Other forces: hydrophobic interactions and hydrogen bonding
Although the binding of proteins to ion exchangers mainly depends on the ionic bond between opposite charges, there may be other forces in the process, such as hydrophobic interaction and hydrogen bond.
Hydrophobic interactions mainly occur in the use of ion exchangers with non-polar skeletons, such as ion exchange resins, especially polystyrene resins, which have strong hydrophobicity and can bind with some hydrophobic amino acid residues in protein molecules through hydrophobic interactions. Although conventional ion exchange resins mentioned above are not commonly used in protein separation due to their properties, there are still some media in modern HPLC using resins as the skeleton. Hydrogen bonds are mainly appeared in the use of the hydrophilic polymer as the skeleton of the ion exchanger, for example, are widely used in Tandex xu glucan (month) or Tanrose xu agarose (month) as the matrix of ion exchanger, skeleton sugar chain of hydroxyl and carboxyl groups with protein molecules in the hydrophilic side chain form hydrogen bonding between amino acid residues. When the two proteins are electrically close, these extra forces play a decisive role in the separation, on which basis separation is often achieved. However, the influence of these forces on chromatographic behavior is often difficult to predict, and different proteins are often very different, so it is not universal.
Ion exchange kinetics
The occurrence and degree of ion exchange, that is, the ion exchange equilibrium depends on the ion interaction, while the ion exchange kinetics depends on the particle structure of the ion exchanger.
The structure of the ion exchanger is a granular gel with meshes. The charged functional groups are evenly distributed on the surface of the gel particles and inside the mesh. The molecules of the protein enter the gel particles in different degrees according to the molecular weight, replace the counter ions on the charged functional groups and combine themselves with the ion exchanger. From the perspective of dynamics, the whole process can be divided into five steps:
1. The diffusion of protein in the solution reaches the surface of gel particles. The hydrophilic gel and water molecules are hydrogen bonded, thus binding a layer of bound water on the surface of the gel to form a water film. The thickness of the water film depends on the hydrophilicity of the gel, the faster the flow rate of the chromatography, the stronger the hydrophilicity, the slower the flow rate, the thicker the water film, and the thinner the water film is. The process of protein diffusion through the water film to the gel surface is called membrane diffusion, and the speed depends on the concentration difference between the two sides of the water film.
2. The protein molecules enter the gel particle mesh and arrive at the location of exchange. This process is called particle diffusion, and its speed depends on many factors such as the size of the gel particle mesh (crosslinking degree), the functional groups of the exchanger, the size of the protein molecule and the number of charges.
3. Ion exchange occurs when protein replaces counter ions on the exchanger;
4. The replacement of counter ion diffusion reaches the surface of gel particles, that is, particle diffusion, and the direction is opposite to step 2.
5. Counter ions diffuse through the water membrane to the solution, i.e. membrane diffusion, in the opposite direction to step 1.
According to the principle of charge balance, a charged protein molecule enters gel particles within a certain period of time, and there is a counter ion diffused out of the gel particles with the net charge of the protein. In other words, in terms of the number of charges, the rate of membrane diffusion and particle diffusion is the same but in the opposite direction.
Therefore, the above five steps are actually three processes: membrane diffusion, particle diffusion and exchange reaction. The diffusion rate of protein in the whole membrane diffusion process is usually the slowest when the concentration of protein is relatively low; When the protein concentration in the solution is high, the particle diffusion process is often the slowest and becomes the limiting step of the whole process.
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