Ion Exchange Chromatography: How it works?
Belongs to the family of chromatography, Ion Exchange Chromatography or sometimes refers to only Ion Chromatography (IC) is also a separation technique, technically a column chromatography that plays by the rule of the ‘opposite attracts’. Slightly difference compares to HPLC or Gas Chromatography, it exploits the interactions of ions and polar molecules, Ion Exchange Chromatography can be used to separate almost all types of molecules as long as these analytes are charged. Widely applied in the fields of protein purification, water analysis, quality control and research laboratories of large proteins to small nucleotides and amino acids.
Under this family of ion exchange chromatography, they are two distinct subgroups namely cation exchange chromatography and anion exchange chromatography. In cation exchange chromatography, the stationary phase uses a resin from negatively charged functional group, thus the cations analytes are immobilized and retained, playing by the game of opposite attracts. The opposite occurrence for anion exchange chromatography whereby the column is packed with positively charged resin thus binding the anionic molecules while positive functional group molecules are eluted from the column.
Factors that affect Ion Exchange Chromatography
pH Buffer: Teamwork of pKa and pI
In order to initiate the coulumbic interactions in Ion Exchange Chromatography, the mobile phase is typically a buffer solution. It is used to wash the solid support until equilibrium is achieved. The rule of thumb says the magic number for the mobile phase’s pH must fall in-between the acid dissociation constant (pKa) and isoelectric point (pI) regardless it is a cation exchange chromatography or an anion exchange chromatography. If you are running a cation exchange chromatography for instance, using a column with functional group of pKa 1.2 and a sample molecule of pI 8.2, then the pH of your mobile phase must be in the range between 2 to 8 such as pH 6.0. Begin with buffer that contains low ionic strength then gradually increase of buffer strength so that the molecules of stronger ionic interactions that formerly bound are also steadily eluted off later.
Ion Exchange Chromatography: Salt Gradient vs Step Gradient
Aside from using buffer pH as mobile phase, salt gradients that provides a linearly increasing salt concentration can be considered to separate the components. Step gradient is an improved method at which employed simpler device yet effective to elute different fractions of sample as long as the suitable salt concentrations are determined from prior experiment of linear gradient.
Ion Exchange Chromatography: pH variation
Varying pH also plays an important role to affect a separation in Ion Exchange Chromatography. By adjusting the ionic strength or net charge of cation or anion species, it allows you to select which analytes to be eluted while the ‘contaminants’ are bound to the resin. In cation exchange chromatography, increasing the pH of the mobile phase buffer will result the molecule to become less positively charged, hence the protein sample starts to mobilize when less ionic interaction occurs with the negatively charged stationary phase. This condition results the molecule to be eluted from the column. Whilst in anion exchange chromatography, lowering the pH of the mobile phase buffer will make the molecule to be more positively charged, thus the protein sample creates dissociation from the positively charged stationary phase, ultimately permitting the molecule to be eluted.