Acronym HPAE | ||
Classification Ion Exchange Chromatography
Column chromatography Related | ||
Anion-exchange chromatography is a process that separates substances based on their charges using an ion-exchange resin containing positively charged groups, such as diethyl-aminoethyl groups (DEAE). In solution, the resin is coated with negatively charged counter-ions (anions). Anion exchange resins will bind to negatively charged molecules, displacing the counter-ion. Anion exchange chromatography is commonly used to purify proteins, amino acids, sugars/carbohydrates and other acidic substances with a negative charge at higher pH levels. The tightness of the binding between the substance and the resin is based on the strength of the negative charge of the substance.
General technique for protein purification
A slurry of resin, such as DEAE-Sephadex is poured into the column. After it settles, the column is pre-equilibrated in buffer before the protein mixture is applied. Unbound proteins are collected in the flow-through and/or in subsequent buffer washes. Proteins that bind to the resin are retained and can be eluted in one of two ways. First, the salt concentration in the elution buffer is gradually increased. The negative ions in the salt solution (e.g. Cl-) compete with protein in binding to the resin. Second, the pH of the solution can be gradually decreased which results in a more positive charge on the protein, releasing it from the resin. As buffer elutes from the column, the samples are collected using a fraction collector.