CN111830144A - A kind of high-performance liquid phase detection method of surfactant Triton X-100 - Google Patents

Abstract

The invention discloses a high-performance liquid phase detection method for Triton X-100, which adopts a reversed-phase C18 chromatographic column and an ELSD detector, the mobile phase is a mixed solution of methanol and water, and isocratic elution is adopted. The method injects 5-20ul of sample, can effectively detect Triton X-100, the minimum detection concentration is 0.5 μg/ml, and the HPLC spectrum baseline is stable and does not drift. The invention can be used for the content determination of triton X-100, has the advantages of high sensitivity, good linear relationship, good reproducibility, high precision and accuracy, and has important research in industrial analysis and product formulation research and the like value.

Description

A kind of high-performance liquid phase detection method of surfactant Triton X-100

technical field

The invention relates to the technical field of industrial analysis, in particular to a high-performance liquid phase detection method of Triton X-100.

Background technique

Triton X-100 (English: Triton X-100) is a non-ionic surfactant, which can dissolve the cell membrane in immunocytochemistry by dissolving the lipid component on the cell membrane, so that the antibody can enter the cell and bind to the antigen. with a hydrophilic polyethylene glycol chain (n is often 9 or 10) and a lipophilic hydrocarbyl group (4-(1,1,3,3-tetramethylbutyl)-phenyl) group). Triton X-100 does not dissociate in water, has high stability in solution, is not easily affected by strong electrolyte inorganic salts, and can combine with lipids such as phospholipids in biological membranes to form soluble complexes; the hydrophobic end can also interact with The hydrophobic regions of membrane proteins bind to form complexes that dissolve in solution.

As a kind of surfactant with high usage rate, the detection of Triton X-100 can be separated by liquid chromatography, and then detected by UV detector. Therefore, the detection sensitivity of this method is not high, and the operation is complicated and the accuracy is average.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a surfactant triton X-100 high-performance liquid phase detection with good separation effect, high precision and accuracy, safe and simple operation, convenient and quick processing, and suitable for high-throughput screening and accurate quantification. The method is used to solve the problems of low detection sensitivity, complicated operation and general accuracy in the above conventional detection methods.

In order to solve the above-mentioned problems, the present invention provides a high-performance liquid phase detection method of surfactant Triton X-100, which is characterized in that, comprising the following steps:

(1) Preparation of reference solution:

Precisely weigh an appropriate amount of Triton X-100 reference substance, dissolve it with mobile phase and dilute to a certain volume of reference substance solution with a certain concentration gradient;

(2) Preparation of the test solution:

Precisely weigh an appropriate amount of the test product, dissolve it with mobile phase ultrasonic and make constant volume, if the solution is cloudy, filter it with a 0.22 μm filter membrane to obtain the test solution;

(3) Determination:

The multiple reference substance solutions and the test solution are subjected to liquid chromatographic determination, the chromatogram is recorded, the linear correlation working curve is prepared according to the spectrum data and the concentration data of the multiple reference substance solutions, and the spectrum data of the test sample is substituted into the calculation and calculated. The concentration of the test solution was obtained, and the determination of the content of Triton X-100 was completed.

As a preferred solution, in the step (1), the quantity of the reference solution is 5, and the concentration of the reference solution is 20, 40, 60, 80, and 100 μg/ml in sequence.

As preferred scheme, in described step (3), the chromatographic condition of mensuration is as follows:

The chromatographic column adopts reversed-phase C18 chromatographic column;

The detector adopts ELSD detector;

The mobile phase includes mobile phase A and mobile phase B; mobile phase A is methanol; mobile phase B is water; and the volume ratio of methanol to water is 90:10.

As a preferred solution, the length of the chromatographic column is 250mm.

As a preferred solution, the column temperature of the chromatographic column is controlled at 30-40°C.

As a preferred solution, the atomization temperature of the ELSD detector is 40-45° C., the evaporation temperature is 55-60° C., and the carrier gas pressure is 25-30 psi.

As a preferred solution, the flow rate of the mobile phase is 1.0 ml/min.

As a preferred solution, the injection volume is 5-20 μl.

As a more preferred solution, in the step (3), the flow rate in the chromatographic conditions is 1.0ml/min, the column temperature is 35°C, the atomization temperature of the ELSD detector is 45°C, and the evaporation temperature of the ELSD detector is 60°C. The carrier gas purge pressure was 26 psi, and the injection volume was 20 μL.

The technical solution has the following beneficial technical effects:

1. The method of the present invention has high sensitivity for the determination of Triton X-100, and the lowest detection limit is 0.5 μg/ml.

2. The method of the present invention is used for the content determination of Triton X-100, has good linear relationship, high reproducibility, realizes the purpose of rapid separation and detection, and has important research value in industrial product analysis and formulation research.

3. The present invention improves and optimizes the detection method in the prior art. Compared with the method in the prior art, the detection method of the present invention not only has high sensitivity, good separation effect, high precision and accuracy, but also operates Safe and simple, convenient and fast processing, suitable for high-throughput screening and accurate quantification. This method has important research value for industrial product analysis and formulation research.

Description of drawings

Fig. 1-Fig. 5 adopts the method of the present invention to measure the HPLC spectrum of Triton X-100 reference substance;

FIG. 6 is a working curve of the triton X-100 drawn by the method of the present invention.

specific implementation

The present invention will be further described below with reference to specific embodiments.

The embodiments provided below are not intended to limit the scope of the present invention, and the described steps are not intended to limit the order of their execution. Those skilled in the art can make obvious improvements to the present invention in combination with the existing common knowledge, which also fall within the protection scope of the present invention.

After extensive research and a lot of experiments, the inventor of the present application has obtained a high-performance liquid phase detection method for Triton X-100, which provides a chromatographic analysis method that can effectively detect Triton X-100, High sensitivity, the lowest detection limit is 0.5μg/ml.

The high-performance liquid phase detection method of Triton X-100 adopted by the inventor, its chromatographic conditions are as follows:

The chromatographic column adopts a reversed-phase C18 chromatographic column, such as a WatersXBridge chromatographic column with octadecylsilane-bonded silica gel as a filler;

The detector adopts evaporative light scattering ELSD detector;

The mobile phase includes mobile phase A and mobile phase B; 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 present invention, the column length of the chromatographic column is 250 mm.

In a preferred embodiment of the present invention, the flow rate is 0.8-1.2 ml/min, more preferably 1.0 ml/min.

In a preferred embodiment of the present invention, the column temperature is controlled at 30-40°C, more preferably 35°C.

In a preferred embodiment of the present invention, the atomization temperature of the ELSD detector is 40-45°C, more preferably 45°C.

In a preferred embodiment of the present invention, the evaporation temperature of the ELSD detector is 55-60°C, more preferably 60°C.

In a preferred embodiment of the present invention, the nitrogen purge pressure of the ELSD detector is 25-30 psi, more preferably 26 psi.

In a preferred embodiment of the present invention, the injection volume is 5-20 μl, more preferably 20 μl.

The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

Example 1

The experimental conditions in the embodiment of the present invention are as follows:

Instrument: high performance liquid chromatograph (Shimadzu LC-20AT);

Chromatographic column: Waters XBridge C18 chromatographic column, the column length is 250mm;

Test solution: Precisely weigh 10.0 mg of Triton X-100 test product, place it in a 10ml volumetric flask, dissolve it with mobile phase (methanol:water=90:10) and dilute to volume to obtain the test solution;

Flow rate: 1.0ml/min;

ELSD detector atomization temperature: 45℃;

ELSD detector carrier gas pressure: 26psi;

Mobile phase: methanol/water=90/10 (v/v).

Based on the above-mentioned experimental conditions, the inventor also provides a high-performance liquid phase method for measuring the content of Triton X-100, which is carried out according to the following steps:

(1) Preparation of reference solution:

Precisely weigh an appropriate amount of Triton X-100 reference substance, dissolve it with mobile phase and dilute to volume to obtain 1mg/ml stock solution, and dilute to volume with mobile phase in turn to obtain 20, 40, 60, 80, 100 μg/ml reference substance solution;

(2) Preparation of the test solution:

Precisely weigh an appropriate amount of the test sample, dissolve it with mobile phase ultrasonic and make constant volume, if the solution is turbid, filter it with a 0.22 μm filter membrane to obtain the test solution;

(3) Determination:

The multiple reference substance solutions and the need testing solution are subjected to liquid chromatographic measurement, record the chromatogram, and prepare a linear working curve according to the logarithm of the Triton X-100 peak area and the logarithm of the concentration logarithm of the 5 reference substance solutions is formula 1

y=bx+a

where y is the logarithm of the peak area; x is the logarithm of the sample concentration in μg/mL; a is the intercept of the working curve; b is the slope of the working curve.

Then substitute the spectral data of the test sample to calculate and obtain the solution concentration of the test sample, and complete the determination of the triton X-100 content. The specific calculation process of triton X-100 in the unknown sample is formula 2:

where 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 constant volume of the sample solution, in mL; n is the sample dilution ratio; a is Intercept of the working curve; b is the slope of the working curve.

It can be known from the working curve of the triton X-100 in Fig. 6 that the working curve has a good linear relationship, and R2=0.9998

Embodiment 2

Instrument: high performance liquid chromatograph (Shimadzu LC-20AT);

Chromatographic column: Waters XBridge C18 chromatographic column, the column length is 250mm;

Blank solution: mobile phase solution;

Flow rate: 1.0ml/min;

ELSD detector atomization temperature: 45℃;

ELSD detector carrier gas pressure: 26psi;

Mobile phase: methanol/water=90/10 (v/v).

Through 10 consecutive blank solution determinations, the peak area mean and standard deviation were obtained, and the value of the 10 blank peak area mean minus 3 times the standard deviation was substituted into the working curve of formula 1, and the lowest detection limit was obtained at 0.5μg/ml, which is relatively The detection sensitivity of the UV detector is doubled.

To sum up, the triton X-100 high-performance liquid phase detection method of the present invention is a method with simple operation, high accuracy/sensitivity and suitable for widespread promotion. The above-mentioned embodiments and accompanying drawings are only preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention , all should be included in the protection 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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