Chromatographic liquid for separating chloroplast pigment from leaf dry powder, product and application

Abstract

The invention discloses a chromatographic solution for separating chloroplast pigment from leaf dry powder, a product and application, and belongs to the technical field of photosynthetic pigment separation, the chromatographic solution is composed of petroleum ether, acetone and benzene, and the volume ratio of the petroleum ether to the acetone to the benzene is 7.5: 4: 1. The chromatographic solution can separate chlorophyll a'and chlorophyll a as well as chlorophyll b 'and chlorophyll b, and compared with the chromatographic solution in the prior art, the chromatographic solution is convenient for middle school students to distinguish chlorophyll a' and chlorophyll a as well as chlorophyll b 'and chlorophyll b respectively.

Description

Technical Field

[0001] This invention belongs to the field of photosynthetic pigment separation technology, and more specifically relates to a chromatography solvent, product and application for separating chloroplast pigments. Background Technology

[0002] "Extraction and Separation of Chloroplast Pigments" is a classic experiment required for high school biology students. While crucial, this experiment is difficult to master, often hindering the achievement of desired teaching results. This is because the success of the experiment hinges on the extraction and chromatographic separation techniques, with the latter depending on the resolution of the chromatographic medium and the formulation of the chromatographic buffer. Using fresh spinach as the extraction material, current techniques can mostly separate four visible pigments: β-carotene, xanthophyll, chlorophyll a, and chlorophyll b, rarely separating more than four. However, by concentrating the extract and combining it with specialized techniques such as thin-layer chromatography, six to nine pigments can be separated.

[0003] One of the purposes of the chloroplast pigment extraction and separation experiment is to enable middle school students to fully understand the types of chloroplast pigments in leaves, so chromatographic separation of chloroplast pigments is crucial. Chromatographic techniques generally used include paper chromatography, thin-layer chromatography, and column chromatography. Column chromatography using chalk instead of silica gel columns is also possible, with paper chromatography and glass thin-layer chromatography being the most common. Thin-layer chromatography offers high resolution and good separation; however, current chromatographic solutions can only separate a maximum of nine photosynthetic pigments from fresh spinach leaves, which is insufficient for students to fully understand all types of chloroplast pigments. Furthermore, chloroplast pigments include chlorophyll a and chlorophyll a′, and chlorophyll b and chlorophyll b′. Chlorophyll a and chlorophyll a′ are difficult to separate in general chromatography due to their similar properties and solubility, as are chlorophyll b and chlorophyll b′.

[0004] Furthermore, traditional chromatography systems suffer from severe problems such as incomplete separation, tailing, overlapping peaks, and blurred peak shapes when separating chloroplast pigments, especially structurally similar isomers (e.g., chlorophyll a and chlorophyll a′). The separation of these isomers has long been considered a problem that cannot be directly separated using conventional organic solvent gradient systems. Existing research has mostly focused on changing the stationary phase material, introducing reversed-phase chromatography, or using high-performance liquid chromatography (HPLC)—high-cost methods—while minute adjustments to the solvent system are often neglected due to poor predictability and high experimental complexity.

[0005] Therefore, how to provide a chromatography solvent or chromatography product that can separate chlorophyll with similar properties is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of existing technologies, the inventors, through extensive experimentation, discovered that slightly increasing the proportion of petroleum ether (from 7 to 7.5) while decreasing the proportion of acetone (from 5 to 4), and keeping the benzene proportion unchanged, can significantly enhance the solubility differences of chlorophyll isomers without drastically altering the overall polarity window, thereby achieving better separation during chromatography. Crucially, this ratio is not a predictable result from linear changes, nor is it an empirical conclusion that can be obtained through simple polarity gradients.

[0007] Therefore, this invention relates to a chromatography solvent for separating chloroplast pigments in leaf powder, particularly suitable for high-resolution separation of chlorophyll a′ with chlorophyll a and chlorophyll b′ with chlorophyll b. Specifically, the chromatography solvent is composed of petroleum ether, acetone, and benzene in a volume ratio of 7.5:4:1. Compared with the widely used petroleum ether:acetone:benzene ratio of 7:5:1 in the prior art, the ratio of this invention achieves effective separation between pigment isomers, especially significantly improving the separation degree between chlorophyll a′ and a, and chlorophyll b′ and b.

[0008] The present invention further provides a product containing a chromatography solvent for separating chloroplast pigments from plant samples and its application.

[0009] Specifically, the present invention provides the following technical solution:

[0010] The first aspect of the present invention provides the application of a chromatography solvent in the separation of chloroplast pigments in plant samples, wherein the separation of chloroplast pigments in the plant samples includes the separation of chlorophyll a′ from chlorophyll a and the separation of chlorophyll b′ from chlorophyll b; the chromatography solvent includes petroleum ether, acetone and benzene, and the volume ratio of each component is petroleum ether:acetone:benzene = 7.5:4:1.

[0011] Furthermore, the separation of chloroplast pigments from plant samples also includes the separation of β-carotene, pheophytin, xanthophyll, ochratoxin, and neoxanthophyll.

[0012] Furthermore, the plant sample includes dried spinach leaf powder.

[0013] A second aspect of the present invention provides a combination of chromatography solvent and silica gel chromatography strip and its application in the separation of chloroplast pigments in plant samples, wherein the separation of chloroplast pigments in plant samples includes the separation of chlorophyll a′ from chlorophyll a and the separation of chlorophyll b′ from chlorophyll b; and also includes the separation of β-carotene, pheophytin, xanthophyll, ochratoxin and neoxanthophyll;

[0014] Furthermore, the chromatography solvent includes petroleum ether, acetone, and benzene, and the volume ratio of each component is petroleum ether:acetone:benzene = 7.5:4:1.

[0015] Furthermore, the surface of the chromatography strip is covered with silica gel, and the silica gel has a particle size of 2000 mesh.

[0016] Furthermore, the chromatography strip is a high-efficiency aluminum foil thin-layer strip.

[0017] Furthermore, the plant sample includes dried spinach leaf powder.

[0018] In a further optimization of the aforementioned basic technical solution, the inventors have innovatively introduced a 2000-mesh silica gel thin-layer plate as the chromatographic separation medium. Compared to conventionally used 300-1000 mesh silica gel thin-layer materials, the 2000-mesh silica gel thin layer used in this invention has a finer, more uniform particle distribution and a higher surface area, thereby providing stronger pigment adsorption selectivity and better chromatographic resolution.

[0019] By combining this ultra-high mesh silica gel thin layer with an optimized chromatography system (petroleum ether: acetone: benzene = 7.5:4:1), not only can the clarity of pigment spots and the degree of tailing be significantly improved, but it is also particularly suitable for the separation, detection and quantitative analysis of structurally similar chlorophyll a′ and a, b′ and b isomers.

[0020] Traditionally, it is believed that increasing the mesh size of thin-layer plates is only applicable to the separation of components in polymers and complex mixtures. In the separation of small molecules such as pigments, "the benefits of increasing the mesh size are not obvious." Therefore, there are few documents or industrial practices in this field that choose to use 2000-mesh thin-layer materials for the separation of plant chlorophyll components.

[0021] However, through numerous comparative experiments, this invention has demonstrated that when the mesh size of the thin-layer medium is increased to 2000, it can truly work synergistically with the solvent system, significantly widening the migration rate differences between pigment isomers and achieving the separation effect of low-abundance pigment spots that were originally considered "almost inseparable" by those skilled in the art.

[0022] Therefore, a third aspect of the present invention provides a product for separating chloroplast pigments from plant samples, the product comprising a chromatography solvent or the product comprising a chromatography solvent and a silica gel chromatography strip.

[0023] Furthermore, the chromatography solvent comprises petroleum ether, acetone, and benzene, and the volume ratio of each component is petroleum ether:acetone:benzene = 7.5:4:1.

[0024] Furthermore, the surface of the chromatography strip is covered with silica gel, and the silica gel has a particle size of 2000 mesh, and the chromatography strip is a high-efficiency aluminum foil thin-layer strip.

[0025] Furthermore, the present invention also provides related products, which include tool components suitable for chloroplast pigment separation and analysis. These components can be reagent kits, experimental kits, pre-assembled separation systems, or analytical devices containing pre-set chromatographic materials and pigment separation solvents. The products can be applied to various scenarios such as teaching experiments, scientific research, and plant pigment analysis, and have good applicability and ease of operation.

[0026] Therefore, a fourth aspect of the present invention provides a method for separating chloroplast pigments from a plant sample, the method comprising at least the following steps:

[0027] Spotting process: After extracting the pigment from the plant sample to be analyzed (e.g., leaf powder) with an extraction solution (such as acetone or methanol), the obtained pigment extract is taken out and spotted onto the chromatography medium, which is preferably a silica gel thin-layer plate or chromatography strip.

[0028] Spotting method: The preferred spotting operation is to spot the sample point by point to form a linear area (i.e., spotting line), and the spotting can be repeated multiple times; preferably, after each spotting, the sample layer is cured by natural air drying or gentle blowing before the next spotting is performed, so as to improve the uniformity of the spots and the clarity of separation.

[0029] Chromatographic development: The spotted chromatography plate (or strip) is placed into a chromatography tank containing the chromatography solvent for development. The chromatography solvent is a three-component mixture as described above (such as a mixture of petroleum ether, acetone, and benzene), and the specific volume ratio can be selected according to the desired separation effect.

[0030] Chromatographic conditions: Preferably, the chromatography process is carried out in a sealed environment to reduce gradient changes caused by solvent evaporation and improve separation stability. The chromatography time can be set according to the solvent system and plate characteristics; for example, under typical conditions, effective pigment separation can be completed in 8 minutes.

[0031] Solvent usage control: Preferably, the amount of added chromatography solvent is controlled at about 1 mL to ensure that the solvent front advances uniformly and to avoid sample diffusion due to excessive liquid level.

[0032] Through the above steps, the isomers of chlorophyll a′ and a, b′ and b in the pigment mixture can be effectively separated. This method has good practical value and stable reproducibility in general teaching, scientific research, plant classification and identification and other applications.

[0033] As can be seen from the above technical solution, the technical effect achieved by the present invention is as follows:

[0034] This invention creatively provides a chromatography solvent that can separate chlorophyll a′ from chlorophyll a and chlorophyll b′ from chlorophyll b. Compared with existing chromatography solvents, this makes it easier for middle school students to distinguish between chlorophyll a′ and chlorophyll a, and chlorophyll b′ and chlorophyll b.

[0035] Meanwhile, in separating chloroplast pigments, the present invention sets the volume ratio of each component in the chromatography solvent to: petroleum ether: acetone: benzene = 7.5: 4: 1. Compared with the chromatography solvent formulation in the prior art, the ratio of the three components can achieve the best separation effect.

[0036] It is important to note that the solvent ratios used in this invention are not the conventional ratios disclosed in the prior art. Instead, they are ratios selected through systematic experimentation and continuous calibration to achieve high resolution in isomer separation. The significant pigment separation effect resulting from this minor ratio variation exceeds the reasonable expectations of those skilled in the art. Importantly, this invention overcomes a long-standing technical bottleneck: for the first time, without relying on expensive equipment or special stationary phase materials, it achieves effective separation of pigment isomers by adjusting the ratios of conventional solvents, providing a low-cost, high-efficiency new approach for plant pigment analysis.

[0037] In fact, the results of this invention exhibit unpredictable and nonlinear effects. Traditionally, it is believed that adjusting the ratio in the petroleum ether-acetone-benzene system primarily affects the overall pigment migration rate in chlorophyll separation, with little impact on isomer resolution. However, the specific ratios in this invention demonstrate highly selective differences in pigment separation behavior, an effect unpredictable in existing literature and empirical knowledge.

[0038] Based on the findings of this invention, the inventors believe the significant pigment separation effect resulting from this slight change in ratio may be due to the following reasons: Petroleum ether has good solubility for non-polar pigments such as carotene, enabling them to move rapidly during chromatography. Acetone is an organic solvent with slightly stronger polarity than petroleum ether, and has good solubility for highly polar pigments such as chlorophyll, facilitating the diffusion of these pigments in the chromatography solvent. Benzene, with polarity between petroleum ether and acetone, can assist in the dissolution of various pigments, ensuring that each pigment has suitable solubility in the chromatography solvent, thus enabling better separation during the separation process. When the ratio of petroleum ether:acetone:benzene is 7.5:4:1, the major photosynthetic pigments in green leaves, namely β-carotene, xanthophyll, chlorophyll a, and chlorophyll, can form clearly separated pigment bands on the silica gel thin layer. The relatively high proportion of petroleum ether causes non-polar pigments such as carotene to move quickly, forming clear bands at the front of the chromatographic spectrum. The presence of acetone and benzene causes more polar pigments such as chlorophyll to move at a moderate speed, keeping them at a distance from pigments such as carotene. The intervals between the pigment bands are obvious, making it easier to observe and distinguish them, resulting in more consistent separation results and ensuring the reproducibility of the experiment.

[0039] In plant pigment analysis, the mainstream technique uses 300-1000 mesh silica gel plates, with some studies even going up to 1500 mesh. 2000 mesh thin-layer plates are generally considered "unsuitable for pigment chromatography" because they are more expensive, more complex to manufacture, and have higher requirements for solvent systems, sample spotting volume, and development control.

[0040] Without altering the chromatography principle and equipment, the inventors unexpectedly used a 2000-mesh silica gel plate and achieved an unexpected separation effect. This innovation involves technical risks and exploration costs, and cannot be accomplished by ordinary technical personnel through experience and intuition alone.

[0041] Importantly, the results of this invention exhibit high selectivity and synergistic enhancement. This is because, when using a 2000-mesh silica gel plate alone, the separation effect is still unsatisfactory under the traditional 7:5:1 solvent ratio. However, when used in conjunction with the optimized solvent ratio (7.5:4:1) of this invention, clear separation of isomers can be achieved. This indicates that the value of the 2000-mesh thin-layer plate does not have an independent linear improvement significance, but rather achieves unexpected technical effects through the synergistic combination of technologies proposed in this invention.

[0042] Furthermore, for those skilled in the art, it is difficult to obtain this preferred solution through conventional experiments. This is because attempting to improve resolution by "increasing the mesh count of the board" is not a conventional method due to the following obstacles:

[0043] Increased background noise due to increased mesh size of chromatography plates;

[0044] The component unfolding speed is reduced, and the unfolding time and ambient humidity need to be re-optimized;

[0045] In pigment separation, high-mesh plates are prone to causing spot diffusion, affecting reproducibility;

[0046] These nonlinear interference factors mean that using 2000-mesh thin-layer plates in conjunction with pigment analysis is not a predictable and successful approach. Only through repeated experiments and adjustment of multiple parameters can the significant separation effect described in this invention be obtained.

[0047] One of the core aspects of this invention lies in the significant improvement in the separation effect between chlorophyll isomers (such as a′ and a, b′ and b) through precise control and optimized combination of the ratio of chromatographic solvent components and the physical parameters of the chromatographic medium. It should be noted that although the preferred chromatographic solvent of this invention is petroleum ether, acetone, and benzene in a volume ratio of 7.5:4:1, in practical applications, this ratio can be finely adjusted within a certain range to adapt to different environmental conditions, sample concentrations, or batch variations of the chromatographic plates. For example, within the range of 7.4-7.6% petroleum ether, 3.8-4.2% acetone, and 0.9-1.1% benzene, essentially equivalent separation effects can still be achieved. Therefore, without changing the separation mechanism and purpose of this invention, linear adjustments to the ratio of the three components within the range of ±0.1-0.2 units should still be considered equivalent to the present invention.

[0048] Similarly, the silica gel particle size of the chromatography plate is 2000 mesh, which was determined through comparative experiments and has the best separation effect. However, those skilled in the art should understand that other silica gel particle sizes between 1900 and 2100 mesh can also achieve essentially the same pigment separation ability under the specific solvent system of this invention. In particular, when the particle size is above 1950 mesh, the separation bandwidth narrows and the spot clarity is improved, achieving similar or the same technical effect. Therefore, without changing the chromatography separation principle and structural characteristics, making a fine adjustment of ±100 mesh to the silica gel particle size should constitute an equivalent variation of this invention and should also be protected by the patent rights of this invention. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort. The impact of extraction effect.

[0050] Appendix Figure 1The results are the chromatographic separation results of Examples 1-3; wherein, A is the separation result of the chromatographic solvent petroleum ether:acetone:benzene = 7.5:4:1 + paper chromatography strip; B is the separation result of the chromatographic solvent petroleum ether:acetone:benzene = 7.5:4:1 + ordinary silica gel thin-layer plate; C is the separation result of the chromatographic solvent petroleum ether:acetone:benzene = 7.5:4:1 + high-performance silica gel thin-layer chromatography strip.

[0051] Appendix Figure 2 The results are the chromatographic separation results of Examples 4 and 5; where A is the separation result of the chromatographic solvent petroleum ether:acetone:benzene = 20:2:1 + ordinary silica gel thin-layer plate; B is the separation result of the chromatographic solvent petroleum ether:acetone:benzene = 20:2:1 + high-efficiency silica gel thin-layer chromatography strip.

[0052] Appendix Figure 3 The results are the chromatographic separation results of Examples 6 and 7; wherein, A is the separation result of the chromatographic solvent petroleum ether:acetone:benzene = 7:5:1 + high-performance silica gel thin-layer chromatography strip; B is the separation result of the chromatographic solvent petroleum ether:acetone:benzene = 7:4:1 + high-performance silica gel thin-layer chromatography strip. Detailed Implementation

[0053] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] To verify the advantages of the chromatography strips and chromatography solvents in this invention and their synergistic effect, the following Examples 1-5 were set up. In Examples 1-3, under the premise that the chromatography solvent was petroleum ether:acetone:benzene = 7.5:4:1, chromatography was performed using paper chromatography strips, ordinary silica gel thin-layer plates (the difference from high-efficiency silica gel thin-layer chromatography strips is that the silica gel particle size is only 400-500 mesh), and high-efficiency silica gel thin-layer chromatography strips (i.e., the chromatography strips disclosed in this invention). It should be understood that the experiments in Examples 1-3 were all set with a single variable selection, and the only variable was the chromatography strip.

[0055] Example 4 involves chromatography using a standard silica gel thin-layer plate with a petroleum ether:acetone:benzene ratio of 20:2:1. Example 5 involves chromatography using a high-efficiency silica gel thin-layer chromatography strip (i.e., the chromatography strip disclosed in this invention) with a petroleum ether:acetone:benzene ratio of 20:2:1. Example 6 involves chromatography using a high-efficiency silica gel thin-layer chromatography strip (i.e., the chromatography strip disclosed in this invention) with a petroleum ether:acetone:benzene ratio of 7:5:1. Example 7 involves chromatography using a high-efficiency silica gel thin-layer chromatography strip (i.e., the chromatography strip disclosed in this invention) with a petroleum ether:acetone:benzene ratio of 7:4:1.

[0056] Example 1: Separation of chloroplast pigments from spinach leaf powder (using chromatography solvent and paper chromatography strips)

[0057] Weigh 1g of dried spinach leaf powder and place it in a mortar. Add 4ml of anhydrous ethanol, a small amount of grinding sand (a 3:1 mixture of low-iron impurities SiO2 and CaCO3, with an amount of 0.15g) and a small amount of sodium EDTA (0.03g). Grind thoroughly for 2 minutes. Place a 55mm diameter white polyester fiber circular cloth into a cone shape on the surface of the grinding liquid. Use a Pasteur pipette to draw up the seeping grinding liquid through the cloth. This is the extract. Pour about 0.5ml into a 2ml small plastic centrifuge tube for later use.

[0058] Using a spotting capillary tube with an inner diameter of 0.5 mm, spot the sample from left to right along the pencil line at the bottom of the paper chromatography strip (the strip dimensions are 90 mm x 10 mm) to form a spotting line. After each spotting, dry the tube with a bulb syringe before spotting again, for a total of 3 spottings.

[0059] Using a Pasteur pipette, pipette 1 ml of chromatography buffer (petroleum ether:acetone:benzene = 7.5:4:1) into the bottom of a 50 ml capped plastic chromatography tube. Carefully place the spotted and dried chromatography strip into the tube, quickly cap it, and place it on a test tube rack for chromatography. After 8 minutes, the pigment molecules will separate. Stop chromatography, remove the chromatography strip, and photograph the separation results. See the attached image for the chromatography results. Figure 1 A in the middle.

[0060] Example 2: Separation of chloroplast pigments from spinach leaf powder (chromatographic solvent + ordinary silica gel thin-layer plate)

[0061] Weigh 1g of dried spinach leaf powder, place it in a mortar, add 4ml of anhydrous ethanol, a little grinding sand and a little sodium EDTA (0.03g), grind thoroughly for 2 minutes, place a white polyester fiber circular cloth with a diameter of 55mm on the surface of the above grinding liquid, and use a Pasteur pipette to draw up the seeping grinding liquid through the cloth, which is the extract. Take about 0.5ml and inject it into a 2ml small plastic centrifuge tube for later use.

[0062] Using a 0.5 mm inner diameter spotting capillary, spot the sample from left to right along the pencil line at the bottom of a standard silica gel thin-layer plate (chromatographic strip dimensions: 100 mm × 25 mm) to form a spotting line. After each spotting, dry the plate with a bulb syringe before spotting again, for a total of 3 spottings.

[0063] Using a Pasteur pipette, pipette 1 ml of chromatography buffer (petroleum ether:acetone:benzene = 7.5:4:1) into the bottom of a 50 ml capped plastic chromatography tube. After spotting and drying, carefully place the thin-layer plate into the tube, quickly cap it, and place it on a test tube rack for chromatography. After 8 minutes, the pigment molecules will separate. Stop chromatography, remove the tube, and photograph the separation results. See the attached image for the chromatography results. Figure 1 B in the middle.

[0064] Example 3: Separation of chloroplast pigments from spinach leaf powder (chromatographic solvent + high-performance silica gel thin-layer chromatography strips)

[0065] Weigh 1g of spinach leaves, place them in a mortar, add 4ml of anhydrous ethanol, a little grinding sand and a little sodium EDTA (0.03g), grind thoroughly for 2 minutes, then place a 55mm diameter white polyester fiber circular cloth on the surface of the above grinding liquid, and use a Pasteur pipette to draw up the seeping grinding liquid through the cloth, which is the extract. Take about 0.5ml and inject it into a 2ml small plastic centrifuge tube for later use.

[0066] Using a spotting capillary tube with an inner diameter of 0.5 mm, spot the sample from left to right along the pencil line at the bottom of the high-performance thin-layer chromatography strip (the strip dimensions are 90 mm x 10 mm) to form a spotting line. After each spotting, dry the tube with a bulb syringe before spotting again, for a total of 3 spottings.

[0067] Using a Pasteur pipette, 1 ml of chromatography buffer (petroleum ether:acetone:benzene = 7.5:4:1) was injected into the bottom of a 50 ml capped plastic chromatography tube. The dried chromatography strip was carefully placed in the tube, and the tube was quickly capped and placed on a test tube rack for chromatographic separation. After 8 minutes, the pigment molecules separated. Chromatography was stopped, the chromatography strip was removed, and the separation results were photographed. The chromatography strip was coated with silica gel with a particle size of 2000 mesh, and the chromatography strip was a high-efficiency aluminum foil thin-layer strip. The chromatographic separation results are shown below. Figure 1 C in the middle.

[0068] From the above Figure 1It is known that, under the premise of keeping the chromatography solvent unchanged, when using paper chromatography strips to separate chloroplast pigments in the extract, all pigments cannot be separated; when using ordinary silica gel thin-layer plates to separate chloroplast pigments in the extract, 7 pigments can be separated, but chlorophyll a′ is not separated from chlorophyll a, and chlorophyll b′ is not separated from chlorophyll b; however, when using the chromatography solvent of the present invention in combination with high-efficiency aluminum foil thin-layer plates to separate chloroplast pigments in the extract, 13 pigments can be separated, and chlorophyll a′ and chlorophyll a, as well as chlorophyll b′ and chlorophyll b, can be clearly separated. This indicates that the chromatography solvent of the present invention and the high-efficiency aluminum foil thin-layer chromatography strips have a synergistic effect, which can effectively separate chlorophyll a′ and chlorophyll a, and chlorophyll b′ and chlorophyll b.

[0069] Example 4: Separation of chloroplast pigments from spinach leaves (using ordinary chromatography solvent and ordinary silica gel thin-layer plate).

[0070] Compared to Example 2, the difference is that a petroleum ether:acetone:benzene ratio of 20:2:1 was used as the chromatographic solvent, and a standard silica gel thin-layer plate was used for spotting. The chromatographic separation results are shown below. Figure 2 A in the middle.

[0071] Example 5: Separation of chloroplast pigments from spinach leaves (using ordinary chromatography solvent and high-performance silica gel thin-layer chromatography strips)

[0072] Compared to Example 2, the difference lies in the use of a petroleum ether:acetone:benzene ratio of 20:2:1 chromatography solvent, the surface of the chromatographic strip used for spotting being covered with silica gel with a particle size of 2000 mesh, and the chromatographic strip being a high-efficiency aluminum foil thin-layer strip. The chromatographic separation results are shown below. Figure 2 B in the middle.

[0073] Depend on Figure 2 It is known that when the chromatographic solvent is petroleum ether:acetone:benzene = 20:2:1, using conventional chromatography and the thin-layer chromatography strips of the present invention, apart from β-carotene, which moves the fastest during chromatography, being separated, other pigments were not effectively separated on either type of thin-layer strip. Therefore, it can be concluded that the known chromatographic solvent of petroleum ether:acetone:benzene = 20:2:1 is not suitable for the effective separation of pigments by conventional and high-performance thin-layer chromatography.

[0074] Example 6: Separation of chloroplast pigments from spinach leaves (using ordinary chromatography solvent and high-performance silica gel thin-layer chromatography strips)

[0075] Compared to Example 2, the difference is that a chromatographic solvent of petroleum ether:acetone:benzene = 7:5:1 was used. The chromatographic separation results are shown below. Figure 3 A in the middle.

[0076] Example 7: Separation of chloroplast pigments from spinach leaves (using ordinary chromatography solvent + high-performance silica gel thin-layer chromatography strips)

[0077] Compared to Example 2, the difference is that a chromatographic solvent of petroleum ether:acetone:benzene = 7:4:1 was used. The chromatographic separation results are shown below. Figure 3 B in the middle.

[0078] Depend on Figure 3 It is known that when the chromatographic solvent is petroleum ether:acetone:benzene = 7:5:1, using the high-efficiency thin-layer chromatography strip of the present invention for chromatography, nine pigments are separated, but chlorophyll a′ is not clearly separated from chlorophyll a, nor is chlorophyll b′ clearly separated from chlorophyll b; when the chromatographic solvent is petroleum ether:acetone:benzene = 7:4:1, using the thin-layer chromatography strip of the present invention for chromatography, the separation effect is similar to the former, and chlorophyll a′ and chlorophyll a, chlorophyll b′ and chlorophyll b cannot be clearly separated.

[0079] Depend on Figure 1 , Figure 2 , Figure 3 As described in the records, the chromatography solvent of this invention has a superior effect on pigment separation compared to the chromatography solvents disclosed in the prior art, and can separate 13 pigments. It can also separate chlorophyll a′ from chlorophyll a and chlorophyll b′ from chlorophyll b to a certain extent. However, the effect is generally poor when using ordinary silica gel thin-layer chromatography strips, separating only 7 pigments, and chlorophyll a′ from chlorophyll a and chlorophyll b′ from chlorophyll b are not easily separated. Furthermore, the high-efficiency thin-layer chromatography strip of this invention also has a better pigment separation effect compared to the chromatography strips of the prior art. However, if only the chromatography solvent is used for separation without using the chromatography strip of this invention, even if more than 9 pigments can be separated, the separation of chlorophyll a′ from chlorophyll a and chlorophyll b′ from chlorophyll b is not significant, making it inconvenient for teaching purposes. Alternatively, if only the chromatography strip is used with the chromatography solvent of the prior art, the chromatography effect will be greatly reduced. Therefore, chlorophyll a′ and chlorophyll a, and chlorophyll b′ and chlorophyll b can only be effectively separated when the chromatography solvent is petroleum ether:acetone:benzene = 7.5:4:1, the chromatography strip is a high-efficiency aluminum foil thin-layer strip, and the surface is covered with silica gel with a particle size of 2000 mesh.

[0080] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of chromatographic fluid for separating chlorophyll pigments in plant samples, characterized in that, The separation of chlorophyll pigments includes or is the separation of chlorophyll a' and chlorophyll a and the separation of chlorophyll b' and chlorophyll b; the chromatographic solution includes petroleum ether, acetone and benzene, and the volume ratio of each component is petroleum ether:acetone:benzene=7.5:4:

1.

2. Use according to claim 1, characterized in that, The separation of chlorophyll pigments in the plant sample also includes the separation of β-carotene, de-magnesium chlorophyll, lutein, violaxanthin and neoxanthin.

3. Use according to any one of claims 1-2, characterized in that, The plant sample is leaf dry powder, such as spinach leaf dry powder.

4. Combination of chromatography fluid and silica gel chromatography strips and use in separating chlorophyll pigments in plant samples, characterized in that, The separation of chlorophyll pigments in the plant sample includes the separation of chlorophyll a' and chlorophyll a and the separation of chlorophyll b' and chlorophyll b; and also includes the separation of β-carotene, de-magnesium chlorophyll, lutein, violaxanthin and neoxanthin; The chromatographic solution includes petroleum ether, acetone and benzene, and the volume ratio of each component is petroleum ether:acetone:benzene=7.5:4:1; The surface of the chromatographic strip is covered with silica gel, and the particle size of the silica gel is 2000 mesh.

5. Use according to claim 4, characterized in that, The chromatographic strip is a high-efficiency aluminum foil thin layer strip.

6. Use according to any one of claims 4-5, characterized in that, The leaf dry powder includes spinach leaf dry powder.

7. A product of separating chlorophyll pigments from leaf blade dry powder, characterized in that, The product includes a chromatographic solution or the product includes a chromatographic solution and a silica gel chromatographic strip; The chromatographic solution includes petroleum ether, acetone and benzene, and the volume ratio of each component is petroleum ether:acetone:benzene=7.5:4:1; The surface of the chromatographic strip is covered with silica gel, and the particle size of the silica gel is 2000 mesh, and the chromatographic strip is a high-efficiency aluminum foil thin layer strip.

8. A product for separating chloroplast pigments from dry powder of leaves according to claim 7, characterized in that, The product includes a kit or an experimental package.

9. A method of separating chloroplast pigments from leaf dry powder, characterized by, The product of any one of claims 7-8 is used for separation, and the specific process includes: first, the chlorophyll pigment extract solution of the leaf dry powder is sucked, and then the sample is spotted on the silica gel chromatographic strip; then the spotted chromatographic strip is placed in the chromatographic solution for chromatography, and after 8 minutes, the separation of chlorophyll pigments is completed.

10. The method of claim 9, wherein, The spotting method is line by point, i.e. line spotting; after each spotting, the next time is spotted after blowing dry; the amount of chromatographic solution added is 1ml; and the chromatography is sealed.

Images