SK50162008A3 - Method of preparation and use poly(2-hydroxy propyl methacrylate) of chromatic phase bounded on silicate - Google Patents

Abstract

The invention relates to a process for preparing a poly (2-hydroxypropyl acrylate) cross-linked with divinyl benzene phase HPLC on activated silica-bound, in which the reaction of toluene with dietoxymetylvinylsilanom initiate a vinyl group and who-ROM, followed by reaction with trimethylchlorosilane blocked reactive free silanols, and thus the modified silica gel treated with a mixture of 2-hydroxypropyl methacrylate and divinylbenzene in the presence of a radical polymerization initiator at a temperature of 100 to 120 � C for 18-24 hours under stirring and in the presence of a suitable solvent, while at the same time-go via polymerization of methacrylate and divinylbenzene cross-linking. The invention relates to the use of poly (2-hydroxypropyl acrylate) crosslinked with divinylbenzene phase chromatography, bound to the activated silica gel for separation Biel-leguminous.

Description

Technical field

The invention relates to the preparation of a silica gel-bound poly (2-hydroxypropyl methacrylate) chromatographic phase (HPLC column packings) with high protein separation selectivity and high alkaline stability.

BACKGROUND OF THE INVENTION

High Performance Liquid Chromatography (HPLC) is currently the most important separation method for substances that cannot be converted to the gas / vapor state. Such substances naturally include macromolecular substances - polymers of natural and synthetic origin, and without exception all proteins. Liquid chromatography separation takes place in most cases in chromatography columns which are packed with a porous bed. The packing of the chromatographic column must:

(a) be mechanically stable to withstand tens of MPa. used in HPLC

(b) be chemically resistant to withstand the effects of mobile phases. eluents and separated samples, did not change its properties and did not dissolve - did not collapse

(c) have a pore structure that allows rapid mass transfer to avoid excessive expansion of the chromatographic zones (wine, peaks) and thus the loss of separation efficiency.

(d) have surface properties which permit controlled interactions with the separated constituents of the mixed samples. The separation of the components of the mixture is carried out on the basis of differences in their interactions with the packed bed. Interactions between the separated substances and the packing of the HPLC column lead to cadsorption. enthalpy distribution (absorption), entropic distribution (exclusion, secretion), or ionic effects (ion exchange, secretion, or inclusion). The result of these processes is controlled, selective, temporary containment, retention of the molecules of the separated substances in the column and ultimately their separation, separation. We are talking about different retention mechanisms in HPLC. leading to separation of sample components. When separating biopolymers, the first three types of interactions, three different retention mechanisms, are sought. The first two retention mechanisms, i. adsorption and enthalpy particionation are based on attractive interactions in the chronigraphic system. In order to suitably adjust the interactivity of the packings of the HPLC columns, they are modified - various low and high molecular groups are chemically bonded to their surface. In the latter case, the macromolecules are inoculated with the surface of the filling.

In the HPLC of both small molecules and biopolymers such as proteins, spherical, porous silica gels are used primarily for column packing. These exceed the other types of column packings (zirconium oxide, titanium oxide and the entire range of column packings prepared from synthetic organic polymers), especially under conditions a) and c) and partly also d). The surface properties of silica gel alone are not suitable for many HPLC applications. However, the free silanols on the surface of the silica gel make it possible to perform various chemical reactions in a simple manner, thereby modifying, modifying its surface properties, interactivity, retentivity to allow the application. Binding of various low molecular weight groups to the surface of silica gel was attempted. Such groups form chemically bonded HPLC phases. For common applications, silica gel is most often applied. on the surface of which there are chemically bonded alkyl groups, usually linear C-18 alkyl. A variety of HPLC column packings having chemically bonded different low molecular weight groups and longer polymer chains have been developed for special applications, including protein separation. Many of them are commercially available, but none of the known materials is universal. Therefore, great attention is paid to the development of new HPLC phases with tailor-made retentivity properties. A serious shortcoming of silica gel-based HPLC packings is their limited chemical resistance. In an alkaline environment above pH ~ 8, the silica gel matrix gradually dissolves. In the first stage of the attack of alkaline mobile phases on the HPLC column packing, there is a gradual hydrolysis of chemically bound groups, bound phases. This gradually changes the retention characteristics of the packings of the HPLC columns, resulting in non-reproducibility of the separation results. Often during the hydrolysis of the bound phase, but definitely after its hydrolysis, the silica gel matrix dissolves and ultimately collapses.

In the field of developing packings for HPLC protein separation columns, two main objectives are pursued - to prepare bound phases. which will be

(a) suitable for the efficient separation of certain types of protein;

(b) exhibit a high degree of hydrolytic stability in an alkaline environment

c) effectively protect the silica gel matrix itself from hydrolysis in an alkaline medium

The invention relates to the preparation and properties of such an HPLC chemically bonded silica gel.

SUMMARY OF THE INVENTION

The essence is a process for the preparation of a poly (2-hydroxypropyl acrylate) HPLC phase cross-linked with divinylbenzene bound to activated silica gel. to which vinyl groups are bound by reaction with diethoxymethylvinylsilane in toluene, and reactive free trimethylsilane is blocked by subsequent reaction with trimethylchlorosilane, and the treated silica gel is treated with a mixture of 2-hydroxypropyl methacrylate and divinylbenzene in the presence of during 18 to 24 hours with stirring and in the presence of a suitable solvent, while simultaneously polymerizing the methacrylate and crosslinking with divinylbenzene.

Azo-bisisobutyronitrile is preferably used as the initiator of the radical polymerization. Toluene is preferably used as the solvent.

The preparation of a new high performance liquid chromatography column based on chemical bonding of poly (2-hydroxy propyl methacrylate) together with divinylbenzene on the silica gel surface has four stages:

1. Wide-porous silica gel is activated with a 1: 4 v / v HC HCl water mixture. at 90 ° C to 100 ° C under reflux. After cooling, it is washed on the filter with deionized water until the filtrate reaches a pH = 7.0 and then dried for 2 to 4 hours at 105 ° C and then for 2 to 4 hours. at 50 ° C under water pump vacuum.

2. The dried silica is reacted with diethoxy-methylvinylsilane in dry toluene. The reaction takes place at 100 to 120 C. ^In the embodiment, reŕluxnom preferably for 18 to 24 hours. Silica gel is formed on which there is a chemically bonded vinyl group capable of reacting with a double bond of vinyl monomers. The material was washed with dry toluene and dried for 1 to 4 hours. at 50 ° C under water pump vacuum.

3. Silica gel with attached vinyl silane groups, product of step no. 2 was treated with trimethylchlorosilane in dry toluene under reflux at 100-120 ° C for 18-24 hours, and the cooled product was washed thoroughly on the filter with toluene followed by methanol. Finally, it is dried for 2 to 4 hours. at 105 ° C and then 4 hours. in a water pump vacuum. Reaction with trimethylchlorosilane chemically blocks reactive free silanols. which remained on silica gel after reaction with the vinvsilane as well as the -4SiOH groups present on the vinylsilane group. Such a process is called 'capping' in the literature.

4. The decisive step is to prepare the original bound phase by reacting the product of step n. 3 with a mixture of 2-hydroxylpropyl methacrylate and divinylbenzene in the presence of a radical polymerization initiator, e.g. 0.5 to 2 wt. % azo-bis-isobutyronitrile (AIBN). The reaction takes place in 10 to 15 wt. excess toluene at 100 to 120 ° C for 18 to 24 hours with continuous gentle stirring. During the reaction, polymerization and crosslinking occur to form a resistant bound phase that effectively separates the proteins. The reaction product was washed well with toluene and methanol and then dried for 2 hours. at 105 ° C and dried for 1 to 2 hours. at 50 ° C under water pump vacuum.

The envisaged bonded phase structure of the present invention is shown in FIG. First

-You are about

CH,

Si _{(CH3) 3}

n = 2-7

DETAILED DESCRIPTION OF THE INVENTION

Example 1:

Activation of silica gel. 10 g of wide-pored silica gel with an effective pore diameter of 30 nm is heated for 2 hours. at 90 ° C in 200 ml of HCI water 1: 4 v / v mixture. The product is cooled, filtered and washed on the filter with deionized water until the filtrate reaches a pH of 7.0. The washed product was dried for 4 hours. at 105 ° C and then under water pump vacuum for 4 hours at 50 ° C.

Preparation of silica gel with chemically bonded vinyl group. 10 g of dried activated silica gel (product from step 1) is reacted with 60 ml of a toluene solution containing 10 ml of diethoxymethylvinylsilane under reflux.

-5 at 100 ° C for 20 hours. The product upon cooling was filtered and washed thoroughly with toluene, then methanol, and dried under a water pump for 4 hours at 50 C. ^The

Capping of silanols. 10 g of the dry product of step 2, i. of silica gel with chemically bonded vinyl silane groups is reacted under reflux with 80 mL of a solution containing 80 mL of dry toluene and 20 mL of dry trimethylchlorosilane for 24 hours at 100 ° C. The product was cooled, filtered and thoroughly washed with toluene and methanol on the filter and dried for 2 hours. at 105 ° C and dried for 4 hours at 50 ° C under water pump vacuum.

Preparation of 2-hydroxypropyl methacrylate chemically bonded phase crosslinked with divinylbenzene. 10 g of the dried product of step 3, i. chemically bonded vinyl silica gel modified with trimethylchlorosilane was treated with 120 mL of a mixture of 10 mL of 2-hydroxypropyl methacrylate and 1.2 mL of divinylbenzene in dry toluene under reflux at 100 ° C for 18 hours. After cooling, the product is filtered off, washed thoroughly on the filter with toluene and methanol and dried for 2 hours. at 105 ° C and dried for 1 hour. at 50 ° C under water pump vacuum.

Example 2

The procedure was as in Example 1 but the starting broad-pored silica gel had an effective pore size of 100 nm

Example 3

The procedure was as in Example 1 but the starting silica gel had an effective pore size of 200 nm, the diethoxymethylvinylsilane concentration increased to 30 ml, the reaction was carried out at 110 ° C for 18 hours, the trimethylchlorosilane content was increased to 25 ml and the reaction was carried out at 110 ° C for 24 hours. and the A1BN concentration was increased to 1%, the divinylbenzene concentration to 1.5 ml and the reaction was carried out at 110 ° C for three hours.

Typical examples of the preparation of the chromatographically bound phase do not limit the scope of the reaction conditions, especially the reaction in the process of the invention.

Physico-chemical properties of products

Elemental analysis of the resulting products showed that the carbon content was 2.16 to 3.69% depending on the specific surface area of the starting broad-pored silica gel. Fourier transmission infrared analysis showed the presence of -SiCH 3 and phenyl

-6-groups in the products as well as that the concentration of free -SiOH groups decreased to less than half their concentration in the starting material. Scanning electron microscopy did not show changes in the porous structure of silica gel due to its activation and other steps (2-4) in which a chromatographic phase was bound to its surface. A photomicrograph of water drops deposited on the surface of the bound phases prepared according to the examples illustrates its water wettability, which creates the prerequisites for the attractive interaction of the phase surface with the water-soluble proteins. Thermogravimetry performed in air in the temperature range of 20 to 850 ° C at a temperature rise of 10 ° C / min showed a significant change in the weight of all materials prepared according to Examples 1 to 3 in the temperature range 321 to 361 ° C. This is the temperature typical for the thermal degradation of vinyl polymers. Exclusion chromatography (gel foam chromatography) performed in tetrahydrofuran using polystyrene standards with a narrow molecular weight distribution showed that the pore diameters of silica gel were reduced by about 5-10% by binding poly (2-hydroxypropyl methacrylate) and crosslinking with divinylbenzene. The results indicate that the bound phase macromolecules lie largely with their area on the inner surface of the pores. This explains the excellent protective ability chemically bonded phase, which is the subject of the invention, before the effects of alkali attack and also shows that the chemical modification can enter its pores and proteins with a molecular weight greater than 300,000 g.mol ¹ for the material described in Example 1, 500,000 gmol ¹ (example 2) and 100,000 gmol ¹ (example 3).

Chromatographic properties of products

The performance-chromatographic important properties of the novel silica-bound 2-hydroxypropyl methacrylate, divinylbenzene crosslinked HPLC phase prepared according to Examples 1 to 3 were tested for these aspects.

(a) Stability in alkaline environment. 10 L of a 0.1 M NaOH solution (pH = 10) was pumped through a column packed with 2 g of bound phases prepared according to Examples 1-3. The column was then emptied and the carbon content was measured by elemental analysis. It was found that even under these drastic conditions there was no measurable change in the carbon content due to hydrolysis. The bound phase silicagels prepared according to Examples 1 to 3 were loaded onto a 150x4.6 mm chromatography column. The columns were washed with a 0.1 mol / L NaOH solution in water / acetonitrile. After 2 overruns each one of the alkali eluent to a column with 50 pL of a solution of bovine serum albumin at a concentration of 1.5 mg ml ^1st Chromatographic waves were detected by a UV photometer

- 7 at 280 nm. The chromatograms obtained did not change their shape or retention volume. This result confirms the high phase stability. which is the subject of the invention, in an alkaline environment.

b) Chromatographic properties in the separation of selected proteins. The new 2-hydroxypropyl methacrylate chemically bonded silica gel crosslinked phase, the preparation of which was described in Examples 1-3, was subjected to basic chromatographic tests. The mixtures were separated on a 150x4.6 mm column

1) papain, insulin, hemoglobin and lysosime in gradient: A-water, Bacetonitrile, linear gradient from 5% B to 40% B at 1 ml / min, 25 ° C

2) yeast ribonuclease, glucose oxidase, lysosin, cytochrome c, ribonuclease, bovine serum albumin and hen albumin. Mobile phase A: 0.1% trifluoroacetic acid in water; B: 0.1% trifluoroacetic acid in acetonitrile, 20 min linear gradient from 95% A to 100% B.

In both cases, the chromatograms were read by a UV detector at 280 nm. Effective separation of model proteins, mostly to the zero line, was achieved. This illustrates the excellent properties of the bound phase of the invention, its extensive utility for protein separation.

Industrial usability

Poly (2-hydroxy propyl methacrylate) silica gel-bound chromatographic phase has particular use in the separation of proteins

Claims (4)

PATENT CLAIMS A process for preparing a poly (2-hydroxypropyl acrylate) HPLC phase cross-linked with divinylbenzene bound on activated silica gel. to which vinyl groups are bound by reaction with diethoxymethylvinylsilanoin in toluene, and in which subsequent reactive free trimethylsilane is blocked by subsequent reaction with trimethylchlorosilane, characterized in that the treated silica gel is treated with a mixture of 2-hydroxypropyl methacrylate and divinylbenzene at 120 ° C for 18 to 24 hours with stirring and in the presence of a suitable solvent, wherein the 2-hydroxypropyl methacrylate polymerizes and crosslinks with divinylbenzene Process according to Claim 1, characterized in that azo-bis-isobutyronitrile is preferably used as the initiator of the radical polymerization. Process according to claim 1, characterized in that toluene is preferably used as the solvent. 4. Use of a poly (2-hydroxypropyl acrylate) chromatographic lazy crosslinked with divinylbenzene bound on activated silica gel for protein separation