RU2638660C1 - Sorbent for determining connections of ion and hydrophilic nature - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: sorbent contains a silica gel matrix with a grafted quaternary ammonium group and nitrogen atoms attached to it with spacers, which are a part of diamine or triamine.

EFFECT: improved operational and chromatographic characteristics, effective for the separation of inorganic anions, weakly retained organic acids, amino acids, sugars, and vitamins.

9 cl, 1 dwg, 1 tbl, 11 ex

Description

Technical field

The invention relates to new separation materials based on a silica gel matrix, which can be used in ion chromatography as sorbents for the determination of organic and inorganic anions, as well as in hydrophilic chromatography to determine polar biologically active compounds.

State of the art

Sorbents for ion chromatography of anions are characterized by the presence of quaternary ammonium groups in the structure, whereas sorbents used in hydrophilic chromatography require the presence of hydrophilic fragments on the matrix surface - hydroxo groups, amino groups, etc. The structure of the functional layer of the sorbent, that is, a set of specific functional groups, the location of these groups on the surface of the matrix and relative to each other, as well as the method of fixing such a layer on the matrix have a significant effect on selectivity sorbent.

The prior art methods for modifying silica gel matrices to obtain sorbents for ion chromatography, usually the option of electrostatic fixing. For example, polyelectrolyte anion-exchange sorbents based on matrices representing silica gel, modified by a water-soluble polymer containing positively charged quaternary ammonium groups in the chain (OV Krokhin, AD Smolenkov, NV Svintsova, ON Obrezkov, OA Shpigun, Modified silica as a phase phase, are known chromatography. // J. Chromatogr. A. 1995. V. 706. P. 93-98). To prepare them, a matrix - silica brand Silasorb C ₈ - was mixed with dodecylbenzylsulfonic acid (to create a negative charge on the surface of the matrix). Modification was then carried out with a polymer containing positively charged quaternary ammonium groups in the chain — for example, with a solution of poly (N-ethyl-4-vinylpyridinium bromide) or poly (dimethyldiallylammonium chloride). The retention of the water-soluble polymer in this case is due to electrostatic interactions between the negatively charged surface of the matrix and the positively charged quaternary ammonium groups in the polymer chain. One of the advantages of polyelectrolyte sorbents is their high efficiency, due to the fact that with this approach to synthesis there is no diffusion of anions deep into the matrix particles, which leads to the absence of smearing of chromatographic peaks. However, a significant drawback of polyelectrolyte sorbents is their low stability, which leads to a gradual decrease in ion exchange capacity as a result of changes in the conformation of the electrostatically fixed water-soluble polymer, as well as its gradual washing off from the matrix surface.

At the same time, when operating in hydrophilic chromatography mode, where mobile phases with a high content of organic solvent are used, only covalent fixation of the functional layer can provide the necessary stability, since electrostatic interactions are significantly weakened in organic media (P. Jandera, Stationary and mobile phases in hydrophilic interaction liquid chromatography: a review. // Anal. Chim. Acta. 2011. V. 692. P. 1-25).

Known adopted for the prototype anion-exchange sorbent for the determination of organic and inorganic anions by ion chromatography, including the anions of weakly retained organic acids and oxyhalides (patent RU 2575454, IPC B01J 20/22, publ. 02.20.2016). The sorbent contains a matrix selected from the series: a divinylbenzene-based polymer that acts as a crosslinking agent for this polymer, polymethacrylate, silicon dioxide, titanium dioxide, zirconia or alumina aminated with a primary amine, and a water-soluble polymer removed from the matrix surface using a spacer containing quaternary ammonium functional groups in the chain, while the general formula of the sorbent corresponds to the formula:

where R = (CH ₂ ) n, n = 2-8,

- четвертичный атом азота.

- Quaternary nitrogen atom.

A method of obtaining such an anion-exchange sorbent includes the following sequence of operations:

- as a starting compound, a matrix selected from a series of divinylbenzene-based polymer in which divinylbenzene is a crosslinking agent, polymethacrylate, silicon dioxide, titanium dioxide, zirconium dioxide or alumina is taken;

- aminate it with a primary amine;

- chemically inoculate a spacer based on a compound from the class of diglycidyl ethers;

- modify the resulting compounds with a water-soluble polymer containing primary, secondary or tertiary amino groups in the chain to obtain quaternary ammonium groups chemically grafted to the diglycidyl ethers in the oxirane ring.

Such a sorbent is intended for ion chromatography of anions and is characterized by high selectivity and efficiency in the ion chromatography mode, but is not universal enough for the selective separation of biologically active substances in the hydrophilic chromatography mode, since it does not provide sufficient retention of these compounds. This is due to the presence of positively charged quaternary ammonium groups in the outer part of the functional layer, which cause repulsion of certain classes of polar substances protonated in an acidic environment by compounds, such as amino acids and water-soluble vitamins, which leads to short retention times of such compounds and, as a result, low selectivity and poor peak resolution. Another disadvantage of this sorbent is the insufficient stability of the grafted layer when working in alkaline and weakly alkaline media, since Quaternary ammonium groups are eliminated according to Hoffmann with cleavage of the amino group, which leads to a gradual loss of its separating ability by the sorbent. The third serious drawback is the need for long-term balancing (about 4 hours) of a chromatographic column with such a sorbent, i.e., a long time for the background signal of the eluent to reach a plateau with a value of no more than 100 units. opt. square * 10 ^-3 , especially in environments with a high content of organic solvent, which is associated with the conformational mobility of the grafted water-soluble polymer.

In order to meet all the requirements for modern sorbents for ionic as well as hydrophilic chromatography, and be suitable for solving a wide range of increasingly complex modern analytical problems requiring the simultaneous highly sensitive determination of an increasing number of compounds of ionic and hydrophilic nature in various objects, sorbents should possess more advanced operational and chromatographic characteristics, as well as be multifunctional, that is, selective towards non how many classes of compounds and suitable for work in several variants of chromatography.

Disclosure of invention

The objective of the invention is the creation of a sorbent for the determination of compounds of ionic and hydrophilic nature, with improved performance and chromatographic characteristics.

The problem is solved by the proposed sorbent for determining organic and inorganic anions by ion chromatography and polar biologically active compounds - amino acids, water-soluble vitamins and sugars - by hydrophilic chromatography containing a silica gel matrix with a chemically grafted quaternary ammonium group, and connected to it using matching spacers R ₁ uncharged nitrogen atoms with substituents R ₂ , which are part of di - or triamine, while the general formula of the sorbent corresponds the formula:

where R ₁ represents an alkyl, alkanol group or an ether-containing group;

R ₂ represents H, Alkyl, Alkyl-OH.

It is preferable to use a matrix with a particle diameter of 1-10 microns as a silica gel matrix.

In the present application, a spacer is understood to mean any radical that can join together the indicated fragments of the structure — a silica gel matrix and a quaternary ammonium group chemically grafted to it; a secondary or tertiary ammonium group of di- or triamine and a quaternary ammonium group chemically grafted to the matrix.

In this case, the spacers connecting the quaternary ammonium functional group grafted to the matrix with secondary or tertiary amino groups that are part of at least one di- or triamine in the proposed sorbent are selected from the number of the most common spacers: an alkyl, alkanol spacer or a spacer containing ether groups (Zatirakha AV, Smolenkov AD, Shpigun O.A. Preparation and chromatographic performance of polymer-based anion exchangers for ion chromatography: a review. // Anal. Chim. Acta. 2016. V. 904. P. 33-50 )

As alkyl spacers, dihaloalkanes such as 1,2-dibromoethane, 1,3-dibromopropane, 1,4-dibromobutane, 1,5-dibromopentane can be used.

As alkanol spacers, 1,3-dibromopropanol-2, 1,5-dibromopentanol-3, 1,4-dibromobutanol-2 and other dihaloalkanes containing hydroxide groups can be used.

Diglycidyl ethers, for example 1,4-butanediol diglycidyl ether, resorcyndiglycidyl ether, 1,2-ethanediol diglycidyl ether, ethylene glycol diglycidyl ether, can be used as spacers containing ether groups.

As di- and triamines, for example, ethylenediamine, diethylene triamine, bis (hexamethylene) triamine, N, N'-bis (2-hydroxyethyl) ethylenediamine can be used.

The selectivity of such a sorbent by anion in ion chromatography mode is ensured by the presence of a quaternary ammonium group in the structure, which is located “inside” the functional layer and is shielded by secondary or tertiary groups of di- or triamines, as a result of which hydroxide ions are difficult to access and the Hoffman elimination probability very small. At the same time, the secondary and tertiary amino groups on the outer part of the functional layer provide the possibility of working in hydrophilic chromatography mode and selectivity for various classes of biologically active compounds, while these groups are not eliminated, which ensures the stability of the sorbent. In addition, due to the absence of a volumetric conformationally mobile polymer on the surface, the chromatographic column with the sorbent quickly (in 30 minutes) equilibrates (i.e., the background eluent signal reaches a plateau with a value of no more than 100 units of opt. Pl. * 10 ^-3 ) before starting the analysis in both aqueous and organic media.

The technical result in this case is the possibility of using sorbents in both ion and hydrophilic chromatography modes, improving the chromatographic characteristics, namely, expanding the selectivity for such classes of polar biologically active compounds as amino acids, sugars and water-soluble vitamins, as well as improving operational characteristics namely, to increase the stability in the mobile phases used in the modes of ionic and hydrophilic chromatography, and to reduce the equilibration time with Bent column chromatography before analysis from 4 hours to 30 minutes.

Brief Description of the Drawings

In FIG. 1 shows chromatograms of the separation of model mixtures of anions, amino acids, sugars and water-soluble vitamins on the resulting sorbent. Peak height was measured in microsiemens.

The implementation of the invention

The inventive sorbents can be obtained by methods known in the literature, in particular by the method described above in the prototype.

Common to all approaches is the need to use an aminated matrix - silica gel with grafted primary amino groups. For the synthesis, you can use commercially available aminated silica gel, as well as ordinary unmodified silica gel with a particle diameter of 1-10 μm, on the surface of which amino groups must be fixed. One of the possible approaches for obtaining aminated silica gel is the modification of the silica gel matrix with aminopropyltrimethoxysilane, which leads to the formation of primary amino groups on the matrix surface (Fuying Dong, Depeng Ma, Shengyu Feng. Aminopropyl-modified silica as cross-linkers of polysiloxane containing γ-chloropropyl groups for preparing heat-curable silicone rubber. // Polymer Testing. 2016. V. 52. P. 124-132).

Other silanes with at least one aminoalkyl substituent can also be used to modify silica gel matrices. An alternative to producing aminated silica gel may be its treatment with glycidyloxypropyltrimethoxysilane or another silylating reagent containing an epoxy group in the structure to produce an epoxidized silica gel, which is then treated with ammonia solution or another amine to open the epoxy rings and inoculate the amino groups (Nesterenko PN, Haddad PR Zwitter II exchangers in liquid chromatography. // Anal. Sci. 2000. V. 16. P. 565-574).

To introduce spacers, the amino groups obtained on the surface are then alkylated with a bifunctional compound, for example, diglycidyl ether, dihaloalkane or dihalogenated alcohols to form quaternary ammonium groups. Then, di- or triamines are fixed on the grafted spacers using the amination reaction, which usually occurs at 50-70 ° С (Shchukina OI, Zatirakha AV, Smolenkov AD, Nesterenko PN, Shpigun OA Anion exchangers with branched functional anion exchange layers of different hydrophilicity for ion chromatography. // J. Chromatogr. A. 2015. V. 1408. P. 78-86).

The following examples confirm, but do not limit, the claimed objects of the invention.

Example 1. Obtaining a sorbent based on silica gel with a particle diameter of 5 μm with a chemically grafted quaternary ammonium group and connected with it using matching spacers containing ether groups, ethylene diamine molecules.

1) matrix selection

Aminated silica gel Diasfer-Amin (BioKhimmak ST, Russia) with a particle diameter of 5 μm was taken as the initial matrix.

2) fixing the diamine to the Quaternary ammonium group through the spacer

1 g of the matrix was placed in a 100 ml flask, 20 ml of water and 1 ml of 1,4-butanediol diglycidyl ether were added. The reaction mixture was stirred for 1.5 h using a mechanical stirrer at a speed of 500 rpm at a temperature of 60 ° C, then was filtered on a glass filter and washed with 100 ml of deionized water. The product was then suspended in an ultrasonic bath in 20 ml of water and 2 ml of ethylenediamine was added. The reaction mixture was stirred for 1 h using a mechanical stirrer at a speed of 500 rpm at a temperature of 60 ° C, then was filtered on a glass filter and washed with 100 ml of deionized water.

The resulting sorbent corresponds to the formula

Example 2. Obtaining a sorbent based on silica gel with a particle diameter of 5 μm with a chemically grafted quaternary ammonium group and ethylene diamine molecules connected to it using identical alkanol spacers.

Obtaining the sorbent was carried out as in example 1, but in stage 2, instead of (1,4-butanediol) diglycidyl ether, 1,5-dibromopentanol-3 in an equivalent amount was taken.

The resulting sorbent corresponds to the formula

Example 3. Obtaining a sorbent based on silica gel with a particle diameter of 5 μm with a chemically grafted quaternary ammonium group and diethylenetriamine molecules connected to it using identical alkanol spacers.

Everything is as in example 1, but in stage 2, instead of 1,4-butanediol diglycidyl ether, 1,3-dibromopropanol-2 in an equivalent amount is taken, and diethylenetriamine in an equivalent amount is taken instead of ethylene diamine.

The resulting sorbent corresponds to the formula

Example 4. Obtaining a sorbent based on silica gel with a particle diameter of 7 μm with a chemically grafted quaternary ammonium group and connected to it using matching spacers containing ether groups with bis (hexamethylene) triamine molecules.

The sorbent was prepared as in Example 1, only instead of an aminated silica gel with a particle diameter of 5 μm, an aminated silica gel with a particle diameter of 7 μm was taken, and in step 3, an equivalent amount of bis (hexamethylene) triamine was taken.

The resulting sorbent corresponds to the formula

Example 5. Obtaining a sorbent based on silica gel with a particle diameter of 5 μm with a chemically grafted quaternary ammonium group and diethylenetriamine molecules connected to it using identical alkyl spacers.

Obtaining the sorbent was carried out as in example 1, only in stage 2, instead of 1,4-butanediol diglycidyl ether, 1,3-dibromopropane was taken, and diethylenetriamine in an equivalent amount was taken in place of ethylene diamine

The resulting sorbent corresponds to the formula

Example 6. Obtaining a sorbent based on silica gel with a particle diameter of 5 μm with a chemically grafted quaternary ammonium group and diethylenetriamine molecules connected to it using identical alkyl spacers.

Obtaining the sorbent was carried out as in example 1, only in stage 2, instead of 1,4-butanediol diglycidyl ether, 1,4-dibromobutane was taken, and diethylenetriamine in an equivalent amount was taken instead of ethylene diamine.

The resulting sorbent corresponds to the formula

Example 7. Obtaining a sorbent based on silica gel with a particle diameter of 5 μm with a chemically grafted quaternary ammonium group and connected with it using matching spacers containing ether groups, diethylene triamine molecules.

Obtaining the sorbent was carried out as in example 1, only at stage 2, instead of 1,4-butanediol diglycidyl ether, resorcindiglycidyl ether was taken, and instead of ethylene diamine, diethylenetriamine in an equivalent amount was taken.

The resulting sorbent corresponds to the formula

Example 8. The separation of organic and inorganic anions using a multifunctional sorbent in ion chromatography mode.

A mixture of 10 anions containing chloride, nitrite, bromide, nitrate, iodide, thiocyanate, acetate, formate, propionate and bromate in a concentration of 50 mg / l of each component was taken as a model mixture. The mixture was prepared by adding 1 ml of a concentrated solution of each component with a content of 5000 mg / L to a 100 ml volumetric flask and then bringing to the mark with deionized water. The mixture was separated using the multifunctional sorbent obtained in Example 1. The sorbent was packed in 100 × 3 mm chromatographic columns under a pressure of 400 bar and the anion mixture was separated in the ion chromatography variant with spectrophotometric detection at a wavelength of 254 nm. To obtain a chromatogram, a Dionex Ultimate 3000 liquid chromatograph (Thermo Fisher Scientific, USA) was used. Potassium hydrophthalate with a pH of 4 and a concentration of 3 mM at a flow rate of 1 ml / min was used as the mobile phase. Peak height was measured in microsiemens. The column equilibration time was 30 minutes. This sorbent made it possible to separate 10 of the indicated anions in approximately 16 minutes with good resolution and peak symmetry. The resulting chromatogram containing 10 peaks shown in FIG. 1 (A), confirms the selective separation of the 10 anions present in the model mixture in ion chromatography mode. It should be noted that there is no erosion and a small width of the peaks, which indicates the high efficiency of the inventive sorbent (50000-80000 tt / m for different anions from the presented series) in the ion chromatography mode.

Example 9. The separation of amino acids using a multifunctional sorbent in hydrophilic chromatography.

A mixture of 7 amino acids containing phenylalanine, methionine, tyrosine, hydroxyproline, asparagine, alanine, series at a concentration of 20 mg / l of each component was taken as a model mixture. The mixture was prepared by adding 0.5 ml of a concentrated solution of each component with a content of 2000 mg / L to a 50 ml volumetric flask and then bringing to the mark with deionized water. The mixture was separated using the multifunctional sorbent obtained in Example 1. The sorbent was packed into 100 × 3 mm chromatographic columns under a pressure of 400 bar and the mixture of amino acids was separated using hydrophilic chromatography with spectrophotometric detection at a wavelength of 210 nm. To obtain a chromatogram, a Dionex Ultimate 3000 liquid chromatograph (Thermo Fisher Scientific, USA) was used. As the mobile phase, we used a solution of the composition 80% CH ₃ CN: 20% phosphate buffer solution, pH 6.5 (2 mM bp) at a flow rate of 1 ml / min. Peak height was measured in microsiemens. The column equilibration time was 30 minutes. This sorbent allowed the separation of 7 of these amino acids in less than 6 minutes with good resolution and peak symmetry. The resulting chromatogram containing 7 peaks shown in FIG. 1 (B), confirms the selective separation of the amino acids present in the model mixture in the hydrophilic chromatography mode. It should be noted that there is no erosion and a small width of the peaks, which indicates the high efficiency of the inventive sorbent (30000-45000 tt / m for different amino acids from the presented series) in the hydrophilic chromatography mode.

Example 10. The separation of water-soluble vitamins using a multifunctional sorbent in hydrophilic chromatography.

Everything is as in example 7, but instead of the mixture of amino acids, as a model mixture, a mixture of water-soluble vitamins containing nicotinic acid (B ₃ ), nicotinamide (B ₃ ), ascorbic acid (C), B ₆ , B ₁₂ , B ₁ , B _{2 was} taken at a concentration of 10 mg / l of each component. The mixture was prepared by adding 1 ml of a concentrated solution of each component with a content of 1000 mg / L to a 100 ml volumetric flask and subsequent adjustment to the mark with deionized water. As the mobile phase, a solution of 100 mM CH ₃ COONH ₄ , pH 5.8 / acetonitrile (gradient elution) is used; detection is carried out at a wavelength of 270 nm. The column equilibration time was 30 minutes. This sorbent allowed the separation of 7 of these vitamins in less than 12 minutes with good resolution and peak symmetry. The resulting chromatogram containing 7 peaks shown in FIG. 1 (B), confirms the selective separation of water-soluble vitamins present in the model mixture in the hydrophilic chromatography mode. It should be noted that there is no erosion and a small peak width, which indicates the high efficiency of the inventive sorbent (30000-45000 tt / m for various water-soluble vitamins from the presented series) in the hydrophilic chromatography mode.

Example 11. The separation of sugars using a multifunctional sorbent in hydrophilic chromatography.

Everything is as in example 7, but instead of the mixture of amino acids, a mixture of sugars containing maltose, glucose, sucrose, lactose, rhamnose, xylose, fructose in a concentration of 50 mg / l of each component was taken as a model mixture. The mixture is prepared by adding 1 ml of a concentrated solution of each component with a content of 5000 mg / L to a 100 ml volumetric flask and then adjusting to the mark with deionized water. As the mobile phase, a solution of water: acetonitrile (15%: 85%) was used at a flow rate of 0.44 ml / min; using refractometric detection at a wavelength of 270 nm. The column equilibration time was 30 minutes. This sorbent allowed to separate 7 of these sugars in about 20 minutes with good resolution and peak symmetry. The resulting chromatogram containing 7 peaks shown in FIG. 1 (D) confirms the selective separation of the sugars present in the model mixture in the hydrophilic chromatography mode. It should be noted that there is no erosion and a rather small peak width, which indicates the high efficiency of the inventive sorbent (25000-35000 tt / m for different sugars from the presented series) in the hydrophilic chromatography mode.

Similar results were obtained when testing all of the claimed multifunctional sorbents.

The chromatographic characteristics obtained in examples 1-7 multifunctional sorbents are presented in table 1.

As can be seen from the above examples and table 1, the obtained sorbents have high efficiency (50000-80000 tt / m in ion chromatography), selectivity (allow simultaneous determination of at least 11 anions in ion chromatography mode, and in hydrophilic chromatography mode - not less 7 amino acids, 7 sugars and 6 water-soluble vitamins) and stability (minimum life without changing retention times of analytes is 5 months). In addition, the proposed production methods make it possible to vary the selectivity of multifunctional sorbents by varying the structure of spacers and fixed di- and triamines.

The approaches to the synthesis of the proposed anion-exchange sorbents are simple and express in execution (synthesis time 2.5 hours) and does not require the use of highly toxic and carcinogenic reagents.

Thus, the present invention confirms the achievement of the stated indicators, namely: increased stability of the sorbents provides a service life of at least 5 months, the efficiency increases to 80,000 tt / m and gives good peak resolution, and increased selectivity to various classes of compounds allows you to work in ionic modes hydrophilic chromatography and confirms the separating ability of the sorbent in the separation of 11 inorganic and organic anions, 7 sugars, 7 amino acids and 7 water-soluble vitamins in one analysis, not a limit. Sorbent allows for one chromatographic analysis to obtain data on the content of compounds of one class in the composition of a multicomponent mixture.

Claims (11)

1. A sorbent for determining ion compounds and / or hydrophilic nature, characterized in that it comprises a silica matrix with chemically grafted quaternary ammonium group and connected thereto via matching spacers R ₁ uncharged nitrogen atoms with substituents R _2, a part of di- or triamine, while the general formula of the sorbent corresponds to the formula

where R ₁ represents an alkyl, alkanol group or an ether-containing group; R ₂ represents H, Alkyl, Alkyl-OH. 2. The sorbent according to claim 1, characterized in that a silica gel matrix with a particle diameter of 1-10 microns is used. 3. The sorbent according to claim 1, characterized in that ethylene diamine, diethylene triamine, bis (hexamethylene) triamine, N, N'-bis (2-hydroxyethyl) ethylene diamine are used as di- and triamines. 4. The sorbent according to claim 1, characterized in that dihaloalkanes are used as alkyl spacers. 5. The sorbent under item 1, characterized in that as alkanol spacers use dihaloalkanes containing hydroxide groups. 6. The sorbent according to claim 1, characterized in that diglycidyl ethers are used as spacers containing ether groups. 7. The sorbent according to claim 4, characterized in that the dihaloalkanes are selected from the group consisting of 1,2-dibromoethane, 1,3-dibromopropane, 1,4-dibromobutane, 1,5-dibromopentane. 8. The sorbent according to claim 5, characterized in that the dihaloalkanes containing hydroxide groups are selected from the group consisting of 1,3-dibromopropanol-2, 1,5-dibromopentanol-3, 1,4-dibromobutanol-2. 9. The sorbent according to claim 6, characterized in that the diglycidyl ethers are selected from the group consisting of 1,4-butanediol diglycidyl ether, resorcyndiglycidyl ether, 1,2-ethanediol diglycidyl ether, ethylene glycol diglycidyl ether.

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