Ion Exchange Chromatography

In ion exchange chromatography, proteins interact with the stationary phase by charge-charge interactions. Proteins with a net positive charge at a given pH adhere to beads with negatively charged functional groups such as carboxylates or sulfates (cation exchangers). Similarly, proteins with a net negative charge adhere to beads with positively charged functional groups, typically tertiary or quaternary amines (anion exchangers). Proteins, which are polyanions, compete against monovalent ions for binding to the support—thus the term "ion exchange." For example, proteins bind to diethylaminoethyl (DEAE) cellulose by replacing the counter-ions (generally Cl- or CH3COO-) that neutralize the protonated amine. Bound proteins are selectively displaced by gradually raising the concentration of monovalent ions in

Figure 4-2. Size-exclusion chromatography. A: A mixture of large molecules (diamonds) and small molecules (circles) are applied to the top of a gel filtration column. B: Upon entering the column, the small molecules enter pores in the stationary phase matrix from which the large molecules are excluded. C: As the mobile phase flows down the column, the large, excluded molecules flow with it while the small molecules, which are temporarily sheltered from the flow when inside the pores, lag farther and farther behind.

the mobile phase. Proteins elute in inverse order of the strength of their interactions with the stationary phase.

Since the net charge on a protein is determined by the pH (see Chapter 3), sequential elution of proteins may be achieved by changing the pH of the mobile phase. Alternatively, a protein can be subjected to consecutive rounds of ion exchange chromatography, each at a different pH, such that proteins that co-elute at one pH elute at different salt concentrations at another pH.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

Get My Free Ebook


Post a comment