text: 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, 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 with no retention. 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 at specific conditions 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.
  Comparatively simple devices are often used to apply counterions of increasing gradient to a chromatography column. Counterions such as copper (II) are chosen most often for effectively separating peptides and amino acids through complex formation.
  A simple device can be used to create a salt gradient. Elution buffer is consistently being drawn from the chamber into the mixing chamber, thereby altering its buffer concentration. Generally, the buffer placed into the chamber is usually of high initial concentration, whereas the buffer placed into the stirred chamber is usually of low concentration. As the high concentration buffer from the left chamber is mixed and drawn into the column, the buffer concentration of the stirred column gradually increase. Altering the shapes of the stirred chamber, as well as of the limit buffer, allows for the production of concave, linear, or convex gradients of counterion.
  A multitude of different mediums are used for the stationary phase. Among the most common immobilized charged groups used are trimethylaminoethyl (TAM), triethylaminoethyl (TEAE), diethyl-2-hydroxypropylaminoethyl (QAE), aminoethyl (AE), diethylaminoethyl (DEAE), sulpho (S), sulphomethyl (SM), sulphopropyl (SP), carboxy (C), and carboxymethyl (CM).
  Successful packing of the column is an important aspect of ion chromatography. Stability and efficiency of a final column depends on packing methods, solvent used, and factors that affect mechanical properties of the column. In contrast to early inefficient dry- packing methods, wet slurry packing, in which particles that are suspended in an appropriate solvent are delivered into a column under pressure, shows significant improvement. Three different approaches can be employed in performing wet slurry packing: the balanced density method (solvent's density is about that of porous silica particles), the high viscosity method (a solvent of high viscosity is used), and the low viscosity slurry method (performed with low viscosity solvents).
  Polystyrene is used as a medium for ion- exchange. It is made from the polymerization of styrene with the use of divinylbenzene and benzoyl peroxide. Such exchangers form hydrophobic interactions with proteins which can be irreversible. Due to this property, polystyrene ion exchangers are not suitable for protein separation. They are used on the other hand for the separation of small molecules in amino acid separation and removal of salt from water. Polystyrene ion exchangers with large pores can be used for the separation of protein but must be coated with a hydrophilic substance.
  Cellulose based medium can be used for the separation of large molecules as they contain large pores. Protein binding in this medium is high and has low hydrophobic character. DEAE is an anion exchange matrix that is produced from a positive side group of diethylaminoethyl bound to cellulose or Sephadex.
  Agarose gel based medium contain large pores as well but their substitution ability is lower in comparison to dextrans. The ability of the medium to swell in liquid is based on the cross-linking of these substances, the pH and the ion concentrations of the buffers used.
  Incorporation of high temperature and pressure allows a significant increase in the efficiency of ion chromatography, along with a decrease in time. Temperature has an influence of selectivity due to its effects on retention properties. The retention factor (k = (t − t)/(t − t)) increases with temperature for small ions, and the opposite trend is observed for larger ions.
  Despite ion selectivity in different mediums, further research is being done to perform ion exchange chromatography through the range of 40–175 °C.
  An appropriate solvent can be chosen based on observations of how column particles behave in a solvent. Using an optical microscope, one can easily distinguish a desirable dispersed state of slurry from aggregated particles.