Question

illustrate the adsorption mechanisms of adsorbent on zeolite, ionic exchange resin and affinity chromatography.

Answer 1

Adsorption mechanism of ionic exchange resin

Before ion-exchange chromatography can be initiated, it must be equilibrated. The stationary phase must be equilibrated to certain requirements that depend on the experiment that you are working with. Once equilibrated, the charged ions in the stationary phase will be attached to its opposite charged exchangeable ions. Exchangeable ions such as Cl- or Na+. Next, a buffer should be chosen in which the desired protein can bind to. After equilibration, the column needs to be washed. The washing phase will help elute out all impurities that does not bind to the matrix while the protein of interest remains bounded. This sample buffer needs to have the same pH as the buffer used for equilibration to help bind the desired proteins. Uncharged proteins will be eluted out of the column at a similar speed of the buffer flowing through the column. Once the sample has been loaded onto to the column and the column has been washed with the buffer to elute out all non-desired proteins, elution is carried out to elute the desired proteins that are bound to the matrix. Bound proteins are eluted out by utilizing a gradient of linearly increasing salt concentration. With increasing ionic strength of the buffer, the salt ions will compete with the desired proteins in order to bind to charged groups on the surface of the medium. This will cause desired proteins to be eluted out of the column. Proteins that have a low net charge will be eluted out first as the salt concentration increases causing the ionic strength to increase. Proteins with high net charge will need a higher ionic strength for them to be eluted out of the column. It is possible to perform ion exchange chromatography in bulk, on thin layers of medium such as glass or plastic plates coated with a layer of the desired stationary phase, or in chromatography columns. Thin layer chromatography or column chromatography share similarities in that they both act within the same governing principles; there is constant and frequent exchange of molecules as the mobile phase travels along the stationary phase. It is not imperative to add the sample in minute volumes as the predetermined conditions for the exchange column have been chosen so that there will be strong interaction between the mobile and stationary phases. Furthermore, the mechanism of the elution process will cause a compartmentalization of the differing molecules based on their respective chemical characteristics. This phenomenon is due to an increase in salt concentrations at or near the top of the column, thereby displacing the molecules at that position, while molecules bound lower are released at a later point when the higher salt concentration reaches that area. These principles are the reasons that ion exchange chromatography is an excellent candidate for initial chromatography steps in a complex purification procedure as it can quickly yield small volumes of target molecules regardless of a greater starting volume.

Adsorption mechanism in affinity chromatography

Affinity chromatography exploits the differences in interactions' strengths between the different biomolecules within a mobile phase, and the stationary phase. The stationary phase is first loaded into a column with mobile phase containing a variety of biomolecules from DNA to proteins (depending on the purification experiment). Then, the two phases are allowed time to bind. A wash buffer is then poured through a column containing both bound phases. The wash buffer removes non-target biomolecules by disrupting their weaker interactions with the stationary phase. Target biomolecules have a much higher affinity for the stationary phase, and remain bound to the stationary phase, not being washed away by wash buffer. An elution buffer is then poured through the column containing the remaining target biomolecules. The elution buffer disrupts interactions between the bound target biomolecules with the stationary to a much greater extent than the wash buffer, effectively removing the target biomolecules. This purified solution contains elution buffer and target biomolecules, and is called elution.

The stationary phase is typically a gel matrix, often of agarose; a linear sugar molecule derived from algae. To prevent steric interference or overlap during the binding process of the target molecule to the ligand, an inhibitor containing a hydrocarbon chain is first attached to the agarose bead (solid support). This inhibitor with a hydrocarbon chain is commonly known as the spacer between the agarose bead and the target molecule.

Usually, the starting point is a crude, heterogeneous group of molecules in a whole cell extract, such as a cell lysate, growth medium or blood serum. The molecule of interest will have a well known and defined property, and can be exploited during the affinity purification process. The process itself can be thought of as an entrapment, with the target molecule becoming trapped on a solid or stationary phase or medium. The other molecules in the mobile phase will not become trapped as they do not possess this property. The stationary phase can then be removed from the mixture, washed and the target molecule released from the entrapment in a process known as dialysis. The desired molecules are eluted with specific substances after washing the non-interacting molecules away. Thus, this results in a highly purified material. Highly specific elution of the desired macromolecule from the stationary phase is usually effected by adding to the eluting buffer a gradient of the same kind on the macromolecule and displaces it.[6] Possibly the most common use of affinity chromatography is for the purification of recombinant proteins. Affinity chromatography is an excellent choice for the first step in purifying a protein or nucleic acid from a crude mixture.

If the molecular weight, hydrophobicity, charge, etc. of a protein is unknown, affinity chromatography can still apply to this situation. An example of this situation is when trying to find an enzyme with a particular activity, where it can be possible to build an affinity column with an attached ligand that is similar or identical to the substrate of choice. The way that the desired enzyme would be eluted would be from the mixture based on the strong interaction of enzyme and the immobilized substrate analog, which would be done selectively through the affinity column. Then, the elution of the enzyme with the appropriate substrate can be done.

Adsorption mechanism of zeolite

Pressure swing adsorption (PSA) and temperature swing adsorption (TSA) are potential techniques for removing carbon dioxide (CO2) from high-pressure fuel gas streams. Zeolites are suitable candidate sorbents for use in these processes; however, the systems would be even more energy efficient if the sorbents were operational at moderate or high temperatures, especially for the removal of CO2 from high-pressure gas streams, such as those from integrated gasification combined-cycle (IGCC) systems. Competitive gas adsorption tests with gas mixtures representing both coal combustion and coal gasification gas streams were conducted in an atmospheric flow reactor with five zeolites at 120 °C. Promising results of preferential adsorption of CO2 were observed with two of these zeolites. However, the CO2 adsorption capacity was significantly lower at 120 °C than at ambient temperature. Volumetric gas adsorption tests of CO2 and nitrogen (N2) on these two zeolites were conducted at 120 °C, up to a pressure of 300 psi (2 × 106 Pa). Both showed high CO2 adsorption capacity at high pressure. High-pressure flow reactor studies also indicated the preferential adsorption of CO2 from gas mixtures at 120 °C. CO2 adsorption rates were measured utilizing thermogravimetric analysis, and the rates were similar for the two zeolites.

Zeolites have same mechanism as that of ion exchange resin.