Ion exchange interaction
The ion exchanger is composed of an insoluble polymer matrix, charged functional groups and counter ions with opposite charges to the functional groups. ions) can be adsorbed on its surface by electrostatic attraction. In this way, there is a competitive relationship between various ions and ion exchangers when combined.
The binding capacity of inorganic ions and exchangers is proportional to the charge of the ions and inversely proportional to the radius of the hydrated ions formed by the ions. That is to say, the higher the valence state of the ion, the stronger the binding force, and when the valence state is the same, the higher the atomic number, the stronger the binding force. On cation exchangers, the order of strength of common ion binding force is:
Li+＜ Na+＜K+ ＜Rb+ ＜Cs+
Mg2+ ＜Ca2+ ＜Sr2+ ＜ Ba2+
Na＋ ＜Ca2+ ＜AI3+ ＜Ti4+
On anion exchangers, the order of binding force is:
F– ＜ Cl– ＜Br– ＜I–
For charged biological macromolecules such as proteins, the binding ability to ion exchangers depends first on the pH of the solution, which determines the charged state of the protein, and then the chromatographically relevant region of the protein, that is, the distribution of charges on the surface of the protein. have been mentioned before. In addition, it also depends on the species and ionic strength of the ions in the solution. Inorganic ions and proteins in the solution competitively bind to the exchanger. Under the initial conditions, the ionic strength in the solution is low. After loading, due to the high number of charges on the protein, the binding ability with the exchanger is stronger. It can replace ions and adsorb on the exchanger. During elution, the ionic strength of the solution is often increased, the competitive binding capacity of ions is increased, and the protein sample is desorbed from the exchanger, which is the essence of ion exchange chromatography.
Effects of pH and ionic strength I
pH and ionic strength I are important factors controlling protein ion exchange behavior, resolution, recovery, etc.
pH determines the charge of proteins and ion exchangers, and is therefore the most important parameter to determine whether proteins are adsorbed. When performing the separation, the pH should be controlled so that the protein and the ion exchanger are oppositely charged, and there are two aspects involved here. On the one hand, the ion exchanger has a working pH range within which it can be ensured that the ion exchanger is sufficiently charged. Generally, cation exchangers are applied with a lower pH limit, below which a large portion of the ion exchange groups lose their negative charge and can no longer bind cations. Anion exchangers are applied with an upper pH limit, above which pH There will be a large fraction of the ion exchange groups that lose their positive charge and can no longer bind anions. On the other hand, the pH of the solution directly determines the type and quantity of the charged protein. Choosing an appropriate pH can ensure that the target protein molecule and the ion exchanger are adsorbed with opposite charges. At the same time, if the pH is too far from the isoelectric point of the protein , the protein and the ion exchanger are bound too strongly and are not easily eluted.
When choosing the operating pH, special attention should be paid to the pH stability range of the target protein. If the pH exceeds this range, the protein activity will be lost and the recovery rate will decrease. Especially due to the Daunan effect, the pH of the ion exchanger surface is not consistent with the pH of the solution. In the microenvironment on the surface of the cation exchanger, H+ is attracted by the cation exchange groups and OH₋ ions are repelled, resulting in the pH of the exchanger surface being 1 pH unit lower than that in the surrounding buffer. In the microenvironment on the surface of the anion exchanger, The OH₋ is attracted by the anion exchange groups and the H+ ions are repelled, causing the pH of the exchanger surface to be 1 pH unit higher than that in the surrounding buffer. For example, a certain protein is adsorbed by a cation exchanger at pH=5. In fact, the protein is in an environment of pH=4 on the surface of the exchanger. If the protein is unstable at this pH, it will be inactivated. Most proteins are less stable and less recoverable below pH=4.
Since other ions in solution compete with proteins for binding to the ion exchanger, ionic species and ionic strength I are another important factor affecting protein binding and elution. Under the condition of low ionic strength I, the protein is bound to the oppositely charged functional group on the ion exchanger through the charged group. When the competing ion concentration, that is, the ionic strength I, gradually increases, the protein is gradually replaced. For a protein with a specific charge number, how high the salt concentration is required to elute it from the ion exchanger, there is no fixed rule, and it needs to be explored from the experiment. Most proteins can be eluted at a salt concentration of 1 mol/L, so in the stage of exploring conditions, people often set the final concentration of eluted salt as 1 mol/L. In fact, salts often play a role in stabilizing the protein structure in solution. In order to prevent protein denaturation or precipitation, the ionic 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 type of ion will also have an impact on the resolution and elution order of different proteins.
Welch ion exchange chromatography packing materials
◌ Q /SP/DEAE/CM Tanrose FF
Fast Flow Agarose Matrix Ion Exchange Media
◌ Q/SP Tanrose HP
High Resolution Agarose Matrix Ion Exchange Media
◌ Q/SP Tanrose XL
High-capacity agarose-based ion exchange media
◌ Q/SP Tanrose BB
Large particle agarose matrix ion exchange medium
◌ DEAE/CM Tandex
Dextran Matrix Ion Exchange Media