Ion exchanger is composed of insoluble polymer matrix, charged functional groups and counter ions with opposite electrical properties to functional groups. In aqueous solution, ions with opposite charges to functional groups (including ions in buffer solution and ions formed by protein) can be adsorbed on its surface by electrostatic attraction. In this way, there is a competitive relationship between various ions and ion exchangers.
The binding ability of inorganic ions to the exchanger is directly proportional to the charge carried by the ions and inversely proportional to the radius of hydrated ions formed by the ions. In other words, the higher the valence state of ions, the stronger the binding force. When the valence state is the same, the higher the atomic number, the stronger the binding force. On cation exchanger, the order of strength of common ion binding force is as follows:
Li+＜Na+＜K+ ＜Rb+ ＜Cs+
Mg2+ ＜Ca2+ ＜Sr2+ ＜ Ba2+
Na＋ ＜Ca2+ ＜Al3+ ＜Ti4+
On the anion exchanger, the order of binding force is:
F– ＜ Cl– ＜Br– ＜I–
For charged biological macromolecules such as proteins, the binding ability with ion exchangers first depends on the pH of the solution, which determines the charged state of the protein, and then the chromatographic related regions of the protein, that is, the distribution of charge on the protein surface, which have been mentioned above. In addition, it also depends on the type and ionic strength of ions in the solution. Inorganic ions and proteins in the solution competitively combine with the exchanger. Under the initial conditions, the ionic strength in the solution is low. After loading, the protein has more charges and stronger binding ability with the exchanger, so it can replace ions and adsorb to the exchanger; During elution, the competitive binding ability of ions is often increased by improving the ionic strength of the solution, so that the protein sample is desorbed from the exchanger, which is the essence of ion exchange chromatography.
pH and ionic strength
PH and ion strength I are important factors controlling ion exchange behavior, resolution and recovery of proteins.
PH determines the charge of proteins and ion exchangers, and is therefore the most important parameter for protein adsorption. During separation, the pH should be controlled so that the protein and the ion exchanger have opposite charges. There are two aspects involved. On the one hand, ion exchangers have a pH range within which to ensure that they carry a sufficient charge. Generally, cation exchanger applications have a lower pH limit, below this pH will have a large part of the ion exchange groups lose negative charge and can no longer bind cations; Anion exchangers have a pH upper limit above which a large proportion of ion exchange groups lose positive charge and can no longer bind anions. Solution pH directly determines the protein, on the other hand, the amount and type of electric charge, select the appropriate pH, protein molecules whose function it is to guarantee purpose by adsorption and ion exchanger with opposite charges, at the same time, if the pH range of protein isoelectric point too far, the protein and too strong and not easy elution ion exchanger.
Special attention should also be paid to the pH stability range of the target protein when selecting the operating pH. If the range is beyond this range, protein activity will be lost and recovery will decrease. In the microenvironment on the surface of cation exchanger, H+ is attracted by cation exchange groups while OH- ions are repelled, resulting in the pH of the surface of the exchanger is 1 pH unit lower than that of the surrounding buffer. In the microenvironment on the surface of anion exchanger, OH- is attracted by anion exchange groups while H+ ions are repelled, resulting in a pH unit higher on the surface of the exchanger than in the surrounding buffer. For example, if a protein is adsorbed by cation exchanger at pH=5, in fact, the protein is in the environment of pH=4 on the surface of the exchanger. If the protein is unstable at this pH condition, it will be inactivated. Most proteins lose stability and recovery below pH 4.
Since other ions in solution compete with proteins to bind to ion exchangers, ion type and ion strength I are another important factors affecting protein binding and elution. At low ionic strength I, proteins bind to functional groups with opposite charges on the ion exchanger through charged groups. As the concentration of competing ions, i. e., ion strength I, increases, proteins are replaced gradually. For proteins with specific charge numbers, there is no fixed rule of how high salt concentration is required to elute them from ion exchangers, which needs to be explored experimentally. Most proteins can be elute at a salt concentration of 1mol/L, so the final concentration of washing and desalting is often set as 1mol/L in the exploratory stage. In fact, salt often plays the role of stabilizing protein structure in solution. In order to prevent protein denaturation or precipitation, ion strength should not be too low. In addition, the type of ion is also an important factor. The ability of different ions to displace proteins from the exchanger is different, and the ion type also affects the resolution and the elution order of different proteins.
Welch Materials ion exchange chromatography packing:
- Q /SP/DEAE/CM Tanrose FF
Fast flow agarose base frame ion exchange medium
- Q/SP Tanrose HP
High resolution agarose base frame ion exchange medium
- Q/SP Tanrose XL
High loading agarose base frame ion exchange medium
- Q/SP Tanrose BB
Large particle agarose base frame ion exchange medium
- DEAE/CM Tandex
A sub-swap medium
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