and filtering the turbid solution by using a 0.22 mu m filter membrane to obtain a test sample solution;
(3) and (3) determination:
performing liquid chromatography determination on the multiple reference substance solutions and the sample solution, recording chromatogram, and preparing a linear working curve with formula 1 according to the logarithm of peak area and the logarithm of concentration of triton X-100 of the 5 reference substance solutions
y＝bx+a
Wherein y is the logarithm of the peak area; x is the logarithm of the concentration of the sample, and the unit is mu g/mL; a is the intercept of the working curve; b is the slope of the working curve.
And then substituting the obtained product into the map data of the test sample to calculate and obtain the concentration of the test sample solution, thereby completing the determination of the content of the triton X-100. The specific calculation process of triton X-100 in the unknown sample is formula 2:
wherein y is the logarithm of the peak area of triton X-100 in the test solution; m is the sample mass in mg; v is the volume of the sample solution with constant volume, and the unit is mL; n is the sample dilution factor; a is the intercept of the working curve; b is the slope of the working curve.
As can be seen from the operation curve X-100 of triton in fig. 6, the operation curve has good linearity, and R2 is 0.9998
Example two
The instrument comprises the following steps: high performance liquid chromatography (Shimadzu LC-20 AT);
a chromatographic column: a Waters XBridge C18 chromatography column, column length 250 mm;
blank solution: is a mobile phase solution;
flow rate: 1.0 ml/min;
ELSD detector atomization temperature: 45 ℃;
ELSD detector carrier gas pressure: 26 psi;
mobile phase: methanol/water 90/10 (v/v).
And (3) obtaining peak area mean value and standard deviation by continuously measuring blank solutions for 10 times, substituting a value obtained by subtracting 3 times of standard deviation from 10 times of blank peak area mean value into a working curve of the formula 1 to obtain the lowest detection limit of 0.5 mu g/ml, and doubling the detection sensitivity compared with an ultraviolet detector.
In conclusion, the high performance liquid detection method of triton X-100 is simple in operation, high in accuracy/sensitivity and suitable for wide popularization. The above embodiments and drawings are only preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (8)
1. A high performance liquid detection method of surfactant triton X-100 is characterized by comprising the following steps:
(1) preparation of control solutions:
precisely weighing a proper amount of Triton X-100 reference substances, dissolving by using a mobile phase and fixing the volume to obtain a plurality of reference substance solutions with a certain concentration gradient;
(2) preparation of a test solution:
precisely weighing a proper amount of a test sample, ultrasonically dissolving the test sample by using a mobile phase, and fixing the volume, and filtering the test sample by using a 0.22 mu m filter membrane if the solution is turbid to obtain a test sample solution;
(3) and (3) determination:
and (3) performing liquid chromatography measurement on the plurality of reference substance solutions in the step (1) and the sample solution in the step (2), recording a chromatogram, preparing a linear working curve according to the logarithm of the X-100 peak area and the logarithm of the concentration of the triton of all the reference substance solutions, substituting the linear working curve into the spectrum data of the sample to calculate and obtain the concentration of the sample solution, and completing the measurement of the content of the triton X-100.
2. The method for detecting the high performance liquid of the surfactant triton X-100 as claimed in claim 1, wherein in the step (1), the number of the plurality of the control solutions is 5, and the concentrations of the control solutions are 20, 40, 60, 80 and 100 μ g/ml in sequence.
3. The high performance liquid chromatography detection method of the surfactant triton X-100 as claimed in claim 1, wherein in the step (3), the liquid chromatography determination conditions are as follows:
the chromatographic column adopts a reversed phase C18 chromatographic column;
the detector adopts an ELSD detector;
the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is methanol; mobile phase B is water; and the volume ratio of methanol to water is 90: 10.
4. the method for detecting the high performance liquid chromatography of the surfactant triton X-100 as claimed in claim 3, wherein the length of the chromatographic column is 250 mm.
5. The high performance liquid chromatography detection method of the surfactant triton X-100 as claimed in claim 3, wherein the column temperature of the chromatographic column is controlled to be 30-40 ℃.
6. The high performance liquid detection method of the surfactant triton X-100 according to claim 3, wherein the ELSD detector has an atomization temperature of 40-45 ℃, an evaporation temperature of 55-60 ℃, and a carrier gas pressure of 25-30 psi.
7. The method for detecting the high performance liquid phase of the surfactant triton X-100 as claimed in claim 3, wherein the flow rate of the mobile phase is 0.8-1.2 ml/min.
8. The high performance liquid chromatography detection method of surfactant triton X-100 as claimed in claim 3, wherein the sample volume is 5-20 μ l.
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Cited By (1)
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| CN115236240A (en) * | 2022-08-10 | 2022-10-25 | 无锡生基医药科技有限公司 | Method for detecting Triton X-100 residue |
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