CN117920164A - High-pH stable reversed phase chromatographic stationary phase and preparation method thereof - Google Patents

Abstract

The invention discloses a preparation method of a high-pH stable reversed phase chromatographic stationary phase, which relates to the field of liquid chromatographic stationary phases, and has the following structural formula:

Description

High-pH stable reversed phase chromatographic stationary phase and preparation method thereof

Technical Field

The invention relates to a liquid chromatography stationary phase, in particular to a high-pH stable reversed phase chromatography stationary phase and a preparation method thereof.

Background

The reversed phase liquid chromatography has the advantages of high column efficiency, strong separation capability, clear retention mechanism and the like, is one of the most widely used liquid chromatography separation modes, and is widely applied to separation analysis of samples such as drug molecules, biological macromolecules, proteins, enzymes and the like. Reverse phase chromatography is a liquid chromatography separation mode in which a surface nonpolar carrier is used as a stationary phase and a solvent having stronger polarity than the stationary phase is used as a mobile phase. The stationary phase of reverse phase chromatography is mostly silica gel with hydrophobic groups bonded to the surface, and is separated based on the difference of hydrophobic interactions between different components in the sample and the hydrophobic groups. However, when the analysis is performed using a reversed-phase immobilized relatively basic compound, electrostatic interaction between the basic compound and silanol remaining on the surface of silica gel is accompanied in addition to the hydrophobic effect, and thus, peak-shape tailing of the basic compound is caused [ Borges, E.M.et al, J.pharm.biomed.Anal,2013,77,100-115]. In order to solve the tailing problem of the alkaline compound, researchers have proposed a series of improvement measures in terms of mobile phase, and in order to reduce the ion exchange effect of silanol groups and the alkaline compound, a low pH mobile phase may be used to inhibit ionization of silanol groups, or a high pH mobile phase may be used to inhibit ionization of alkaloids, and an organic amine modifier may be added to compete for occupying ionized silanol groups, thereby blocking the interaction between the alkaline compound and silanol groups. When the alkali compound is analyzed by a high pH method, when the mobile phase pH is >8, silica gel surface erosion occurs due to dissolution of silica particles, so that column efficiency is reduced, column pressure is increased, and column bed of the silica gel matrix packing chromatographic column is collapsed, so that the tolerance of the silica gel matrix packing under the alkali condition is a challenging problem [Kirkland,J.J.;Henderson,J.W.;De Stefano,J.J.;van Straten,M.A.;Claessens,H.A.J.Chromatogr.A 1997,762,97-112]., besides the hybrid silica gel and the core-shell silica gel, the conventional silica gel packing can enhance alkali resistance of the packing from the reduction of the silanol group exposed on the surface. Yarita and the like are subjected to high-temperature tail sealing on C18 bonded silica gel by a supercritical fluid reaction medium method, and the prepared stationary phase has higher surface coverage rate [ Yarita T, ihara T, horimoto Y, et al, anal. Sci, 1999,15,377-380] than a liquid phase method, but the method has the advantages of high device cost and inconvenient operation, and compared with the traditional liquid phase method, the tail sealing effect is not obviously improved, and the requirement of the industry on high-pH stable reverse phase silica gel chromatographic packing cannot be met. Sudo reports that C18 bonded silica gel and a micromolecule silanization reagent are sealed and thermally blocked in an ampoule bottle at high temperature [ Sudo Y., J.chromatogrA, 1997,757,21-28], and the result shows that the activity of silanol groups on the surface of the silica gel is weakened, but the reaction temperature is usually higher than 250 ℃, and certain danger exists in closed reaction equipment, which is unfavorable for industrial production. The invention prepares a reversed phase stationary phase stable under the high pH condition by simple and mild liquid phase silanization reaction, bonding and end capping on the surface of silica gel, and can tolerate a mobile phase system with pH=1-12.

Disclosure of Invention

The invention aims to at least solve one of the technical problems in the prior art and provides a preparation method of a high-pH stable reversed phase chromatographic stationary phase.

The technical scheme of the invention is as follows:

A high pH stable reversed phase chromatographic stationary phase having the structural formula:

Wherein SILICA GEL is silica gel, R is phenyl, oxyphenyl or alkyl chain with 5-27 carbon atoms, n=0-5.

The invention also discloses a preparation method of the high-pH stable reversed phase chromatographic stationary phase, which comprises the following steps:

S1: pretreatment of silica gel: adding silica gel into strong acid solution with the concentration of 10-40 wt%, heating, refluxing and stirring for 1-48 hours, filtering, washing with water to pH=6-7, and vacuum drying the obtained solid at 80-160 ℃ for 8-24 hours to obtain acidified silica gel;

S2: and (3) silanization reaction bonding: under the protection of nitrogen or argon, adding a silane coupling agent, an alkaline catalyst and acidified silica gel into an organic solvent, reacting for 3-24 hours at 40-150 ℃, filtering, washing with dimethylbenzene, methanol water, methanol and tetrahydrofuran in sequence, and vacuum drying the obtained solid at 40-80 ℃ for 8-24 hours to obtain an intermediate;

S3: the first step of liquid phase silanization reaction end capping: adding a silylation reagent, an alkaline catalyst and an intermediate into an organic solvent under the protection of nitrogen or argon, reacting for 3-24 hours at 30-180 ℃, filtering, washing with dimethylbenzene, methanol water, methanol and tetrahydrofuran in sequence, and vacuum drying the obtained solid at 40-80 ℃ for 8-24 hours to obtain a preliminary end-capped stationary phase;

S4: and the second step of liquid phase silanization reaction end capping: under the protection of nitrogen or argon, adding two or more trimethyl silanization reagents, an alkaline catalyst and a primary end-capped stationary phase into an organic solvent, reacting for 3-24 hours at 30-180 ℃, filtering, washing with dimethylbenzene, methanol water, methanol and tetrahydrofuran in sequence, and vacuum drying the obtained solid at 40-80 ℃ for 8-24 hours to obtain the chromatographic stationary phase.

Further, in step S1, the strong acid is at least one of hydrochloric acid, nitric acid, and sulfuric acid.

Further, in step S2, the silane coupling agent has the following structure:

wherein X is one of chlorine, methoxy or ethoxy, and R is phenyl, oxyphenyl or alkyl chain with 5-27 carbon atoms.

Further, in step S2, the organic solvent is at least one of dichloromethane, toluene, xylene, n-heptane, and isooctane that are not miscible with water.

Further, in the step S2, the basic catalyst is at least one of diisopropylethylamine, triethylamine, 1, 8-diazabicyclo [5,4,0] undec-7, 1, 5-diazabicyclo [4.3.0] non-5-ene, N-lutidine, pyridine, and imidazole.

Further, in step S3, the silylating agent is at least one of n-octyl dimethyl chlorosilane, n-heptyl dimethyl chlorosilane, n-hexyl dimethyl chlorosilane, n-pentyl dimethyl chlorosilane, n-butyl dimethyl chlorosilane and n-propyl dimethyl chlorosilane.

Further, in step S3,

The basic catalyst is at least one of diisopropylethylamine, triethylamine, 1, 8-diazabicyclo [5,4,0] undecene-7, 1, 5-diazabicyclo [4.3.0] non-5-ene, N-lutidine, pyridine and imidazole;

the organic solvent is at least one of toluene, xylene, n-heptane, isooctane and diphenyl ether.

Further, in step S4,

Two or more of the silylating agents trimethylchlorosilane, hexamethyldisilazane, trimethylimidazole, trifluoromethylsilane triflate, bis (trimethylsilicon) acetamide, bis (trimethylsilicon) trifluoroacetamide and dimethylaminotrimethylsilane;

the organic solvent is at least one of toluene, xylene, n-heptane, isooctane and diphenyl ether;

The basic catalyst is at least one of diisopropylethylamine, triethylamine, 1, 8-diazabicyclo [5,4,0] undecene-7 and 1, 5-diazabicyclo [4.3.0] non-5-ene, N-lutidine, pyridine and imidazole.

Further, in the step S1, the dosage of the strong acid solution is 5-20 mL/g silica gel;

in the step S2 of the process,

The dosage of the organic solvent is 5-20mL of the organic solvent per gram of silica gel;

the dosage of the silane coupling agent is 1-10mmol of the silane coupling agent per gram of silica gel;

The dosage of the alkaline catalyst is 3-30mmol of the alkaline catalyst per gram of silica gel;

in the step S3 of the process,

The dosage of the organic solvent is 5-20mL of the organic solvent per gram of silica gel;

the dosage of each silanization reagent is 1-20mmol per gram of silica gel;

the dosage of the alkaline catalyst is 1-40mmol of the alkaline catalyst per gram of silica gel;

in the step S4 of the process,

The dosage of the organic solvent is 5-20mL of the organic solvent per gram of silica gel;

the dosage of each silanization reagent is 1-20mmol per gram of silica gel;

the amount of the alkaline catalyst is 1-40mmol per gram of silica gel.

The beneficial effects of the invention are as follows:

(1) The preparation method of the liquid chromatography stationary phase is simple and mild, the bonding and end sealing are carried out on the surface of the silica gel through simple and reliable liquid phase silanization reaction, the preparation process is simple and reliable, the reaction condition is mild, and the industrialization is facilitated.

(2) The liquid chromatography stationary phase provided by the invention has good tolerance under the high pH condition, and can be widely used for separation analysis of various samples.

Drawings

FIGS. 1 and 2 are chromatograms of example 4;

FIG. 3 is a chromatogram of example 5;

FIG. 4 is a chromatogram of example 6.

In the figure ■ is labeled as stationary phase in example 1, x is labeled as conventional C18 chromatographic column.

Detailed Description

The invention is further illustrated by the following examples. The examples are only illustrative of the invention and are not intended to be limiting.

Example 1

10G of silica gel is added into a 250mL flask, 100mL of 30wt% hydrochloric acid solution is added, the mixture is heated, refluxed and stirred for 5 hours, filtered, washed with water until the pH value is between 6 and 7, and the obtained solid is dried for 24 hours at 160 ℃ in a drying box, so that the acidified silica gel is obtained.

10G of acidified silica gel (particle size 10 μm, pore diameter 10 nm) dried at 160 ℃ for 16 hours, 8g of octadecyl dimethyl chlorosilane, 3g of N, N-lutidine and 60mL of xylene are added into a 250mL flask under the protection of nitrogen, reacted for 3 hours at 115 ℃, filtered, washed with xylene, methanol water, methanol and tetrahydrofuran in sequence, and the obtained solid is dried for 16 hours at 80 ℃ in a vacuum drying oven to obtain octadecyl bonding intermediate;

Under the protection of nitrogen, adding 10g of octadecyl bonding intermediate, 3g of n-butyldimethyl chlorosilane, 3g of N, N-lutidine and 80mL of diphenyl ether into a 250mL flask, reacting for 3 hours at 180 ℃, filtering, washing sequentially with dimethylbenzene, methanol water, methanol and tetrahydrofuran, and vacuum drying the obtained solid in a drying oven at 80 ℃ for 24 hours to obtain an octadecyl bonding phase after preliminary end capping;

Under the protection of nitrogen, 10g of octadecyl bonding intermediate, 6g of trimethylchlorosilane, 5g of hexamethyldisilazane, 3g of N, N-lutidine and 80mL of diphenyl ether are added into a 250mL flask to react for 3 hours at 180 ℃, the mixture is filtered, the mixture is washed with xylene, methanol water, methanol and tetrahydrofuran in turn, the obtained solid is dried in vacuum for 24 hours at 80 ℃ in a drying box, a chromatographic stationary phase 1 is obtained, and the carbon content of the stationary phase is 20.73wt% through an elemental analysis test, and the structure is as follows:

Example 2

10G of silica gel is added into a 250mL flask, 100mL of nitric acid solution with the concentration of 12wt% is added into the flask, the mixture is heated, refluxed and stirred for 2 hours, filtered, washed with water until the pH value is between 6 and 7, and the obtained solid is dried for 24 hours at 160 ℃ in a drying box, so that the acidified silica gel is obtained.

10G of acidified silica gel (particle size 10 μm, pore diameter 10 nm) dried at 160 ℃ for 16 hours, 6g of dodecyl dimethyl chlorosilane, 2g of triethylamine and 60mL of xylene are added into a 250mL flask under the protection of nitrogen, reacted for 3 hours at 130 ℃, filtered, washed sequentially with xylene, methanol water, methanol and tetrahydrofuran, and the obtained solid is dried for 16 hours at 80 ℃ in a vacuum drying oven to obtain a dodecyl bonding intermediate;

Under the protection of nitrogen, adding 10g of dodecyl bonding intermediate, 1g of propyl dimethyl chlorosilane, 3g of pyridine and 80mL of diphenyl ether into a 250mL flask, reacting for 6 hours at 180 ℃, filtering, washing with dimethylbenzene, methanol water, methanol and tetrahydrofuran in sequence, and vacuum drying the obtained solid in a drying oven at 80 ℃ for 24 hours to obtain a dodecyl bonding phase after preliminary end capping;

Under the protection of nitrogen, 10g of dodecyl bonding intermediate, 6g of trimethylchlorosilane, 5g of hexamethyldisilazane, 3g of triethylamine and 80mL of diphenyl ether are added into a 250mL flask to react for 6 hours at 180 ℃, the mixture is filtered, the mixture is washed with dimethylbenzene, methanol water, methanol and tetrahydrofuran in turn, the obtained solid is dried in vacuum for 24 hours at 80 ℃ in a drying box, a chromatographic stationary phase 2 is obtained, and the carbon content of the stationary phase is 15.26 weight percent through an elemental analysis test, and the structure is as follows:

Example 3

10G of silica gel is added into a 250mL flask, 100mL of hydrochloric acid solution with the concentration of 20wt% is added, the mixture is heated, refluxed and stirred for 5 hours, filtered, washed with water until the pH value is between 6 and 7, and the obtained solid is dried for 24 hours at 160 ℃ in a drying box, so that the acidified silica gel is obtained.

10G of acidified silica gel (particle size of 10 mu m, aperture of 10 nm) dried at 160 ℃ for 16 hours, 20g of normal triacontyl dimethyl chlorosilane, 5g of imidazole and 80mL of xylene are added into a 250mL flask under the protection of nitrogen, reacted for 6 hours at 150 ℃, filtered, washed with xylene, methanol water, methanol and tetrahydrofuran in sequence, and the obtained solid is dried for 16 hours at 80 ℃ in a vacuum drying oven to obtain a triacontyl bonding intermediate;

Under the protection of nitrogen, adding 10g of a triacontyl bonding intermediate, 4g of n-butyldimethyl, 2g of trimethylsilylimidazole, 3g of triethylamine and 80mL of diphenyl ether into a 250mL flask, reacting for 16 hours at 180 ℃, filtering, washing sequentially with xylene, methanol water, methanol and tetrahydrofuran, and vacuum drying the obtained solid in a drying oven at 80 ℃ for 24 hours to obtain a triacontyl bonding phase after preliminary end capping;

10g of a triacontyl bonding intermediate, 4g of n-butyldimethyl, 2g of trimethylsilylimidazole, 3g of triethylamine and 80mL of diphenyl ether are added into a 250mL flask under the protection of nitrogen, the mixture is reacted for 16 hours at 180 ℃, filtered, washed by xylene, methanol water, methanol and tetrahydrofuran in sequence, and the obtained solid is dried in vacuum for 24 hours at 80 ℃ in a drying box to obtain a chromatographic stationary phase 3, and the carbon content of the stationary phase is 23.12 weight percent through an elemental analysis test, and the structure is as follows:

Example 4

The chromatographic stationary phase 1 obtained in example 1 was used for packing 4.6X1150 mm column and 4.6X1150 mm standard conventional C18 stationary phase column for evaluation of packing tolerance under high pH conditions. As shown in fig. 1 and 2, the stationary phase in the present invention can tolerate 6720 column volumes and the changes in column efficiency and retention time are small under the condition of ph=12, whereas the conventional C18 column can only tolerate 4560 column volumes, which indicates that the stationary phase has better tolerance under the high pH condition, and the chromatographic conditions are:

chromatographic column: 4.6X105 mm;

Sample: sinomenine 1mg/mL;

Solvent: a:0.125% ammonia/water (pH 12), B: methanol;

Eluting: a: b=60: 40, a step of performing a;

Flow rate: 1.0mL/min; the running time is 120min, and the retention and column effect of sinomenine are recorded as a destruction cycle.

Column temperature: 30 ℃;

and (3) detection: PDA (190 nm-400 nm) & UV (254 nm);

And (3) sample injection: 2. Mu.L.

Example 5

The chromatographic stationary phase 1 obtained in example 1 was used to pack a 4.6X150 mm column for separation analysis of nonpolar compounds in liquid chromatography mode, and the stationary phase had good selectivity to polar compounds. As shown in fig. 3, the chromatographic conditions are:

Chromatographic column: 4.6X150 mm;

sample: mixing (uracil 1mg/mL, nitrobenzene 1mg/mL, naphthalene 2mg/mL, fluorene 2.2 mg/mL);

Solvent: a: acetonitrile, D: water;

eluting: a: d=70:30;

flow rate: 1.0mL/min;

Column temperature: 30 ℃;

and (3) detection: PDA (190 nm-400 nm) & UV (254 nm);

And (3) sample injection: 1.0. Mu.L.

Example 6

The chromatographic stationary phase 1 obtained in example 1 was used to pack a 4.6X150 mm column for separation analysis of polar and nonpolar compounds in liquid chromatography mode, and had good separation selectivity.

As shown in fig. 4, the chromatographic conditions are:

Chromatographic column: 4.6X150 mm;

sample: mixing (uracil 1mg/mL, amitriptyline 2mg/mL, butyl p-hydroxybenzoate 1 mg/mL);

Solvent: a: acetonitrile, B: water, D:100mM ammonium formate (ph=3.2);

Eluting: a: b: d=45: 45:10;

flow rate: 1.0mL/min;

Column temperature: 30 ℃;

and (3) detection: DAD (190 nm-400 nm) & UV (254 nm);

And (3) sample injection: 1 mul.

The foregoing is only a preferred embodiment of the present invention, and all technical solutions for achieving the object of the present invention by substantially the same means are included in the scope of the present invention.

Claims (10)

1. The high pH stable reversed phase chromatographic stationary phase is characterized by having the following structural formula: Wherein SILICA GEL is silica gel, R is phenyl, oxyphenyl or alkyl chain with 5-27 carbon atoms, n=0-5.

2. The preparation method of the high-pH stable reversed phase chromatographic stationary phase is characterized by comprising the following steps of:

S1: pretreatment of silica gel: adding silica gel into strong acid solution with the concentration of 10-40 wt%, heating, refluxing and stirring for 1-48 hours, filtering, washing with water to pH=6-7, and vacuum drying the obtained solid at 80-160 ℃ for 8-24 hours to obtain acidified silica gel;

S2: and (3) silanization reaction bonding: under the protection of nitrogen or argon, adding a silane coupling agent, an alkaline catalyst and acidified silica gel into an organic solvent, reacting for 3-24 hours at 40-150 ℃, filtering, washing with dimethylbenzene, methanol water, methanol and tetrahydrofuran in sequence, and vacuum drying the obtained solid at 40-80 ℃ for 8-24 hours to obtain an intermediate;

S3: the first step of liquid phase silanization reaction end capping: adding a silylation reagent, an alkaline catalyst and an intermediate into an organic solvent under the protection of nitrogen or argon, reacting for 3-24 hours at 30-180 ℃, filtering, washing with dimethylbenzene, methanol water, methanol and tetrahydrofuran in sequence, and vacuum drying the obtained solid at 40-80 ℃ for 8-24 hours to obtain a preliminary end-capped stationary phase;

S4: and the second step of liquid phase silanization reaction end capping: under the protection of nitrogen or argon, adding two or more trimethyl silanization reagents, an alkaline catalyst and a primary end-capped stationary phase into an organic solvent, reacting for 3-24 hours at 30-180 ℃, filtering, washing with dimethylbenzene, methanol water, methanol and tetrahydrofuran in sequence, and vacuum drying the obtained solid at 40-80 ℃ for 8-24 hours to obtain the chromatographic stationary phase.

3. The method according to claim 2, wherein in the step S1, the strong acid is at least one of hydrochloric acid, nitric acid and sulfuric acid.

4. The method for preparing a high pH stable reversed phase chromatography stationary phase according to claim 2, wherein in step S2, the silane coupling agent has the following structure:

wherein X is one of chlorine, methoxy or ethoxy, and R is phenyl, oxyphenyl or alkyl chain with 5-27 carbon atoms.

5. The method for preparing a high pH stable reversed phase chromatography stationary phase according to claim 2, wherein in step S2, the organic solvent is at least one of dichloromethane, toluene, xylene, n-heptane, isooctane, which is not miscible with water.

6. The method for preparing a high pH stable reversed phase chromatographic stationary phase according to claim 2, wherein in step S2, the basic catalyst is at least one of diisopropylethylamine, triethylamine, 1, 8-diazabicyclo [5,4,0] undecene-7, 1, 5-diazabicyclo [4.3.0] non-5-ene, N-lutidine, pyridine, imidazole.

7. The method for preparing a high pH stable reversed phase chromatography stationary phase according to claim 2, wherein in step S3, the silylating agent is at least one of n-octyl dimethyl chlorosilane, n-heptyl dimethyl chlorosilane, n-hexyl dimethyl chlorosilane, n-pentyl dimethyl chlorosilane, n-butyl dimethyl chlorosilane, and n-propyl dimethyl chlorosilane.

8. The method for preparing a stationary phase for high-pH stationary phase reversed-phase chromatography according to claim 2, wherein in step S3,

The basic catalyst is at least one of diisopropylethylamine, triethylamine, 1, 8-diazabicyclo [5,4,0] undecene-7, 1, 5-diazabicyclo [4.3.0] non-5-ene, N-lutidine, pyridine and imidazole;

the organic solvent is at least one of toluene, xylene, n-heptane, isooctane and diphenyl ether.

9. The method for preparing a stationary phase for high-pH stationary phase reversed-phase chromatography according to claim 2, wherein in step S4,

Two or more of the silylating agents trimethylchlorosilane, hexamethyldisilazane, trimethylimidazole, trifluoromethylsilane triflate, bis (trimethylsilicon) acetamide, bis (trimethylsilicon) trifluoroacetamide and dimethylaminotrimethylsilane;

the organic solvent is at least one of toluene, xylene, n-heptane, isooctane and diphenyl ether;

The basic catalyst is at least one of diisopropylethylamine, triethylamine, 1, 8-diazabicyclo [5,4,0] undecene-7 and 1, 5-diazabicyclo [4.3.0] non-5-ene, N-lutidine, pyridine and imidazole.

10. The method for preparing a stationary phase for high-pH stable reverse phase chromatography according to claim 2, wherein in step S1, the amount of the strong acid solution used is 5-20mL per gram of silica gel;

in the step S2 of the process,

The dosage of the organic solvent is 5-20mL of the organic solvent per gram of silica gel;

the dosage of the silane coupling agent is 1-10mmol of the silane coupling agent per gram of silica gel;

The dosage of the alkaline catalyst is 3-30mmol of the alkaline catalyst per gram of silica gel;

in the step S3 of the process,

The dosage of the organic solvent is 5-20mL of the organic solvent per gram of silica gel;

the dosage of each silanization reagent is 1-20mmol per gram of silica gel;

the dosage of the alkaline catalyst is 1-40mmol of the alkaline catalyst per gram of silica gel;

in the step S4 of the process,

The dosage of the organic solvent is 5-20mL of the organic solvent per gram of silica gel;

the dosage of each silanization reagent is 1-20mmol per gram of silica gel;

the amount of the alkaline catalyst is 1-40mmol per gram of silica gel.