Magnetic Support Particle Characterisation

2.3.1

Determination of amine group

density

2.3.1.1 TNBS assay

Reactive amino groups were determined by the assay involving TNBS (2,4,6- trinitrobenzene sulphonic acid) as described by Dean et al. (1970). TNBS is thought to react by nucleophillic substitution with free NH4 groups on the support surface to

produce an immobilised trinitrophenyl derivative, which when hydrolysed produces picric acid which is released into solution (Habeeb, 1966).

The amine compound to be tested (silanized BioMag®) was suspended in 0.1 M sodium tetraborate pH 9.3 to a volume o f 1 mL. 25 pL o f 30 mM TNBS was added, mixed well, and allowed to mix on a vibrax shaker at room temperature for 30 minutes. Following magnetic separation the absorbance o f the aspirated supernatant was read at 420 nm (Beckman DU 64 spectrophotometer, Beckman Instruments, High Wycombe, U.K.). A standard curve was constructed with known concentrations o f L-alanine and a reagent blank consisted o f the TNBS solution mixed with 1 mL o f 0.1 M sodium tetraborate.

2.3.1.2 SPDP assay

The assay method used was an adaptation o f the method described by Carlsson et al. (1978). Amine terminated magnetic particles were treated with an excess o f SPDP (N- succinimdyl 3-[2-pyridyldithio] propionate) in a buffer/solvent mixture to convert all amino groups to 2-pyridyldithiopropionamide functions. Treatment with excess dithiotreitol (DTT) reduces the included disulphide bond and releases pyridine-2-thione into solution as a chromophore and its concentration was measured spectrophotometrically.

20 pL (1 mg) amine-terminated particles were washed with 20 mM sodium phosphate buffer, pH 6 . 8 and the volume was adjusted to 1 mL with the same buffer. 0.3 mL o f 20

mM SPDP solution (in 99.5% ethanol) was added dropwise and left for 30 minutes shaking at room temperature. Excess reagent was removed by washing with buffer. The volume was adjusted to 1 mL with buffer and 0.1 mL o f 50 mM DTT was added. The mixture was shaken for a few minutes to release the pyridine-2-thione. The support was magnetically separated and the absorbance at 340 nm o f the aspirated supernatant was measured against a buffer blank. Pyridine-2-thione has G^ax o f 8080 at 340 nm.

2.3.2

Determination of epoxide group density

Reactive epoxide groups were determined by a procedure adapted from the methods described by Sundberg and Porath (1974) for use with agarose supports. The reaction involves the oxirane ring and sodium thiosulphate resulting in the release o f OH” which can be followed by titration with hydrochloric acid in a pH-stat.

A sample o f epoxide activated support (approximately 1 mg) was resuspended to 1 mL with distilled water and transferred to a purpose built pH titration vessel designed for small sample volumes. The pH was adjusted to 7. 1 ml o f 2 M sodium thiosulphate pH 7 was added to the stirred epoxy-activated preparations and the pH was kept constant until the reaction was complete (typically 15-40 minutes) by the addition o f 0.01 M HCl using a pH-stat autoburette (Radiometer, Copenhagen, Denmark). The amount o f oxirane present in solution was calculated from the amount o f HCl needed in order to maintain neutrality.

2.3.3

Determination of ligand group density

Iminodiacetic acid (IDA) densities on supports were determined by inference from measurements o f immobilised metal ion (M^^) concentrations. Hochuli et al. (1987) and others have shown that there is a 1 : 1 stoichiometry for the binding ofM^^ to immobilised

IDA when is in excess. Metal ion concentration was measured by atomic absorption spectrophotometry using the appropriate lamps.

The sample o f support was washed twice with 1 mL 0.1 M EDTA which strips the support o f metal ions by chelation. The support was magnetically separated and the supernatant was diluted as appropriate and aspirated into the flame. Copper was determined at 320.4 nm and zinc at 213.9 nm using single element lamps, operated at 10 mA and 16 mA currents respectively, with an air-acetylene flame in a atomic absorption spectrophotometer AA3100 (Perkin Elmer, Seer Green, U.K.). Metal ion concentration in parts per million (ppm) was determined from an absorption curve o f samples o f known concentration (1 ppm = 1 pg/mL).

2.3.4

Quantitative support measurement

An estimated quantity o f magnetic support was completely hydrolysed by dissolution in 1 mL o f concentrated hydrochloric acid. The ferrous/ferric ion content was measured by

absorb at 248.3 nm at a current setting o f 16 mA. In addition dry weight measurements o f supports were determined on an analytical balance after 24 hour desiccation in a 100® C oven.

An equal volume o f suspended support samples were quantitatively determined (in quadruplicate) by atomic absorption spectrophotometry (AAS) and by dry weight measurement. Support quantity determined by dry weight was found to be 1.15 times lower than by atomic absorption analysis. Support quantities were routinely determined by AAS but quoted as dry weight equivalents by dividing by a factor of 1.15.

2.3.5

Measurement of pH stability

2.3.5.1 Potassium thiocyanate test

This test determined the pH stability o f the supports by measuring the effect o f pH on ferric ion (Fe^^) liberation from amine-terminated iron oxide supports. It was adapted from the method used by Munro (1976). The test was employed over a pH range o f 0 (1 M HCl) to 14 (1 M NaOH). To 50 |iL (2.5 mg) o f amine-terminated BioMag® suspension was added 25 ^iL o f pH test solution (two sets). One set was stationary and the other was shaken on a vibrax shaker at room temperature for 24 hours. The particles were separated magnetically, the supernatant was aspirated and 50 were mixed with 50 |iL o f 1 M potassium thiocyanate. The appearance of a colour (pink to brick red in intensity) indicated Fe^^ ion leakage, which was measured spectrophotometrically at 480 nm (1 cm light path). Molar concentrations of liberated ferric ion were calculated against a standard curve o f ferric chloride solutions o f known concentration. The test is sensitive to ferric ion concentrations as low as 10"^ M.

2.3.5.2 Effect of successive coating and coupling chemistries

The effect o f coating and coupling reactions on the pH stability or corrosion resistance o f supports at the various stages in the preparation o f magnetic chelator supports was tested, i.e., from the core particle -> amine terminated particle -> polyglutaraldehyde coated particle -> epoxy-activated particle -> IDA coupled particle.

Known quantities o f magnetic support particles (~ 2.5 mg/test) were subjected to a range o f HCl concentrations, 10 M, 1 M, 0.1 M, 0.01 M and 0.001 M, corresponding to pH values o f 0, 1, 2, and 3, for 9 time intervals ranging between 1-360 minutes. The magnetic particle preparations were mixed vigerously with the acid solution and shaken for a given time period. Following magnetic separation the supernatant was assayed for

ferric ion content by atomic absorption spectroscopy at 248.3 nm (section 2.3.3). Ferric ion concentration in solution was expressed as a percentage o f the total used per test.