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Magnetic separation has been an emerging technology in the recent decades for biomedical science and the medical industry. In magnetic separation, the magnetic property and behavior of micro- or nano-sized particles, known as magnetism, is employed for the separation of macromolecules (e.g. nucleic acids, proteins, peptides) from biological samples or chemical suspensions.

Download our Free Guide on Biomagnetic Separation Scale-up HERE.

The casual term “magnetic materials” generally refers to ferro- or ferrimagnetic substances that show a large response to applied magnetic fields, allowing magnetic interaction at various distances. In their standard form, all these materials have a “memory” and remain magnetized when the applied magnetic field is removed. If the size of these magnetic particles is reduced, the capacity of the memory increases. The magnetic memory of these materials has historically been exploited to store data for items like hard drives, floppy disks, and magnetic tapes. However, if we decrease the magnetic particle size well below a certain threshold the magnetic memory is completely lost. This state, when magnetic particles exhibit a high response to applied magnetic field but zero magnetisation when the field is removed, is called superparamagnetism.

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Magnetic beads and particles have been a breakthrough technique for the separation and purification of macromolecules in the realm of biomedical sciences. Even properties of the magnetic beads have improved. For example, some magnetic beads have highly specialized surface affinity ligands to more readily isolate target molecules. Magnetic separation technologies have evolved in recent decades to satisfy the need for quicker and more efficient processes to replace conventional non-magnetic filtration, centrifugation, and separation techniques.

Magnetic beads surface-coated with specific affinity ligands, bind their biomolecules of interest, for example antigens, antibodies, catalyzers, proteins or nucleic acids. After incubation, the containment vessel holding the sample solution is placed inside a magnetic separation rack. The magnetic bead-biomolecule complexes are attracted to and retained by an externally applied magnetic force, emanating from the magnetic rack. After the separation phase, the supernatant (which is not affected by the magnetic field) can be discarded and the biomolecules (attached to the magnetic beads) can easily be isolated from the original sample solution and eluted.

Magnetic separation is the only feasible method, among all well established separation techniques, for the recovery of small particles with the diameter 0.05–1 μm in the presence of biological debris and other material of similar size. Furthermore, the efficiency of magnetic separation is especially well suited for large-scale biomolecular purification endeavors. Commercial magnetic separation racks function effectively at many scales, in low- to high-throughput procedures, to save time and costs. Using magnetic racks for separation offers a simple procedure, does not require centrifugation, minimizes cross-contamination, and has the ability to operate with a variety of magnetic beads and particles in the manual, semi-manual, and fully automated mode.

The Interaction of Magnetic Beads and The Magnetic Separation Rack

Before starting with a separation process, the first step is to select the most suitable separation strategy. This means thinking about the separation system required, process as a whole, and taking into account all relevant parameters including the target biomolecule, the beads’ adsorbents, and separator design. The magnetic separation strategy chosen depends on the target molecule and includes two factors: the magnetic separation process, and the interaction between the magnetic material and the biomolecules.

Copious research and studies exist in the literature regarding particular characteristics of various magnetic beads and particles (e.g. diameter, density, magnetic pigment content, surface activation) and how these affect the efficiency of certain magnetic separation processes. This overwhelming focus on magnetic beads and particles, however, partially ignores another key element of importance; the magnetic separation rack. Not to be understated, the functional characteristics of the magnetic rack have great potential in impacting the outcome of an experiment. A faulty or inadequate magnetic separation rack may lead to costly and time consuming issues, such as high molecular or bead losses, slow separation times, aggregation or clump formation, and inconsistent or unreliable results.

Hence, it is vital to consider that the magnetic bead separation process is as important as magnetic beads and particles. Magnetic carriers, as in the beads and particles, must interact with a magnetic field in order to move. The magnetic field must generate a magnetic force strong enough to overcome the opposing drag force generated by the viscosity of the buffer. If the magnetic separation rack does not generate the appropriate magnetic force, which is different from the magnetic field intensity, the entire process will be inefficient, regardless of the characteristics of the magnetic beads. Despite the importance of this factor, detailed technical literature describing how the magnetic field generated by the magnetic rack interacts with beads is very thin on the ground.

Magnetic Separation Racks and Systems

A simple magnetic separation rack has a straightforward design and utilizes a permanent magnet. Calculating the value of a magnetic field at each point in space and then determining the gradient is not too complex. The magnetic field gradient strength decreases as magnetic beads move away from the center of the surface of the magnet. In the best case scenario (assuming the magnetic beads are always saturated) the magnetic force changes very quickly according to the distance from the surface. When different magnet sizes are compared, it may seem surprising that small magnets generate bigger forces near the surface than larger ones. This is because larger magnets generate a more constant magnetic field and, as we have just learned, a constant magnetic field does not generate force.

Some magnetic separation racks and devices try to overcome this by using two permanent magnets with opposite polarity. These separation devices generate a field that is never constant near the center of the assembly, and instead have greater forces near the surface of the permanent magnet where the magnetic beads travel to at the end of the process.

However, the resulting magnetic force for these separation devices is extremely uneven, and the value of the magnetic force changes quickly with the distance between the magnet and the magnetic beads. This means that the force exerted on the beads closer to the magnet is very strong, while the force on the beads farther away from the surfaces of the magnet is very weak.

Magnetic racks and devices with this force profile have two undesired practical drawbacks on the separation process:

  1. Very long separation times or high sample losses: Because the magnetic force exerted on the most distant beads is very weak, it takes a longer time to recover these beads and their attached biomolecules. In the case where  vessels are larger than a few milliliters, the separation time can be up to several hours. Though the process can be halted early, this will mean sacrificing a significant loss of sample.
  2. High risk of irreversible aggregation: Because the nearest beads are subjected to a greater magnetic force, they move very quickly. Once they reach the retention area directly outside of the magnet within the magnetic rack, they experience an even greater force and build up against their neighboring beads. Given that the separation process for the beads farthest from the magnet suffer an extensive separation time, beads that quickly reach their final position are exposed to extremely high forces for a very long time and may form clumps.

Magnetic separation rack

Solving Aggregation and Distant Bead Loss

To make the process efficient, the magnetic separation rack must be modified to solve both problems simultaneously. By increasing the force using, for example, larger assemblies of magnet pieces, loss of the farthest beads is reduced but there is far more irreversible bead aggregation near the surface of the magnet. In contrast, reducing the magnetic force in the retention area palliates the irreversible aggregation problem but significantly increases the potential loss of magnetic beads and their attached biomolecules.

To solve both problems simultaneously, more magnetic force must be exerted on the farthest beads while a lesser magnitude of force is exerted in the retention area. It is difficult to find a solution with a magnetic force profile that changes with distance, characteristic of classical magnetic separation racks. Because of this, an ideal separation system is one that has a magnetic force that does not change with distance. When the force is homogenous, an essential attribute of Sepmag’s Magnetic Bead Separation Systems, it is possible to have a higher magnetic force farther from the retention area and a lower force near the magnet at the same time.

In these magnetic bead separation systems the most distant beads within the vessel travel faster towards the magnet, ensuring that the entire separation process occurs more rapidly. The beads reach the retention area at a constant pace because all the beads move at the same speed and are subject to a gentle force, just strong enough to retain them when the supernatant is extracted. Techniques using such magnetic fields limit loss of captured beads and biomolecules and minimize clumping issues, avoiding the need for sonication and greatly simplifying the scaling up process.

Homogeneous and Inhomogeneous Magnetic Fields

The basic and oft-forgotten concept of homogenous magnetic fields is that they generate magnetic torque but not magnetic force. When beads are successfully separated with a simple neodymium-iron-boron magnet, this is not because of the strong magnetic field generated; it is due only to the significant change in the magnetic field and distance. To generate a magnetic force, an inhomogeneous magnetic field is required.

Magnetic separation rack

The second important concept is that the strength of the magnetic force exerted over a bead depends on both the magnetic moment of the bead and on the magnetic field profile of the magnetic separation rack. The magnetic moment of the bead can be visualized by a magnetization curve. For superparamagnetic materials, when the magnetic field is very low, the magnetic moment is proportional to the intensity of the applied magnetic field (termed having a “constant susceptibility”). When the field applied is high enough, the magnetic properties become “saturated” and the bead magnetic moment is constant. The exact “saturation field” value depends on the specific material used for the magnetic pigment, a core component of the beads. For example, for typical nanosized iron oxide commercial beads, the applied magnetic field (B) should be >0.1 Tesla (80 kA/m).

Magnetic Separation Rack vs Constant Magnetic Force Separation Systems

The difference between a magnetic separation rack and a constant magnetic force separation system is evident. Using the same number of permanent magnets, the separation time is much shorter even though the constant magnetic force separation system is far gentler on the beads throughout the process to avoid clumps.

The exact value of the force depends not only on the magnetic gradient provided by the magnetic separation rack but also on the magnetic moment of the beads and buffer viscosity. Therefore, there is no universal magnetic field gradient value that can be recommended . Fortunately, companies that develop and manufacture advanced Magnetic Bead Separation systems have amassed a great deal of experience in the field and can help you determine the best process parameters for your desired product.

Magnetic separation rack

Choice of the right magnetic gradient is of premier importance. Too low a value makes it impossible to separate small beads or beads in high viscosity suspensions (e.g., whole blood). In contrast, too high a magnetic field gradient is not effective for large beads in water-based suspensions because the resultant excessive magnetic force can generate clumps. With most life science processes, there is no single suitable system for all Magnetic Bead Separation processes.

Using devices with different magnetic forces at each point of the working volume makes it difficult to validate a separation process. Given that magnetic separation conditions for each bead are different, we cannot assume that all separation systems, parameters, and components for each experiment should be identical. However, working with a constant magnetic force makes it possible to test the process in well-known conditions for the entire suspension, allows for testing different magnetic forces as necessary, and will help to determine the right value for the magnetic bead separation process.

SEPMAG® Magnetic Bead Separation Racks

A variety of magnetic separators are available on the market, ranging from very simple, one-tube-only concentrators to complicated fully automated devices. Molecules with a diameter larger than 1 micrometer can be separated easily using simple magnetic separators, though separation of smaller particles, like magnetic colloids that have a particle size from 10 to hundreds of nanometres, may require the use of high-gradient magnetic separators. These magnetic separation racks are designed to accommodate test tubes at a range of sizes, for a range of samples. Test tube magnetic separators allow to separate magnetic particles from volumes.

Typical applicationsSmall volumes

(0.5-50 ml)

Medium volumes
(250ml- 500 ml)
Large volumes

(1 -50L)

Standard magnetic forceImmuno and Molecular diagnostic Magnetic beads production,

Protein purification

Sepmag A with MA adaptorsSepmag ASepmag Q & N
Large magnetic forceCell separation

Small magnetic beads

Viscous media

High magnetic beads concentration

Sepmag LABSepmag QF & NFSepmag QS & NS

 

Processes are monitored and recorded using the MONITOR hardware and software and QUALITANCE system, enabling operators to monitor the entire magnetic separation process. The Sepmag LAB model is and R&D oriented setup that will allow you to determine the effect of changes in the suspension (type of beads, bead concentration, buffer composition, temperature) over the magnetic separation behavior. In short, the Sepmag LAB magnetic separation rack allows you to develop processes that can be later directly scaled-up (or down) to other volumes using the same magnetic force.

The QUALITANCE systems help guarantee Quality Assurance throughout production scale applications. The QUALITANCE system works by comparing the separation dynamics of the current batch with referenced separation curves. This capability allows early detection of problems regarding bead size, distribution, concentration or buffer conditions, so that immediate corrective actions can be taken, rather than having to wait hours, days or weeks later.

Don’t forget to check these posts from our blog in order to get a deeper insight into the scaling-up capabilities of Magnetic Bead Separation processes:

Check www.sepmag.eu/ebooks for access to FREE eBooks on the subject, or contact us. We will be glad to help you to achieve an efficient Magnetic Bead Separation process!

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Lluis M. Martínez | SEPMAG Chief Scientific Officer

Founder of SEPMAG, Lluis holds a PhD in Magnetic Materials by the UAB. He has conducted research at German and Spanish academic institutions. Having worked in companies in Ireland, USA and Spain, he has more than 20 years of experience applying magnetic materials and sensors to industrial products and processes. He has filed several international patents on the field and co-authored more than 20 scientific papers, most of them on the subject of magnetic particle movement.

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