CN114720621A - Thin-layer chromatographic analysis method for distinguishing fermented cordyceps sinensis powder from fermented cordyceps sinensis powder - Google Patents
Thin-layer chromatographic analysis method for distinguishing fermented cordyceps sinensis powder from fermented cordyceps sinensis powder Download PDFInfo
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Abstract
The invention relates to a thin-layer chromatography analysis method, in particular to a thin-layer chromatography analysis method for distinguishing fermented cordyceps sinensis powder from fermented cordyceps sinensis powder. The technical problems that fermented cordyceps sinensis powder and fermented cordyceps sinensis powder are very easy to be confused for selling and using due to similar appearance, and no quick and effective method can be used for distinguishing the fermented cordyceps sinensis powder and the fermented cordyceps sinensis powder at present are solved. The method comprises the following steps: 1) preparing a standard control solution and a sample solution; 2) carrying out thin-layer sample application; 3) preparing a developing agent; 4) unfolding; 5) and (5) developing and identifying. The thin-layer chromatography identification method has the advantages of rapidness, high efficiency, simple operation, low cost, visual and reliable detection result and the like, can quickly and efficiently identify and distinguish the fermented cordyceps sinensis powder and the fermented cordyceps sinensis powder, effectively ensures the quality of the two bacteria powders, and fills the gap that the fermented cordyceps sinensis powder and the fermented cordyceps sinensis powder are not quickly and effectively identified and distinguished by the existing method.
Description
Technical Field
The invention relates to a thin-layer chromatography analysis method, in particular to a thin-layer chromatography analysis method for distinguishing and distinguishing fermented cordyceps sinensis powder and fermented cordyceps sinensis powder.
Background
Fermented Cordyceps powder (Cs-C-Q80) is dried powder of mycelium obtained by submerged fermentation of hirsutella sinensis (Hirstellasinesis L), and can be used for TLC identification and analysis under BAOKE Capsule item in Chinese pharmacopoeia. The fermented Cordyceps powder (Cs-4) is prepared by deep fermentation and culture of Cordyceps sinensis Paecilomyces hepiali (Paecilomyces hepialichen) Cs-4 separated from Cordyceps (Cordycepsinensis (Berk) Sacc), filtering the fermentation product, and drying to obtain powder for thin layer chromatography analysis under Jinshuibao tablet in Chinese pharmacopoeia.
The fermented cordyceps sinensis powder and the fermented cordyceps sinensis powder belong to liquid fermentation powder, are sold as cordyceps sinensis on the market, and are widely applied to the industries of medicines and health care products. However, the difference between the two is very large, firstly, the fermented strains are different, the strains for fermenting the cordyceps sinensis powder are derived from hirsutella sinensis, and the strains for fermenting the cordyceps sinensis powder are derived from cordyceps sinensis-paecilomyces hepiali separated from cordyceps sinensis. Secondly, the price of the cordyceps sinensis powder and the cordyceps sinensis powder is different, and the price of the fermented cordyceps sinensis powder is higher and is usually 40-50 times of the price of the fermented cordyceps sinensis powder. Finally, the two are different in component content regulation and pharmaceutical method dosage regulation, the fermented cordyceps sinensis bacterial powder is regulated in component content, and the adenosine content of the fermented cordyceps sinensis bacterial powder is not lower than 0.08%; the fermented cordyceps sinensis powder is orally taken 1-3 g each time for 3 times a day according to the dosage of a medical method, and the component content and the dosage of the medical method of the fermented cordyceps sinensis powder are not specified.
Although the fermented cordyceps sinensis powder and the fermented cordyceps sinensis powder are substantially greatly different, in appearance, the fermented cordyceps sinensis powder and the fermented cordyceps sinensis powder are brown fine powder, and the two kinds of powder are difficult to distinguish by naked eyes, so that the phenomenon that the fermented cordyceps sinensis powder is used as the fermented cordyceps sinensis powder is caused in the market, the quality credit of medicines and health-care products produced by using the fermented cordyceps sinensis powder is influenced, and the fermented cordyceps sinensis powder are not taken according to corresponding specified dosage because the fermented cordyceps sinensis powder and the fermented cordyceps sinensis powder are greatly different in dosage, so that potential harm can be caused to the body health of a user. So far, no quick and effective method can be used for distinguishing the two bacterium powders.
Disclosure of Invention
The invention aims to solve the technical problems that fermented cordyceps sinensis powder and fermented cordyceps sinensis powder are extremely easy to be confused for sale and use due to the appearance and similarity of the fermented cordyceps sinensis powder and the fermented cordyceps sinensis powder, and no quick and effective method can be used for distinguishing the fermented cordyceps sinensis powder and the fermented cordyceps sinensis powder at present, and provides a thin-layer chromatography analysis method for distinguishing the fermented cordyceps sinensis powder and the fermented cordyceps sinensis powder. The thin-layer chromatography has the advantages of good separation effect, quick identification, high sensitivity, low cost, simple operation, visual spectrum and the like, and is widely applied to the separation and analysis of the traditional Chinese medicinal materials. However, no research report for distinguishing the fermented cordyceps sinensis powder from the fermented cordyceps sinensis powder by applying thin-layer chromatography is available at present.
The technical solution of the invention is as follows:
a thin-layer chromatography analysis method for distinguishing and distinguishing fermented cordyceps sinensis powder and fermented cordyceps sinensis powder is characterized by comprising the following steps:
1) preparation of Standard control solutions and sample solutions
1.1) taking a fermented cordyceps sinensis powder reference substance raw material and a fermented cordyceps sinensis powder reference substance raw material, respectively placing the fermented cordyceps sinensis powder reference substance raw material and the fermented cordyceps sinensis powder reference substance raw material in a sealable test container, respectively adding ethanol with the mass concentration of 70-100% in an amount which is 5-10 times that of the fermented cordyceps sinensis powder reference substance raw material and the fermented cordyceps sinensis powder reference substance raw material, performing ultrasonic extraction for 30-60 minutes, placing the mixture to room temperature, filtering the mixture by adopting a microporous filter membrane, and collecting filtrate to respectively serve as a fermented cordyceps sinensis powder standard reference solution and a fermented cordyceps sinensis powder standard reference solution;
1.2) taking to-be-identified and distinguished bacteria powder to be detected, placing the bacteria powder in a sealable test container, adding ethanol with the mass concentration of 70-100% and the mass of 5-10 times that of the bacteria powder to be detected, performing ultrasonic extraction for 30-60 minutes, placing the mixture to room temperature, then filtering the mixture by adopting a microporous filter membrane, and collecting filtrate as sample liquid;
2) performing thin layer sample application
Spotting the fermented cordyceps sinensis bacterial powder standard reference solution and the fermented cordyceps sinensis bacterial powder standard reference solution prepared in the step 1.1) and the sample solution prepared in the step 1.2) on different positions of the same silica gel GF thin layer plate at the same height to form spotting origins;
3) preparation of developing agent
3.1) uniformly mixing ethyl acetate, formic acid, acetic acid and water in a volume ratio of 8-12: 0.8-1.2: 1.8-2.2 to obtain a developing agent;
3.2) placing the developing solvent into an expansion cylinder, sealing the expansion cylinder, and presaturating for 15-20 minutes;
4) is unfolded
4.1) opening the developing cylinder after the presaturation in the step 3), and quickly putting the silica gel GF thin-layer plate subjected to the sample application in the step 2) into the developing cylinder to enable the silica gel GF thin-layer plate to lean against the developing cylinder;
4.2) when the front edge of the developing agent ascends to the position 6-10 cm away from the origin of the sample application, taking out the silica gel GF thin-layer plate;
5) color development and identification
5.1) after the reagent on the silica gel GF thin-layer plate developed in the step 4.2) is dried, spraying 5-10% sulfuric acid ethanol solution by mass ratio, and then heating for 3-5 minutes at the temperature of 100-;
5.2) after the temperature is reduced to the room temperature, the silica gel GF thin-layer plate is inspected under the illumination of an inspection light source to obtain an inspection view;
5.3) comparing spots formed at the sample application origin of the sample solution of the bacteria powder to be detected on the inspection view with spots formed at the sample application origins of the fermented cordyceps sinensis bacteria powder standard reference solution and the fermented cordyceps sinensis bacteria powder standard reference solution respectively;
if the spots of the to-be-detected bacterium powder are consistent with the spots of the fermented cordyceps sinensis bacterium powder standard reference liquid, the to-be-detected bacterium powder is fermented cordyceps sinensis bacterium powder, if the spots of the to-be-detected bacterium powder are inconsistent with the spots of the fermented cordyceps sinensis bacterium powder standard reference liquid, and the spots of the to-be-detected bacterium powder are inconsistent with the spots of the fermented cordyceps sinensis bacterium powder standard reference liquid, the to-be-detected bacterium powder is other bacterium powder.
Further, in step 5.2), the inspection light source is one of 365nm ultraviolet light, 254nm ultraviolet light and sunlight.
Further, the step 5.2) is specifically as follows: and (4) after the temperature is reduced to room temperature, inspecting the silica gel GF thin-layer plate under 365nm ultraviolet light, 254nm ultraviolet light and sunlight respectively to obtain an inspection view.
Further, the step 1.1) is specifically as follows: respectively placing a fermented cordyceps sinensis fungus powder reference substance raw material and a fermented cordyceps sinensis fungus powder reference substance raw material into triangular flasks with plugs, respectively adding ethanol with the mass concentration of 70% which is 6 times that of the fermented cordyceps sinensis fungus powder reference substance raw material and the fermented cordyceps sinensis fungus powder reference substance raw material, performing ultrasonic extraction for 60 minutes, placing the mixture to room temperature, filtering the mixture by adopting a microporous filter membrane, and collecting filtrate as standard reference solution;
the step 1.2) is specifically as follows: putting to-be-identified bacterium powder into a triangular flask with a plug, adding ethanol with the mass concentration of 70% and the mass which is 6 times that of the to-be-identified bacterium powder, performing ultrasonic extraction for 60 minutes, cooling to room temperature, filtering by adopting a microporous filter membrane, and collecting filtrate as sample liquid.
Further, in the step 2), the distance between the sample application origin position and the bottom of the silica gel GF thin-layer plate is 1.0-1.5 cm.
Further, in step 3.1), the volume ratio of ethyl acetate, formic acid, acetic acid and water is 8:0.8:0.8: 1.8.
Further, in step 3.2), the developing cylinder is a double-groove developing cylinder, the size of the double-groove developing cylinder is 10cm × 10cm, 20cm × 10cm or 10cm × 20cm, the developing agent dosage in the double grooves is kept consistent, and the developing agent is presaturated for 15 minutes.
Further, the step 4.2) is specifically that when the front edge of the developing agent ascends to 8-10 cm away from the sample application origin, the silica gel GF thin-layer plate is taken out.
Further, the step 5.1) is specifically as follows: blowing the reagent on the silica gel GF thin-layer plate developed in the step 4.2) dry, spraying a sulfuric acid ethanol solution with the mass ratio of 10%, and then heating for 3-5 minutes at 105 ℃ by using an electric heating plate.
The invention has the beneficial effects that:
1. the invention adopts the thin-layer chromatography to distinguish the fermented cordyceps sinensis powder from the fermented cordyceps sinensis powder, has the advantages of rapidness, high efficiency, good separation effect, quickness in distinguishing, high sensitivity, simplicity, economy and intuition, and well solves the distinguishing problem of the fermented cordyceps sinensis powder and the fermented cordyceps sinensis powder.
2. According to the invention, ethyl acetate, formic acid, acetic acid and water are mixed according to the volume ratio of 8-12: 0.8-1.2: 1.8-2.2 to prepare the developing agent, the obtained spot separation effect is good, the characteristic spots are prominent, the phenomena of spot trailing and spot diffusion are avoided, the result is visual and obvious, and the judgment is easy.
3. The invention adopts the silica gel GF thin layer plate, and on the basis of the inspection under the ultraviolet light 365nm and sunlight, the inspection under the ultraviolet light 254nm is added, and the result has more reference value while the detection mode is added.
4. The invention adopts 5 to 10 percent of sulfuric acid ethanol as the color developing agent, is convenient and quick, and has a plurality of color developing spots and obvious spot colors.
5. According to the invention, a proper amount of ethanol is selected to directly carry out ultrasonic treatment and filtration on the raw medicinal materials, and the filtrate is used as a detection solution, so that the pretreatment of the sample is simple, the cost is low, and the operation is easy.
Drawings
FIG. 1 is an examination view of a GF thin layer plate under 254nm illumination in an embodiment of the present invention;
FIG. 2 is an examination view of a GF thin layer plate under 365nm light in an embodiment of the present invention;
fig. 3 is an examination view of a GF sheet in an embodiment of the invention under daylight illumination.
Detailed Description
The invention is described in further detail below with reference to the figures and the specific embodiments.
According to the embodiment of the invention, the fermented cordyceps sinensis bacterial powder and the fermented cordyceps sinensis bacterial powder raw materials for preparing the standard control solution are from China food and drug testing research institute, and the bacterial powder raw materials to be tested to be identified and distinguished come from random purchase in the market.
Example one
1) Preparation of Standard control solutions and sample solutions
1.1) taking a fermented cordyceps sinensis fungus powder reference substance raw material and a fermented cordyceps sinensis fungus powder reference substance raw material, respectively placing the fermented cordyceps sinensis fungus powder reference substance raw material and the fermented cordyceps sinensis fungus powder reference substance raw material into triangular flasks with plugs, respectively adding ethanol with the mass concentration of 70% in an amount which is 6 times that of the fermented cordyceps sinensis fungus powder reference substance raw material and the fermented cordyceps sinensis fungus powder reference substance raw material, performing ultrasonic extraction for 60 minutes, placing the mixture to room temperature, then filtering the mixture by adopting a microporous filter membrane, and collecting filtrate as a standard reference solution; the standard reference solution numbers of the reference product raw material of fermented Cordyceps sinensis powder and the reference product raw material of fermented Cordyceps sinensis powder are respectively marked as No. 1 and No. 4.
1.2) taking two parts of fermented cordyceps sinensis powder and two parts of fermented cordyceps sinensis powder which are purchased from the market and are to be identified and distinguished as raw materials of the powder to be detected, respectively placing the raw materials into triangular bottles with plugs, respectively adding ethanol with the mass concentration of 70 percent and the mass which is 6 times that of the raw materials of the powder to be detected, carrying out ultrasonic extraction for 60 minutes, placing the mixture to room temperature, then filtering the mixture by adopting a microporous filter membrane, and collecting filtrate as sample liquid; the sample liquids of the two portions of fermented cordyceps sinensis powder to be identified and distinguished are sequentially numbered as 2 and 3, and the sample liquids of the two portions of fermented cordyceps sinensis powder to be identified and distinguished are sequentially numbered as 5 and 6.
2) Performing thin layer sample application
Sequentially dotting the standard reference solution and the sample solution prepared in the step 1) on different positions of the same silica gel GF thin-layer plate at the same height according to the number to form six point sample origins; and the distance from the sample application origin to the bottom of the silica gel GF thin-layer plate is 1.0 cm;
3) preparation of developing solvent
3.1) uniformly mixing ethyl acetate, formic acid, acetic acid and water in a volume ratio of 8:0.8:0.8:1.8 to prepare 20mL of developing agent, wherein the ethyl acetate, the formic acid and the acetic acid are pure products;
3.2) placing the developing solvent into a double-groove developing cylinder with the size of 10 multiplied by 10cm, wherein the amount of the developing solvent in the two grooves is consistent, then sealing the developing cylinder, and presaturating for 15 minutes;
4) is unfolded
4.1) opening the developing cylinder pre-saturated in the step 3), and quickly putting the silica gel GF thin-layer plate subjected to sample application in the step 2) into the developing cylinder, so that the silica gel GF thin-layer plate is obliquely leaned against the developing cylinder and the developing agent in the developing cylinder cannot exceed the original point of sample application;
4.2) when the developing agent spreads upwards along the thin-layer plate until the front edge of the developing agent moves upwards to 8cm away from the origin of the sample application, taking out the silica gel GF thin-layer plate;
5) color development and identification
5.1) blowing dry the reagent on the silica gel GF thin-layer plate developed in the step 4.2), spraying a sulfuric acid ethanol solution with the mass ratio of 10%, and heating for 3-5 minutes at 105 ℃ by using an electric heating plate;
5.2) after the temperature is reduced to room temperature, the silica gel GF thin layer plate is inspected under 365nm ultraviolet light, 254nm ultraviolet light and sunlight in sequence to obtain an inspection view;
and 5.3) comparing spots formed by sample application origins of the bacteria powder to be detected ( numbers 2, 3, 5 and 6) on the detection view with spots formed by sample application origins of numbers 1 (fermented cordyceps sinensis bacteria powder standard control liquid) and 4 (fermented cordyceps sinensis bacteria powder standard control liquid), respectively.
The experimental results are shown in fig. 1 to 3, and on the three inspection views, the spots of numbers 2 and 3 are identical to the spots of number 1 in number and Rf, and the spots of numbers 5 and 6 are identical to the spots of number 4 in number and Rf, so that it can be determined that the fungus powder to be inspected of numbers 2 and 3 is fermented cordyceps sinensis fungus powder, and the fungus powder to be inspected of numbers 5 and 6 is fermented cordyceps sinensis fungus powder. The inspection views obtained under three different inspection light sources are consistent, the spots in each view are obviously separated, the view result is visual and easy to judge, and the variety of the bacterial powder to be inspected can be distinguished only by using one of 365nm ultraviolet light, 254nm ultraviolet light and sunlight. In other embodiments, the inspection light source may be used for inspection to obtain an inspection view.
Example two
1) Preparation of Standard control solutions and sample solutions
1.1) taking a fermented cordyceps sinensis fungus powder reference substance raw material and a fermented cordyceps sinensis fungus powder reference substance raw material, respectively placing the fermented cordyceps sinensis fungus powder reference substance raw material and the fermented cordyceps sinensis fungus powder reference substance raw material into triangular flasks with plugs, respectively adding ethanol with the mass concentration of 90% in an amount which is 5 times that of the fermented cordyceps sinensis fungus powder reference substance raw material and the fermented cordyceps sinensis fungus powder reference substance raw material, performing ultrasonic extraction for 30 minutes, placing the mixture to room temperature, then filtering the mixture by adopting a microporous filter membrane, and collecting filtrate as a standard reference solution; the reference solutions of fermented Cordyceps powder and fermented Cordyceps powder are respectively numbered as No. 1 and No. 4.
1.2) taking two parts of fermented cordyceps sinensis powder and two parts of fermented cordyceps sinensis powder which are purchased from the market and are to be identified and distinguished as raw materials of the powder to be detected, respectively placing the raw materials into triangular bottles with plugs, respectively adding ethanol with the mass concentration of 90 percent and the mass which is 5 times that of the raw materials of the powder to be detected, carrying out ultrasonic extraction for 30 minutes, placing the mixture to room temperature, then filtering the mixture by adopting a microporous filter membrane, and collecting filtrate as sample liquid; the sample liquids of the two portions of fermented cordyceps sinensis powder to be identified and distinguished are sequentially numbered as 2 and 3, and the sample liquids of the two portions of fermented cordyceps sinensis powder to be identified and distinguished are sequentially numbered as 5 and 6.
2) Performing thin layer sample application
Sequentially dotting the standard reference solution and the sample solution prepared in the step 1) on different positions of the same silica gel GF thin-layer plate at the same height according to the number to form six point sample origins; and the distance between the sample application origin and the bottom of the silica gel GF thin layer plate is 1.5 cm;
3) preparation of developing agent
3.1) uniformly mixing ethyl acetate, formic acid, acetic acid and water in a volume ratio of 10:1:1:2 to prepare 20mL of developing agent, wherein the ethyl acetate, the formic acid and the acetic acid are pure products;
3.2) placing the developing solvent into a double-groove developing cylinder with the size of 20 multiplied by 10cm, wherein the amount of the developing solvent in the two grooves is consistent, then sealing the developing cylinder, and presaturating for 20 minutes;
4) is unfolded
4.1) opening the developing cylinder pre-saturated in the step 3), and quickly putting the silica gel GF thin-layer plate subjected to sample application in the step 2) into the developing cylinder, so that the silica gel GF thin-layer plate is obliquely leaned against the developing cylinder and the developing agent in the developing cylinder cannot exceed the original point of sample application;
4.2) when the developing agent spreads upwards along the thin-layer plate until the front edge of the developing agent moves upwards to be 6cm away from the origin of the sample application, taking out the silica gel GF thin-layer plate;
5) color development and identification
5.1) blowing the reagent on the silica gel GF thin-layer plate developed in the step 4.2) by a blower, spraying 5% sulfuric acid ethanol solution by mass ratio, and heating for 3-5 minutes at 100 ℃ by an electric heating plate;
5.2) after the temperature is reduced to room temperature, the silica gel GF thin layer plate is inspected under 365nm ultraviolet light, 254nm ultraviolet light and sunlight in sequence to obtain an inspection view;
and 5.3) comparing spots formed by sample application origins of the bacteria powder to be detected ( numbers 2, 3, 5 and 6) on the detection view with spots formed by sample application origins of numbers 1 (fermented cordyceps sinensis bacteria powder standard control liquid) and 4 (fermented cordyceps sinensis bacteria powder standard control liquid), respectively.
The resulting viewing images are substantially identical to the first embodiment.
EXAMPLE III
1) Preparation of Standard control solutions and sample solutions
1.1) taking a fermented cordyceps sinensis fungus powder reference substance raw material and a fermented cordyceps sinensis fungus powder reference substance raw material, respectively placing the fermented cordyceps sinensis fungus powder reference substance raw material and the fermented cordyceps sinensis fungus powder reference substance raw material into triangular flasks with plugs, respectively adding ethanol with the mass concentration of 100% in an amount which is 10 times that of the fermented cordyceps sinensis fungus powder reference substance raw material and the fermented cordyceps sinensis fungus powder reference substance raw material, performing ultrasonic extraction for 50 minutes, placing the mixture to room temperature, then filtering the mixture by adopting a microporous filter membrane, and collecting filtrate as a standard reference solution; the reference solutions of fermented Cordyceps powder and fermented Cordyceps powder are respectively numbered as No. 1 and No. 4.
1.2) taking two parts of fermented cordyceps sinensis powder and two parts of fermented cordyceps sinensis powder which are purchased from the market and are to be identified and distinguished as raw materials of the powder to be detected, respectively placing the raw materials into triangular bottles with plugs, respectively adding ethanol with the mass concentration of 100 percent and the mass which is 10 times that of the raw materials of the powder to be detected, carrying out ultrasonic extraction for 50 minutes, placing the mixture to room temperature, then filtering the mixture by adopting a microporous filter membrane, and collecting filtrate as sample liquid; the sample liquids of the two portions of fermented cordyceps sinensis powder to be identified and distinguished are sequentially numbered as 2 and 3, and the sample liquids of the two portions of fermented cordyceps sinensis powder to be identified and distinguished are sequentially numbered as 5 and 6.
2) Performing thin layer sample application
Sequentially spotting the standard reference solution and the sample solution prepared in the step 1) on different positions of the same height on the same silica gel GF thin-layer plate according to the number to form six spotting origin points; and the distance from the sample application origin to the bottom of the silica gel GF thin-layer plate is 1.3 cm;
3) preparation of developing agent
3.1) uniformly mixing ethyl acetate, formic acid, acetic acid and water in a volume ratio of 12:1.2:1.2:2.2 to prepare 20mL of developing agent, wherein the ethyl acetate, the formic acid and the acetic acid are pure products;
3.2) placing the developing solvent into a double-groove developing cylinder with the size of 10 multiplied by 20cm, wherein the amount of the developing solvent in the two grooves is consistent, then sealing the developing cylinder, and presaturating for 17 minutes;
4) is unfolded
4.1) opening the developing cylinder pre-saturated in the step 3), and quickly putting the silica gel GF thin-layer plate subjected to sample application in the step 2) into the developing cylinder, so that the silica gel GF thin-layer plate is obliquely leaned against the developing cylinder and the developing agent in the developing cylinder cannot exceed the original point of sample application;
4.2) when the developing agent spreads upwards along the thin-layer plate until the front edge of the developing agent moves upwards to a position 10cm away from the origin of the sample application, taking out the silica gel GF thin-layer plate;
5) color development and identification
5.1) blowing the reagent on the silica gel GF thin-layer plate developed in the step 4.2) by a blower, spraying a 7% sulfuric acid ethanol solution in mass ratio, and then heating for 3-5 minutes at 110 ℃ by using an electric heating plate;
5.2) after the temperature is reduced to room temperature, the silica gel GF thin layer plate is inspected under 365nm ultraviolet light, 254nm ultraviolet light and sunlight in sequence to obtain an inspection view;
and 5.3) comparing spots formed by sample application origins of the bacteria powder to be detected ( numbers 2, 3, 5 and 6) on the detection view with spots formed by sample application origins of numbers 1 (fermented cordyceps sinensis bacteria powder standard control liquid) and 4 (fermented cordyceps sinensis bacteria powder standard control liquid), respectively.
The resulting viewing images are substantially identical to the first embodiment.
Example four
1) Preparation of Standard control solutions and sample solutions
1.1) taking a fermented cordyceps sinensis fungus powder reference substance raw material and a fermented cordyceps sinensis fungus powder reference substance raw material, respectively placing the fermented cordyceps sinensis fungus powder reference substance raw material and the fermented cordyceps sinensis fungus powder reference substance raw material into triangular flasks with plugs, respectively adding ethanol with the mass concentration of 70% in an amount which is 6 times that of the fermented cordyceps sinensis fungus powder reference substance raw material and the fermented cordyceps sinensis fungus powder reference substance raw material, performing ultrasonic extraction for 60 minutes, placing the mixture to room temperature, then filtering the mixture by adopting a microporous filter membrane, and collecting filtrate as a standard reference solution; the reference solutions of fermented Cordyceps powder and fermented Cordyceps powder are respectively numbered as No. 1 and No. 4.
1.2) taking two parts of fermented cordyceps sinensis powder and two parts of fermented cordyceps sinensis powder which are purchased from the market and are to be identified and distinguished as raw materials of the powder to be detected, respectively placing the raw materials into triangular bottles with plugs, respectively adding ethanol with the mass concentration of 70 percent and the mass which is 6 times that of the raw materials of the powder to be detected, carrying out ultrasonic extraction for 60 minutes, placing the mixture to room temperature, then filtering the mixture by adopting a microporous filter membrane, and collecting filtrate as sample liquid; the sample liquids of the two portions of fermented cordyceps sinensis powder to be identified and distinguished are sequentially numbered as 2 and 3, and the sample liquids of the two portions of fermented cordyceps sinensis powder to be identified and distinguished are sequentially numbered as 5 and 6.
2) Performing thin layer sample application
Sequentially dotting the standard reference solution and the sample solution prepared in the step 1) on different positions of the same silica gel GF thin-layer plate at the same height according to the number to form six point sample origins; and the distance from the sample application origin to the bottom of the silica gel GF thin-layer plate is 1.0 cm;
3) preparation of developing agent
3.1) uniformly mixing ethyl acetate, formic acid, acetic acid and water in a volume ratio of 10:1:1:2 to prepare 20mL of developing agent, wherein the ethyl acetate, the formic acid and the acetic acid are pure products;
3.2) placing the developing solvent into a double-groove developing cylinder with the size of 10 multiplied by 10cm, wherein the amount of the developing solvent in the two grooves is consistent, then sealing the developing cylinder, and presaturating for 15 minutes;
4) is unfolded
4.1) opening the developing cylinder after the presaturation in the step 3), quickly putting the silica gel GF thin-layer plate subjected to the sample application in the step 2) into the developing cylinder, and enabling the silica gel GF thin-layer plate to lean against the developing cylinder in an inclined manner and the developing agent in the developing cylinder not to exceed the original point of the sample application;
4.2) when the developing agent spreads upwards along the thin-layer plate until the front edge of the developing agent moves upwards to 8cm away from the origin of the sample application, taking out the silica gel GF thin-layer plate;
5) color development and identification
5.1) blowing dry the reagent on the silica gel GF thin-layer plate developed in the step 4.2), spraying a sulfuric acid ethanol solution with the mass ratio of 10%, and heating for 3-5 minutes at 105 ℃ by using an electric heating plate;
5.2) placing the silica gel GF thin layer plate to room temperature, and inspecting the silica gel GF thin layer plate under 365nm ultraviolet light illumination to obtain an inspection view;
and 5.3) comparing spots formed by sample application origins of the bacteria powder to be detected ( numbers 2, 3, 5 and 6) on the detection view with spots formed by sample application origins of numbers 1 (fermented cordyceps sinensis bacteria powder standard control liquid) and 4 (fermented cordyceps sinensis bacteria powder standard control liquid), respectively.
The resulting viewing pattern substantially corresponds to that of figure 2.
EXAMPLE five
1) Preparation of Standard control solutions and sample solutions
1.1) taking a fermented cordyceps sinensis fungus powder reference substance raw material and a fermented cordyceps sinensis fungus powder reference substance raw material, respectively placing the fermented cordyceps sinensis fungus powder reference substance raw material and the fermented cordyceps sinensis fungus powder reference substance raw material into triangular flasks with plugs, respectively adding ethanol with the mass concentration of 70% in an amount which is 6 times that of the fermented cordyceps sinensis fungus powder reference substance raw material and the fermented cordyceps sinensis fungus powder reference substance raw material, performing ultrasonic extraction for 60 minutes, placing the mixture to room temperature, then filtering the mixture by adopting a microporous filter membrane, and collecting filtrate as a standard reference solution; the reference solutions of fermented Cordyceps powder and fermented Cordyceps powder are respectively numbered as No. 1 and No. 4.
1.2) taking two parts of fermented cordyceps sinensis powder and two parts of fermented cordyceps sinensis powder which are purchased from the market and are to be identified and distinguished as raw materials of the powder to be detected, respectively placing the raw materials into triangular bottles with plugs, respectively adding ethanol with the mass concentration of 70 percent and the mass which is 6 times that of the raw materials of the powder to be detected, carrying out ultrasonic extraction for 60 minutes, placing the mixture to room temperature, then filtering the mixture by adopting a microporous filter membrane, and collecting filtrate as sample liquid; the sample liquids of the two portions of fermented cordyceps sinensis powder to be identified and distinguished are sequentially numbered as 2 and 3, and the sample liquids of the two portions of fermented cordyceps sinensis powder to be identified and distinguished are sequentially numbered as 5 and 6.
2) Performing thin layer sample application
Sequentially dotting the standard reference solution and the sample solution prepared in the step 1) on different positions of the same silica gel GF thin-layer plate at the same height according to the number to form six point sample origins; and the distance from the sample application origin to the bottom of the silica gel GF thin-layer plate is 1.0 cm;
3) preparation of developing agent
3.1) uniformly mixing ethyl acetate, formic acid, acetic acid and water in a volume ratio of 10:1:1:2 to prepare 20mL of developing agent, wherein the ethyl acetate, the formic acid and the acetic acid are pure products;
3.2) placing the developing solvent into a double-groove developing cylinder with the size of 10 multiplied by 10cm, wherein the amount of the developing solvent in the two grooves is consistent, then sealing the developing cylinder, and presaturating for 15 minutes;
4) is unfolded
4.1) opening the developing cylinder pre-saturated in the step 3), and quickly putting the silica gel GF thin-layer plate subjected to sample application in the step 2) into the developing cylinder, so that the silica gel GF thin-layer plate is obliquely leaned against the developing cylinder and the developing agent in the developing cylinder cannot exceed the original point of sample application;
4.2) when the developing agent spreads upwards along the thin-layer plate until the front edge of the developing agent moves upwards to 8cm away from the origin of the sample application, taking out the silica gel GF thin-layer plate;
5) color development and identification
5.1) blowing dry the reagent on the silica gel GF thin-layer plate developed in the step 4.2), spraying a sulfuric acid ethanol solution with the mass ratio of 10%, and heating for 3-5 minutes at 105 ℃ by using an electric heating plate;
5.2) placing the silica gel GF thin layer plate to room temperature, and inspecting the silica gel GF thin layer plate under 254nm ultraviolet light illumination to obtain an inspection view;
and 5.3) comparing spots formed by sample application origins of the bacteria powder to be detected ( numbers 2, 3, 5 and 6) on the detection view with spots formed by sample application origins of numbers 1 (fermented cordyceps sinensis bacteria powder standard control liquid) and 4 (fermented cordyceps sinensis bacteria powder standard control liquid), respectively.
The resulting viewing pattern substantially corresponds to that of figure 1.
Example six
1) Preparation of Standard control solutions and sample solutions
1.1) taking a fermented cordyceps sinensis fungus powder reference substance raw material and a fermented cordyceps sinensis fungus powder reference substance raw material, respectively placing the fermented cordyceps sinensis fungus powder reference substance raw material and the fermented cordyceps sinensis fungus powder reference substance raw material into triangular flasks with plugs, respectively adding ethanol with the mass concentration of 70% in an amount which is 6 times that of the fermented cordyceps sinensis fungus powder reference substance raw material and the fermented cordyceps sinensis fungus powder reference substance raw material, performing ultrasonic extraction for 60 minutes, placing the mixture to room temperature, then filtering the mixture by adopting a microporous filter membrane, and collecting filtrate as a standard reference solution; the reference solutions of fermented Cordyceps powder and fermented Cordyceps powder are respectively numbered as No. 1 and No. 4.
1.2) taking two parts of fermented cordyceps sinensis powder and two parts of fermented cordyceps sinensis powder which are purchased from the market and are to be identified and distinguished as raw materials of the powder to be detected, respectively placing the raw materials into triangular bottles with plugs, respectively adding ethanol with the mass concentration of 70 percent and the mass which is 6 times that of the raw materials of the powder to be detected, carrying out ultrasonic extraction for 60 minutes, placing the mixture to room temperature, then filtering the mixture by adopting a microporous filter membrane, and collecting filtrate as sample liquid; the sample liquids of the two portions of fermented cordyceps sinensis powder to be identified and distinguished are sequentially numbered as 2 and 3, and the sample liquids of the two portions of fermented cordyceps sinensis powder to be identified and distinguished are sequentially numbered as 5 and 6.
2) Performing thin layer sample application
Sequentially dotting the standard reference solution and the sample solution prepared in the step 1) on different positions of the same silica gel GF thin-layer plate at the same height according to the number to form six point sample origins; and the distance from the sample application origin to the bottom of the silica gel GF thin layer plate is 1.0 cm;
3) preparation of developing agent
3.1) uniformly mixing ethyl acetate, formic acid, acetic acid and water in a volume ratio of 10:1:1:2 to prepare 20mL of developing agent, wherein the ethyl acetate, the formic acid and the acetic acid are pure products;
3.2) placing the developing solvent into a double-groove developing cylinder with the size of 10 multiplied by 10cm, wherein the amount of the developing solvent in the two grooves is consistent, then sealing the developing cylinder, and presaturating for 15 minutes;
4) is unfolded
4.1) opening the developing cylinder pre-saturated in the step 3), and quickly putting the silica gel GF thin-layer plate subjected to sample application in the step 2) into the developing cylinder, so that the silica gel GF thin-layer plate is obliquely leaned against the developing cylinder and the developing agent in the developing cylinder cannot exceed the original point of sample application;
4.2) when the developing agent spreads upwards along the thin-layer plate until the front edge of the developing agent moves upwards to 8cm away from the origin of the sample application, taking out the silica gel GF thin-layer plate;
5) color development and identification
5.1) blowing dry the reagent on the silica gel GF thin-layer plate developed in the step 4.2), spraying a sulfuric acid ethanol solution with the mass ratio of 10%, and heating for 3-5 minutes at 105 ℃ by using an electric heating plate;
5.2) after the temperature is reduced to room temperature, inspecting the silica gel GF thin-layer plate under the illumination of 2 suns to obtain an inspection view;
and 5.3) comparing spots formed by sample application origins of the bacteria powder to be detected ( numbers 2, 3, 5 and 6) on the detection view with spots formed by sample application origins of numbers 1 (fermented cordyceps sinensis bacteria powder standard control liquid) and 4 (fermented cordyceps sinensis bacteria powder standard control liquid), respectively.
The resulting viewing pattern is substantially identical to that of FIG. 3.
While the invention has been described with reference to specific embodiments, the invention is not limited thereto, and various equivalent modifications or substitutions can be easily made by those skilled in the art within the technical scope of the present disclosure.
Claims (9)
1. A thin-layer chromatography analysis method for distinguishing and distinguishing fermented cordyceps sinensis powder and fermented cordyceps sinensis powder is characterized by comprising the following steps of:
1) preparation of Standard control solutions and sample solutions
1.1) taking a fermented cordyceps sinensis powder reference substance raw material and a fermented cordyceps sinensis powder reference substance raw material, respectively placing the fermented cordyceps sinensis powder reference substance raw material and the fermented cordyceps sinensis powder reference substance raw material in a sealable test container, respectively adding ethanol with the mass concentration of 70-100% in an amount which is 5-10 times that of the fermented cordyceps sinensis powder reference substance raw material and the fermented cordyceps sinensis powder reference substance raw material, performing ultrasonic extraction for 30-60 minutes, placing the mixture to room temperature, filtering the mixture by using a microporous filter membrane, and collecting filtrate to respectively serve as a fermented cordyceps sinensis powder standard reference solution and a fermented cordyceps sinensis powder standard reference solution;
1.2) placing the to-be-identified and distinguished powder of the to-be-detected bacterium in a sealable test container, adding 5-10 times of ethanol with the mass concentration of 70-100% by mass of the powder of the to-be-detected bacterium, performing ultrasonic extraction for 30-60 minutes, placing the solution to room temperature, then filtering the solution by adopting a microporous filter membrane, and collecting filtrate as sample solution;
2) performing thin layer sample application
Spotting the fermented cordyceps sinensis bacterial powder standard reference solution and the fermented cordyceps sinensis bacterial powder standard reference solution prepared in the step 1.1) and the sample solution prepared in the step 1.2) on different positions of the same silica gel GF thin layer plate at the same height to form spotting origins;
3) preparation of developing agent
3.1) uniformly mixing ethyl acetate, formic acid, acetic acid and water in a volume ratio of 8-12: 0.8-1.2: 1.8-2.2 to obtain a developing agent;
3.2) placing the developing agent into an expansion cylinder, sealing the expansion cylinder, and presaturating for 15-20 minutes;
4) is unfolded
4.1) opening the developing cylinder after the presaturation in the step 3), and quickly putting the silica gel GF thin-layer plate subjected to the sample application in the step 2) into the developing cylinder to enable the silica gel GF thin-layer plate to lean against the developing cylinder;
4.2) taking out the silica gel GF thin-layer plate when the front edge of the developing agent ascends to a position 6-10 cm away from the sample application origin;
5) color development and identification
5.1) after the reagent on the silica gel GF thin-layer plate developed in the step 4.2) is dried, spraying 5-10% sulfuric acid ethanol solution by mass ratio, and then heating for 3-5 minutes at the temperature of 100-;
5.2) after the temperature is reduced to the room temperature, the silica gel GF thin-layer plate is inspected under the illumination of an inspection light source to obtain an inspection view;
5.3) comparing spots formed at the sample application origin of the sample solution of the bacteria powder to be detected on the inspection view with spots formed at the sample application origins of the fermented cordyceps sinensis bacteria powder standard reference solution and the fermented cordyceps sinensis bacteria powder standard reference solution respectively;
if the spots of the to-be-detected bacterium powder are consistent with the spots of the fermented cordyceps sinensis bacterium powder standard reference liquid, the to-be-detected bacterium powder is fermented cordyceps sinensis bacterium powder, if the spots of the to-be-detected bacterium powder are inconsistent with the spots of the fermented cordyceps sinensis bacterium powder standard reference liquid, and the spots of the to-be-detected bacterium powder are inconsistent with the spots of the fermented cordyceps sinensis bacterium powder standard reference liquid, the to-be-detected bacterium powder is other bacterium powder.
2. The TLC analysis method for distinguishing and distinguishing fermented cordyceps sinensis powder from fermented cordyceps sinensis powder according to claim 1, which is characterized in that:
in the step 5.2), the inspection light source is one of 365nm ultraviolet light, 254nm ultraviolet light and sunlight.
3. The TLC analysis method for distinguishing and distinguishing the fermented cordyceps sinensis powder and the fermented cordyceps sinensis powder according to claim 1, wherein the step 5.2) is specifically as follows: and (4) after the temperature is reduced to room temperature, inspecting the silica gel GF thin-layer plate under 365nm ultraviolet light, 254nm ultraviolet light and sunlight respectively to obtain an inspection view.
4. The TLC analysis method for distinguishing and distinguishing the fermented cordyceps sinensis powder from the fermented cordyceps sinensis powder according to claim 1, 2 or 3, wherein the step 1.1) is specifically as follows: respectively placing a fermented cordyceps sinensis fungus powder reference substance raw material and a fermented cordyceps sinensis fungus powder reference substance raw material into triangular flasks with plugs, respectively adding ethanol with the mass concentration of 70% which is 6 times that of the fermented cordyceps sinensis fungus powder reference substance raw material and the fermented cordyceps sinensis fungus powder reference substance raw material, performing ultrasonic extraction for 60 minutes, placing the mixture to room temperature, filtering the mixture by adopting a microporous filter membrane, and collecting filtrate as standard reference solution;
the step 1.2) is specifically as follows: putting the to-be-identified bacterium powder into a triangular flask with a plug, adding ethanol with the mass concentration of 70% which is 6 times that of the to-be-identified bacterium powder, performing ultrasonic extraction for 60 minutes, putting the mixture to room temperature, filtering the mixture by adopting a microporous filter membrane, and collecting filtrate as sample liquid.
5. The TLC analysis method for distinguishing and distinguishing fermented cordyceps sinensis powder from fermented cordyceps sinensis powder according to claim 4, wherein the TLC analysis method comprises the following steps:
in the step 2), the distance between the sample application origin position and the bottom of the silica gel GF thin-layer plate is 1.0-1.5 cm.
6. The TLC analysis method for distinguishing and distinguishing fermented cordyceps sinensis powder from fermented cordyceps sinensis powder according to claim 5, wherein the TLC analysis method comprises the following steps:
in step 3.1), the volume ratio of ethyl acetate, formic acid, acetic acid and water is 8:0.8:0.8: 1.8.
7. The TLC analysis method for distinguishing and differentiating the fermented cordyceps sinensis bacterial powder and the fermented cordyceps sinensis bacterial powder according to claim 6, wherein the TLC analysis method comprises the following steps:
in the step 3.2), the developing cylinder adopts a double-groove developing cylinder, the size of the double-groove developing cylinder is 10cm multiplied by 10cm, 20cm multiplied by 10cm or 10cm multiplied by 20cm, the developing agent dosage in the double grooves is kept consistent, and the developing agent is presaturated for 15 minutes.
8. The TLC analysis method for distinguishing and differentiating the fermented cordyceps sinensis bacterial powder and the fermented cordyceps sinensis bacterial powder according to claim 7, wherein the TLC analysis method comprises the following steps:
and step 4.2) specifically, taking out the silica gel GF thin-layer plate when the front edge of the developing agent ascends to 8-10 cm away from the origin of the sample application.
9. The TLC analysis method for distinguishing and distinguishing the fermented cordyceps sinensis powder and the fermented cordyceps sinensis powder according to claim 8, wherein the step 5.1) is specifically as follows: blowing the reagent on the silica gel GF thin-layer plate developed in the step 4.2) dry, spraying a sulfuric acid ethanol solution with the mass ratio of 10%, and then heating for 3-5 minutes at 105 ℃ by using an electric heating plate.
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