Anion-Exchange Chromatography: Videos & Practice Problems

Anion exchange chromatography is a technique used to purify negatively charged proteins by utilizing a positively charged stationary phase. This method is essentially the reverse of cation exchange chromatography, where the roles of positive and negative charges are swapped. Understanding one type of ion exchange chromatography allows for a seamless transition to the other by simply reversing the charge interactions.

In anion exchange chromatography, the stationary phase is composed of positively charged resins, such as those containing diethylaminoethyl (DEAE) functional groups. These DEAE groups bind negatively charged anions, including the target proteins that need to be purified. When a protein mixture is introduced into the chromatography column, the negatively charged proteins interact with the positively charged resin, while neutral and positively charged proteins do not bind and thus elute more quickly.

The elution order is determined by the charge of the proteins: positively charged proteins elute first, followed by neutral proteins, while negatively charged proteins remain bound to the resin and move through the column very slowly. The rate of movement for negatively charged proteins is influenced by their net charge; those with a higher negative charge interact more strongly with the resin and elute more slowly than those with a lower negative charge.

To effectively collect the negatively charged proteins, a common strategy involves the addition of salt to the mobile phase. Salt reduces the strength of ionic interactions between the negatively charged proteins and the positively charged resin, facilitating their elution from the column. This method not only speeds up the process but also conserves resources by minimizing the need for excessive mobile phase.

In summary, anion exchange chromatography is a powerful technique for the purification of negatively charged proteins, leveraging the principles of charge interactions to achieve effective separation and collection.

In anion exchange chromatography, the goal is to purify negatively charged proteins. At a physiological pH of around 7, proteins can be analyzed based on their net charge, which is determined by their ionizable groups. To estimate the net charge of a peptide, it is essential to consider the seven amino acids with ionizable R groups, which can be remembered using the mnemonic: "yucky crazy dragons eat knights riding horses." These amino acids include aspartic acid, glutamic acid, lysine, arginine, histidine, cysteine, and tyrosine.

Each peptide will inherently have an ionizable amino group (positively charged at physiological pH) and an ionizable carboxyl group (negatively charged). The backbone of a peptide at this pH exists as a zwitterion, possessing both positive and negative charges. For example, when analyzing two peptides, the net charge is calculated by summing the contributions from the R groups of the amino acids present.

For the first peptide, glycine and alanine do not contribute to the charge as they are not among the seven ionizable amino acids. However, aspartic acid contributes a negative charge, and lysine contributes a positive charge. The total net charge for this peptide is calculated as follows: 2 positive charges (from the amino group and lysine) and 3 negative charges (from the carboxyl group and aspartic acid), resulting in a net charge of -1.

In the second peptide, similarly, only the ionizable R groups are considered. Histidine and arginine contribute positive charges, while the other amino acids do not affect the net charge. The total for this peptide is 3 positive charges (from the amino group, histidine, and arginine) and 1 negative charge (from the carboxyl group), leading to a net charge of +2.

Ultimately, in anion exchange chromatography, the peptide with the greatest negative charge elutes last. Therefore, the first peptide, with a net charge of -1, will elute last, while the second peptide, with a net charge of +2, will elute first. Understanding these concepts is crucial for effectively utilizing anion exchange chromatography in protein purification.