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pH is an important factor in the operation of ion exchange chromatography, and pH stabilization and changes are usually achieved with buffers, so the choice of buffer is an important factor affecting the separation. When selecting a buffer, pH and ionic strength are two key factors that affect not only the separation of the target product from the impurities, but also the yield of the product. The pH chosen depends on the isoelectric point, stability and solubility of the target product, not only to make the separated material an ion that can be exchanged, but also to maintain its high activity. The pK value of the ion exchanger should also be considered. Since the buffer itself is charged, it is also combined with the  ion exchange chromatography medium . This combination will cause two kinds of interference, on the one hand, reducing the concentration of the buffer, thereby reducing the buffering capacity; on the other hand, it is exchanged with the separation medium to compete w...

In the industrial products of ion exchangers, a small amount of organic oligomers and some inorganic impurities are often contained, which will gradually dissolve and release at the initial stage of use, affecting the quality of the target product. Therefore, industrial grade ion exchangers must be pretreated prior to use. The general-purpose ion exchange resin is usually treated with acid or alkali, and can be treated with 1~2 mol/L hydrochloric acid and sodium hydroxide solution in an alternate 4~6 times resin bed volume, between acid and alkali treatment. Wash with deionized water until neutral. For macroporous resins, it is also necessary to treat them with an organic solvent such as ethanol or acetone to remove organic residues used in the production process. Pretreatment of the separation medium not only improves its working capacity, but also improves the purity of the separated product. The pretreated resin should eventually be converted to the applicable ionic form during the ...

When the chromatographic medium is exhausted, it should be eluted. The basic principle is to desorb the target product by a more active ion or group. Different sorbents are different in their activity. Therefore, a suitable eluent should be selected to elute the protein from  chromatography media . There are roughly three ways to elute ion exchange chromatography: One is for simultaneous elution. The eluent is a dilute acid, alkali or salt solution may be used, and an appropriate organic solvent may also be used, wherein the salt solution is mainly used, and the dosage form according to the nature of the target product and the final product is selected. Since the substance to be absorbed is often not a single species, the charge of each substance is different, and the binding strength to the medium is different. Even if the same eluent is used, the easily replaced substance will flow out of the medium first, and the binding force is strong. After the material is discharged, as long...

Polypeptide synthesis on a solid support, a concept of originality pioneered by Robert Bruce M Merrifield in 1963, is a major breakthrough in peptide chemistry.  In solid phase synthesis, it is conventional to assemble peptide chains starting from the C-terminus. First, a linker is introduced on the polymer carrier; then, the N-terminal protected amino acid reacts with the functional group of the linker; then the temporary protecting group is removed and condensed with the next amino acid, and this step is repeated until synthesis is required The sequence; finally, cleaves the covalent bond between the tether and the peptide chain, and removes the insoluble carrier from the dissolved product. The  solid phase synthesis  method effectively avoids the cumbersome and time-consuming separation and purification steps necessary for liquid phase synthesis, and has remarkable advantages, and its application value is irreplaceable.

The Boc/Bzl (tert-butoxycarbonyl/ /benzyl) method is a classical  peptide solid phase synthesis method  in which Boc is used as a temporary protecting group in combination with a benzylic semi-permanent side chain protecting group. Boc is unstable to acid and is usually removed with 20% to 50% TFA. The main strategy of the oc synthesis method is that the Boc which can be removed by TFA is an α-amino protecting group, and the side chain is protected by a benzyl alcohol. A Boc-amino acid derivative is covalently cross-linked to a Merrifield or M BH A resin during synthesis, Boc is removed by TFA, the free amino terminus is neutralized with triethylamine, and then the next amino acid is activated by DCC. Finally, the HF method or the TFM SA method is used for the deprotection. Although the Boc method is gradually replaced by the Fm oc method, since the Boc-amino acid is easily crystallized, it is easy to be acid-decomposed, has stability, and is easy to store for a long time. It ...

The Fmoc method is a new method for  solid phase synthesis of peptides  developed by Carpino and Hart based on the Boc method. The fundamental difference between the Fmoc/t-Bu(9-fluorenylmethoxycarbonyl/tert-butoxy) strategy and the Boc/Bzl strategy is the use of alkali-removable Fmoc is a protecting group for α-amino group, the side chain is protected by t-Bu which can be removed by TFA, and the solid phase carrier Wang resin with acid-sensitive linking arm is used, and the final step of the synthesis is removed by TFA. Han Xiang et al. successfully synthesized the 32-peptide thymosin α 1 by Fm oc solid phase method, starting from Fm oc-Asn(Trt)-W angResin, Boc protecting the side chain amino group of Lys, and tert-butyl ester group (O-t-Bu Protecting the side chain carboxyl group of Asp and Glu, t -Bu protects the side chain hydroxyl group of Ser and Thr, and trityl group (Trt) protects the side chain amide group of Asn, and the total synthesis yield is 33.2%, and the purity...

The immobilized enzyme is a biological reagent with high efficiency, high selectivity and mild reaction conditions. The immobilized enzyme immobilizes the enzyme onto an inert, low-water-soluble substance, so that the enzyme is immobilized in the reaction, and thus can be easily separated from the reaction system and used again. This technology has great commercial value and has been widely used in industrial enzymatic reactions. Enzyme immobilization method: Affinity-tag binding: The enzyme is adsorbed on the outside of the inert substance. In general, this method is the slowest of the three methods listed here. The adsorption reaction is not a chemical reaction, so the active site of the immobilized enzyme may be blocked by the matrix, thereby greatly reducing the enzyme activity. Entrapment: Enzymes are embedded in insoluble columns or microspheres, such as calcium alginate columns. However, this insolubility hinders substrate entry or product removal. Cross-linking method: ...