Globular packing agent for liquid chromatography

The invention relates to a spherical filler material, which is suitable in a special way for the separation of materials, such as monosaccharides and oligosaccharides, by liquid chromatography.

be fillers for liquid chromatography usually made of silica gel, chemically modified Silica gel, the synthetic gels of high molecular weight, natural gels of high molecular weight, Kohlenstoffgelen, etc. produced. There are also porous Filler materials known to those of porous silica chemically modified, porous silica, porous Polymers, etc. produced.

Compounds based on calcium phosphate, in particular synthetic hydroxyapatite (Ca₁₀ [PO₄] ₆ [OH] ₂), have the same Composition, such as the substantially inorganic Components of teeth and bones; using the superior biocompatibility were artificial Tooth roots and bone prosthesis materials synthetic hydroxyapatite developed. For many years Experiments were conducted to filling materials for the Liquid chromatography of this hydroxyapatite, the called close relationship with the living body has, produce; the results have been for the separation of Substances such as proteins and enzymes employed. In the recent years have been suggested filler materials either through their chrakterisiert Manufacturing process or by their shape; these will z. B. Unexamined Japanese Laid-Open Patent Application Nos. 1 43 762/1985.

The conventional base compounds of Calcium phosphate are used mainly for Separation of materials such as proteins and enzymes; around to achieve a higher resolution of the separation process, are the most connections in the porous state with a large surface area used.

Porous fillers are also in the Gel permeation chromatography is not used (see, eg. B. unexamined published Japanese patent application no. 1 55 290/1975). In the gel permeation chromatography Materials similar chemical composition in Depending separated on their molecular weight, wherein the components with the higher molecular weight first are eluted.

The inventors have now found that, when a porous filling material, consisting of a compound on the Based on calcium phosphate consists, for the separation of Saccharides is used, the separation performance of the Filler by its porosity adversely is affected, whereby a separation of the components in decreasing order of their molecular weight takes place, as in the gel permeation chromatography the case is.

Object of the present invention is, therefore, a to provide filler material available, which consists of a Compound produced on the basis of calcium phosphate , and which is suitable for the separation of saccharides suitable.

The inventors have made further investigations for separation of components that is of proteins or enzymes differ, with filling materials from compounds based on calcium phosphate, such as. for example, hydroxyapatite, performed. As a result, it was found that certain tight connections based on calcium phosphate for the separation of saccharides are suitable, wherein a Separation patterns is produced in which the components lower molecular weight are eluted first.

The spherical filler for liquid chromatography according to the present invention includes spherical Particles selected from at least one material from the group consisting of Ca₁₀ (PO₄) ₆ (OH) ₂,Ca₃ (PO₄) ₂, Ca₂P₂O₇, Ca (PO₃) ₂,Ca₁₀ (PO₄) ₆F₂, and Ca₁₀ (PO₄) ₆Cl₂ exist,said filler material or a packing material dense structure with a porosity of not more than 5% having.

<b>Fig.</b> 1 illustrates an electron micrograph of (× 15,000), which the structure of a particle in the filler material, as in the example was prepared according to the invention, reproduces, and

<b>Fig.</b> 2 is a chromatogram with the results of the Separation of glucose from sucrose by Liquid chromatography shows, said in the produced example of the invention Filler was used.

Since the filling material according to the invention of dense spherical or spherical particles with a composed porosity of not more than 5%, it has less capacity for separating components according to the elution profile of Gel permeation chromatography, and is therefore to Application of the separation of saccharides in increasing suitable order of their molecular weight.

The filler according to the invention consists of at least one material selected from the group consisting of Ca₁₀ (PO₄) ₆ (OH) ₂, Ca₃ (PO₄) ₂,Ca₂P₂O₇, Ca (PO₃) ₂, Ca₁₀ (PO₄) and ₆F₂Ca₁₀ (PO₄) ₆Cl₂. These compounds based on of calcium phosphate can be prepared by various known Methods are synthesized, such. As after Wet process in which a water-soluble Phosphate salt with a water soluble calcium salt in aqueous solution is reacted, and after Dry method in which a phosphoric acid compound with a calcium compound at elevated temperatures is implemented.

The compounds based on calcium phosphate, as they are listed above, by various Methods, such as, for example, by spray drying and by a Rolling-drum combination to spheric Particles granulated. In the granulation process by spray drying is a compound based on Calcium phosphate dispersed in water; the resulting Slurry is at a temperature of about 100 to 250 ° C to form spherical granules spray dried. In the granulation process by Rolling and tumbling are particles of a Compound based on calcium phosphate from above a central part of an inclined rotating disk added and the rotating action of the disc agglomerated.

The granules are then dried at a temperature of 900 to fired to 1400 ° C to form a filler material, wherein The latter spherical particles having a porosity of not more than 5% comprising. When the firing temperature is less than 900 ° C, results in an often Filler material having a porosity of more than 5%; the Porosity of such a filler leads to a Separation of the components in decreasing order their molecular weight, as in the Gel permeation chromatography is the case; this Circumstance is for the separation of saccharides not suitable. When the firing temperature exceeds 1400 ° C, so experiences the connection on the basis of Calcium phosphate, a decomposition reaction and it gives a filler having a low resolution.

The particles of the filling material according to the invention must not completely and perfectly spherical in shape be, provided that it is generally a have spherical or spherical shape, and egg-shaped structures or those having the form of a Rugby ball also by the term "spherical or spherical, "as used herein, with are included.

The average size (average Diameter) of the particles according to the invention is not limited to a specific value; However, it is advantageous that this is in the range of about 1 to about 100 microns. If the average size less is than 1 .mu.m, so has the filler material a increasing resistance to the passage of the mobile Phase through a column. On the other hand the average size exceeds 100 microns, so has the filling material give less resolution.

The filling material according to the present invention is particularly effective in the separation of Monosaccharides such as glucose, or oligosaccharides such as Sucrose, lactose and raffinose, whereby a separation pattern arises, in which the components having the lower Molecular weight elute first.

example

A solution of a phosphate salt and a solution of a calcium salt were by a known method reacted to form a hydroxyapatite slurry. The slurry was a by spray drying with Mobile Minor type spray granulated. The obtained granules having a nearly spherical exhibited form 4 were fired at 1100 ° C one hour to Filler of the type as according to the invention is desired to obtain. The particles of this Filler had an average size of 5 microns and an average porosity of from 1% to. An electron micrograph (× 15,000) of a single particle of this filler is in <b>Fig.</b> 1 reproduced.

The filler material was placed in a column (diameter 7.5 mm, L 100 mm) stainless steel for High performance liquid chromatography packed. there has been a Separation of glucose and sucrose on this column with a liquid solution of 80% acetonitrile as Eluent with a flow rate of 1.5 ml / min. was applied is performed. The resulting Chromatogram in <b>Fig.</b> 2 again, keeping the vertical axis represents the absorbance at 195 nm, the horizontal Axis the number of minutes available. In<b>Fig.</b> 2 means G represents the glucose peak, S the sucrose peak. As from<b>Fig.</b> 2 apparent, the filling material is in accordance with the Invention for separation of saccharides with high Resolution.

As described above, the filling material is out according to the invention is generally virtually spherical particles of a compound on the basis of Calcium phosphate together, whereby the same has a density Structure with a porosity of not more than 5% exhibit. This filler has decreased Tendency to a separation of the components in decreasing order of their molecular weight bring as in the case of Gel permeation chromatography is the case. Therefore, the said material for separation of saccharides according to the Order of their molecular weight suitable, and the components of lower molecular weight first elute.