Inorganic Matrices

A large selection of inorganic matrices are commercially available for incorporating homogenous catalysts, and some have been applied to chromatography separations. These matrices include silica, alumina, glass, zeolite, clay, celite, zerconia, titania, magnesium oxide, and carbon. Only a limited number of these matrices with suitable pore structure, particle size, and mechanical stability can be used for chromatography separation of biomolecules. Silica, alumina, and glass are available with a large variety of surface areas, pore structure, and particle sizes, and some have been used extensively in both analytical and preparative chromatography of biological molecules.

Controlled Pore Glass

Controlled pore glass (CPG) has excellent mechanical stability and can be manufactured with a wide range of pore diameters, a high surface area, and a range of particle sizes. The material is almost pure silica and has a very narrow pore size distribution. Surface-modified CPG with the trade name PROSEP manufactured by Bioprocessing Limited has been extensively used in large-scale purification of biomolecules. (See later sections on CPG for a more detailed discussion of this material.)

Silica

Silica is extensively used in the analytical field (HPLC) and to a much lesser extent for preparative chromatogra-phy of biomolecules. Silica matrices for chromatography applications are generally produced by a wet process, that is, by acidification of sodium silicate in water (4,5), and are available in particle sizes of up to 40 im and pore sizes of up to 30 nm. In most cases, these materials are unsuitable for large-scale purification processes. The small particle size causes excessive backpressure in preparative chro-matography columns, and the pore size of 30 nm is not large enough for the separation of most proteins, especially in the field of affinity chromatography, where both the li-gand and the ligate could be large proteins.

In our laboratories, we have used Matrex® silica (Ami-con) for the large-scale purification of biomolecules. This material is available in a range of pore diameters and particle sizes (Table 1) and is manufactured by first forming a hydrosol from organosilicic acid. Hydrosol is then allowed to set to a jelly-like mass called hydrogel. After setting, the hydrogel is thoroughly washed to remove salts resulting from the reaction. Temperature and pH of the washing medium and the washing time influence the specific surface area, pore volume, and purity of the hydrogel. The hydro-gel is subsequently dried to give a structurally stable xero-gel. This material is 99.5% silica, with impurities including Na + , Ca2 + , Fe3 + , SO2", and Cl" ranging from 600 to 60 ppm.

Alumina

Alumina-based materials have a much wider pH stability than silica matrices and therefore are of great interest in chemical separations and industrial-scale chromatogra-phy. Commercially available alumina matrices are all aluminium oxides prepared by thermal dehydration of aluminum hydroxide (6,7). The origin of the starting material together with other factors, such as the heating rate, drying time, and temperature, influence the properties of the final product.

Alumina matrices can be obtained in a wide range of particles and pore sizes (Table 2). The physical stability of alumina particles in chromatography columns, even under moderate flow rates and pressure, has been a major problem in our laboratories. Generally, the particles break up and cause a substantial increase in the backpressure generated by the column. Furthermore, any risk of contamination of the purified product with aluminum may not be acceptable to the pharmaceutical and biotechnology industries, particularly when the reports relating to Alzheimer's disease to aluminum are considered (8).

Zirconia and Titania

Recently, there has been an increasing interest in zirconia-and titania-based matrices in chromatography applications, mainly because of their chemical stability over a relatively wide pH range and their physical characteristics. Zirconia or titania supports with properties suitable for preparative chromatography or HPLC are not commercially available, although zirconia and titania powders can be obtained commercially from a number of sources and have been used by researchers to prepare matrices with a small particle size (1-10 im) and pore sizes of up to 100 nm, suitable for HPLC applications (9). More recently, larger porous zirconia particles (30-150 im) have been prepared and used in packed and fluidized beds for protein purification (10-13).

Zirconia and titania as base stable matrices not only enable separation of biomolecules at high pH values, but they can also be sanitized with alkaline media, which is the standard sanitization procedure in downstream processing of biological molecules. Various adsorbents such as affinity, ion-exchange, and reversed phase can be prepared from these matrices for protein purification by either polymer coating or reaction with organosilanes (14-17).

Table 1. Properties of Matrex® Silica Table 2. Properties of Alumina Matrix (Selecto Scientific)

Nominal pore

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