Question

Explain a different way of eluting a second protein in an ion-exchange column (other than by the addition of salt). Explain why this method is not often used with proteins.

Answer 1

Ion exchange chromatography is the most frequently used chromatographic technique for the separation and purification of proteins, polypeptides, nucleic acids,etc. Ion exchange chromatography utilizes both a stationary and mobile phase in which the stationary phase carries either a positive or negative charge. For anion exchange chromatography, the stationary phase consists of a synthetic polymer (resin) which has many positively charged functional groups, typically quaternary ammonium ions. Negatively charged species are attracted to the resin and travel more slowly down the column. Similarly, positively charged species are attracted to a cation exchange resin's negatively charged functional groups. E.g. sulfonate groups.

Proteins are charged molecules and so electrostatic forces will therefore allow proteins to bind to other molecules of opposite charge. In many cases, small differences in charge can result in significant separations on ion exchange columns. Ion exchange columns are typically loaded at low ionic strength, and the protein removed by raising the ionic strength. The particles that bind to the stationary phase can be released by changing the buffer conditions i.e. changing the pH of the buffer solution. By changing the pH of the buffer solution, the pI of a protein becomes the pH of a solution, the net charge of the protein will be zero. The protein will no longer bind to the stationary phase and will be released and washed out. Change of pH in the column can be aimed to decrease the net absolute value of the charges of adsorbed proteins, decrease their attraction to the stationary phase, and accelerate the elution.

In practice, pH changes in the column are difficult to control, as they do not reliably correspond to pH changes of the applied eluting buffer because of the buffering power of proteins adsorbed to the column. For weak ion exchangers, buffering power of the adsorbent functional groups themselves. Resolution of proteins by pH elution is achieved in a separate technique called “Chromatofocusing.”