pH and concentration of buffer
Ion exchange chromatography usually uses at least two different buffers: one is used for protein loading and washing away non adsorbed impurities, which is called starting buffer; The other is used to elute the protein adsorbed on the column, which is called elution buffer.
The elution buffer that reaches the final pH and salt concentration is called the limit buffer. There are two cases here. There are two methods to elute the target protein after adsorption: one is to change the charged state of the protein by changing the pH, which can not be combined with the exchanger. This method will use two different pH starting and elution buffers; The other is to increase the ionic strength, which will not change the charged state of the protein, but the high ionic strength will reduce the electrostatic attraction between the protein and the ion exchanger, so as to elute the protein. This method will use two buffers with the same pH and different ionic strength. Usually, the limit buffer is formed by adding a specific concentration of salts to the starting buffer.
In order to ensure that the target protein can bind to the exchanger in the adsorption stage, the concentration of the initial buffer is generally low, ranging from 0.01-0.05mol/l. However, too low initial concentration may cause the protein to adsorb too firmly on the ion exchanger and make it difficult to elute. At the same time, the time of adsorption stage should also be controlled. The research shows that it is obviously more difficult to elute the protein after being adsorbed on the ion exchange column overnight than directly. This is because the long-time adsorption will cause the conformation of the protein to change slowly, which makes it more firmly combined with the exchanger, and this conformation change will generally lead to the decline of protein activity. The appropriate initial buffer concentration can be determined by small sample test.
When using the method of changing pH for elution, the types of buffer components in the elution buffer are often the same as those in the initial buffer, but the final pH is different due to different control ratio. The elution buffer has no special requirements for concentration and is often the same as the starting buffer. When eluting with the method of increasing ionic strength, the elution buffer used is often the same as the starting buffer in terms of buffer material type, pH and concentration. Usually, the ionic strength is increased by adding other kinds of salts (such as NaCl) with specific concentration to the starting buffer.
Therefore, in fact, no matter what kind of buffer, the concentration of buffer substance is usually small. In order to have sufficient buffer capacity, certain conditions must be met. According to Henderson hasselbalch formula:
When the pH of the buffer solution is close to the pKa of the buffer material, it has good buffer capacity. The maximum buffering capacity appears at pKa, and the buffering capacity of one pH unit deviated from pKa value will decrease by 5 times. Generally speaking, the effective pH range of buffer is about pKa ± 2ph unit, and to obtain strong enough buffer capacity, the pH is preferably between pKa ± 0.5ph unit. When the ratio of weak acid (or weak base) to the amount of corresponding salt is close to 1:1, the buffer capacity of this buffer system is the strongest. Therefore, when selecting the buffer solution, first select the buffer substance with pKa value close to it according to the pH required for the initial buffer solution, so that the ratio of the amount of weak acid (or weak base) to the corresponding salt substance is close to 1. It should be noted that the pKa value of buffer material will change with temperature, which will have a significant impact on the pH of the solution when the temperature difference is large. For example, Tris buffer prepared at room temperature is 0.05 pH units lower than that in refrigeration chamber (4 ℃).
Effect of ions on chromatographic behavior
When using the method of increasing ionic strength to elute protein, some non buffer salt is usually selected, the most commonly used is NaCl, which is added to the initial buffer to form the elution buffer. During chromatographic separation, the buffer substances in the elution buffer will certainly affect the chromatographic effect. In fact, the types and properties of non buffer salts will also affect the resolution and selectivity of chromatography. When using different non buffer salt ions, the order of elution of different substances may change. Therefore, the selection of non buffer salts is also of great significance.
As discussed in the previous part of ion exchange theory, ions with high valence have stronger binding force with the exchanger, so in general, multivalent ions are better replacement ions than monovalent ions, which can make the protein elute earlier (the retention value is small). Therefore, in the cation exchanger, the relationship between the retention value of protein and the replacement ion is: Ba2 + < Ca2 + < Mg2 + < NH4 + < K + < Na + < Li +, but there are exceptions. For example, for lysozyme, NH4 + is a more effective replacement ion than Mg2 +. Many factors should be considered when selecting replacement ions. Strong replacement ions can effectively shorten the chromatographic time, but often reduce the protein recovery. In general, it is more appropriate to select the salt ion with medium replacement ability. When eluting the protein firmly bound to the exchanger, it is advantageous to select the strong replacement ion.
Not only replace ions, but also the ions in the elution salt that have the same charge with the functional groups and do not participate in the ion exchange process will also affect the chromatographic behavior of proteins for a variety of reasons. Some are caused by the interaction between ions and proteins. In addition, divalent ions such as Ca2 + and Mg2 + can form complexes with amide groups in protein molecules and affect their chromatographic behavior.
In short, the influence of ions on chromatographic behavior is multifaceted and has no certain law. In the first chromatographic separation, some common buffer substances and salts can be preferentially selected and further optimized on this basis.
In some cases, some other compounds will be added during ion exchange. Their main functions are: increasing the solubility of proteins, improving chromatographic resolution, protecting substances to be separated, etc. Most proteins are soluble in water, but some proteins have strong hydrophobicity and low solubility in water, which makes chromatographic operation difficult to achieve. A specific example is the separation of membrane proteins. Membrane proteins exist in biofilms, and their surface is a hydrophobic region. They bind with hydrophobic lipid components in biofilms by hydrophobic interaction. These proteins are insoluble or have low solubility in water. Adding non-ionic or facultative ionic detergent to the solution can dissolve and disperse the membrane protein in the aqueous solution, which is convenient for chromatography. Organic solvents (such as chloroform, methanol, etc.) can also increase the solubility of hydrophobic proteins, and because organic solvents reduce the dielectric constant of the solution, the electrostatic force between protein and exchanger is stronger and the adsorption is more firm.
In some cases, the addition of specific substances can also improve the resolution of chromatography. For example, when separating some proteins, the addition of betaine or taurine can reduce the formation of protein aggregates and their combination with exchanger, so as to improve the resolution. Some neutral polymers such as polyethylene glycol (PEG) can compete with proteins for water molecules in solution, which will increase the interaction between proteins and ion exchangers and improve the resolution. Enzyme inhibitors sometimes need to be added in the separation process. For example, in the process of protein separation, if there is protease in the sample system, the target protein will be hydrolyzed and the recovery rate will be reduced. The addition of protease inhibitor can effectively protect the target protein. Benzenesulfonyl fluoride is the common inhibitor of serine protease, iodoacetamide is the common inhibitor of sulfhydryl protease, and metal chelating agent can be used as the inhibitor of metalloproteinase.
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