Ion exchange how to test

I. Basic principle Ion exchange chromatography (ion exchange chromatography, referred to as IEC) is one of the methods to separate macromolecules of similar nature from complex mixtures, based on the principle of the acidity and alkalinity of the substance, polarity, that is, the different anions and cations. Substances with different charges have different affinities for the ion exchanger on the column, and by changing the ionic strength and pH of the rinsing solution, the substances can be separated from the chromatography column in turn. Ion exchange chromatography is roughly divided into 5 steps: 1. Ions diffuse to the surface of the resin. 2. Ions diffuse through the resin to the exchange sites. 3. Ions are exchanged at the exchange site; the more charge the exchanged molecule has, the more tightly it binds to the resin, and the less likely it is to be replaced by other ions. 4. The exchanged ions diffuse to the surface of the resin. 5. 5. The rinsing solution passes through, and the exchanged ions diffuse into the external solution. The exchange reaction of the ion exchange resin is reversible, following the law of chemical equilibrium, when the quantitative mixture passes through the column, the ions are exchanged continuously, the concentration decreases gradually, and almost all of them can be adsorbed on the resin; in the process of rinsing, due to the continuous addition of a new exchange solution, so it will be moved in the direction of the positive reaction direction, and thus the ions on the resin can be rinsed down. If the purified substance is an amino acid molecule, the net charge on the molecule depends on the isoelectric point of the amino acid and the pH of the solution, so when the pH of the solution is low, the amino acid molecule is positively charged, and it will be bonded to the strongly acidic cation exchange resin; with the gradual increase of the buffer pH through the buffer solution, the amino acid will gradually lose its positive charge, and the bonding force will weaken, and finally be washed down. Since different amino acids have different isoelectric points, these amino acids will be washed out sequentially, the first to be washed out are acidic amino acids such as apartic acid and glutamic acid (at about pH 3~4), followed by neutral amino acids such as glycine and alanine. basic amino acids such as arginine and lysine still carry a positive charge in the pH of the very high buffer. Basic amino acids such as arginine and lysine are still positively charged in a very high pH buffer, so these do not appear until about pH 10~11. The most common ion exchange resin material is polystyrene-benzene (polystyrenedivinylbenzene), which is a three-dimensional mesh structure produced by the polymerization of styrene and benzene (divinylbenzene), for example, Dow Chemical Company produces the resin Dowex 50×8, which contains 8% benzene. For example, Dowex 50×8, a resin produced by Dow Chemical, contains 8% divinylbenzene. Exchange resins are categorized into cationic and anionic exchange resins based on their properties. 1. Cation exchange resins are classified into three types: strong acid type, medium-strong acid type, and weak acid type; strong acid type resins contain -R-SO3H; medium-strong acid type resins contain -PO3H2, -PO2H2, or -O The strong acid resins contain -R-SO3H, the medium-strong acid resins contain -PO3H2, -PO2H2 or -OO2H2, and the weak acid resins contain -COOH or -OH. The cation exchange resins carry out the following reactions Anion exchange resins are classified into three categories: strong, medium-strong, and weak bases, and they contain ammonium salts, with the tetrakisodium salt [ -N+(CH3)3] being the strong base type. ) 3] is a strong base type resin, the ammonium salts [-N(CH3)2], [-NHCH3], [-NH2] below the third grade are all weak base type resins, and the ones with both strong base and weak base groups are medium strong base type resins. The reaction of anion exchange resin is as follows: In addition to resin, cellulose, dextran gel (Sephadex gel) and agarose gel (Sepharose) are also commonly used ion exchange materials, characterized by hydrophilicity and a large surface area, which is a milder environment for biologically active substances, and also suitable for the separation and purification of macromolecules. It is also suitable for the separation and purification of large molecules. Commonly used types are as follows: Experiments have proved that the fiber material is quite effective in the separation and purification of proteins and nucleic acids, because there are many types of ion exchange fibers, which can be used according to different separation purposes, and the functional groups are mainly on the surface, which is very favorable to the exchange of biological macromolecules. Third, the choice of ion exchanger Ion exchanger selection, the first to maintain the biological activity of the substance to be separated, as well as in different pH environments, the substance with the charge and the strength of the electrical properties. 1. 1. Selection of anion and cation exchanger If the substance to be separated is positively charged, such as polymyxin, cytochrome C, these alkaline proteins, which are more stable in acidic solution and have strong affinity, therefore, the cation exchanger is used; other acidic substances such as heparin, nucleic acid, which are more stable in alkaline solution, the anion exchanger is used; If the substance to be separated is an amphoteric ion, generally consider what kind of charge it carries in its stable pH range, as the choice of exchange agent. In the case of insulin, for example, its isoelectric point is pH 5.3, so in a pH<5.3 (acidic) solution, a cation exchanger is used, and in an alkaline solution of pH>5.3, an anion exchanger is used. In short, substances with a known isoelectric point should be exchanged anionically under pH conditions above the isoelectric point because of their negative charge, and cationically under pH below the isoelectric point. Substances with unknown isoelectric point are electrophoresed under certain pH conditions, and substances moving faster to the anode can be adsorbed by anion exchanger under the same conditions, and substances moving faster to the cathode can be adsorbed by cation exchanger. 2. Selection of buffer The selection of buffer acidity and alkalinity is determined by the isoelectric point, stability, solubility and pK value of the exchanger ion of the separated substance. The use of anion-exchange fiber should be selected below the pK value of the buffer, if the substance to be separated belongs to the acidic, the pH value of the buffer should be higher than the isoelectric point of the substance; cation-exchange fiber should be selected higher than the pK value of the buffer, the purpose of the substance belongs to the alkaline substances, the buffer should be lower than the isoelectric point of the pH value of the substance. Buffer ion does not interfere with the separation of the activity of the determination, does not affect the solubility of the substance to be measured, does not precipitate as a principle, such as the use of UV absorption method to measure the sample, then pyridine or barbital such substances that will absorb UV is not applicable. Pre-treatment of ion exchange resin Before using ion exchange resin, first remove the impurities with distilled water and process the resin with NaOH and HCl to make the functional group on it completely exposed. The anion exchange resin is first soaked in 15 times the amount of 0.5 N HCl for 30 to 120 minutes, then washed with water to pH 7.0, followed by soaking in 0.5 N NaOH for 30 to 120 minutes, also washed with water to pH 7.0, and finally soaked in the buffer solution to be used. The cation exchange resin is soaked in 15 times the amount of 0.5 N NaOH for 30 to 120 minutes, rinsed with water to pH 7.0, then soaked in 0.5 N HCl for 30 to 120 minutes, rinsed with water to pH 7.0, and finally soaked in the buffer solution to be used.