Color development method of thin-layer chromatogram

Abstract

The thin-layer chromatography color developing agent provided by the invention comprises phosphoric acid, ethanol and vanillin, is thin in liquid, good in fluidity and low in water absorption, cannot be carbonized when a thin-layer plate is heated after color development, is relatively low in dangerousness, is not influenced by humidity, and is high in operational safety and good in stability; in addition, the color development and the background can form a relatively obvious contrast, and the chromatographic color is richer. The invention also provides application of the thin-layer chromatography color developing agent in thin-layer identification of reference extracts of spina date seed formula granules and common vladimiria root formula granules, and the thin-layer chromatography color developing agent has a relatively good application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of quality control of traditional Chinese medicines, in particular to a thin layer chromatography developer and application thereof. Background Art

[0002] At present, a certain proportion of vanillin sulfuric acid solution is used as a color developer in the [Identification] item of some varieties included in the 2020 edition of the "Chinese Pharmacopoeia". It was found that this solution has certain disadvantages during use. Sulfuric acid is a colorless, clear, oily liquid with a relative density of 1.831-1.849. It is corrosive and has strong water absorption. It is miscible with water or ethanol and releases a large amount of heat. After a large number of experiments, it is concluded that the solution with sulfuric acid as the solvent is viscous and not easy to spray. It has strong water absorption and high requirements for the humidity of the experimental environment, and the stability of the color development results is poor. It is corrosive and will produce SO2 gas that pollutes the atmosphere after heating. The risk factor is relatively high, and after the color is developed, the thin layer plate will be heated and the thin layer chromatographic spots will carbonize and turn black, which reduces the identification and aesthetics of the thin layer plate surface.

[0003] The developed 10% ethanol phosphoric acid solution of vanillin can reproduce the color development effect of vanillin sulfuric acid solution, and the liquid is thin, has good fluidity, low water absorption, and will not carbonize when the thin layer plate is heated after color development, and the danger is also low. It has the advantages of high operational safety and good stability. Therefore, it is recommended that the 10% ethanol phosphoric acid solution of vanillin can be used to replace the vanillin sulfuric acid solution. Summary of the invention

[0004] The technical problem solved by the present invention is to provide a thin layer chromatography color developer, which has a thin liquid, good fluidity and low water absorption, and will not be carbonized when the thin layer plate is heated after color development, and is also less dangerous, not affected by humidity, and has high operability safety and good stability. The present invention also provides the application of the thin layer chromatography color developer in thin layer identification of control extracts of Chinese date seed formula granules and Sichuan costusroot formula granules.

[0005] The invention provides a thin layer chromatography developer, which comprises phosphoric acid, ethanol and vanillin.

[0006] Preferably, the volume ratio of the phosphoric acid to ethanol is 1:12 to 1:6, and the weight-to-volume ratio of the vanillin to phosphoric acid is 1:15 to 1:5 g / ml.

[0007] Further preferably, the volume ratio of the phosphoric acid to ethanol is 1:9, and the weight-to-volume ratio of the vanillin to phosphoric acid is g / ml 1:10.

[0008] The invention also provides a method for preparing the thin layer chromatography developer, comprising the following steps: slowly adding phosphoric acid into ethanol, shaking well, cooling, adding vanillin, dissolving, and shaking well to obtain the developer.

[0009] The invention also provides application of the thin layer chromatography developer in thin layer identification of control extracts of the jujube seed formula granules and the Sichuan costusroot formula granules.

[0010] The present invention provides a thin layer identification method for the jujube seed formula granules, comprising the following steps:

[0011] (1) Sample preparation:

[0012] Preparation of control extract solution: Take 0.4 g of the control extract of the jujube seed formula granule, add 2 ml of methanol, and perform ultrasonic treatment for 30 minutes at an ultrasonic power of 300 W and a frequency of 40 kHz, and filter through a medium-speed double-circle qualitative filter paper to obtain a formula granule solution;

[0013] Preparation of reference medicinal material solution: Take 1g of Chinese date seed reference medicinal material, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 30ml of methanol to the residue, ultrasonically treat for 30 minutes, the ultrasonic power is 300W, the frequency is 40kHZ, filter with medium-speed double-circle qualitative filter paper, evaporate the filtrate to dryness, add 1ml of methanol to the residue to dissolve, and obtain;

[0014] Preparation of reference solution: Take appropriate amount of jujuboside A reference substance and jujuboside B reference substance, add methanol to make a mixed solution containing 0.8 mg of jujuboside A and 0.5 mg of jujuboside B per 1 ml;

[0015] Preparation of formula granule solution: Take 0.4g of three batches of jujube seed formula granules respectively, grind them into powder, add 2ml of methanol, and treat them with ultrasound for 30 minutes at an ultrasound power of 300W and a frequency of 40kHZ, and filter them with medium-speed double-circle qualitative filter paper to obtain the solution;

[0016] (2) Thin layer chromatography identification, the conditions are:

[0017] Thin layer plate: TLC G60 prefabricated silica gel thin layer plate;

[0018] Spotting: 5 μl of reference solution and 10 μl of other solutions, spotting in strip form;

[0019] Developing solvent: water-saturated n-butanol;

[0020] Inspection: Spray with the TLC developer and heat at 105°C until the spots are clear.

[0021] The present invention provides a thin layer identification method of a reference extract of Sichuan woody root formula granules, comprising the following steps:

[0022] (1) Sample preparation:

[0023] Preparation of control extract solution: Take 0.5g of the control extract of Sichuan woody root formula granules, add 20ml of ether, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, and add 1ml of methanol to dissolve the residue to obtain;

[0024] Preparation of reference medicinal material solution: Take 2g of Sichuan costusroot reference medicinal material, add 50ml of water, heat and reflux for 30 minutes, filter, concentrate the filtrate to about 20ml, shake and extract twice with ether, 20ml each time, combine the ether solution, evaporate to dryness, add 1ml of methanol to the residue to dissolve, and obtain;

[0025] (2) Thin layer chromatography identification, the conditions are:

[0026] Thin layer plate: TLC G60 prefabricated silica gel thin layer plate;

[0027] Spotting: 4μl, strip-shaped spotting;

[0028] Developing solvent: toluene-ethyl acetate, volume ratio of 19:1;

[0029] Inspection: Spray with 5% vanillin in 10% phosphoric acid ethanol solution and heat at 105°C until the spots are clear.

[0030] The present invention has the following beneficial effects:

[0031] The thin layer chromatography developer provided by the present invention has a thin liquid, good fluidity and low water absorption. After color development, the thin layer plate will not be carbonized when heated, and the danger is also low. It is not affected by humidity, has high operability safety and good stability. Moreover, the color development of the present invention can form a relatively obvious contrast with the background, and the chromatographic color is richer. The present invention also provides the application of the thin layer chromatography developer in the thin layer identification of the control extracts of the jujube seed formula granules and the Sichuan costus root formula granules, which has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description in conjunction with the accompanying drawings, in which:

[0033] Figure 1 In order to use the thin layer chromatography color developer of the present invention to perform thin layer chromatography identification on the Chinese jujube seed formula granules, 1 to 3 correspond to the Chinese jujube seed formula granules, 4 corresponds to the Chinese jujube seed formula granule control extract, 5 corresponds to the Chinese jujube seed control medicinal material, and 6 corresponds from bottom to top: Chinese jujube seed saponin A and Chinese jujube seed saponin B.

[0034] Figure 2 The thin layer chromatography color developer of the present invention is used to perform thin layer chromatography identification on the control extract of the formula granules of Sichuan costusroot. 1 is the control extract of the formula granules of Sichuan costusroot, and 2 is the control medicinal material of Sichuan costusroot.

[0035] Figure 3 In order to use conventional thin layer chromatography colorimetric agents to perform thin layer identification on the control extract of the Chinese jujube seed formula granules under experimental environments with different humidity, and compare it with the color development effect of Example 2 of the present invention, experimental humidity: 61%, 1: from bottom to top: Chinese jujube seed saponin A, Chinese jujube seed saponin B, 2: Chinese jujube seed control medicinal materials, 3-8: Chinese jujube seed formula granule control extract.

[0036] Figure 4 In order to use conventional thin layer chromatography colorimetric agents to perform thin layer identification on the control extract of the Chinese jujube seed formula granules under experimental environments with different humidity, and compare the color development effect with that of Example 2 of the present invention, experimental humidity: 90%, 1: from bottom to top: Chinese jujube seed saponin A, Chinese jujube seed saponin B, 2: Chinese jujube seed control medicinal material, 3-8: Chinese jujube seed formula granule control extract.

[0037] Figure 5 In order to use conventional thin layer chromatography colorimetric reagents to perform thin layer chromatography identification on the control extract of Sichuan wood root formula granules, 1 is the control extract of Sichuan wood root formula granules, and 2 is the control medicinal material of Sichuan wood root. DETAILED DESCRIPTION

[0038] Embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Reagents or instruments used that do not indicate the manufacturer are conventional products that can be obtained commercially. The term "and / or" as used herein includes any and all combinations of one or more related listed items.

[0039] The sources of the instruments and materials used in the following examples are as follows:

[0040] ATS 4 TLC automatic sample spotter (CAMAG, Switzerland), TLC double-slot developing cylinder, TLCvisualizer (CAMAG, Switzerland), balance (Sartorius, Germany), and thin layer silica gel preformed plate (Merck).

[0041] Experimental reagents:

[0042] Phosphoric acid, sulfuric acid, ethanol, methanol, n-butanol, petroleum ether II, chloroform, formic acid, ethyl acetate, ether, and toluene were all analytical grade (Guangzhou Chemical Reagent Factory); vanillin (Shanghai Aladdin, catalog number: V100115-100g, I1501028)

[0043] Reference medicinal material of Chinese jujube seed: China Food and Drug Inspection Institute, batch number 121517-201604.

[0044] Jujuboside A: China Food and Drug Inspection Institute, batch number 110734-201713.

[0045] Jujuboside B: China Food and Drug Inspection Institute, batch number 110814-201809.

[0046] Control extract of Suanzaorensis seed formula granules: Guangzhou Keman Biotechnology Co., Ltd., batch number ESZRKL-20220701.

[0047] The three batches of Chinese jujube seed formula granules came from: Guangdong Yifang Pharmaceutical Co., Ltd., CG1805014; China Resources Sanjiu Co., Ltd., batch number 1805003Y; Beijing Kangrentang Pharmaceutical Co., Ltd., batch number 20000702.

[0048] The ratios of the developing agents involved in the present invention are all volume ratios.

[0049] Example 1

[0050] This example provides a thin layer identification method for the jujube seed formula granules.

[0051] 1. Preparation of thin layer color developer (10% phosphoric acid ethanol solution containing vanillin, which can be used to replace vanillin sulfuric acid solution): Take 10 ml of phosphoric acid solution and slowly add it to 90 ml of ethanol solution, shake well, cool, then add 1 g of vanillin powder, dissolve, and shake well.

[0052] 2. Sample Preparation

[0053] Preparation of control extract solution: Take 0.4 g of the control extract of the Chinese jujube seed formula granules (Guangzhou Keman Biotechnology Co., Ltd., batch number ESZRKL-20220701), add 2 ml of methanol, and ultrasonically treat for 30 minutes at an ultrasonic power of 300 W and a frequency of 40 kHz. Filter with medium-speed double-circle qualitative filter paper (filter pore size of 30 to 50 microns) to obtain.

[0054] Preparation of reference medicinal material solution: take 1g of Chinese jujube seed reference medicinal material, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate, add 30ml of methanol to the residue, ultrasonically treat for 30 minutes, the ultrasonic power is 300W, the frequency is 40kHZ, filter with medium-speed double-circle qualitative filter paper (filter pore size is 30-50 microns), evaporate the filtrate, add 1ml of methanol to the residue to dissolve it.

[0055] Preparation of reference solution: Take appropriate amount of jujuboside A reference substance and jujuboside B reference substance, add methanol to make a mixed solution containing 0.8 mg of jujuboside A and 0.5 mg of jujuboside B per 1 ml.

[0056] Preparation of formula granule solution: Take 0.4g of each of 3 batches of Chinese jujube seed formula granules, grind them into powder, add 2ml of methanol, and treat them ultrasonically for 30 minutes with an ultrasonic power of 300W and a frequency of 40kHZ. Filter them through medium-speed double-circle qualitative filter paper (with a filter pore size of 30-50 microns).

[0057] 3. Chromatographic conditions

[0058] Thin layer plate: TLC G60 prefabricated silica gel thin layer plate;

[0059] Spotting: 5 μl of reference solution and 10 μl of other solutions, spotting in strip form;

[0060] Developing solvent: water-saturated n-butanol

[0061] Inspection: Spray with 1% vanillin in 10% phosphoric acid ethanol solution and heat at 105°C until the spots are clear.

[0062] Experimental humidity: 90%

[0063] 4. The results are as follows Figure 1 Shown: 1-3. Ziziphus jujuba seed formula granules, 4. Ziziphus jujuba seed formula granules control extract, 5. Ziziphus jujuba seed control medicinal material, 6. From bottom to top: Ziziphus jujuba seed saponin A, Ziziphus jujuba seed saponin B.

[0064] It can be seen that in the chromatogram of the formula particles, clear spots can be seen at the corresponding positions of the chromatograms of the control extract, the control medicinal material and the control substance. In a high humidity environment, the thin layer plate can keep the background clear, the spots clear and rich in color, and can accurately identify the formula particles of the spiny jujube seed.

[0065] Example 2

[0066] This example provides a thin layer identification method for the control extract of the Sichuan costusroot formula granules.

[0067] 1. Preparation of thin layer color developer: Take 10 ml of phosphoric acid solution and slowly add it into 90 ml of ethanol solution, shake well, let cool, then add 5 g of vanillin powder, dissolve, and shake well.

[0068] 2. Sample Preparation

[0069] Preparation of control extract solution: Take 0.5g of the control extract of Sichuan costusroot formula granules, add 20ml of ether, ultrasonically treat for 30 minutes, the ultrasonic power is 300W, the frequency is 40kHZ, filter with medium-speed double-circle qualitative filter paper (filter pore size is 30-50 microns), evaporate the filtrate, add 1ml of methanol to the residue to dissolve it, and obtain the solution.

[0070] Preparation of reference medicinal material solution: Take 2g of Sichuan costusroot reference medicinal material, add 50ml of water, heat and reflux for 30 minutes, filter through medium-speed double-circle qualitative filter paper (filter pore size is 30-50 microns), concentrate the filtrate to about 20ml, shake and extract twice with ether, 20ml each time, combine the ether solutions, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and obtain the solution.

[0071] 3. Chromatographic conditions

[0072] Thin layer plate: TLC G60 prefabricated silica gel thin layer plate;

[0073] Spotting: 4μl, strip-shaped spotting;

[0074] Developing solvent: toluene-ethyl acetate (19:1);

[0075] Inspection: Spray with 5% vanillin in 10% phosphoric acid ethanol solution and heat at 105°C until the spots are clear.

[0076] 4. Figure 2 :1. Sichuan wood fragrance formula granule control extract 2. Sichuan wood fragrance control medicinal material. In the formula granule chromatogram, clear spots can be seen at the corresponding position of the control medicinal material chromatogram. After color development and heating, the thin layer plate can keep the background clear, the spots clear and the color rich, which can accurately identify the Sichuan wood fragrance formula granules.

[0077] Comparative Example 1

[0078] According to [Identification] (2) under the item of Ziziphus jujuba seed in the 2020 edition of the Chinese Pharmacopoeia, a colorimetric method using 1% vanillin sulfuric acid solution was used to perform thin layer identification of the Ziziphus jujuba seed formula granule control extract under experimental environments with different humidity levels, and the colorimetric effect was compared with that of Example 2 of the present invention.

[0079] 1. Sample Preparation

[0080] Preparation of control extract solution: Take 0.4g of the control extract of the Chinese jujube seed formula granules, add 2ml of methanol, ultrasonically treat for 30 minutes, the ultrasonic power is 300W, the frequency is 40kHZ, and filter with medium-speed double-circle qualitative filter paper (filter pore size is 30-50 microns) to obtain.

[0081] Preparation of reference medicinal material solution: take 1g of Chinese jujube seed reference medicinal material, add 50ml of water, boil for 30 minutes, filter, evaporate the filtrate, add 30ml of methanol to the residue, ultrasonically treat for 30 minutes, the ultrasonic power is 300W, the frequency is 40kHZ, filter with medium-speed double-circle qualitative filter paper (filter pore size is 30-50 microns), evaporate the filtrate, add 1ml of methanol to the residue to dissolve it.

[0082] Preparation of reference solution: Take appropriate amount of jujuboside A reference substance and jujuboside B reference substance, add methanol to make a mixed solution containing 0.8 mg of jujuboside A and 0.5 mg of jujuboside B per 1 ml.

[0083] 2. Chromatographic conditions

[0084] Thin layer plate: TLC G60 prefabricated silica gel thin layer plate;

[0085] Spotting: 5 μl of reference solution and 10 μl of other solutions, spotting in strip form;

[0086] Developing solvent: water-saturated n-butanol;

[0087] Inspection: Spray with 1% vanillin sulfuric acid solution and take photos immediately.

[0088] 4. Figure 4 :Experimental humidity: 61%, 1: From bottom to top: Jujube seed saponin A, Jujube seed saponin B 2: Jujube seed control medicinal material 3-8: Jujube seed formula granule control extract

[0089] Figure 5 :Experimental humidity: 90%, 1: From bottom to top: Jujube seed saponin A, Jujube seed saponin B 2: Jujube seed control medicinal material 3-8: Jujube seed formula granule control extract

[0090] 5. Result analysis: Figure 3 and Figure 4 Comparison of color development effects shows that when the experimental humidity is 90%, the green spots on the thin layer plate fade due to water absorption, which lacks specificity. This shows that the color development effect of the color developer is unstable and the reproducibility is poor. Figure 3 The color development effect of the color developer prepared by the present invention ( Figure 1 ) compared, the color rendering effect is roughly the same, Figure 1 Not affected by humidity, the developer is safer to operate.

[0091] Comparative Example 2

[0092] 1. Sample Preparation

[0093] Preparation of control extract solution: Take 0.5g of the control extract of Sichuan costusroot formula granules, add 20ml of ether, ultrasonically treat for 30 minutes, the ultrasonic power is 300W, the frequency is 40kHZ, filter with medium-speed double-circle qualitative filter paper (filter pore size is 30-50 microns), evaporate the filtrate, add 1ml of methanol to the residue to dissolve it, and obtain the solution.

[0094] Preparation of reference medicinal material solution: Take 2g of Sichuan costusroot reference medicinal material, add 50ml of water, heat and reflux for 30 minutes, filter through medium-speed double-circle qualitative filter paper (filter pore size is 30-50 microns), concentrate the filtrate to about 20ml, shake and extract twice with ether, 20ml each time, combine the ether solutions, evaporate to dryness, add 1ml of methanol to the residue to dissolve it, and obtain the solution.

[0095] 2. Chromatographic conditions

[0096] Thin layer plate: TLC G60 prefabricated silica gel thin layer plate;

[0097] Spotting: 4μl, strip-shaped spotting;

[0098] Developing solvent: toluene-ethyl acetate (19:1)

[0099] Inspection: Spray with 5% vanillin sulfuric acid solution and heat at 105°C until the spots are clear.

[0100] 3. Result analysis: Figure 5 The color development effect of the color developer prepared by the present invention ( Figure 2 ) compared, the color rendering effect is roughly the same, Figure 2 The chromatographic background is clearer, the spot colors are richer, and the developer is safer to operate.

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A thin layer chromatography developer, characterized in that Includes phosphoric acid, ethanol and vanillin.

2. The thin layer chromatography developer according to claim 1, characterized in that The volume ratio of the phosphoric acid to the ethanol is 1:12 to 1:6, and the weight-to-volume ratio of the vanillin to the phosphoric acid is 1:15 to 1:5 (g / ml).

3. The thin layer chromatography developer according to claim 2, characterized in that The volume ratio of the phosphoric acid to ethanol is 1:9, and the weight-volume ratio of the vanillin to phosphoric acid is 1:10 (g / ml).

4. The method for preparing a thin layer chromatography developer according to claim 3, characterized in that: The following steps are involved: Slowly add phosphoric acid to ethanol, shake well, let cool, then add vanillin, dissolve, and shake well.

5. Use of the thin layer chromatography developer according to any one of claims 1 to 3 in thin layer identification of control extracts of Suanzaoren Semen Granules and Chuanmuxiang Granules.

6. A thin layer identification method for the formula granules of Semen Ziziphi Spinosae, characterized in that: The steps include: (1) Sample preparation: Preparation of control extract solution: Take 0.4 g of the control extract of the jujube seed formula granule, add 2 ml of methanol, and perform ultrasonic treatment for 30 minutes at an ultrasonic power of 300 W and a frequency of 40 kHz, and filter through a medium-speed double-circle qualitative filter paper to obtain a formula granule solution; Preparation of reference medicinal material solution: Take 1g of Chinese date seed reference medicinal material, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 30ml of methanol to the residue, ultrasonically treat for 30 minutes, the ultrasonic power is 300W, the frequency is 40kHZ, filter with medium-speed double-circle qualitative filter paper, evaporate the filtrate to dryness, add 1ml of methanol to the residue to dissolve, and obtain; Preparation of reference solution: Take appropriate amount of jujuboside A reference substance and jujuboside B reference substance, add methanol to make a mixed solution containing 0.8 mg of jujuboside A and 0.5 mg of jujuboside B per 1 ml; Preparation of formula granule solution: Take 0.4g of three batches of jujube seed formula granules respectively, grind them into powder, add 2ml of methanol, and treat them with ultrasound for 30 minutes at an ultrasound power of 300W and a frequency of 40kHZ, and filter them with medium-speed double-circle qualitative filter paper to obtain the solution; (2) Thin layer chromatography identification, the conditions are: Thin layer plate: TLC G60 prefabricated silica gel thin layer plate; Spotting: 5 μl of reference solution and 10 μl of other solutions, spotting in strip form; Developing solvent: water-saturated n-butanol; Inspection: Spray with the TLC developer according to any one of claims 1 to 3, and heat at 105°C until the spots are clear.

7. A thin layer identification method for a control extract of Sichuan woody root formula granules, characterized in that: The steps include: (1) Sample preparation: Preparation of control extract solution: Take 0.5g of the control extract of Sichuan woody root formula granules, add 20ml of ether, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, and add 1ml of methanol to dissolve the residue to obtain; Preparation of reference medicinal material solution: Take 2g of Sichuan costusroot reference medicinal material, add 50ml of water, heat and reflux for 30 minutes, filter, concentrate the filtrate to about 20ml, shake and extract twice with ether, 20ml each time, combine the ether solution, evaporate to dryness, add 1ml of methanol to the residue to dissolve, and obtain; (2) Thin layer chromatography identification, the conditions are: Thin layer plate: TLC G60 prefabricated silica gel thin layer plate; Spotting: 4μl, strip-shaped spotting; Developing solvent: toluene-ethyl acetate, volume ratio of 19:1; Inspection: Spray with any of the TLC developer described in claim 1-3 and heat at 105°C until the spots are clear.

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