First, don't confuse MW with size. BSA monomer is ~ 7.1 nm.

Whatman 1 'pore'* size is ~22 um (u = mu), so ~10,000 bigger. The smallest 'pore'* size I know of in filter paper is ~2um (Whatman 602/1575), still 1,000 fold to big.

(* in paper filters there is no "pore").

What you need is a dialysis membrane or something similar. If you want fast separation you can try Vivafree/Vivaspin or Centricon.

See:

https://www.vivaproducts.com/vivafree-centrifugal-filters/vivafree-2/

https://www.sigmaaldrich.com/IL/en/product/mm/ufc8030

Here is a table that summarizes the advantages and disadvantages of using different methods to separate BSA from secondary metabolites, including centrifuge:

Method Pros Cons

Whatman filter paper grade 0 Inexpensive and easy to use Not efficient Dialysis membrane Gentle method for separation Slow process Ultrafiltration membrane Fast method for separation More forceful Centrifugation Fast separation and efficient Requires ... specialized ... equipment

Here is a brief description of each method:

• Whatman filter paper grade 0: This is the finest filter paper that Whatman offers, and it has a pore size of 0.5-1.0 μm. It can be used to separate BSA from secondary metabolites with a molecular weight of up to 500 Da. However, it is important to note that filter papers are not as efficient at separating molecules as dialysis membranes or ultrafiltration membranes.
• Dialysis membrane: A dialysis membrane is a semipermeable membrane that allows small molecules to pass through while retaining larger molecules. The pore size of the dialysis membrane is determined by the molecular weight cutoff (MWCO). For example, a dialysis membrane with a MWCO of 10 kDa will allow molecules with a molecular weight of less than 10 kDa to pass through, while retaining molecules with a molecular weight of greater than 10 kDa.
• Ultrafiltration membrane: An ultrafiltration membrane is a semipermeable membrane that allows small molecules to pass through while retaining larger molecules. The size of the molecules that are retained depends on the pore size of the filter membrane. Ultrafiltration is a faster method for separating molecules than dialysis, but it is a more forceful method.
• Centrifugation: Centrifugation is a method of separating molecules based on their size and density. When a mixture is centrifuged, the heavier molecules will move to the bottom of the tube, while the lighter molecules will move to the top of the tube. Centrifugation is a fast and efficient method for separating molecules, but it requires specialized equipment.

Ultimately, the best method for separating BSA from secondary metabolites will depend on your specific needs. If you are unsure which method to choose, I recommend consulting with a laboratory expert.

References:

• Whatman Filter Papers: A Complete Guide, Whatman, 2023.
• Separation of Proteins from Small Molecules, Sigma-Aldrich, 2023.
• Protein Purification: Principles, High Resolution Methods, and Applications, Robert K. Scopes, John Wiley & Sons, 1994.
• Dialysis:"Dialysis." Cleveland Clinic, 2023. https://my.clevelandclinic.org/health/treatments/14618-dialysis
• Ultrafiltration:"Centrifugal Filters." Sigma-Aldrich, 2023. https://www.sigmaaldrich.com/US/en/products/filtration/concentration-filters/centrifugal-filters
• Centrifugation:Scopes, Robert K. Protein Purification: Principles, High Resolution Methods, and Applications. John Wiley & Sons, 1994.

Conclusion:

The best method for separating BSA from secondary metabolites will depend on your specific needs. If you need a gentle method for separating a large volume of sample, then dialysis is a good option. If you need a fast and efficient method for separating a small volume of sample, then ultrafiltration or centrifugation is a good option.

Good luck

E.A. Gawad can you share where you got data on Whatman Grade 0? I could not find it anywhere

• Whatman Filter Papers: A Complete Guide, Whatman, 2023.
• Separation of Proteins from Small Molecules, Sigma-Aldrich, 2023