CN111001188A - Reversed phase separation medium and preparation method and application thereof - Google Patents
Reversed phase separation medium and preparation method and application thereof Download PDFInfo
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- CN111001188A CN111001188A CN201911382992.8A CN201911382992A CN111001188A CN 111001188 A CN111001188 A CN 111001188A CN 201911382992 A CN201911382992 A CN 201911382992A CN 111001188 A CN111001188 A CN 111001188A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/32—Bonded phase chromatography
- B01D15/325—Reversed phase
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention provides a reverse phase separation medium and a preparation method and application thereof, wherein the reverse phase separation medium comprises a stationary phase and a neutral hydrophilic coating layer coated on the surface of the stationary phase; wherein, the neutral hydrophilic coating is chemically bonded with reversed-phase hydrophobic groups through-O-CO-NH-. The reversed phase separation medium provided by the invention has the characteristics of good water resistance, high stereoselectivity and low interference of ion exchange action.
Description
Technical Field
The invention belongs to the technical field of liquid chromatographic separation, and relates to a reverse phase separation medium, and a preparation method and application thereof.
Background
High Performance Liquid Chromatography (HPLC) is an important analytical tool, suitable for separating various compounds with different charges, hydrophobicity, polarity, structure, size, etc., and therefore requires different stationary phases. Among these, the most commonly used are C18 or C18-like stationary phases.
Under neutral conditions, the interaction of the basic compound with underivatized silicon hydroxyl groups on the surface of the stationary phase leads to peak tailing. The solution commonly used at present is to minimize the residual of surface silicon hydroxyl groups by high density bonding of the silica gel surface with C18 silanization reagent, followed by multiple treatments with excess capping reagent. However, the reversed phase stationary phase obtained by this method has strong hydrophobicity, resulting in "hydrophobic collapse" to greatly reduce the retention of the analyte and affect reproducibility, and thus is not suitable for use in 100% aqueous phase. Hydrophilic end capping techniques are commonly used to overcome the problem of "hydrophobic collapse", but the reversed-phase stationary phase generated by this method is less chemically stable and thus, in many cases, a tailing phenomenon of basic compounds under neutral conditions occurs.
Another solution is to introduce polar intercalating groups in the reverse bonding phase. These bonding phases comprise hydrophobic groups and hydrophilic intercalating groups (between the hydrophobic groups and the bonding sites to the silica gel surface). Commonly used polar intercalating groups include amide, urea, ether, and carbamate groups. In general, a polar intercalated reversed phase stationary phase has the following advantages:
1. compared with the traditional Cl8 filler, the alkaline compound has good peak shape;
2. compared with the traditional Cl8 filler, the filler has good compatibility with a pure water phase mobile phase;
3. different (complementary) selectivity compared to conventional C18;
4. compared with the traditional C18, the method has higher stereoselectivity and can better separate the structurally related compounds.
Small amounts of anion exchange groups can be easily introduced during the synthesis of the reversed-phase polar intercalated stationary phase, resulting in tailing of acidic compounds during use. This problem can be alleviated by the following synthetic method: 1) firstly, synthesizing a polar embedded reverse phase silanization reagent; 2) and then carrying out silica gel surface bonding. This requires corresponding measures in the synthesis of the polar built-in reverse phase silylating agent to avoid the formation of by-products which can lead to poor ion exchange, or methods for the effective removal of such materials. These measures undoubtedly increase the technical difficulty and the cost.
CN1421693A discloses an alkyl silica gel bonded chromatographic stationary phase and a preparation method thereof, wherein a bonded chain is embedded into an amide polar group and has a bidentate structure, and the preparation method thereof is also provided and comprises the following steps:
a. reacting silica gel particles with a silane coupling agent in an organic solvent at 70-130 ℃ for 4-10 hours; b. then reacting with acyl chloride at 20-130 ℃ for 8-48 hours to obtain the stationary phase; the internal amide group of this patent application forms hydrogen bond or ion pair with remaining silanol on the particulate silica gel, can shield silanol activity better, eliminates the influence of remaining silanol, but selectivity is relatively poor.
Accordingly, it is desirable to develop a reverse phase separation medium that addresses the challenges encountered in the current production and use of water-resistant reverse phase separation media and related chromatography columns, including water resistance, ion exchange interference, peak shapes of acidic and basic compounds, and synthetic methods, among others.
Disclosure of Invention
The invention provides a reverse phase separation medium and a preparation method and application thereof. The reversed phase separation medium provided by the invention has the characteristics of good water resistance, high stereoselectivity and low interference of ion exchange action.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a reverse phase separation medium, comprising a stationary phase and a neutral hydrophilic coating layer coated on the surface of the stationary phase;
wherein, the neutral hydrophilic coating is chemically bonded with reversed-phase hydrophobic groups through-O-CO-NH-.
The invention provides a reverse phase separation medium, which comprises three parts:
(A) a stationary phase with surface silicon hydroxyl groups;
(B) a neutral hydrophilic coating layer coated on the surface of the stationary phase in a covalent bond mode;
(C) and a reverse-phase hydrophobic group introduced through-oc (o) -NH-chemical bonding on the neutral hydrophilic coating layer.
The neutral hydrophilic coating layer is introduced to the surface of the stationary phase, so that the silicon hydroxyl group carried by the stationary phase is effectively covered, and therefore, the reversed phase separation medium provided by the invention can avoid the unfavorable cation exchange phenomenon in application, thereby improving the peak shape of the alkaline analyte.
The reversed phase separation medium provided by the invention can avoid the unfavorable anion exchange phenomenon of the polar embedded reversed phase packing commonly used at present, thereby improving the peak shape of the acidic analyte.
The invention introduces a compact neutral hydrophilic coating layer on the surface of the stationary phase, so the synthesized reverse phase separation medium has excellent water resistance and does not have the problems of 'hydrophobic collapse' and the like.
The reversed phase separation medium provided by the invention has higher stereoselectivity, and can separate a mixture with higher structural similarity.
In the present invention, the hydrophobic groups that provide the reversed phase retention can be linked to the hydroxyl groups on the neutral hydrophilic coating by an excess of isocyanate bearing hydrophobic groups in an-OC (O) -NH-linkage.
The neutral hydrophilic coating layer refers to a neutral hydrophilic coating layer, for example, C-OH does not ionize and has neutral charge, and-OH has hydrophilicity, so the neutral hydrophilic coating layer can be a coating layer with C-OH.
In the invention, the chemical bonding amount of the hydrophobic group on the neutral hydrophilic coating layer is 1 mu mol/m2Above, e.g. 1.2. mu. mol/m2、1.5μmol/m2、2μmol/m2、3μmol/m2、4μmol/m2And the like.
Preferably, the hydrophobic group comprises any one or a combination of at least two of a substituted or unsubstituted alkyl group of C1-C30, a substituted or unsubstituted aryl group of C6-C30, preferably an octadecyl group, an octyl group, a butyl group, or a phenyl group.
The C1-C30 comprises C2, C4, C6, C8, C10, C12, C15, C18, C20, C22, C25, C28 and the like.
The C6-C30 comprises C8, C10, C12, C15, C18, C20, C22, C25, C28 and the like.
In the present invention, the stationary phase is a solid matrix with Si-OH groups.
In the present invention, the solid substrate may also be a non-porous material.
In the present invention, the solid matrix may be in various shapes such as granular shape, block shape, lamellar shape, etc. which are commonly used in the prior art.
In the present invention, the solid substrate may be spherical, square, or irregular in shape.
Preferably, the solid matrix is selected from silica/organic hybrid microspheres and/or silica microspheres.
In the present invention, the silica/organic hybrid microspheres refer to composite microspheres composed of silica and organic hybrids.
Preferably, the solid matrix has an average particle size of 1.5-50 μm, such as 3 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, and the like.
Preferably, the specific surface area of the solid matrix is 0.5 to 300m2G, e.g. 1m2/g、5m2/g、10m2/g、50m2/g、100m2/g、150m2/g、200m2/g、250m2And/g, etc.
Preferably, the solid matrix is a porous material having an average pore size of
For example
And the like.
In the present invention, the solid substrate may also be a non-porous material.
In the present invention, the solid matrix may be in various shapes such as granular shape, block shape, lamellar shape, etc. which are commonly used in the prior art.
In the present invention, the solid substrate may be spherical, square, or irregular in shape.
In a second aspect, the present invention provides a method of preparing a reverse phase separation medium according to the first aspect, the method comprising the steps of:
(1) carrying out crosslinking reaction on the fixed phase with the epoxy group and polyhydric alcohol to form a neutral hydrophilic coating layer;
(2) and (2) reacting the product obtained in the step (1) with isocyanate to obtain the reversed phase separation medium.
The stationary phase selected by the invention is silica gel microspheres. The silica gel microsphere has excellent mechanical strength and mature and diversified surface chemical bonding processes. In the step (1), the stationary phase with epoxy groups and a proper amount of polyol compound are subjected to ring-opening crosslinking under the action of a catalyst to form a compact neutral hydrophilic layer rich in alcoholic hydroxyl groups. This step is critical to reduce the unfavorable cation exchange properties of the target separation medium and to improve the water resistance of the target separation medium. In the step (2), the hydrophobic group is introduced by the addition reaction of isocyanate and the dense neutral hydrophilic layer rich in alcoholic hydroxyl group synthesized in the step (1). The reaction condition is mild and controllable, the operation is simple and convenient, no by-product is generated, the conversion rate is high, and no unfavorable anion exchange group is formed. Therefore, the preparation method of the invention does not need strong alkali or strong acid condition, and effectively maintains the stability of the silica gel matrix. Therefore, the preparation method has obvious superiority compared with the preparation method of the polar embedded reversed phase separation medium which is commonly used at present.
In the present invention, the mass ratio of the product of the step (1) to the isocyanate is 1 (0.15-10), for example, 1:0.2, 1:0.5, 1:1, 1:2, 1:4, 1:5, 1:5.5, 10:6, 10:6.5, 10:7, 10:7.5, 10:8, 10:8.5, 10:9, 10:9.5, etc.
In the preparation process of the present invention, the isocyanate should be used in an excess amount, and an excessively small amount results in insufficient retention of hydrophobicity (reverse phase).
Preferably, the isocyanate is selected from C1-C30 alkyl isocyanate or C6-C30 aryl isocyanate, further preferably any one or a combination of at least two of octadecyl isocyanate, octyl isocyanate, butyl isocyanate or phenyl isocyanate.
The C1-C30 can be C2, C4, C6, C8, C10, C12, C14, C16, C20, C24, C26, C28 and the like, the C6-C30 can be C8, C10, C12, C14, C16, C20, C24, C26, C28 and the like, and the C does not include the number of carbon atoms in isocyanate, for example, methyl isocyanate is isocyanate of C1.
The alkyl isocyanate in the present invention refers to an isocyanate having an alkyl group, such as a butane-based isocyanate; the aryl isocyanate refers to an isocyanate having an aryl group, such as phenyl isocyanate.
Preferably, the reaction in step (2) is carried out at a temperature of 0 to 100 deg.C, such as 5 deg.C, 10 deg.C, 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C, etc., for a period of 1 to 24 hours, such as 2 hours, 5 hours, 10 hours, 12 hours, 15 hours, 20 hours, etc.
Preferably, the polyol of step (1) comprises any one of ethylene glycol, diethylene glycol or triethylene glycol or a combination of at least two thereof.
According to the invention, polyol and epoxy are introduced to react and crosslink, so that a compact neutral hydrophilic film is formed on the surface of the stationary phase, and the exposure of silicon hydroxyl can be avoided, thereby reducing the influence of the silicon hydroxyl on the adverse charge interaction of alkaline substances to be separated.
Preferably, the mass ratio of the epoxy-bearing stationary phase to the polyol is (1-10): (10-1), e.g., 10:1, 8:1, 5:1, 2:1, 1:2, 1:5, 1:8, 1:10, etc.
Preferably, the catalyst for the crosslinking reaction in step (1) is a boron trifluoride etherate catalyst selected from boron trifluoride diethyl etherate.
Preferably, the crosslinking reaction is carried out in a solvent which is tetrahydrofuran and/or 1, 4-dioxane.
Preferably, the temperature of the crosslinking reaction is 0 to 120 ℃ (e.g., 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 100 ℃, 110 ℃, etc.) or the reflux temperature of the solvent for 1 to 24 hours, e.g., 2 hours, 5 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, etc., preferably 8 hours.
Preferably, the preparation method of the stationary phase with the epoxy group comprises the following steps:
(A) and reacting the stationary phase with a silylation reagent with epoxy groups to obtain the stationary phase with the epoxy groups.
The silanization reagent with epoxy group in the step (A) can be effectively bonded on the surface of the silica gel, and lays a foundation for the formation of the neutral hydrophilic layer in the step (1).
Preferably, the mass ratio of the stationary phase to the epoxy-bearing silylation agent is (1-5): (5-1), e.g., 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, etc.
Preferably, the silylating agent with epoxy groups is selected from 3- (2, 3-glycidoxy) propyltrimethoxysilane.
Preferably, the reaction in step (a) is carried out in a solvent which is any one of toluene, xylene or 1, 4-dioxane or a combination of at least two thereof.
Preferably, the reaction in step (A) is carried out at a temperature of 0 to 140 deg.C, such as 10 deg.C, 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C, 100 deg.C, 120 deg.C, 130 deg.C, etc., for a period of 8 to 48 hours, such as 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, etc.
In a third aspect, the present invention provides liquid chromatography performance evaluation of the reverse phase separation medium according to the first aspect and use in a pure water phase.
The reversed phase separation medium provided by the invention has good peak shapes for neutral, acidic and alkaline compounds; the product has good compatibility with a pure water phase mobile phase; the stereoselectivity is higher, and the structure can be better separated; with different (complementary) selectivity compared to conventional C18.
Compared with the prior art, the invention has the following beneficial effects:
(1) the neutral hydrophilic coating layer is introduced to the surface of the stationary phase, so that the silicon hydroxyl group carried by the stationary phase is effectively covered, and therefore, the reversed phase separation medium provided by the invention can avoid the unfavorable cation exchange phenomenon in application, thereby improving the peak shape of the alkaline compound.
(2) The reversed phase separation medium provided by the invention can avoid the common unfavorable anion exchange phenomenon of polar embedded reversed phase packing, thereby improving the peak shape of the acidic analyte.
(3) According to the invention, a compact neutral hydrophilic coating layer is introduced on the surface of the stationary phase, so that the synthesized reverse phase separation medium has excellent water resistance and can generate the problems of 'hydrophobic collapse' and the like.
(4) The reversed phase separation medium provided by the invention has higher stereoselectivity, and can separate a mixture with higher structural similarity.
(5) The invention introduces different hydrophobic groups through the addition reaction of isocyanate and a compact neutral hydrophilic layer rich in alcoholic hydroxyl. The reaction condition is mild and controllable, the operation is simple and convenient, no by-product is generated, the conversion rate is high, and no unfavorable anion exchange group is formed. Therefore, the preparation method of the invention does not need strong alkali or strong acid condition, and effectively maintains the stability of the stationary phase. Therefore, the preparation method has obvious superiority compared with the preparation method of the polar embedded reversed phase separation medium which is commonly used at present.
Drawings
FIG. 1 is a graph of the results of the retention of hydrophobicity of the reverse phase separation medium provided in example 1.
FIG. 2 is a graph showing the results of stereoselectivity of the reversed phase separation medium provided in example 1.
FIG. 3 is a graph of the results of the silicon hydroxyl activity of the reverse phase separation media provided in example 1.
FIG. 4 is a graph of the results of acid activity of the reverse phase separation medium provided in example 1.
FIG. 5 is a graph showing the results of separation of nucleic acid bases in an aqueous mobile phase using the reverse phase separation medium provided in example 1.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Some of the materials and manufacturer information relating to the following examples and comparative examples are as follows
Example 1
A reverse phase separation medium is prepared by the following steps:
(1) preparation of stationary phase with epoxy group
Adding 50g of 3- (2, 3-epoxypropoxy) propyltrimethoxysilane to 50g of a toluene (300mL) dispersion of silica gel A-1 under the protection of dry nitrogen, stirring at 100 ℃ for 24 hours, filtering the reaction mixture, washing with toluene, 1, 4-dioxane and acetone in sequence, and then drying in vacuum at 50 ℃ for 8 hours to obtain an intermediate I-a;
(2) preparation of intermediate with neutral hydrophilic coating
40g of ethylene glycol were added to 50g of a tetrahydrofuran (300mL) dispersion of intermediate I-a under a blanket of dry nitrogen, and after stirring at room temperature for 1 hour, 2mL of boron trifluoride diethyl etherate were added to the reaction mixture, which was then heated to reflux temperature and stirring continued for 8 hours. After the reaction is finished, filtering the reaction mixture and washing the reaction mixture by using acetone, deionized water and acetone in sequence; drying the filter cake in vacuum at 50 ℃ for 8 hours to obtain an intermediate II-a;
(3) introduction of hydrophobic groups
Under the protection of dry nitrogen, 10g of the intermediate II-a is dispersed in 30mL of toluene, and the dispersion is continuously stirred at room temperature; 10mL of a toluene solution of 10g of octadecyl isocyanate was dropped into the dispersion; stirring at 100 deg.C for 8 hr, and stirring at room temperature for 1 hr; after the reaction is finished, filtering the reaction mixture and washing the reaction mixture by using acetone, deionized water and acetone in sequence; the filter cake was dried under vacuum at 50 ℃ for 8 hours to give the reverse phase separation medium 1. The surface bond and density calculated from the elemental analysis data (19.93% C, 0.58% N) of the bonding phase were 2.1. mu. mol/m2。
Example 2
The only difference from example 1 is that 10g of octadecyl isocyanate in step (3) was replaced with 10g of octyl isocyanate to give a reversed phase separation medium 2.
Example 3
The only difference from example 1 is that 10g of octadecyl isocyanate in step (3) was replaced by 10g of butyl isocyanate to give reversed phase separation medium 3.
Example 4
The only difference from example 1 was that 10g of octadecyl isocyanate in step (3) was replaced with 10g of phenyl isocyanate to give a reversed phase separation medium 4.
Example 5
The only difference from example 1 was that silica gel A-1 in step (1) was replaced with silica gel A-2 to give a reversed phase separation medium 5.
Example 6
A reverse phase separation medium is prepared by the following steps:
(1) preparation of stationary phase with epoxy group
Referring to example 1, the difference from example 1 is that 3- (2, 3-glycidoxy) propyltrimethoxysilane was added in an amount of 30g, and silica gel A-1 was replaced with silica gel A-3;
(2) preparation of intermediate with neutral hydrophilic coating
Referring to example 1, the difference from example 1 is that ethylene glycol was added in an amount of 30 g;
(3) introduction of hydrophobic groups
Referring to example 1, the difference from example 1 is that octadecyl isocyanate was added in an amount of 8g, to obtain reverse phase separation medium 6.
Example 7
The difference from example 6 is that silica gel A-3 was replaced with silica gel A-4 to give a reversed phase separation medium 7.
Example 8
A reverse phase separation medium is prepared by the following steps:
(1) preparation of stationary phase with epoxy group
Referring to example 1, the difference from example 1 is that 3- (2, 3-glycidoxy) propyltrimethoxysilane was added in an amount of 20g, and silica gel A-1 was replaced with silica gel A-5;
(2) preparation of intermediate with neutral hydrophilic coating
Referring to example 1, the difference from example 1 is that ethylene glycol was added in an amount of 20 g;
(3) introduction of hydrophobic groups
Referring to example 1, the difference from example 1 is that octadecyl isocyanate was added in an amount of 5g, and a reverse phase separation medium 8 was obtained.
Example 9
A reverse phase separation medium is prepared by the following steps:
(1) preparation of stationary phase with epoxy group
Referring to example 1, the difference from example 1 is that 3- (2, 3-glycidoxy) propyltrimethoxysilane was added in an amount of 10g, and silica gel A-1 was replaced with silica gel A-6;
(2) preparation of intermediate with neutral hydrophilic coating
Referring to example 1, the difference from example 1 is that ethylene glycol was added in an amount of 20 g;
(3) introduction of hydrophobic groups
Referring to example 1, the difference from example 1 is that octadecyl isocyanate was added in an amount of 5g, and a reverse phase separation medium 9 was obtained.
Example 10
The only difference from example 1 is that the ethylene glycol in step (2) was replaced with diethylene glycol to give a hydrophobic chromatography medium 1B.
Example 11
The only difference from example 1 is that ethylene glycol in step (2) was replaced with triethylene glycol to give a hydrophobic chromatography medium 1C.
Example 12
The only difference from example 1 is that 3- (2, 3-glycidoxy) propyltrimethoxysilane was replaced with 3- (2, 3-glycidoxy) propyl-dimethyl-methoxysilane in step (1), yielding hydrophobic chromatography medium 1D.
Comparative example 1
The only difference from example 1 is that 40g of ethylene glycol in step (2) was replaced with 40g of octadecanol, and step (3) was omitted, yielding a hydrophobic chromatography medium 11.
Comparative example 2
50g of octadecane-dimethyl-chlorosilane and 10g of imidazole were added to a 50g dispersion of silica A-1 in toluene (300mL) under dry nitrogen, and after stirring for 24 hours at 100 ℃, the reaction mixture was filtered, washed successively with toluene, 1, 4-dioxane/water, and acetone, and then dried for 8 hours under vacuum at 50 ℃ to give an intermediate. Then 50g of trimethylchlorosilane and 10g of imidazole were added to a toluene (300mL) dispersion of the above intermediate under dry nitrogen, and after stirring at 100 ℃ for 24 hours, the reaction mixture was filtered, washed with toluene, 1, 4-dioxane/water, and acetone in that order, and then dried under vacuum at 50 ℃ for 8 hours to give a C18 bound phase (hydrophobic chromatography medium 12).
Comparative example 3
50g of octadecyl carbamate-dimethyl-methoxysilane and 1g of imidazole were added to a toluene (300mL) dispersion of 50g of silica A-1 under dry nitrogen, and after stirring for 24 hours at 100 deg.C, the reaction mixture was filtered, washed with toluene, 1, 4-dioxane/water, and acetone in that order, and then dried under vacuum at 50 deg.C for 8 hours to give an intermediate. Then 50g of trimethylchlorosilane and 1g of imidazole were added to a toluene (300mL) dispersion of the above intermediate under dry nitrogen, and after stirring for 24 hours at 100 ℃, the reaction mixture was filtered, washed with toluene, 1, 4-dioxane/water, and acetone in that order, and then dried under vacuum at 50 ℃ for 8 hours to give the octadecanocarbamate polar intercalated bonded phase (hydrophobic chromatography medium 13).
Performance testing
The reversed phase separation media provided in examples 1-12 and comparative examples 1-3 were subjected to performance testing as follows:
the samples were packed into 4.6X 150mm stainless steel columns using conventional high pressure slurry technology and then tested as follows:
(1) hydrophobic retention test:
this test is intended to evaluate the retention of neutral hydrophobic compounds by the separation medium under reverse phase chromatographic conditions. The test standard contained a mixture of uracil (10. mu.g/mL, Peak 1), dimethyl phthalate (0.1. mu.L/mL, Peak 2), and naphthalene (300. mu.g/mL, Peak 3). Wherein the retention time of a neutral hydrophobic compound naphthalene is used as an index of hydrophobic retention capacity of a bonding phase, and longer retention time indicates stronger hydrophobic retention capacity.
And (3) testing conditions are as follows: eluent, CH3CN/H2O (60:40 v/v); flow rate: 1 mL/min; the sample injection amount is 5 mu L; the temperature is 30 ℃; the detection wavelength is 254 nm.
FIG. 1 is a graph of the results of the retention of hydrophobicity of the reverse phase separation medium provided in example 1. As can be seen, the reversed-phase separation medium 1 provided by the present invention has reversed-phase retention capacity (retention time of 4.1 minutes). Under the same conditions, the separation medium retention times in comparative example 1 (ether bond-intercalated C18 synthesized by direct epoxy ring opening), comparative example 2 (synthesized by conventional method C18) and comparative example 3 (carbamate intercalated C18 synthesized by conventional method) were 2.2 minutes, 6.5 minutes and 5.5 minutes, respectively. It can be seen that the hydrophobicity of the reversed phase separation medium provided in example 1 is lower than that of C18 and carbamate intercalated into C18 synthesized by conventional methods, but is significantly improved compared with that of C18 intercalated with ether linkage synthesized by direct epoxy ring opening.
(2) And (3) stereoselectivity testing:
the test sample was a mixture of uracil (40. mu.g/mL, Peak 1), ortho-terphenyl (140. mu.g/mL, Peak 2), and triphenylene (40. mu.g/mL, Peak 3) (ortho-terphenyl and triphenylene were similar in composition and size, but the former was in a stereoconfiguration and the latter was in a planar configuration).
Stereoselectivity is expressed as the ratio of the retention factors of triphenylene and ortho-terphenyl αTO＝(t3-t0)/(t2-t0)，
And (3) testing conditions are as follows: eluent MeOH/H2O (90:10 v/v); the flow rate is 1 mL/min; the sample injection amount is 5 mu L; the temperature is 30 ℃; the detection wavelength is 254 nm.
FIG. 2 is a graph showing the results of stereoselectivity of the reversed phase separation medium provided in example 1. As can be seen from fig. 2, the stereoselectivity of the separation medium in the example was very high, 3.0. Under the same conditions, the stereoselectivity of the separation media in comparative example 2 (the conventional method synthesized C18) and comparative example 3 (the carbamate synthesized by the conventional method is inserted into C18) is 1.6 and 2.2 respectively, namely, the reversed-phase separation media provided by the invention is more favorable for separating the substances to be separated with higher structural similarity.
(3) Silicon hydroxyl activity test
The test standard contained a mixture of uracil (10 μ g/mL, Peak 1), toluene (1 μ L/mL, Peak 2), ethylbenzene (1 μ L/mL, Peak 3), amitriptyline (240 μ g/mL, Peak 4), and quinixaline (240 μ g/mL, Peak 5). Amitriptyline is a hydrophobic basic drug; it is positively charged at neutral pH and interacts with the free silicon hydroxyls (now negatively charged) on the stationary phase surface, resulting in peak tailing; therefore, the degree of peak asymmetry of amitriptyline is an indicator of the silicon hydroxyl activity.
And (3) testing conditions are as follows: eluent, MeOH/30mM phosphate buffer, pH 7.0(80:20 v/v); flow rate, 1 mL/min; the sample injection amount is 5 mu L; the temperature is 30 ℃; the detection wavelength is 254 nm.
FIG. 3 is a graph of the results of the silicon hydroxyl activity of the reverse phase separation media provided in example 1. As can be seen from the figure, amitriptyline is eluted in a symmetrical peak (tailing factor is 1.10) at neutral pH, which shows that the reverse phase separation medium provided by the invention has very low activity of silicon hydroxyl, namely, the neutral hydrophilic coating layer effectively shields unreacted silicon hydroxyl on the surface of the stationary phase. Under the same conditions, the tailing factors of the separating medium in comparative example 1 (ether bond-intercalated C18 synthesized by direct epoxy ring opening), comparative example 2 (synthesized by a conventional method C18) and comparative example 3 (carbamate intercalated by a conventional method C18) to amitriptyline were 2.3, 1.15 and 1.20, respectively. It can be seen that the reverse phase separation medium provided in example 1 has superior (minimal) silicon hydroxyl activity and therefore has excellent versatility for the separation of different compounds.
(4) Acid activity test:
the test standard contained a mixture of uracil (10. mu.g/mL, Peak 1), dimethyl phthalate (0.1. mu.L/mL, Peak 2), and 4-chlorocinnamic acid (300. mu.g/mL, Peak 3).
And (3) testing conditions are as follows: eluent, methanol/10 mM phosphate buffer, pH 2.7(45/55 v/v); flow rate: 1 mL/min; the sample injection amount is 5 mu L; the temperature is 30 ℃; the detection wavelength is 254 nm.
At pH 3, chlorocinnamic acid can interact with the remaining anion exchange sites on the stationary phase (during synthesis), showing a peak tail in the figure, and thus, the acid activity test can determine whether the separation medium carries unwanted anion exchange sites.
FIG. 4 is a graph of the results of acid activity of the reverse phase separation medium provided in example 1. In fig. 4, the clethonium silicate has a perfect symmetrical peak shape (tailing factor of 1.05), and no tailing occurs, so that it can be seen that the reverse phase separation medium provided by the present invention has no anion exchange sites affecting the peak shape of the acidic analyte.
Under the same conditions, the tailing factors of the separation media of comparative example 1 (ether bond-intercalated C18 synthesized by direct epoxy ring opening), comparative example 2 (synthesized by a conventional method C18) and comparative example 3 (carbamate intercalated by a conventional method C18) to chlorocinnamic acid were 1.05, 1.10 and 1.30, respectively. It can be seen that the reversed phase separation medium provided in example 1 does not exhibit the defect of anion exchange sites and has good universality for separation of acidic compounds, compared with the carbamate-intercalated C18 separation medium synthesized by the conventional method.
(5) Water solubility test:
and (3) testing conditions are as follows: eluent, 100mM ammonium acetate buffer, pH 5.2 (aqueous solution); flow rate, 1 mL/min; the sample volume is 10 mu L; the temperature is 30 ℃; the detection wavelength is 254 nm.
The test standard was a mixture of nucleobases including cytosine (50. mu.g/mL, Peak 1), uracil (10. mu.g/mL, Peak 2), thymine (50. mu.g/mL, Peak 3), guanine (62.5. mu.g/mL, Peak 4) and adenine (50. mu.g/mL, Peak 5).
FIG. 5 is a graph showing the results of separation of nucleic acid bases in an aqueous mobile phase using the reverse phase separation medium provided in example 1. As can be seen, all 5 nucleobases were separated at baseline under 100% aqueous conditions. The reversed-phase separation medium provided in example 1 exhibited excellent reproducibility of retention time and had reproducible reproducibility of retention time after repeated sample injection 200 times for a long period of time, indicating that the reversed-phase separation medium provided in the present invention has excellent water compatibility (water resistance).
The separation medium of comparative example 1 (direct epoxide ring opening synthesis ether bond-embedded C18) also showed excellent reproducibility of retention time under the same conditions, but the five nucleobases had lower retention times and did not achieve efficient separation. The separation medium in comparative example 2 (conventional synthesis C18) initially had good retention time and separation efficiency, but the retention time and separation efficiency continued to decrease over time, with retention time decreasing to 65% of the initial time after 200 injections, which is typical of the "hydrophobic collapse" phenomenon. The separation medium of comparative example 3 (carbamate intercalated C18 synthesized by the conventional method) exhibited good retention time and reproducibility of the separation effect, with no significant change in retention time and separation degree after 200 injections.
The applicant states that the present invention is illustrated by the above examples of the present invention and the method of preparation and application thereof, but the present invention is not limited to the above process steps, i.e. it is not meant that the present invention must rely on the above process steps to be carried out. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (10)
1. The reversed phase separation medium is characterized by comprising a stationary phase and a neutral hydrophilic coating layer coated on the surface of the stationary phase;
wherein, the neutral hydrophilic coating is chemically bonded with reversed-phase hydrophobic groups through-O-CO-NH-.
2. The reverse-phase separation medium of claim 1, wherein the hydrophobic group is chemically bonded to the neutral hydrophilic coating in an amount of 1 μmol/m2The above;
preferably, the hydrophobic group comprises any one or a combination of at least two of a substituted or unsubstituted alkyl group of C1-C30, a substituted or unsubstituted aryl group of C6-C30, preferably an octadecyl group, an octyl group, a butyl group, or a phenyl group.
3. A reverse phase separation medium according to claim 1 or 2 wherein the stationary phase is a solid matrix with Si-OH groups selected from silica/organic hybrid microspheres and/or silica microspheres;
preferably, the solid matrix has an average particle size of 1.5 to 50 μm;
preferably, the solid substrate is a non-porous or porous material;
preferably, the specific surface area of the solid matrix is 0.5 to 300m2/g；
Preferably, the porous material has an average pore diameter of
4. A method of preparing a reverse phase separation medium according to any one of claims 1 to 3, comprising the steps of:
(1) carrying out crosslinking reaction on the fixed phase with the epoxy group and polyhydric alcohol to form a neutral hydrophilic coating layer;
(2) and (2) reacting the product obtained in the step (1) with isocyanate to obtain the reversed phase separation medium.
5. The method according to claim 4, wherein the isocyanate is selected from C1-C30 alkyl isocyanate or C6-C30 aryl isocyanate, further preferably any one or a combination of at least two of octadecyl isocyanate, octyl isocyanate, butane isocyanate or phenyl isocyanate;
preferably, the mass ratio of the product of the step (1) to the isocyanate is 1 (0.15-10).
6. The method according to claim 4 or 5, wherein the reaction in the step (2) is carried out at a temperature of 0 to 100 ℃ for 1 to 24 hours.
7. The production method according to any one of claims 4 to 6, wherein the polyol of step (1) comprises any one of ethylene glycol, diethylene glycol or triethylene glycol or a combination of at least two thereof;
preferably, the mass ratio of the fixing phase with the epoxy groups to the polyol is (1-10): (10-1);
preferably, the catalyst for the crosslinking reaction in the step (1) is a boron trifluoride etherate catalyst selected from boron trifluoride diethyl etherate;
preferably, the crosslinking reaction is carried out in a solvent which is tetrahydrofuran and/or 1, 4-dioxane;
preferably, the temperature of the crosslinking reaction is 0 to 120 ℃ or the reflux temperature of the solvent for 1 to 24 hours.
8. The preparation method according to any one of claims 4 to 7, wherein the preparation method of the stationary phase with epoxy groups comprises the following steps:
(A) and reacting the stationary phase with a silylation reagent with epoxy groups to obtain the stationary phase with the epoxy groups.
9. The production method according to claim 8, wherein the mass ratio of the stationary phase to the epoxy-containing silylation agent is (5-1): (1-5);
preferably, the silylating agent with epoxy groups is selected from 3- (2, 3-glycidoxy) propyltrimethoxysilane;
preferably, the reaction in step (a) is carried out in a solvent which is any one of toluene, xylene or 1, 4-dioxane or a combination of at least two thereof;
preferably, the reaction in step (A) is carried out at a temperature of 0 to 140 ℃ for a time of 8 to 48 hours.
10. Use of a reverse phase separation medium according to any one of claims 1 to 3 in a reverse phase chromatographic separation.
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| CN112755985A (en) * | 2020-12-28 | 2021-05-07 | 纳谱分析技术（苏州）有限公司 | Liquid chromatography separation medium and preparation method and application thereof |
| CN113694907A (en) * | 2020-05-22 | 2021-11-26 | 中国科学院大连化学物理研究所 | Pure water-resistant chromatographic stationary phase and preparation method and application thereof |
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| CN112755985A (en) * | 2020-12-28 | 2021-05-07 | 纳谱分析技术（苏州）有限公司 | Liquid chromatography separation medium and preparation method and application thereof |
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