Editing Chromatography (section)

==Techniques by separation mechanism==

=== Affinity chromatography ===
{{further|Affinity chromatography}}
Affinity chromatography<ref>{{cite book |title=Affinity Chromatography |vauthors=Wilchek M, Chaiken I |publisher=Humana Press |year=2000 |isbn=978-1-60327-261-2 |veditors=Bailon P, Ehrlich GK, Fung WJ, Berthold W |series=Methods in Molecular Biology |volume=147 |pages=1–6 |chapter=An Overview of Affinity Chromatography |doi=10.1007/978-1-60327-261-2_1 |pmid=10857080}}</ref> is based on selective non-covalent interaction between an analyte and specific molecules. It is very specific, but not very robust.<ref>{{cite book |last1=Urh |first1=Marjeta |title=Guide to Protein Purification, 2nd Edition |last2=Simpson |first2=Dan |last3=Zhao |first3=Kate |year=2009 |isbn=9780123745361 |series=Methods in Enzymology |volume=463 |pages=417–438 |chapter=Chapter 26 Affinity Chromatography |doi=10.1016/S0076-6879(09)63026-3 |pmid=19892186 |chapter-url=https://pubmed.ncbi.nlm.nih.gov/19892186/}}</ref> It is often used in biochemistry in the purification of [[protein]]s bound to tags. These [[fusion protein]]s are labeled with compounds such as [[His-tag]]s, [[biotin]] or [[antigen]]s, which bind to the stationary phase specifically. After purification, these tags are usually removed and the pure protein is obtained.

Affinity chromatography often utilizes a biomolecule's affinity for the [[cations]] of a metal (Zn, Cu, Fe, etc.).  Columns are often manually prepared and could be designed specifically for the proteins of interest. Traditional affinity columns are used as a preparative step to flush out unwanted biomolecules, or as a primary step in analyzing a protein with unknown physical properties.<ref>{{cite journal |last=Markwell |first=John |date=September 2009 |title=Fundamental laboratory approaches for biochemistry and biotechnology, 2nd edition |journal=Biochemistry and Molecular Biology Education |volume=37 |issue=5 |pages=317–318 |doi=10.1002/bmb.20321 |issn=1470-8175 |doi-access=free}}</ref>

However, liquid chromatography techniques exist that do utilize affinity chromatography properties.  Immobilized metal affinity chromatography (IMAC)<ref>{{cite book |title=Affinity Chromatography |vauthors=Singh NK, DSouza RN, Bibi NS, Fernández-Lahore M |year=2015 |isbn=978-1-4939-2447-9 |veditors=Reichelt S |series=Methods in Molecular Biology |volume=1286 |pages=201–12 |chapter=Direct Capture of His6-Tagged Proteins Using Megaporous Cryogels Developed for Metal-Ion Affinity Chromatography |doi=10.1007/978-1-4939-2447-9_16 |pmid=25749956}}</ref><ref>{{cite journal |vauthors=Gaberc-Porekar V, Menart V |date=October 2001 |title=Perspectives of immobilized-metal affinity chromatography |journal=Journal of Biochemical and Biophysical Methods |volume=49 |issue=1–3 |pages=335–60 |doi=10.1016/S0165-022X(01)00207-X |pmid=11694288}}</ref> is useful to separate the aforementioned molecules based on the relative affinity for the metal.  Often these columns can be loaded with different metals to create a column with a targeted affinity.<ref>{{cite journal |last1=Mahmoudi Gomari |first1=Mohammad |last2=Saraygord-Afshari |first2=Neda |last3=Farsimadan |first3=Marziye |last4=Rostami |first4=Neda |last5=Aghamiri |first5=Shahin |last6=Farajollahi |first6=Mohammad M. |date=December 2020 |title=Opportunities and challenges of the tag-assisted protein purification techniques: Applications in the pharmaceutical industry |url=https://www.sciencedirect.com/science/article/abs/pii/S0734975020301555 |journal=Biotechnology Advances |language=en |volume=45 |pages=107653 |doi=10.1016/j.biotechadv.2020.107653 |issn=0734-9750 |pmid=33157154 |s2cid=226276355}}</ref>

===Ion exchange chromatography===
{{further|Ion exchange chromatography}}
Ion exchange chromatography (usually referred to as ion chromatography) uses an ion exchange mechanism to separate analytes based on their respective charges. It is usually performed in columns but can also be useful in planar mode. Ion exchange chromatography uses a charged stationary phase to separate charged compounds including [[anion]]s, [[cation]]s, [[amino acid]]s, [[peptide]]s, and [[protein]]s. In conventional methods the stationary phase is an [[ion-exchange resin]] that carries charged [[functional group]]s that interact with oppositely charged groups of the compound to retain. There are two types of ion exchange chromatography: Cation-Exchange and Anion-Exchange. In the Cation-Exchange Chromatography the stationary phase has negative charge and the exchangeable ion is a cation, whereas, in the Anion-Exchange Chromatography the stationary phase has positive charge and the exchangeable ion is an anion.<ref>{{cite book|title=Fundamental Laboratory Approaches for Biochemistry and Biotechnology|last=Ninfa|first=Alexander J |year=2009|isbn=978-0-470-47131-9}}</ref> Ion exchange chromatography is commonly used to purify proteins using [[fast protein liquid chromatography|FPLC]].

===Size-exclusion chromatography===
{{further|Size-exclusion chromatography}}
Size-exclusion chromatography (SEC) is also known as ''gel permeation chromatography'' (GPC) or ''gel filtration chromatography'' and separates molecules according to their size (or more accurately according to their hydrodynamic diameter or hydrodynamic volume).
Smaller molecules are able to enter the pores of the media and, therefore, molecules are trapped and removed from the flow of the mobile phase. The average residence time in the pores depends upon the effective size of the analyte molecules. However, molecules that are larger than the average pore size of the packing are excluded and thus suffer essentially no retention; such species are the first to be eluted. It is generally a low-resolution chromatography technique and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining the [[tertiary structure]] and [[quaternary structure]] of purified proteins, especially since it can be carried out under native solution conditions.

===Expanded bed adsorption chromatographic separation===
{{further|Expanded bed adsorption}}
An expanded bed chromatographic adsorption (EBA) column for a biochemical separation process comprises a pressure equalization liquid distributor having a self-cleaning function below a porous blocking sieve plate at the bottom of the expanded bed, an upper part nozzle assembly having a backflush cleaning function at the top of the expanded bed, a better distribution of the feedstock liquor added into the expanded bed ensuring that the fluid passed through the expanded bed layer displays a state of piston flow. The expanded bed layer displays a state of piston flow. The expanded bed chromatographic separation column has advantages of increasing the separation efficiency of the expanded bed.

Expanded-bed adsorption (EBA) chromatography is a convenient and effective technique for the capture of proteins directly from unclarified crude sample. In EBA chromatography, the settled bed is first expanded by upward flow of equilibration buffer. The crude feed, which is a mixture of soluble proteins, contaminants, cells, and cell debris, is then passed upward through the expanded bed. Target proteins are captured on the adsorbent, while particulates and contaminants pass through. A change to elution buffer while maintaining upward flow results in desorption of the target protein in expanded-bed mode. Alternatively, if the flow is reversed, the adsorbed particles will quickly settle and the proteins can be desorbed by an elution buffer. The mode used for elution (expanded-bed versus settled-bed) depends on the characteristics of the feed. After elution, the adsorbent is cleaned with a predefined cleaning-in-place (CIP) solution, with cleaning followed by either column regeneration (for further use) or storage.