Ion Exchange Chromatography Pdf
The degree of swelling decreases with increasing ionic strength. Further information on controlling software, gradient programmers and related pump systems is available from Amersham Biosciences.
These groups remain charged and maintain consistently high capacities over the working pH ranges of and respectively. If maximum separation of the curves occurs at the position of sample application i. With any chromatographic method, it helps to know some basic rules for predicting retention order. This will only happen if a phthalate ion replaces it in the resin. Work by amersham Biosciences, Uppsala, Sweden.
The conductivity of a solution is a measure of the extent to which the solution conducts electricity. These groups are attached to glucose units in the matrix by stable ether linkages. Repacking between experiments is thus eliminated, improving reproducibility. Continuous pH gradients are difficult to produce at constant ionic strength, since simultaneous changes in ionic strength, although small, also occur.
Further information on techniques and media for chromatofocusing is available on request. Detergents can be used if they are non-ionic or have the same charge as the gel. The resolution which can be achieved on any chromatographic matrix is a result of a combination of the efficiency and selectivity of the system. If it is not, it can be changed by gel filtration on Sephadex G using e.
Ion exchange chromatography overview
Approximate relative viscosities can be quickly estimated by comparing emptying times from a pipette. If stability is high over a wide pH range on both sides of pI, either type of ion exchanger can be used.
As resolution and efficiency are maintained with increasing column diameter and sample load, separations using these media are easy to scale up. An analysis of this composite plot for the point of maximum separation will indicate at what pH and on which type of exchanger maximum resolution between any two or more components can be expected. As with other weak ion exchangers the capacity varies with pH. The fixed ions are attached through a derivatization of the phenyl rings of the polystryene.
They are stable in water, salt solutions, and organic solvents. The particles are kept in suspension by the balance between upward flow rate and particle sedimentation velocity.
On application of a gradient the increasing salt concentration will cause the most weakly bound molecules to migrate and leave the column first. Some very thin capillary tubing is attached to a syringe and the free end is flared by gentle heating.
The handbook is illustrated with examples of different types of biological molecules which have been separated using ion exchange chromatography and different ways the technique can be used. These counter-ions can be reversibly exchanged with other ions of the same charge without altering the matrix. Decant and re-suspend the ion exchanger in this buffer several times. The gel can also be packed in a column after the washing step and be eluted stepwise in the same way as during normal column chromatography.
Contact your local Amersham Biosciences supplier for further information. The question to consider is which ion would be the most disruptive of the three to the hydrogen bond network? If better resolution is required then the separation can be improved by altering the shape or slope of the gradient. Once the separation parameters have been determined, scale-up is easily achieved by increasing the column diameter. Electrophoretic titration curves Fig.
Capacity As is the case with all ion exchangers the capacity is dependent upon the accessibility of the charged groups and their number. In contrast the trailing edge of the peak is exposed to continuously increasing eluting power. Repulsion between charged groups is greatest at pH values where the ion exchanger is fully dissociated, and decreases at pH values close to the pK of the charged groups. This is a result of an optimized pore size distribution, a high substitution level and a large binding surface area. The influence of increasing sample load on resolution.
Add sample, mix and assay the supernatant to determine the maximum ionic strength which permits binding of the substance of interest and the minimum ionic strength required for complete desorption. These are reservoirs which, used in combination with a suitable valve, e. The electrophoretic titration curve of chicken breast muscle using zymogram detection for creatine kinase. Thus Sepharose ion exchangers can be regenerated and re-equilibrated repeatedly in the column. The column can then be connected for elution.
Oxidizing agents should be avoided. The properties of the protein.
Ion Exchange Chromatography - PDF Free Download
The high rigidity of the cross-linked agarose matrix eliminates volume variations due to changes in pH or ionic strength. In addition, the extremely high charge density gives very strong binding and the hydrophobic matrix tends to denature labile biological materials. The flow rate is of particular importance with respect to dynamic capacity, which decreases as the flow rate is increased. The charged groups are associated with mobile counterions.
The stationary phase effects are more important in this case in determining the retention order. The conditions used column type, buffers, pH, etc. Choosing the direction of the gradient for elution. These in turn depend upon the pH of the eluent.
The separation is based on the reversible interaction between a charged protein and an oppositely charged chromatography resin. High available capacity is obtained by having a matrix which is macroporous and highly substituted with ionic groups which maintain their charge over a wide range of experimental conditions. If we use high capacity resins, 3 phase squirrel cage induction motor pdf the retention times will be much too long. The solubility of the sample components at the pH and salt concentrations to be used during separation should always be tested in advance. Hydrolysis may occur in strongly acidic solutions and the macromolecular structure is broken down in strongly alkaline solutions.
The capacity may be expressed as total ionic capacity, available capacity or dynamic capacity. Polystyrene polymer derivatized with fixed ionic groups. In a specified salt gradient, the retention of particular molecular species is dependent upon the molecules net charge and charge density. Equipment Amersham Biosciences also supply a full range of equipment for operating all of the ion exchangers covered in this handbook. Increasing the ionic strength increases competition and reduces the interaction between the ion exchanger and the sample substances, resulting in their elution.
Ion Exchange Chromatography - GE Healthcare Life Sciences
The required resolution When choosing an ion exchanger it is important to decide the degree of resolution required from the separation. Sepharose Fast Flow and Sepharose High Performance ion exchangers, have good flow properties and stable bed volumes that are largely insensitive to changes in ionic strength and pH. If turbid solutions are injected onto the column, the column lifetime, resolution and capacity can be reduced. Ion exchange thus serves as a useful means of concentrating a sample in addition to fractionating it.
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