Introduction to Ion Exchange Chromatography (IEX)

0:00-0:50 | What is ion exchange chromatography (IEX)?
0:50-3:03 | How does IEX work?
3:03-4:24 | Ion exchange chromatography workflow
4:24-5:38 | What are the pros & cons of IEX?
Ion-exchange chromatography or IEX is to purify proteins based on charge. Pure proteins are vital for successful experiments. Ion-exchange chromatography separates proteins (or any biomolecules) based on the differences in their net charge at a particular pH. Protein charge depends on the number and type of ionizable amino acid side chain groups. Each protein has an isoelectric point (pI), which is the pH where its overall net charge is equal to zero. (If you are confused by isoelectric point I have a video on the topic which is linked by the end of this video) In short, if the pH of the environment is below its pI, the protein is positively charged and if it is above its pI, the protein is negatively charged.
Two fundamental concepts need to be understood before performing IEX.
1. In a buffered solution, below the protein’s pI, the protein is positively charged or protonated and will bind to the negatively charged functional groups of a cation exchange resin.
2. In a buffered solution above the protein’s pI, the protein is negatively charged or deprotonated and will therefore bind to the positively charged functional groups of an anion exchange resin.
The reason why has to do with the structure of proteins and by extension amino acids and when these accept versus donate protons. Again, this is further explained in my video about the isoelectric point.
Another essential instrument to understand how IEX works is ion-exchange resin beads. Ion-exchange resins have charged functional groups bound to resin beads that are either positively or negatively charged. These resins are called cation (+) or anion (-) exchange resins depending on if they are negatively or positively charged. These can either be weak or strong, depending on what functional groups are used.
Imagine a cat and a sheep walking across a grassy field. (In this case the cat is the more negatively charged protein, the sheep is the more positively charged protein and the grassy field is the negatively charged anion exchange resins) The sheep will be constantly distracted by all the tasty grass and stop and eat constantly while the cat just keeps on walking. If we measure how far they have traveled after 10 minutes, the cat has probably walked a lot further than the sheep. (Unless the cat just decides to lay in the sun ofc…) In a similar way, the proteins that have the strongest electromagnetic attraction to the surrounding resins beads will move the slowest.
Let us look at the workflow of ion exchange chromatography. In this example, the ion-exchange chromatography column is filled with anion exchange resins or negatively charged resins. Therefore, the more positive the protein, the greater the attraction to these beads and the slower it will move.
1. Insert the sample into the column
2. Proteins collect at the top of the column
3. The different proteins separate from each other based on charge
4. Proteins can be collected, in order of charge, from negative → positive
5. Elution can be used to speed up the collection of the final protein which is the one that is the most attracted to the negatively charged beads
This can be a salt solution that disrupts the attraction between our protein and the negatively charged beads, effectively displacing the protein so that it can continue moving through the column.

[@user-rw5ul]

Musta tuntuu, että tässä on selitetty vähän väärin. Tossa esimerkissä toi aineiden eluutiojärjestys menee niinkuin kationinvaihtajilla, mutta sanottiin kuitenkin, että kyseessä on anioninvaihtajat, jolloin negatiivisten pitäis eluoitua viimeisenä, koska anioninvaihtaja sitoo negatiivisia ioneja. Ja vähän harhaanjohtavasti selitetty noi ioninvaihtajat, kun kationinvaihtajahan on negatiivinen ja anioninvaihtaja on positiivinen, niin tosta selityksestä sai nyt sen käsityksen, että anioninvaihtaja olis negatiivinen ja kationinvaihtaja positiivinen. Mutta siis tosi hyviä videoita sulla, yksinkertasesti ja selkeesti selitetty sekä visuaalisesti hyvin havainnollistettu eri aiheita!

[@biotechlucas4126]

Kiitos palautteesta. Tää video on aika vanha niin voi kyllä olla että teen uusi versio tulevaisuudessa ja sitten pystyn korjaa nuo asiat!