Chromatography is the science behind separating molecules to quantify, purify, or identify them. Understanding their properties will help you perform tests and determine the issue and recommended treatment plan.
Ion-exchange chromatography is one class of LC (liquid chromatography) used to separate inorganic molecules from organic ones. It’s particularly useful for separating charged biomolecules, such as nucleic acids, carbohydrates, proteins, and amino acids.
Protein molecules have a unique structure and particular ionisable groups on the surface, so the pH is specific to that molecule. You can exploit that characteristic to separate molecules based on how they interact with oppositely charged particles.
The Ion-exchange Chromatography Process
There are five steps in the IEC (Ion-exchange Chromatography) process:
1. Equilibration – You’ll wash the stationary phase with a start buffer to stabilise the baseline and keep the pH constant. This ensures that your ionisable groups can interact freely with the analyte molecules.
2. Sample Loading – You will then dissolve samples in the start buffer or a buffer with the same pH levels. The ionic and pH strength of your buffer must be adjusted so the analytes will bind while the impurities don’t.
3. Wash – Once again, you’ll wash the column with your starting buffer, removing any uncharged molecules and molecules featuring the same charge as your stationary phase.
4. Elution – You’ll use a salt gradient to elute your bound analytes as the ions compete for or replace the analytes on the column surface at charged sites. Typically, you must increase the salt concentration periodically until you have strongly bound molecules.
5. Column Regeneration – Finally, you must restore the column capacity for the next run, which includes washing away the molecules bound to the column.
Appropriate Tools to Help You
Trinity Biotech offers its Premier Resolution software. This haemoglobin fractionation system utilises the principles of high-performance liquid chromatography and ion-exchange chromatography to give you more information in a short time frame. Learn more today.