Anion-exchange chromatography

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Anion exchange chromatography
Acronym HPAE[1]
Classification Ion Exchange Chromatography
Column chromatography
Other techniques
Related Chromatography
High performance liquid chromatography
Aqueous Normal Phase Chromatography
Size exclusion chromatography
Micellar liquid chromatography

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).[2] In solution, the resin is coated with positively charged counter-ions (cations). 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 [3] 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[edit]

A slurry of resin, such as DEAE-Sephadex is poured into the column. After settling, 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 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.[4][5]

References[edit]

  1. ^ Lee, Y.C. (1990). "High-performance Anion-exchange Chromatography for Carbohydrate Analysis.". Analytical Biochemistry 189 (2): 151–62. doi:10.1016/0003-2697(90)90099-u. 
  2. ^ Fotsis, T., H. Adlercreutz; Järvenpää, Paula; Setchell, K.D.R.; Axelson, M.; Sjövall, J. (1981). "Group Separation of Steroid Conjugates by DEAE-Sephadex Anion Exchange Chromatography.". Journal of Steroid Biochemistry 14 (5): 457–63. doi:10.1016/0022-4731(81)90357-5. 
  3. ^ Rocklin, Roy D.; Pohl, Christopher A. (1983). "Determination of Carbohydrates by Anion Exchange Chromatography with Pulsed Amperometric Detection.". Journal of Liquid Chromatography 6 (9): 1577–590. doi:10.1080/01483918308064876. 
  4. ^ Duong-Ly, Krisna C.; Gabelli, Sandra B. (2014). "Chapter Eight - Using Ion Exchange Chromatography to Purify a Recombinantly Expressed Protein". Methods in Enzymology (Academic Press) 541: 95–103. doi:10.1016/b978-0-12-420119-4.00008-2. 
  5. ^ "FPLC Ion Exchange and Chromatofocusing: Principles and Methods". Pharmacia Biotech (Pharmacia Biotech AB). 1985.