Introduction

The Starting material selected for the preparation o f magnetic chelators was an amine- terminated non-porous superparamagnetic iron oxide (BioMag®, PerSeptive Biosystems, Framingham, Mass, U.S.A.) with a particle size o f 0.5-1.5 pm diameter, density o f ~ 2.5 g/cm^ and available surface area o f between 100 and 150 mVg (Josephson, 1987). These iron oxide core particles comprise clusters o f superparamagnetic crystals o f dimensions roughly 300 Â or less. Superparamagnetism is the magnetic behaviour which is characterised by responsiveness to a magnetic field without resultant permanent magnetization. The property is lost when the crystal size is increased much beyond 300 Â (e.g. > 500 Â) and the material then becomes ferromagnetic. In the absence o f an externally applied magnetic field, because they are small and only weakly magnetic, they can be dispersed very easily as slow settling suspensions possessing extremely high surface areas. Magnetic separation times o f less than about ten minutes can be achieved by contacting a vessel containing a dispersion of the particles with a pole face of a permanent magnet no larger in volume than the volume o f the vessel, where the magnetic separation time is defined to be the time for the turbidity o f the dispersion to fall by 95%

(Josephson, 1987). Once a field is applied they become magnetic, agglomerating readily due to inter-particle forces and are separated easily.

The BioMag® iron oxide core particles were surrounded by a stable silane coat. Silane coupling agents form metal siloxane bonds (M -O -Si) where M is the metal and Si is the coupling agent, silica (Plueddemanns, 1991), and this bond retains composite properties through harsh environments (Wang and Jones, 1993). Silanisation procedures carried out in the past employed air and / or oven drying in the dehydration step. When applied to silanisation of magnetic carrier particles, such dehydration methods allowed the silanised surfaces o f the particles to contact each other, which tended to result in interparticle bonding, including, e.g., cross-linking between particles by siloxane formation, van der Waals interactions or physical adhesion between adjacent particles. This interparticle bonding yielded covalently or physically bonded aggregates o f silanised carrier particles o f considerably larger diameter ( > 1 0 pm) than individual carrier particles o f diameter range 1.5-10 pm (Hersh and Yaverbaum, 1976).

In the silanisation procedure followed according to the BioMag® patent (Josephson, 1987), silane (3-aminopropyl trimethoxysilane) was deposited on the metal oxide core fi*om acidic organic solution. The silanisation reaction occured in two steps. First, a trimethoxysilane was placed in an organic solvent (methanol), water and an acid (glacial acetic acid). It condensed to form silane polymers;

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R — Si(OCH]) — - HO — Si — Q — Si — O —

I

I

Secondly, these polymers associated with the metal oxide, perhaps by forming a covalent bond with surface OH groups through dehydration:

R R OH OH I. I I I HO — Si — O — Si — O — --- I I OH OH — H2O HO - Si - O — Si - O - I I

0

o

Adsorption o f silane polymers to the metal oxide is also possible. However, good silane coats are only observed when there is evidence o f chemical rather than purely secondary bonding between the silane primer and the metal oxide surface (Gettings and Kinlock, 1977). Good bonding across an interface requires maximal initial formation o f M -O -Si, a minimum equilibrium concentration of water at the interface and polymer structures that hold silanols at the interface. The reaction o f metal-siloxane bonds with water is of major importance for the stability o f the metal-silane interface; the reaction below must not procees too far to the right.

M — O — Si + H2O — ^ M — O — H + H — O — Si

Adsorptive or covalent binding o f the silane polymer to the metal oxide was accomplished by heating the silane polymer and metal oxide in the presence o f a wetting agent miscible in both the organic solvent and water. Glycerol, with a boiling temperature o f about 290°C, is a suitable wetting agent. Heating to about 160°-170°C in the presence o f glycerol served two purposes. It insured the evaporation o f water, the organic solvent (methanol) and any excess silane monomer. Moreover, the presence o f glycerol prevented the aggregation or clumping and potential cross-linking o f particles, that is an inherent problem o f other silanisation techniques when dehydration is brought about by heating to dryness.

The presence o f silane on iron oxide particles was indicated by the observation that after treatment with 6N hydrochloric acid, the iron oxide was dissolved and a white, amorphous residue was left which is not present if the unsilanised iron oxide is similarly digested. These results suggest that the acid insoluble residue was silane, though no direct evidence in the form o f a test on the residue was presented (Josephson, 1987).

The starting point for work with non-porous magnetic supports was begun in the 1970s, in this department, where Robinson et al. (1973) were the first to develop a non-porous magnetic support for immobilised enzymes. The majority o f published work on magnetic supports since then concerns their use with immobilised enzymes and in cell sorting applications. Comparatively little has been published on the application o f magnetic affinity adsorbents in protein purifications and most o f these have involved porous support structures (Dunnill and Lilly, 1974; Ennis and Wisdom, 1991; Mosbach and Andersson, 1977; Whitesides et al., 1983).

Two decades on, in this department, silane-coated BioMag® iron oxide particles were stabilised by grafting on a layer o f polyglutaraldehyde as described by Hailing and Dunnill (1979a). The subsequent chemical reactions that were adapted and developed to produce metal chelating ligands on magnetic supports, were based on the work carried out by Porath et al. (1975) and others who chemically derivatised agarose to invent the technique known as immobilised metal affinity chromatography (section 1.3)