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At Sepmag, when we review magnetic beads protocols, we are often surprised by the lack of detail given in the description of the magnetic separation process. Contrastingly, the same documents often provide detailed descriptions of how to prepare the buffers, add the beads, and incubate the suspensions.

Here are some examples of magnetic separation descriptions that we often encounter:

  • Magnetic Separation: 20 minutes in a magnetic separator. This is a real protocol example, where not even the specific magnet rack, or use thereof, is described.
  • Magnetic Separation: 20 minutes in the magnetic separator (including the specified device number or name). A more detailed protocol such as this one (also a real example) specifies the specific magnetic rack, as well as the time to perform this step for. But is this enough?

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There are five key factors, as discussed in previous chapters, that a good magnetic separation protocol should cover. Let’s review the typical approaches users take when describing these factors in their protocols.

Quality control

When describing how to ensure quality control of their protocol, users should often consider how to check if the magnetic separation has been completed. A common way to do this is by eye: when the suspension is ‘clear’ or ‘translucent’, the process is considered to be completed. Alternatively, the total separation time can be defined by picking samples at different times and measuring the transmittance and/or absorbance in a spectrophotometer. By comparing the values against a calibration curve, we can determine when the concentration of magnetic beads is below the desired threshold. It’s worth bearing in mind that this process should be repeated for each separate magnetic suspension experiment. The separation time determined in this way is also linked to the specific volume of sample and magnetic rack used.

 Scale Up

Scaling up is one of the main bottlenecks in Magnetic Separation Processes. Many Magnetic Bead Separation protocols lack sufficient detail on the magnetic rack, and also on how to specify the magnetic strength used at different volumes. Often, if it is only described to the effect of: “users might consider using a larger or stronger magnet at larger volumes”.

However, traditional magnetic racks don’t scale well and using a larger or stronger magnet won’t correctly scale up the protocol. This is because using a larger magnet will increase the separation time exponentially, exacerbate aggregation problems, and increase losses of magnetic beads. This is a common problem for IVD manufacturers struggling to scale up their magnetic beads reagent production to cope with the increasing demand from chemiluminescent immunoassay analyzers.

Cost

The direct cost of the separation process is relatively simple to obtain, and can be therefore easily incorporated into the protocol description. Typical magnetic separation racks use a permanent magnet, which makes them relatively low cost for each year of their operational life.  Magnetic separation, additionally, has fewer steps and requires less consumables than traditional methods. Furthermore, magnetic separation systems require little to no electrical energy to operate efficiently, lack parts that are prone to wear overtime, and the magnetic force strength is stable for decades of use.

However, their indirect costs can be significant, and many protocols fail to take this into account. Large magnets may need large clear benchtop areas, requiring valuable space. Poorly specified magnetic racks also generate bead clumps, adding cost through the need for re-suspensions, on top of potential losses of beads and ligands.

Safety

The basic safety measures for working with magnets are well known and easily achieved. No pacemakers or computers should be inside the region with a magnetic field higher than 0.5 mT (5 Gauss), and no ferromagnetic objects should be inside the region with a magnetic field over 3 mT (30 Gauss).

However, these measures increase the ‘magnetic floor-print’ size. Space is a critical factor in clean rooms or laminar flow hoods, where it can be difficult to achieve the required safe distance.

Standardization

Most of the difficulties in developing protocols using magnetic beads we have described so far are caused by a lack of understanding of how to correctly standardize the magnetic separation process.

It follows that if we can find a way to standardize the strength of a magnetic separation experiment, then we would have a simple method for a well-documented, reproducible, efficient and easy way to use a magnetic separation protocol. In the next chapter, we will cover how to achieve just that, and how to apply the concepts to any batch volume.

In summary, we have explored the limitations in magnetic beads separation protocols, highlighting the common lack of specificity in protocol descriptions. Instances like “Magnetic separation: 20 minutes in a magnetic separator” reveal a deficiency in detailing the magnetic rack used. Despite some improvement with device names, comprehensive coverage remains elusive. The chapter revisits five crucial factors—quality control, scale up, cost, safety, and standardization—showcasing prevalent shortcomings. Protocols often lack precision in quality control methods, overlook scaling challenges, and underestimate indirect costs. Basic safety measures are acknowledged, but spatial constraints in controlled environments are downplayed. The present chapter sets the stage for the next, promising insights into achieving standardized, well-documented, and efficient magnetic separation protocols.

Classical magnetic separator has not well defined magnetic separation conditions. Force is very strong near the magnets, but decreases very quickly with the distance.

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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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