Preparation method of filler for amino acid enantiomer separation and application of filler

Abstract

The invention relates to a preparation method of a filler for amino acid enantiomer separation and application of the filler, the preparation method comprises the following steps: (1) uniformly dispersing graphene oxide nanosheets in a PBS buffer solution to form a mixed solution I, the pH range of the mixed solution I is 6-7, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide into the mixed solution I after ultrasonic treatment, uniformly stirring to form a mixed solution II, then adding N-hydroxysuccinimide into the mixed solution II, and stirring until the solution becomes black from yellowish-brown to form a mixed solution III; and (2) adding an L-type or D-type amino acid compound into the mixed solution III, uniformly stirring at room temperature to form a mixed solution IV, alternately centrifugally cleaning the mixed solution IV for multiple times by using ultrapure water and diluted hydrochloric acid with the concentration of not more than 10%, and then transferring the obtained chiral nanosheet solution into a dialysis bag for dialysis to obtain the filler. The separation effect is good.

Description

Technical Field

[0001] The present invention relates to the technical field of chiral compound separation, and in particular to a preparation method of a filler for amino acid enantiomer separation and application of the filler. Background Art

[0002] Chirality is a mirror-image structure characteristic of molecules, and there are many differences in their physical and chemical properties. Chiral molecules are closely related to human health and daily life; for example, enantiomeric drugs, food additives, biocatalytic enzymes, etc. Due to the high cost, low yield, and precise and harsh synthesis conditions of direct synthesis of single chiral molecules, people currently obtain single chiral molecules mainly through purification and separation. The chiral screening of the enantiomeric mixture synthesized in the previous order is used to capture the single chiral molecule. The current methods used for chiral screening include chromatographic separation and membrane separation; the former has the disadvantages of high instrument cost, long analysis time, high pressure conditions, and low separation flux; the latter is particularly difficult to achieve in-situ and real-time online detection and monitoring. In addition, the above two methods are highly dependent on separation materials (separation columns and separation membranes). In order to overcome the above difficulties, the innovation of chiral separation materials is the key.

[0003] In recent years, two-dimensional carbon-based materials have attracted the interest of researchers due to their unique structure and properties. Graphene-based materials are a typical two-dimensional carbon-based material. Since the first isolation of single-layer graphene in 2004, graphene-based materials have received extensive research and attention. Graphene-based materials have a unique atomic structure, excellent mechanical properties and a large specific surface area. These properties give graphene-based materials excellent loading space and low mass transfer resistance, enabling them to carry a large amount of external materials.

[0004] For example, according to the Chinese invention patent with patent number CN202010543991.3 (publication number CN111735897A), "A graphene oxide-based chiral nanocomposite material and its preparation method", S1. Disperse the left-handed amino acid derivative gel factor in a metal ion solution, and obtain a composite hydrogel after heating and cooling; S2. Add the graphene oxide solution dropwise to the composite hydrogel, shake and mix evenly, and let stand at room temperature to separate into layers; S3. Filter to obtain a chiral solid composite material coated with graphene oxide, that is, the graphene oxide-based chiral nanocomposite material.

[0005] The chiral function of this patent is imparted by different metal ions, and it can only perform chiral screening on one substance, with poor universality; and the main body of the material of this patent is still the traditional coating gel system, in which graphene oxide only plays the role of an additive, and the separation effect is limited. Summary of the invention

[0006] The first technical problem to be solved by the present invention is to provide a method for preparing a filler for amino acid enantiomer separation with good separation effect in view of the above-mentioned prior art.

[0007] The second technical problem to be solved by the present invention is to provide an application of the above-mentioned filler in a chiral chromatographic column in view of the above-mentioned prior art.

[0008] The technical solution adopted by the present invention to solve the above-mentioned first technical problem is: a method for preparing a filler for amino acid enantiomer separation, characterized in that it comprises the following steps:

[0009] (1) The graphene oxide nanosheets are uniformly dispersed in a PBS buffer solution to form a mixed solution 1, and the pH range of the mixed solution 1 is 6 to 7. After ultrasonication for 50 to 70 minutes, 1-ethyl-(3-dimethylaminopropyl)carbodiimide is added to the mixed solution 1 and stirred to form a mixed solution 2, and then N-hydroxysuccinimide is added to the mixed solution 2 and stirred until the solution changes from yellow brown to black to form a mixed solution 3;

[0010] The purpose of ultrasonication for 50 to 70 minutes in this step is to stretch the curled or stacked graphene oxide nanosheets into a single layer as much as possible without damaging the structure of the sheet.

[0011] The purpose of adding PBS is that the pH value of PBS is usually between 7.2 and 7.4, which helps to maintain the stability of the NHS-EDC reaction system. Before using EDC-NHS, PBS is used to wash and disperse the graphene oxide nanosheets to remove impurities and unnecessary ions that may affect the reaction and ensure the purity of the reaction. At the same time, PBS buffer does not contain any primary amines or carboxyl groups, which can avoid competitive reactions during the activation process and improve the activation efficiency.

[0012] The NHS-EDC activation process involves two steps: first, EDC acts as a zero-length crosslinker to react with the carboxyl groups on graphene oxide to form a highly active ester intermediate. In this step, EDC reacts with the carboxyl groups to form carboxyl esters, preparing for the next coupling reaction. Next, NHS reacts with the carboxyl esters generated by the reaction of EDC to form NHS esters. This NHS ester is very active and can quickly react with the amine groups on the added chiral amino acids to form stable amide bonds, ultimately forming a graphene oxide nanosheet solution with chiral functions.

[0013] The preparation method of PBS buffer solution is as follows: add 7.8-8.2 g NaCl, 0.2-0.25 g KCl, 1.42-1.45 g Na2HPO4, and 0.22-0.25 g KH2PO4 into 1 L ultrapure water and mix well.

[0014] (2) Adding L-type or D-type amino acid compounds to the mixed solution three (the size and electrical properties of the stacked two-dimensional channels can be regulated by changing the type of amino acids), stirring for 12 to 18 hours at room temperature and a stirring speed of 800 to 1200 r / min to form a mixed solution four, and washing the mixed solution four with ultrapure water and dilute hydrochloric acid with a concentration of no more than 10% by alternating centrifugation for multiple times, and then transferring the obtained chiral nanosheet solution to a dialysis bag with a molecular cutoff of 8000-12000 kDa and dialyzing for 5 to 7 days to remove excess acid and remaining impurities, thereby obtaining the filler.

[0015] The mass ratio of the graphene oxide nanosheets to the amino acid compound with a purity of 99% is 0.8:1 to 1.2:1;

[0016] The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) is 4:1-5:1;

[0017] The volume ratio of graphene oxide nanosheets with a concentration of 4 mg / mL to PBS buffer was 1:6 to 1:8;

[0018] The mass ratio of graphene oxide nanosheets to EDC is 1.6:1 to 2:1.

[0019] In the step (1), the method for preparing the graphene oxide nanosheets comprises the following steps:

[0020] (a) adding 2.4-2.6 g K2SO4 and 2.4-2.6 g P2O5 to 11-13 mL H2SO4, stirring and ultrasonicating until the solution is clear and transparent, adding 2.8-3.2 g 8000 mesh graphite powder to the mixed solution, stirring in a water bath at 80-85° C. for 4.5-5.5 h, to obtain a pre-oxidized graphite raw material;

[0021] (b) drying the pre-oxidized graphite raw material at 60-70° C. under vacuum for 8-12 h to obtain a pre-oxidized graphite block, then grinding the obtained pre-oxidized graphite block using an agate mortar, gradually dissolving it in 118-122 mL of concentrated H2SO4 under stirring at 4-6° C., then placing it in a water bath controlled at 55-60° C., then gradually adding 14.7-15.2 g of KMnO4 to the solution, and finally continuing to stir at room temperature for 2.0-2.5 h to obtain a mixed solution V, at which the solution appears dark green;

[0022] (c) slowly diluting the mixed solution 5 with 200-250 mL of deionized water in a water bath at 75-80° C., stirring at room temperature for 2.0-2.5 h after the dilution, and finally adding 18-22 mL of 30% H2O2, stirring for 1-1.5 h, and ultrasonicating for 20-40 min to obtain a mixed solution 6;

[0023] (d) After the mixed solution VI is allowed to stand, the bottom precipitate is taken and dissolved in 1000-1200 mL ultrapure water. After centrifugal washing, the precipitate at the bottom of the cup is crushed with a glass rod while adding a 1:10 HCl aqueous solution for dilution and washing to ensure that the precipitated graphene oxide nanosheets are fully washed. Then, the precipitate is centrifuged and washed with pure water, and 1000 mL ultrapure water is used to dilute and fix the volume of the centrifuged precipitate. The solution is stirred for 1-1.5 hours, ultrasonicated for 20-40 minutes, and then allowed to stand for 5-7 days to obtain a graphene oxide nanosheet solution with a concentration of 3.5-4.2 mg / mL.

[0024] The temperature for adding KMnO4 in step (b) is 55-60°C, and the temperature for diluting with water in step (c) is 75-80°C. The above conditions will make the strong acid peel off the graphene oxide more intense, and the obtained nanosheet layer has more carboxyl groups at the edge, thereby greatly increasing the sites of the chiral modifier, thereby improving the chiral separation performance of the filler.

[0025] The technical solution adopted by the present invention to solve the above-mentioned second technical problem is: an application of the above-mentioned filler in a chiral chromatographic column, characterized in that: the chiral chromatographic column includes a column tube and an inlet tube and a sample outlet tube connected to the column tube, the column tube is filled with the above-mentioned filler, and the sample outlet tube of the column tube is connected to a vacuum pump, so that the filler is filtered to form a stacked channel structure.

[0026] In order to make the graphene oxide nanosheets form a stacked channel structure, the packing method of the chiral chromatographic column is:

[0027] (1) Ultrasonicate the filler to make it evenly dispersed, and ensure that the water temperature is always below 30° C. during the ultrasonication process to avoid curling and stacking of graphene oxide nanosheets;

[0028] (2) Extend and position the column tube vertically, add the ultrasonic filler into the column tube, and then let it stand for 10 to 30 minutes;

[0029] (3) A vacuum pump is connected to the sample outlet tube, and the vacuum degree of the vacuum pump is controlled between 0.1 MPa and 0.3 MPa for filtration to ensure uniform deposition of the graphene oxide nanosheet layer. Too high a pressure will cause excessive compression between the layers of the graphene oxide nanosheet, thus affecting the permeability and separation performance.

[0030] In order to further form a stacked channel structure of graphene oxide nanosheets, the mass of filler to be loaded in the column tube is M, which is added in N times, and each time the filler is added, the above steps (1), (2), and (3) are sequentially adopted. In this way, adding filler in small amounts and multiple times can improve the filtration effect and ensure the stability and uniformity of the formed channel structure.

[0031] In the above scheme, there are at least two tube bodies, and each tube body is detachably connected, the sample inlet tube is connected to the top tube body, and the sample outlet tube is connected to the bottom tube body. Each tube body is provided with an upper gasket and a lower gasket, the upper gasket is located above the lower gasket and the two are arranged at an interval, and the area between the upper gasket and the lower gasket is a filling area for filling fillers. In this way, the filler in a tube body is added each time, the amount added is small, and the filler is uniform, which is convenient for forming a stacked channel structure.

[0032] Preferably, a diverter plate is provided above each of the upper gaskets, the diverter plate is provided with a plurality of vertically penetrating liquid holes, and a liquid passage is provided on the upper surface of the diverter plate to connect the liquid passage holes. If the mobile phase flows unevenly, it may cause partial collapse of the packing and destroy the stacked channel structure formed by it. Therefore, the present application sets a diverter plate so that the mobile phase flows evenly through the packing, so that the impact force on the packing is uniform.

[0033] The diverter plate can have various structural forms. Preferably, the liquid-passing hole located in the central part of the diverter plate is used as the central hole, and a plurality of liquid-passing hole groups are evenly spaced radially around the central hole. Each liquid-passing hole group includes a plurality of liquid-passing holes evenly spaced circumferentially along the central hole.

[0034] The liquid passage includes a plurality of first passages and a plurality of second passages, each of the first passages is annular and extends along the circumference of the central hole, and the plurality of second passages are radially distributed with the central hole as the center, and the liquid passage holes in the same group are connected through the first passages, and the liquid passage holes in different groups are connected through the second passages. In this way, the liquid is transmitted through the liquid passage, so that liquid flows at each liquid passage hole, and the liquid passage holes are evenly arranged on the diverter plate and flow downward evenly.

[0035] Compared with the prior art, the advantages of the present invention are: the filler of the present invention is a carbon-based nanomaterial, the present invention uses two-dimensional graphene oxide nanosheets as a matrix, and is modified by a chiral single enantiomer (D / L) amino acid compound to prepare a chiral filler that can be used to screen amino acid enantiomers, that is, the chiral function of the present application is determined by the introduced chiral modifier (chiral single enantiomer (D / L) amino acid compound), the chiral function is more efficient, and the chiral screening of amino acids can be performed at the same time, and it has certain universality, and it shows efficient selectivity for various amino acids such as D / L-tyrosine, D / L-phenylalanine, and D / L-threonine;

[0036] The chiral filler prepared by the present invention has two-dimensional nano-transport channels stacked layer by layer, and the structures of chiral sites and water channels are stable. Therefore, it does not need to be further coupled to a supporting material, and can be used as an adsorbent or filler used in an extraction column or a chromatographic column, thereby improving the separation efficiency per unit volume.

[0037] The filler of the present application has pH response to mobile phases of different acidity and alkalinity, and has good repeatability, and can be adapted to a variety of mobile phases (such as ultrapure water). The specific reasons are as follows:

[0038] 1. Traditional and currently mainstream chromatographic columns require organic solvents as mobile phases to provide a suitable dissolving environment for the sample, ensuring that the sample can be fully dissolved and move in the chromatographic system, but chiral amino acids also have good solubility in water.

[0039] 2. Since graphene oxide has a large specific surface area and chiral sites, a good separation effect can still be achieved without changing the type of organic solvent (such as methanol, acetonitrile, isopropanol, etc.) and thus changing the interaction between the solvent and the sample.

[0040] 3. The pH value of the mobile phase has an inevitable and significant impact on the separation effect. Acids and bases are directly used without other complex organic solvents to adjust the pH value of the mobile phase. For example, after the mobile phase is adjusted to pH = 12 by adding NaOH, in addition to the change in hydrogen bond formation, the chiral amino acid is deprotonated. Due to the different degrees of deprotonation of amino acids with different chiralities, the difference increases, the retention time difference is greater, and the separation effect is best. Under neutral conditions, there are only chiral sites provided by chiral modifiers, so the chiral chromatographic column has the effect of pH regulation.

[0041] 4. The chiral chromatographic column filler prepared by the present invention has a good stacking arrangement between the chiral graphene oxide nanosheet layers due to the appropriate pressure driving size and multiple small-scale filling methods during preparation, and the structures of the chiral sites and water channels are stable. Therefore, it does not need to be further coupled to a supporting material, and can be used as an adsorbent or filler used in an extraction column or a chromatographic column, thereby improving the separation efficiency per unit volume.

[0042] The novel carbon-based chiral separation filler provided by the present invention is safe and environmentally friendly, can be adapted to a variety of filtering devices (such as suction filtration, cross flow, filter press, chromatography (such as HPLC, FPLC), etc.), and can be used in a variety of separation phase environments (such as ultrapure water), solving important technical limitations of traditional fillers such as high energy consumption, high cost, low efficiency, and difficulty in preparation. The filler can self-assemble into a membrane material, relying on the current mainstream membrane technology equipment and means to achieve high-throughput and high-selectivity separation of chiral enantiomer mixtures. When the filler is used, it is simple to operate, low in energy consumption, low in cost, and has high practicality and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is a SEM cross-sectional view of the filler of Example 1 of the present invention;

[0044] Figure 2 FTIR-ATR diagram of the filler of Example 1 of the present invention;

[0045] Figure 3 This is a chromatogram of the separation of tyrosine enantiomers using the filler of Example 3 of the present invention;

[0046] Figure 4 The filler of Example 3 of the present invention is used for the separation chromatogram of single-component D-tyrosine;

[0047] Figure 5 The filler of Example 3 of the present invention is used for the separation chromatogram of single-component L-tyrosine;

[0048] Figure 6 The figure is a comparison of the retention rates of D- / L-tyrosine in tyrosine enantiomers by the filler of Example 4 of the present invention under different pH mobile phases;

[0049] Figure 7 This is a chromatogram of the separation of phenylalanine enantiomers using the filler of Example 5 of the present invention;

[0050] Figure 8 is a cross-sectional view of a separation unit according to Embodiment 3 of the present invention;

[0051] Fig. 9 For the feed pipe and the discharge pipe to be installed Figure 8 A schematic diagram of the structure of a separation unit;

[0052] Fig.10 for Fig. 9 A schematic diagram of the structure in another direction;

[0053] Fig.11 for Fig.10 A cross-sectional view of

[0054] Fig.12 for Fig.10 Schematic diagram of the decomposition of

[0055] Fig.13 for Fig.10 Schematic diagram of the structure of the manifold. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below with reference to the accompanying drawings.

[0057] Example 1

[0058] A method for preparing a filler for amino acid enantiomer separation comprises the following steps:

[0059] (1) Graphene oxide nanosheets were dispersed uniformly in a PBS buffer solution to form a mixed solution 1, and the pH of the mixed solution 1 was adjusted to 6.5. After ultrasonic treatment for 60 minutes, 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added to the mixed solution 1 and stirred uniformly to form a mixed solution 2. N-hydroxysuccinimide was then added to the mixed solution 2, and the solution was stirred until it changed from yellow-brown to black to form a mixed solution 3.

[0060] (2) adding an L-type or D-type amino acid compound (specifically L-proline) to the mixed solution three, stirring for 16 hours at room temperature and a stirring speed of 900 r / min to form a mixed solution four, washing the mixed solution four alternately by centrifugation for 3 times with ultrapure water and dilute hydrochloric acid with a concentration of not more than 10% (i.e., washing by centrifugation with water, then washing by centrifugation with dilute hydrochloric acid, then washing by centrifugation with water, and so on, alternately 3 times), and then transferring the obtained chiral nanosheet solution to a dialysis bag with a molecular cutoff of 8000 to 12000 kDa and dialyzing for 5 days to remove excess acid and remaining impurities, thereby obtaining the filler;

[0061] The mass ratio of the graphene oxide nanosheets to the amino acid compound with a purity of 99% is 1:1;

[0062] The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) is 4:1;

[0063] The added volume ratio of graphene oxide nanosheets with a concentration of 4 mg / mL to PBS buffer was 1:5;

[0064] The mass ratio of graphene oxide nanosheets to EDC is 1.6:1.

[0065] In this embodiment, the method for preparing graphene oxide nanosheets comprises the following steps:

[0066] (a) 2.5 g K2SO4 and 2.5 g P2O5 were added to 12 mL H2SO4, and the mixture was stirred and ultrasonicated until the solution became clear and transparent, and then 3.0 g 8000 mesh graphite powder was added to the mixed solution, and the mixture was stirred in a water bath at 80° C. for 5 h to obtain a pre-oxidized graphite raw material;

[0067] (b) The pre-oxidized graphite raw material was dried at 60° C. under vacuum for 12 h to obtain a pre-oxidized graphite block, and then the pre-oxidized graphite block was fully ground using an agate mortar, and gradually dissolved in 120 mL of concentrated H2SO4 under stirring at 5° C., and then placed in a water bath controlled at 60° C., and then 15.0 g of KMnO4 was gradually added to the solution, and finally stirred at room temperature for 2 h to obtain a mixed solution V, at which time the solution was dark green;

[0068] (c) slowly diluting the mixed solution 5 with 250 mL of deionized water in a water bath at 80° C., stirring at room temperature for 2.5 h after the dilution, and finally adding 20 mL of 30% H2O2, stirring for 1 h, and ultrasonicating for 30 min to obtain a mixed solution 6;

[0069] (d) After the mixed solution VI was allowed to stand, the bottom precipitate was taken and dissolved in 1000 mL of ultrapure water. After centrifugal washing, the precipitate at the bottom of the cup was crushed with a glass rod while adding a 1:10 HCl aqueous solution for dilution and washing, and then centrifuged and washed again with pure water. The precipitate after centrifugation was diluted and fixed to volume with 1000 mL of ultrapure water. The solution was stirred for 1 hour, ultrasonicated for 30 minutes, and then allowed to stand for 5 days to obtain a graphene oxide nanosheet solution with a concentration of 4.1 mg / mL.

[0070] The performance test results of the filler in this embodiment are as follows:

[0071] one, Figure 1 This is a SEM cross-sectional view of the filler of this embodiment. It can be seen from the figure that the transport channel inside the filler is a layered structure that is stacked layer by layer and is relatively flat.

[0072] two, Figure 2 The FTIR-ATR spectrum of the filler in this example shows that the infrared data at 1728 cm -1 and 1210cm -1 Represent carboxyl and epoxy groups, respectively. Compared with pure GO (graphene oxide), the peak of proline-GO is reduced, indicating that the group is consumed by the grafted amino acid, proving that the chiral functionalization is successful; 1622cm -1 and 1478cm-1 They represent the methylene stretching vibrations unique to aromatic groups and proline, respectively, which are enhanced and appear compared with pure GO, and also prove that it is endowed with chiral functionalization.

[0073] Example 2

[0074] The filler prepared in Example 1 is applied to a chiral chromatographic column. The filling method of the filler in the chiral chromatographic column is as follows:

[0075] (1) 1 mL of filler with a concentration of 1 mg / mL was ultrasonicated for 1 h, and the water in the ultrasonic machine was replaced every 10 min during the ultrasonication process to ensure that the water temperature was always below 30 °C during the ultrasonication process to avoid thermal reduction of the graphene oxide chiral filler and ensure that the nanosheets were evenly dispersed;

[0076] (2) Extend and position the column tube vertically, add the ultrasonic filler into the column tube, and then let it stand for 20 minutes;

[0077] (3) A vacuum pump is connected to the sample outlet tube. After the vacuum degree of the vacuum pump is controlled at 0.1 MPa, segmented filtration is performed. Specifically, 0.25 mL of filler solution is slowly added along the inner wall of the chiral column using a pipette. When the solution in the column is drained, 0.25 mL is added again. This is repeated 4 times. In other words, the mass of filler to be loaded in the column tube is M, which is added in N times. Each time the filler is added, the above steps (1), (2), and (3) are sequentially adopted.

[0078] Example 3

[0079] The chiral chromatographic column filled with filler in Example 2 was used to separate tyrosine enantiomers, and the specific operation was as follows:

[0080] 1. Use an analytical balance to weigh 10.8 mg of 99.9% pure D-tyrosine and L-tyrosine powder respectively, and then transfer them into 100 mL of pure water and stir for 30 minutes;

[0081] 2. Then, take 10 mL of the prepared D-Tyr (D-tyrosine) and L-Tyr (L-tyrosine) aqueous solutions and pour them into 50 mL centrifuge tubes, then add 10 mL of ultrapure water, dilute and shake for 10 minutes to obtain D-Tyr and L-Tyr aqueous solutions with a concentration of 0.3 mM;

[0082] 3. Take 10 mL of the prepared D-Tyr and L-Tyr aqueous solutions respectively and pour them into a 50 mL centrifuge tube and shake for 10 minutes to obtain a 0.3 mM tyrosine racemic aqueous solution;

[0083] 4. The filler prepared in Example 1 was loaded according to the method in Example 2 and connected to a fast protein purification instrument (FPLC) for testing.

[0084] The chiral chromatographic column can adopt an existing chromatographic column. In this embodiment, the structure of the chiral chromatographic column is as follows:

[0085] like Figures 8 to 13 As shown, the combined chromatographic column of this preferred embodiment includes a plurality of separation units which are detachably connected in sequence from top to bottom.

[0086] Each separation unit includes a tube body (not shown in the figure), which can be a vertically penetrating glass tube or plastic tube. The tube bodies of all separation units together constitute a column tube, and the tube body is provided with an upper joint 1, a diverter plate 7, an upper support plate 2, an upper diaphragm 3, a lower diaphragm 4, a lower support plate 5 and a lower joint 6 from top to bottom.

[0087] The upper part of the tube body is provided with an upper diaphragm 3 through which liquid can flow, and the lower part of the tube body is provided with a lower diaphragm 4 through which liquid can flow, and the area between the upper diaphragm 3 and the lower diaphragm 4 is a packing area 0 for accommodating packing and preventing the packing 01 from leaking out. The packing can be silica gel or graphene.

[0088] In each tube body, a diverter plate 7 is provided above the upper diaphragm 3 , and a plurality of vertically penetrating liquid holes 71 are provided on the diverter plate 7 , and a liquid passage is provided on the upper surface of the diverter plate 7 to connect the liquid holes 71 .

[0089] In this embodiment, the liquid passage hole 71 located in the central part of the diverter plate 7 is used as the central hole ( Fig.13 A hole labeled a in the middle), with the central hole as the center, is provided with a plurality of liquid-passing hole groups spaced evenly in sequence along the radial direction, and each liquid-passing hole group includes a plurality of liquid-passing holes 71 spaced evenly along the circumference of the central hole.

[0090] The liquid passage includes a plurality of first passages 72 and a plurality of second passages 73. Each first passage 72 is annular and extends along the circumference of the central hole. The plurality of second passages 73 are radially distributed with the central hole as the center. The liquid passage holes 71 of the same group are connected through the first passages 72, and the liquid passage holes 71 of different groups are connected through the second passages 73. In this way, the liquid is transmitted through the liquid passage, so that liquid flows in each liquid passage hole 71, and the liquid passage holes 71 are evenly arranged on the diverter plate 7, and flow downward evenly.

[0091] This embodiment realizes the disassembly and assembly of two adjacent separation units in the following manner:

[0092] An upper joint 1 is provided above the diverter plate 7 in the tube body, and a lower joint 6 is provided below the lower diaphragm 4 in the tube body. In two adjacent separation units, the upper joint 1 of one separation unit is detachably connected to the lower joint 6 of the other separation unit, and the upper joint 1 has a first liquid flow channel 13 for liquid to flow through, and the lower joint 6 has a second liquid flow channel 61 for liquid to flow through.

[0093] In this embodiment, the upper joint 1 includes a plate body 11 and a screw 12 vertically mounted on the plate body 11, the plate body 11 and the screw 12 are integrally formed, the first liquid flow channel 13 vertically penetrates the screw 12 and the plate body 11, the lower joint 6 is a nut, the second liquid flow channel 61 vertically corresponds to the hollow area 62 of the nut, the screw 12 can be threadedly connected with the nut, and when the screw 12 is connected with the nut, the first liquid flow channel 13 and the second liquid flow channel 61 are connected. The threaded connection between the upper joint 1 and the lower joint 6 not only makes the two easy to disassemble and assemble, but also makes them difficult to loosen in the connected state.

[0094] Of course, it is also possible that the lower joint 6 includes a plate body 11 and a screw rod 12 vertically installed on the plate body 11, the upper joint 1 is a nut, the screw rod 12 can be threadedly connected to the nut, the first liquid flow channel 13 vertically penetrates the nut, and the second liquid flow channel 61 vertically penetrates the screw rod 12 and the plate body 11.

[0095] In order to prevent liquid from leaking out from between the upper joint 1 and the tube body, the upper joint 1 is in sealed contact with the tube body, and in order to prevent liquid from leaking out from between the lower joint 6 and the tube body, the lower joint 6 is in sealed contact with the tube body. The upper joint 1 and the lower joint 6 not only play the role of connecting two adjacent separation units, but also play the role of sealing. The upper joint 1 and the lower joint 6 can directly use flexible materials as sealing parts, or sealing rings can be set between the upper joint 1 and the tube body and between the lower joint 6 and the tube body.

[0096] In order to prevent the upper diaphragm 3 from being insufficiently strong to press the filler, an upper support sheet 2 through which fluid can flow is provided between the diverter plate 7 and the upper diaphragm 3. The strength of the upper support sheet 2 is greater than that of the upper diaphragm 3. The interiors of the upper support sheet 2 and the upper diaphragm 3 are both mesh structures. In this embodiment, the aperture on the upper support sheet 2 is greater than the aperture of the upper diaphragm 3. Of course, there is no restriction on the aperture size between the two.

[0097] In order to prevent the lower diaphragm 4 from being insufficiently strong to support the filler, a lower support sheet 5 through which the fluid can flow is provided between the lower joint 6 and the lower diaphragm 4. The strength of the lower support sheet 5 is greater than that of the lower diaphragm 4. The interiors of the lower support sheet 5 and the lower diaphragm 4 are both mesh structures. In this embodiment, the aperture on the lower support sheet 5 is greater than the aperture of the lower diaphragm 4. Of course, there is no restriction on the aperture size between the two.

[0098] The central part of the lower support sheet 5 is concave, so that the separated sample can quickly form a water drop shape and gather in the middle to flow down. In addition, the concave structure in the middle makes the middle filler gather thicker, avoiding cracking due to continuous pressure driving the middle filler, making the structure smoother.

[0099] The chiral chromatographic column of this embodiment also includes a feed pipe 8 and a discharge pipe 9. The feed pipe 8 allows the eluent and the mixture to be separated to enter the tube body, and the discharge pipe 9 allows the eluent and the compounds eluted by the eluent to flow out. When there is only one separation unit, the feed pipe 8 is connected to the upper joint 1 of the separation unit and is fluidically connected, and the discharge pipe 9 is connected to the lower joint 6 of the separation unit and is fluidically connected.

[0100] When there are at least two separation units, the feed pipe 8 is connected to the upper joint 1 of the uppermost separation unit and is in fluid communication, and the discharge pipe 9 is connected to the lower joint 6 of the lowermost separation unit and is in fluid communication.

[0101] Specifically in this embodiment, the feed pipe 8 is threadedly connected to the screw 12 of the upper joint 1, and the feed pipe 8 is connected to the first liquid flow channel 13 to achieve fluid connectivity; the discharge pipe 9 is threadedly connected to the lower joint 6, and the discharge pipe 9 is connected to the second liquid flow channel 61 to achieve fluid connectivity.

[0102] When using the chiral chromatographic column of this embodiment, when a large amount of mixture needs to be separated, a larger number of separation units can be used; when a small amount of mixture needs to be separated, a smaller number of separation units can be used.

[0103] The following scenarios can also be applied: when the eluent reaches the sample outlet, or when it is observed or detected from the outside that the mixture has been separated during the elution process, the elution can be stopped, and then each separation unit can be disassembled, and then the separation units can be rinsed separately with the eluent, and the corresponding components can be obtained after drying.

[0104] Separation performance evaluation

[0105] L-proline grafted graphene oxide chiral filler was used, ultrapure water was used as the mobile phase, and tyrosine racemate was selected as the target analyte for enantiomer separation. The feed volume was 500 μL, pH = 12 water was used as the mobile phase, the flow rate was 0.3 mL / min, the column pressure was 0.4 MPa, the detection wavelength was 240 nm, and the column temperature was 25 ° C. Fast protein liquid chromatography was performed under the conditions. The results are shown in Figure 3 It was shown that L-proline grafted graphene oxide chiral filler had a high separation degree for tyrosine racemate, with the retention time of D-Tyr being 2.1-5.2 min and the retention time of L-Tyr being 6.1-9.2 min.

[0106] In addition, the target analyte was changed from tyrosine racemate to D-tyrosine or L-tyrosine standard solution. Under the same experimental conditions, D-Tyr showed a retention time of 2.5 to 5.0 min ( Figure 4 ). Similarly, the retention time of the chromatogram of the L-Tyr standard sample is 6.2~8.0min( Figure 5). In summary, it is shown that the filler using the self-assembled chromatographic column has a significant separation effect on tyrosine and tyrosine enantiomers.

[0107] Example 4

[0108] The response of the filler prepared in Example 1 to different mobile phase pH values ​​is specifically performed as follows:

[0109] Use a measuring cylinder to measure 3 cups of 1L ultrapure water each. First, use 0.1mol / L NaOH to adjust the pH of one cup to 12, then use 0.1mol / L HCl to adjust the pH of another cup to 2, and keep the last cup unchanged. These three cups of solution are used as mobile phases for chiral separation experiments.

[0110] The feed volume was 500 μL, the above liquids were mobile phases, the flow rate was 0.3 ml / min, the column pressure was 0.4 MPa, the detection wavelength was 240 nm, the column temperature was 25 °C, and the test was performed three times. Figure 6 The reason why the separation effect is best under the mobile phase of pH = 12 is that tyrosine will be deprotonated under alkaline conditions, but the degree of deprotonation of the two is different, which leads to an increase in the difference in adsorption strength of the chiral filler to the two, achieving a better separation effect.

[0111] In this example, the chiral chromatographic column of Example 2 was used, and the preparation methods of D-Tyr, L-Tyr aqueous solution, and Tyr racemic aqueous solution were the same as those of Example 3.

[0112] Example 5

[0113] The filler prepared in Example 1 is used to separate the phenylalanine enantiomers, and the specific operation is as follows:

[0114] 1. Use an analytical balance to weigh 9.9 mg of D-phenylalanine and L-phenylalanine powder respectively, and then transfer them into 100 mL of pure water and stir for 30 minutes;

[0115] 2. Then, take 10 mL of the prepared D-Phe (D-phenylalanine) and L-Phe (L-phenylalanine) aqueous solutions and pour them into 50 mL centrifuge tubes, then add 10 mL of ultrapure water, dilute and shake for 10 minutes to obtain D-Phe and L-Phe aqueous solutions with a concentration of 0.3 mM;

[0116] 3. Take 10 mL of the prepared D-Phe and L-Phe aqueous solutions respectively and pour them into a 50 mL centrifuge tube and shake for 10 minutes to obtain a 0.3 mM aqueous solution of the phenylalanine racemate;

[0117] 4. The L-proline grafted graphene oxide chiral filler was loaded according to the method in Example 2 and connected to a fast protein purification instrument (FPLC) for testing.

[0118] Separation performance evaluation

[0119] L-proline grafted graphene oxide chiral filler was used, ultrapure water was used as the mobile phase, and phenylalanine racemate was selected as the target analyte for enantiomer separation. The feed volume was 500 μL, pH = 12 water was used as the mobile phase, the flow rate was 0.3 mL / min, the column pressure was 0.4 MPa, the detection wavelength was 245 nm, and the column temperature was 25 ° C. Fast protein liquid chromatography was performed under the conditions. The results are shown in Figure 7 It was shown that the L-proline grafted graphene oxide chiral filler had a high separation degree for the phenylalanine racemate, with the retention time of D-Phe being 1.2-2.1 min and the retention time of L-Phe being 2.7-3.5 min.

[0120] In summary, it can be seen that the filler prepared in the present application and the self-assembled chromatographic column in the present application have significant separation effects on various amino acids.

Claims

1. A method for preparing a filler for amino acid enantiomer separation, characterized in that: The steps include: (1) The graphene oxide nanosheets are uniformly dispersed in a PBS buffer solution to form a mixed solution 1, and the pH range of the mixed solution 1 is 6 to 7. After ultrasonication, 1-ethyl-(3-dimethylaminopropyl)carbodiimide is added to the mixed solution 1 and stirred to form a mixed solution 2. Then, N-hydroxysuccinimide is added to the mixed solution 2 and stirred until the solution changes from yellow brown to black to form a mixed solution 3. (2) adding L-type or D-type amino acid compounds to the mixed solution three, stirring evenly at room temperature to form a mixed solution four, washing the mixed solution four with ultrapure water and dilute hydrochloric acid with a concentration of no more than 10% by alternating centrifugation for multiple times, and then transferring the obtained chiral nanosheet solution to a dialysis bag for dialysis to remove excess acid and remaining impurities, thereby obtaining the filler.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the graphene oxide nanosheets to the amino acid compound with a purity of 99% is 0.8:1 to 1.2:1; The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 4:1 to 5:1; The volume ratio of graphene oxide nanosheets with a concentration of 4 mg / mL to PBS buffer was 1:6 to 1:8; The mass ratio of graphene oxide nanosheets to 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1.6:1 to 2:

1.

3. The preparation method according to claim 1, characterized in that: In the step (1), the method for preparing the graphene oxide nanosheets comprises the following steps: (a) adding 2.4-2.6 g K2SO4 and 2.4-2.6 g P2O5 to 11-13 mL H2SO4, stirring and ultrasonicating until the solution is clear and transparent, adding 2.8-3.2 g graphite powder to the mixed solution, stirring in a water bath at 80° C. to 85° C. for 4.5-5.5 h, to obtain a pre-oxidized graphite raw material; (b) drying the pre-oxidized graphite raw material at 60-70° C. under vacuum for 8-12 h to obtain a pre-oxidized graphite block, then grinding the obtained pre-oxidized graphite block, gradually dissolving it in 118-122 mL of concentrated H2SO4 under stirring at 4-6° C., then placing it in a water bath controlled at 55-60° C., then gradually adding 14.7-15.2 g of KMnO4 to the solution, and finally continuing to stir at room temperature for 2.0-2.5 h to obtain a mixed solution V, at which the solution appears dark green; (c) slowly diluting the mixed solution 5 with 200-250 mL of deionized water in a water bath at 75-80° C., stirring at room temperature for 2.0-2.5 h after the dilution, and finally adding 18-22 mL of 30% H2O2, stirring for 1-1.5 h, and ultrasonicating for 20-40 min to obtain a mixed solution 6; (d) After the mixed solution is allowed to stand, the bottom precipitate is taken out and dissolved in 1000-1200 mL of ultrapure water. After centrifugal washing, the precipitate at the bottom of the cup is crushed and diluted and washed with a 1:10 HCl aqueous solution, and then centrifuged and washed again with pure water. The precipitate after centrifugation is diluted and fixed to volume with 1000 mL of ultrapure water; the solution is stirred for 1-1.5 h, ultrasonicated for 20-40 min, and then allowed to stand for 5-7 days to obtain a graphene oxide nanosheet solution with a concentration of 3.5-4.2 mg / mL.

4. Use of the filler according to any one of claims 1 to 3 in a chiral chromatographic column, characterized in that: The chiral chromatographic column comprises a column tube and an inlet tube and an outlet tube connected to the column tube. The column tube is filled with the filler according to claim 1. The outlet tube of the column tube is connected to a vacuum pump so that the filler is filtered to form a stacked channel structure.

5. The use according to claim 4, characterized in that: The packing method of the chiral chromatographic column is: (1) Ultrasonicate the filler to make it evenly dispersed, and ensure that the water temperature is always below 30° C. during the ultrasonication process; (2) extending and positioning the column tube vertically, adding the ultrasonicated filler into the column tube, and then allowing it to stand; (3) A vacuum pump is connected to the sample outlet tube, and the vacuum degree of the vacuum pump is controlled between 0.1 MPa and 0.3 MPa for filtration.

6. The use according to claim 5, characterized in that: The mass of filler to be loaded in the column tube is M, which is added in N times, and each time the filler is added, the above steps (1), (2), and (3) are sequentially adopted.

7. The use according to claim 6, characterized in that: The column tube includes at least two tube bodies that are detachably connected, the sample inlet tube is connected to the top tube body, and the sample outlet tube is connected to the bottom tube body. An upper gasket and a lower gasket are provided in each of the tube bodies. The upper gasket is located above the lower gasket and the two are spaced apart. The area between the upper gasket and the lower gasket is a filling area for filling fillers.

8. The use according to claim 7, characterized in that: A flow divider plate is arranged above each of the upper gaskets, the flow divider plate is provided with a plurality of vertically penetrating liquid holes, and a liquid passage channel is arranged on the upper surface of the flow divider plate to connect the liquid holes.

9. The use according to claim 8, characterized in that: The liquid-passing hole located at the central part of the diverter plate is used as the central hole, and a plurality of liquid-passing hole groups are evenly spaced in sequence along the radial direction with the central hole as the center, and each liquid-passing hole group includes a plurality of liquid-passing holes evenly spaced along the circumference of the central hole; The liquid passage includes a plurality of first channels and a plurality of second channels, each of the first channels is annular and extends along the circumference of the center hole, and the plurality of second channels are radially distributed with the center hole as the center, the liquid passage holes in the same group are connected through the first channels, and the liquid passage holes in different groups are connected through the second channels.

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