About Seplife® Ion Exchange Chromatography

How to use ion exchange chromatography resins?

1. Operation method: 

Since the sample, buffer and eluent of biochemical separation are all mobile phases, they can be separated while flowing through the column. Therefore, ion exchange can be performed in column operation and separation in chromatographic form. During the separation process, the unadsorbed substances continue to flow out of the reaction system, which makes the balance shift to the right continuously, which is a kind of dynamic balance, so it is also called dynamic operation. The dynamic operation mode has good separation effect, is suitable for all kinds of samples, and can realize continuous operation. In the operation of chromatographic separation, the loading condition of the chromatographic column has a certain influence on the separation. The resins should be evenly distributed in the column, the existence of air bubbles is not allowed, and the stratification of the resins should also be prevented.

For some samples with high viscosity, the "static" treatment method can also be used for preliminary extraction and separation. The ion-exchange resins and the working liquid to be treated are stirred in the reaction vessel. When the adsorption equilibrium is reached, separate the resins and the raffinate and load them into a column for elution.

This static batch operation method has simple process equipment and easy operation. For example, the preliminary separation of some natural products such as heparin sodium often adopts this static separation method. 

In the operation of static separation mode, the stirring speed of the ion exchanger in the working fluid should be properly controlled. If the stirring speed is too fast and the shear force is too large, the ion exchange particles will be broken and it will be difficult to filter and separate; If the speed is too slow, it will affect the contact between the resins and the working fluid, and also affect the exchange rate.

2. The impact of the sample on the separation effect:

In order to achieve high resolution and high load capacity for biochemical separation, the preparation and performance of the working solution are also very important factors. The viscosity and clarity of the working fluid not only affect the separation effect of the ion exchange resins, but also affect the service life of the separation medium.

Biochemical separation is often a relatively complex system, in which there are many kinds of impurities, not only small molecules, but also some colloidal substances, lipid substances, etc. In particular, some irreversibly adsorbed macromolecules can cover the functional groups of the medium, or block the pores of the medium, causing irreversible contamination and shortening the service life of the separation medium. Therefore, before the separation operation, the working fluid should be properly pretreated as much as possible to ensure the separation effect.

In the process of biochemical separation and purification, some target products are taken away by the elution process, or the target product is retained on the medium due to incomplete elution, resulting in product loss, which is an important factor affecting product yield.

At the same time, structural changes in the protein cause inactivation, which will also affect the yield. Adding some stabilizers or protective agents in the ion exchange process can not only increase the yield, but also improve the selectivity of the separation medium for proteins.

3. The impact of flow rate on the separation effect:

In ion exchange chromatography separation, the flow rate is an important factor affecting the separation effect. In order to obtain an excellent separation effect, experiments should be carried out based on factors such as the type of ion exchange resin, particle size, and molecular structure of the active ingredients in the working fluid to establish better experimental parameters.

If the molecular weight of the target product is relatively small and the pore size of the medium is relatively large, a higher flow rate can be used because it is conducive to mass transfer.

However, when the target product is a biomacromolecule, and the pore size of the medium is smaller than that of the separated substance molecule, a slower flow rate should be adopted due to the slower diffusion rate of the molecule.

When the viscosity of the working fluid is high, a lower flow rate should also be used due to the lower mass transfer rate.

The flow rate not only affects the effect of exchange adsorption, but also affects the effect of elution. Usually, the flow rate during elution is slower than that during the ion exchange adsorption.

4. The elution methods of Ion exchange chromatography:

When the target protein in the sample is completely bound to the ion exchanger, it should be eluted. The basic principle is to use an ion or group that is more active than the adsorption substance to desorb the target product that is exchanged and adsorbed to the outer surface and interior of the medium particle. Different target proteins have different binding abilities to ion-exchange resins. Therefore, a suitable eluent should be selected to elute the protein from the medium and collect the separated and purified products. There are roughly three elution methods for ion-exchange chromatography:

1) Simultaneous elution: The eluent is the same substance, and dilute acid, alkali or salt solution can be used, or an appropriate organic solvent can be used, among which salt solution is the main one, and the choice is made according to the properties of the target product and the dosage form of the final product. 

Since the adsorbed substances are often not of a single type, the charges carried by various substances are different, and the binding strength with the medium is different. Even if the same eluent is used, the easily replaced substances will flow out of the medium first, and the binding force will be stronger. After the substances flow out, as long as they are collected by classification, various substances can be separated to obtain relatively pure products.

This method is mostly used for the separation when the properties of the target product are well known, or for the separation of analytical kinds.

2) Stepwise elution: that is, elution is carried out with different concentrations of salt solutions. During the exchange adsorption process of the separation medium, various proteins are adsorbed. If a constant elution condition is used, sometimes all the components cannot be properly separated, and the elution condition needs to be changed.

The change can be a staged change meaning different eluents or eluents with different pH values are selected for elution in stages, and different elution peaks can be obtained according to different concentrations and different acidities of the eluent. That is, one kind of salt concentration can get a kind of target protein, and different salt concentration can get different target proteins.

This step-by-step elution method is suitable for the separation of proteins with known properties, especially for large-scale production, and is easy to operate and control.

3) Gradient elution, that is, changing the ionic strength or pH value of the eluent according to a certain linear change (generally only in special cases, the elution method of changing the pH value is used). During the gradual change of the eluent, different proteins can be replaced one by one, and various protein components can be obtained. 

At the same time, proteins generally do not tail. Gradient elution is the most commonly used elution method in ion exchange chromatography, and it is also the elution method with the strongest elution ability, which is suitable for the elution of components with similar charge properties.

In the elution process, both co-current elution or counter-current elution can be used. In co-current elution, the flow direction of the eluent is the same as that of the working fluid. In counter-current elution, or reverse elution, the flow direction of the eluent is opposite to that of the working solution.

If the feed liquid is exchanged and adsorbed through the exchange column from top to bottom, the concentration of the adsorbate in the upper layer of the exchange column is higher than that in the lower layer, and the reverse desorption of the eluent from bottom to top can achieve the purpose of elution more efficiently. However, since the operation of reverse elution is much more complicated than that of co-current elution, co-current elution is mostly used now.

Disinfection of ion exchange chromatography resins:

In the preparation process of some biochemical products with high purity requirements, it is often required to sterilize the separation media to prevent impurities such as microorganisms from mixing into the target product.

High-temperature disinfection is the most commonly used method. At present, most ion exchangers have stable physical and chemical properties and can be subjected to high-temperature disinfection. However, when using polysaccharide media, it must be noted that the media must be in the salt type, and high-temperature disinfection must be carried out under neutral conditions, otherwise it will lead to the degradation of the polysaccharide macromolecular matrix, which will seriously affect the service life of the media.

NaOH is also a good disinfectant. However, the appropriate concentration of NaOH should be selected according to the alkali resistance of the medium and the type and degree of microbial contamination. When using NaOH disinfection, column soaking can also be used, that is, pass a certain concentration of NaOH into the column, close the liquid outlet valve, and soak for several hours to achieve the purpose of disinfection. If NaOH is used in combination with ethanol, better results can be obtained. When using NaOH disinfection, disinfection and CIP can be combined. 

Storage of ion exchange chromatography resins:

All kinds of chromatography resins should be cleaned before storage after use. This is especially important for polysaccharide separation media.

After the separation media is used, wash it with 2CV of water, and then pass through the column with 2 bed volumes of 20% ethanol. For SP strongly acidic cationic media, wash with 20% ethanol solution containing 0.2mol/L sodium acetate, and then wash with degassed ethanol-water solution at a slower flow rate.

After treatment, it can be stored at room temperature, or at 4-8°C for a long time. The chromatographic column must be completely sealed during storage to prevent moisture volatilization and dry column.

The medium that is not used for the time being must be stored in 20% ethanol solution. All ion exchange separation media should be stored at 4°C to 30°C and protected from freezing.

The process of separation and purification of biological macromolecules by ion exchange chromatography is mainly based on the dissociation of various molecules, the net charge of ions, and the electrical difference in surface charge distribution for selective separation. It has become one of the most frequently used purification techniques in the separation and purification of biochemical products, proteins, peptides and other substances.

Seplife® Dextran-based Ion Exchange Chromatography Resins:

The Seplife® dextran ion exchange chromatography resins use the dextran matrix of G series gel filtration chromatography resins (Seplife G-25 and Seplife G-50), and the ion exchange functional ligands of different properties are firmly bonded to the cross-linked dextran matrix.

Dextran ion exchange resins are usually stored in the form of dry powder, which must be swollen before use. It is widely used in low molecular weight proteins such as prothrombin and low molecular weight heparin.

Sunresin’s dextran ion exchange chromatography resins:

DEAE Seplife® A25/A50

Q Seplife® A25/A50

CM Seplife® C25/C50

SP Seplife® C25/C50

Seplife® Ultra-Fast Flow Agarose-based Ion Exchange Chromatography Resins (BB):

This series of Seplife® ion-exchange chromatography resins are prepared by bonding ion-exchange ligands to agarose microspheres with a particle size of 100-300um. The back pressure is relatively small at the flow rate. For samples with high viscosity and turbidity, the use of this series of resins can improve the efficiency.

Sunresin’s Ultra-fast flow rate agarose ion exchange chromatography resins:

DEAE Seplife® BB

Q Seplife® BB

CM Seplife® BB

SP Seplife® BB

Seplife® Fast Flow Agarose-based Ion Exchange Chromatography Resins (FF):

This series of Seplife® resins use 45-165um agarose microspheres as the matrix, bonding with different functional groups. The suitable particle size range allows it to have a wider application range. It is widely used in various stages of capture, intermediate purification and polishing of biological products.

Sunresin’s Fast Flow Agarose ion exchange chromatography resins:

DEAE Seplife® FF

Q Seplife® FF

CM Seplife® FF

SP Seplife® FF

Seplife® High Resolution Agarose-based Ion Exchange Chromatography resins (HP):

This series ses 25-45um agarose microspheres as the matrix and is prepared by bonding different functional groups.

The small particle size allows the resins to have higher resolution, and it is widely used in fine separation and preparation of small amount of samples.

Sunresin’s High Resolution Agarose ion exchange chromatography resins:

DEAE Seplife® HP

Q Seplife® HP

CM Seplife® HP

SP Seplife® HP

Ultra High Capacity Agarose Ion Exchange Chromatography Resins (XL): 

The special "tentacle" design on the agarose microspheres reduces the influence of steric hindrance when binding to biomolecules, and the ligands are more reasonably distributed, giving it ultra-high loading and very cost-effective.

Sunresin’s Ultra High Capacity agarose ion exchange chromatography resins:

DEAE Seplife® XL

Q Seplife® XL

CM Seplife® XL

SP Seplife® XL

Seplife® High Rigidity Agarose Ion Exchange Chromatography Resins (Large Scale):

Sunresin’s high-rigidity (Large Scale) agarose ion exchange medium has a maximum pressure resistance of 0.5 MPa, a maximum flow rate of 1000 cm/h, and a faster mass transfer rate, allowing a significantly improved efficiency for large-scale production.

According to the particle size of the matrix, Sunresin’s high rigidity agarose ion exchange medium is divided into high rigidity + high flow rate medium (Large Scale) and high rigidity + high resolution medium (Large Scale HP). 

Sunresin’s High Rigidity agarose ion exchange chromatography resins (Large Scale): 

DEAE Large Scale /HP

Q Large Scale /HP

CM Large Scale /HP

SP Large Scale /HP

Seplife® Uniform Particle Size polystyrene Ion Exchange Chromatography Resins (LXMS):

Seplife® IEX LXMS type ion exchange chromatography resins provide two kinds of pore sizes (50nm, 150nm) and three particle sizes (15, 30 and 50um) of polystyrene uniform particle size resins. Its high cross-linking properties allow the resins to withstand higher operating pressure (3MPa).

The two pore sizes of 50nm and 150nm cover the application of the capture, intermediate purification and fine purification of antibodies, proteins, peptides, nucleic acids, antibiotics, natural products and other products with different molecular weights.

Sunresin’s Uniform Particle Size polystyrene Ion Exchange Chromatography Resins:

Seplife® LXMS 15Q/15S (particle size 15um, pore size 50nm)

Seplife® LXMS 30Q/30S (particle size 30um, pore size 50nm)

Seplife® LXMS 50Q/50S (particle size 50um, pore size 100nm)

Seplife® LXMS 50HQ/50HS (particle size 50um, pore size 150nm)

Seplife® Polymethylacrylate Ion Exchange Chromatography Resins (LXPM):

This group of ion exchange chromatography resins are microspheres with polymethacrylate as the matrix using Sunresin’s unique polymer synthesis technology. The microspheres are modified by precise pore-making technology and surface hydrophilic long-chain macromolecules, and are coupled with different ion-exchange groups.

Due to its good hydrophilicity, good chemical and physical stability and rigid structure, the ion exchange chromatography resins have good biocompatibility and service life, and improve the purification efficiency. They cover the application of production and purification stages such as capture, intermediate purification, and fine purification of molecules such as antibodies, proteins, peptides, nucleic acids, antibiotics, and natural products, and provides customers with an overall solution for the industrial production of biological samples.

Sunresin’s Polymethylacrylate ion exchange chromatography resins:

Seplife® LXPM CM/DEAE/SP/Q 650M (strong hydrophilicity, particle size 80um）

Seplife® LXPM CM/DEAE/SP/Q 650S (strong hydrophilicity, particle size 50um）

Seplife® LXPM CM/DEAE/SP/Q 706 (strong hydrophobicity，strong ionic multimodal, particle size 80um）

Seplife® LXPM CM/DEAE/SP/Q 5504（strong hydrophobicity，high resolution, strong ionic multimodal, particle size 80um)

For more information about the different types of ion exchange chromatography resins, please contact us at (info.lifescience@sunresin.com).