Ion Exchange Chromatography: Separation and Purification of Molecules

Ion exchange chromatography separates molecules based on charge attraction, widely used for protein purification and influenced by isoelectric pH.

### Ion Exchange Chromatography: Separation and Purification of Biomolecules Based on Charge
* 🧪 Ion exchange chromatography relies on the attraction between **opposite charges** to separate and purify sample molecules.
* 🔄 The stationary phase, known as the ion exchanger, is chosen to have a **specific charge that attracts molecules with the opposite charge**.
* This chromatography method is commonly used for the **separation and purification** of proteins, peptides, nucleic acids, and polynucleotides.
* 🔋 Two types of ion exchangers are cation exchangers (negatively charged, attracting **positively charged cations**) and anion exchangers (positively charged, attracting **negatively charged anions**).
* 🔵 Cation exchange buffers use functional groups like **carboxyl, carboxymethyl, sulfur, metal**, while anion exchangers use functional groups like **amino, ethyl, aminoethyl**.
* 🔀 "The buffers used with an ion exchanger have a **charge opposite** to that of the stationary phase."
* Understanding the **isoelectric pH of proteins** is crucial for selecting the appropriate ion exchange chromatography method based on the protein's **charge state relative to the isoelectric pH**.

heroin quit ba chemistry basics here. Let's talk about ion exchange chromatography. Ion exchange chromatography is based on the phenomenon of attraction between opposite charges. The stationary phase is selected such that it has a particular charge and attracts the sample molecules with the opposite charge. The stationary phase is also known as the ion exchanger. Ion exchange chromatography is widely used for the separation and purification of proteins, peptides, nucleic acids, polynucleotides, etc. The ion exchanger can be of two types: cation exchanger and the anion exchanger. A cation exchanger has a negative charge and attracts positively charged cations, while an anion exchanger is positively charged and attracts negatively charged anions. Different functional groups such as carboxyl, carboxymethyl, sulfur, metal, etc., can be used as a cation exchange buffers. Having negatively charged ions like acetate, phosphate, etc., can be used with cation exchanger. On the other hand, functional groups like amino, ethyl, aminoethyl, etc., can be used as

an ion exchanger. The buffers used with an ion exchanger has a positive charge. For example, Tris buffer. Let's try to understand the separation of proteins with ion exchange chromatography and isoelectric pH. The net charge and protein is zero, that is, proteins have equal number of positive and negative charge. If the pH is shifted one unit above the isoelectric pH, then the protein will have net negative charge. [Music] And if a pH is shifted one unit below the isoelectric pH, then the protein will have net positive charge. Before proceeding further, it must be checked at which pH the protein is stable. If the protein is stable at pH one unit above isoelectric pH, then an anion exchange chromatography is preferred as the protein will have a negative charge. And if the protein is stable one unit below the isoelectric pH, then cation exchange chromatography is preferred as the protein will have a positive charge. Once the protein binds the stationary phase in the column, elution of protein can be done by changing in the pH or by changing the

Ion exchange chromatography

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Ion exchange chromatography is based on the attraction between opposite charges, using a stationary phase to attract sample molecules with the opposite charge.

It is widely used for separating and purifying proteins, peptides, and nucleic acids, with cation and anion exchangers being the two types used.

The choice between the two types depends on the net charge of the protein, which is determined by its isoelectric pH, and the elution of the protein from the column can be achieved by changing the pH or the ionic strength of the buffer.

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