In the past 30 years, various techniques and methods for the separation of biological macromolecules have been continuously developed. With the improvement and commercialization of various media, column chromatography based on fillers plays an important role in the purification of natural products after crude extraction.
1. Hydrophobic Interaction Chromatography (HIC)
HIC is based on protein hydrophobicity. Specifically, it is a column chromatography method that separates proteins by differences in surface hydrophobicity.
The formation of hydrophobic bonds has no specific requirements for non-polar groups, and the number, size, strength, distribution, and steric hindrance of hydrophobic zones on the surface and inside of different proteins are different, which is also the fundamental reason for HIC protein separation. Among the 20 amino acids, 8 are hydrophobic amino acids, and their strength order is Trp, Ile, Phe, Pro, ValLeu, Met, and Ala. When the medium and sample protein concentration is unchanged, more factors are affecting HIC, mainly salt, its role is to expose the non-polar base of the interface so that it is easy to form hydrophobic effect, followed by the increase of water polarity, improve the surface tension of the interface, the higher the salt concentration, promote hydrophobic effect is stronger. Generally, (NH4)2SO4 is commonly used to promote salt thinning. ② Miscible organic solvent, the function is to weaken the interfacial tension, reduce the hydrophobic effect. ③ Temperature, when the system temperature rises from 4℃ to 25℃, the hydrophobic force increases by 20% ~ 30%, which is mainly due to the increase of kinetic energy, improve the surface tension, generally when the temperature is reduced, can increase the salt concentration, so that the hydrophobic effect is not reduced. (4) pH, when pH≈pI can eliminate the repulsion of surface charge, and surfactants and denaturing agents also have an effect on it.
In principle, HIC can be used for all protein purification with a large processing capacity. In particular, it has obvious advantages in extracting low content of target protein from large-volume samples and can replace the traditional salting-out precipitation technology. HIC has a high removal rate of DNA and calf serum (albumin, immunoglobulin, amino acid, and other components), especially since the one-step removal rate of cell DNA can reach more than 90%. In principle, the HIC can be placed anywhere in the purification process, but mostly in the front. Because it also belongs to adsorption column chromatography, especially the hydrophobic effect on the interior of protein space can make the loss of this step larger, and also has a fine effect on the structure of the protein. 2 Gel Filtration column Chromatography (GFC)
GFC is based on molecular size and shape. Specifically, it is a column chromatography method that uses the difference of gel retention time between molecules of different sizes to separate mixtures. Another view is that the separation is based on the size of the molecular volume, which is the solvated volume of the molecule and is affected by the shape of the molecule and the relative molecular mass. Sometimes denatured agents (urea, guanidine hydrochloride, etc.) are added to destroy the third and fourth-order structure of proteins, which can make the separation mainly controlled by the size of the relative molecular weight.
GFC is especially suitable for the separation of macromolecule compounds with a molecular weight greater than 2000 and mixtures with large molecular weight differences and oligomers. The separation of GFC does not depend on the interaction between mobile and stationary phases, so gradient elution is not necessary. However, due to the charge shielding factor, to reduce the interaction between fillers and biomolecules, the salt concentration is often appropriately increased, with a minimum of more than 0.05 mol/L NaCl. Since the retention time of the sample in the column does not exceed the total volume (Vt) of the solvent in the column, the capacity of the detection limit GFC, which often occurs in other liquid chromatography due to dispersion of chromatographic peaks, is usually controlled by the mobile phase effect. Since the separation of GFC depends on the difference of the length of the process path of the big and small components, the column should have sufficient length (75 ~ 100 cm), but the column should not be too long and the loading quantity should not be too much (< 20% Vt). Since the elution time of GFC can be roughly estimated, continuous injection at certain intervals can improve the efficiency of the column and shorten the purification cycle. In addition to the above characteristics, GFC also has high recovery, mild elution conditions, less adverse reactions, long service life, and can be used for salt exchange and other characteristics. However, due to the small processing capacity, relatively weak protein separation ability, and long column chromatography time, it is generally not suitable to be put in front of the purification process design.
3 Ion Exchange Chromatography (IEC)
IEC is based on the separation of net charge and surface charge distribution of protein molecules. Specifically, it is a column chromatography method that makes use of the difference of protein electrification, different electrostatic adsorption capacity on ion adsorbent, and different pH/ ion strength eluent elution, to separate proteins. Ion exchange is divided into anion exchange and cation exchange. Ion exchange agent and strong points, strong ion exchange agent in the whole pH range is ionized, and weak ion exchange agent is usually only between pH6 ~ 9 is the ionized state, so the latter to the applicable pH range and has a further limit.
In terms of elution mode, cation exchange mainly changes pH, which is mainly applied to proteins with an isoelectric point greater than 5. Anion exchange, which changes salt concentration, applies to most proteins. Most of the 20 amino acids are positively or negatively charged, which accounts for IEC's wide range of applications. IEC processing capacity, high loading coefficient, one step to reduce the sample volume. IEC has a strong ability to remove impurities, such as thermogenesis, nucleic acids, exotoxins, most proteins in calf serum, and proteins with different charges can be removed. In addition, IEC makes use of the different charged properties of proteins under different pH and salt concentrations and applies the same type or different types of media (such as DEAE and CM) under different conditions to achieve purification of target proteins in two steps. This is a separate ability that cannot be achieved by other column chromatography except affinity column chromatography. In principle, IEC can be placed in any step of the purification process. It is adsorptive chromatography and has a large loss in one step. When expanding the purification process, it is especially necessary to consider the diameter of amplification instead of the column height alone. Generally speaking, the column height can not be greater than 4 times the diameter of the column, otherwise, the purification results and small test will be more different.
IEC elution is generally divided into 3 kinds: ① synchronous elution (Isocratic), that is, the salt concentration is unchanged, which is generally applied to the properties of the sample is known, elution repeatability is good, and most of it is used for analytical separation. (2) Stepwise, that is several times of synchronous elution, several times of salt concentration Stepwise elution. This method is mostly used to harvest a target protein in one of the salt concentration elution peaks, but there are some problems with this method, for example, one elution peak can contain two proteins or one protein is scattered between two peaks, so the general step elution method is best used for protein separation with known properties. (3) Gradient, the way of elution by continuously changing salt concentration according to a certain concentration Gradient. For example, the increasing speed is called Line Gradient, which has the strongest response among the three elutions. The protein peak shape generally does not tail, and there is rarely one protein appearing in several peaks. Therefore, it is better to use this method for the separation of components with similar charge properties and components with larger proteins and wide surface charge distribution. 4 Affinity Chromatography (AC)
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