Magnetic separation

Magnetic Separation Technology General

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Techniques for separation and purification are essential in biomedical research, disease diagnostics and drug development. In recent years substantial progress has been made in developing separation methods through magnetic particles. Using these magnetic particles offer unique possibilities for the simple, effective and rapid concentration of purified specific proteins, cells, and other bio-molecules without exposing them to harmful chemical and physical treatment.

Magnetic separation technology offers a distinct advantage by subjecting the analytic to very little mechanical stress (compared to the centrifuge or chromatography membrane). In general MS methods are non-laborious, inexpensive and often highly scalable. Moreover, techniques employing magnetism are more amenable to automation and miniaturization.

Magnetic beads are now widely used for the isolation and detection of bio-molecules in life sciences, microbiology, immunology, molecular biology, drug discovery, research routine laboratories and in diagnostics. 3 Generations in Magnetic Separation

1. First Generation: A magnet is activated outside the well plate while liquids are pumped out of the well plate. Coated particles stay in the well plate. In general, all first generation separator contact vessels contain magnetic particles suspensions- attracting them to the inner surface of the vessel.

Disadvantage:

The separation is inefficient since the magnets are not in direct contact with particles due to the thickness of the plate (the vessel side thickness). There is an inefficient contact between the magnet and vessel.

During the “liquid separation step” a portion of the magnetic particles is pumped out together with the liquid.

2. Second Generation:

Magnetic Rods protected by plastic sleeves or tips: These rods or pins move into the liquid suspension that contains the particles and are then pulled out from a well plate; they can then be transferred to another plate.

Disadvantage:

All 24 / 96 magnets are moving together in and out from Well Plate, thereby allowing no flexibility in processing.

All current tips that cover the magnet during the separation are very thick and do not efficiently collect the particles.

3. Third Generation CMS:

Third generation

In the third generation the magnets can move with CMS allows the user to select and move any combination of desired magnetic pins into a standard 96 and 384 well plat. This freedom is impotent when conducting continuation tests on positive detections, and for rechecking positive reactions. The working volumes are now between 1 µl and 2500µl. The costs of disposable tips decrease the costs of a separation.

The disposable tip eliminate the cross-contamination of the separation.The method is not limited to the special form of reaction vessels. The gap between the tip (with the magnet inside) can be minimized and held constant, special washing methods unique to CMS.

Another problem that was solved with magnetic bead-based separation technologies was the previous insufficient, and/ or ineffective, re-suspension of the magnetic bead pellets to enable sufficient washing and elution steps (which results in higher yields and purity of the separated product compared to other purification methods).

The Flip-Flop process The Flip-Flop (FF) is defined as a stream of magnetic particles in a static liquid suspension moving from one magnet to another:

When one magnet is close to the suspension particles the magnetic particles move towards this, creating an aggregate (button) near the magnetic pole. The magnet is now removed and a second magnet, directly opposite it, is activated; as a result, particles detach from the aggregate (button) and move towards the magnetic pole. The magnetic field creates a stream (flow) of particles moving to the second magnet- while the first aggregate (button) becomes smaller and smaller until all the magnetic beads are moved. This process can be activated as many times as necessary within a given time period. The user can regulate the system and insure that the gap between the Microtip (magnet inside) and the beads is at an absolute minimum.

During this FF process the liquid remains static and, as a result, the FF process becomes a very efficient method of washing of the particles with a maximum of the particle’s surface in contact with the liquid. Furthermore, if the particles have bound DNA, there is less risk for the DNA to become damaged.

If the liquid contains a chemical reagent that binds to the particles, then the binding reaction may be accelerated during an FF process due to a faster interaction of the reagent and particles. Therefore, if the reaction is a detection step, it may be accelerated by the FF.

Applications for magnetic bead separation technology

The major applications are:

1. DNA/RNA separation and purification:

a.PCR amplifications

b.Sequencing

c.Drug discovery and biomedicine

2. Cell separation:

a.Concentration of rare cancer cells (early cancer diagnosis)

b.Cell sorting (various white blood cells)

c.Flow Cytometry

d.Bacteria detection

e.Virus detection

f.Sub cellular organelles

g.Biological quality control in cell cultures

3. Immunodiagnostics:

a.HIV/FIV/SIV monitoring in virus research

b.Immunoprecipitation

c.Protein purification

d.Therapy of human and animal AIDS

e.Antiviral inhibitor screening

High Gradient magnetic Separation

In the recent past the problem of removing the deleterious iron particles from a process stream had a few alternatives. Magnetic separation was typically limited and moderately effective. Magnetic separators that used permanent magnets could generate fields of low intensity only. These worked well in removing ferrous tramp but not fine paramagnetic particles. Thus high intensity magnetic separators that were effective in collecting paramagnetic particles came into existence. These focus on the separation of very fine particles that are paramagnetic.

The current is passed through the coil, which creates a magnetic field, which magnetizes the expanded steel matrix ring. The matrix material being paramagnetic behaves like a magnet in the magnetic field and thereby attracts the fines. The ring is rinsed when it is in the magnetic field and all the non-magnetic particles are carried with the rinse water. Next as the ring leaves the magnetic zone the ring is flushed and a vacuum of about – 0.3 bars is applied to remove the magnetic particles attached to the matrix ring.