Editing High-performance liquid chromatography (section)

===Size-exclusion chromatography===
{{further|Size-exclusion chromatography}}
'''Size-exclusion chromatography''' ('''SEC''')<ref>{{Citation |last1=Kazakevich |first1=Yuri |title=Size-Exclusion Chromatography |date=2007-01-22 |url=https://onlinelibrary.wiley.com/doi/10.1002/9780470087954.ch6 |work=HPLC for Pharmaceutical Scientists |pages=263–279 |editor-last=Kazakevich |editor-first=Yuri |access-date=2023-10-10 |edition=1 |publisher=Wiley |language=en |doi=10.1002/9780470087954.ch6 |isbn=978-0-471-68162-5 |last2=LoBrutto |first2=Rosario |editor2-last=LoBrutto |editor2-first=Rosario}}</ref> separates polymer molecules and [[biomolecule]]s based on differences in their molecular size (actually by a particle's [[Stokes radius]]). The separation process is based on the ability of sample molecules to permeate through the pores of gel spheres, packed inside the column, and is dependent on the relative size of analyte molecules and the respective pore size of the absorbent. The process also relies on the absence of any interactions with the packing material surface.

Two types of SEC are usually termed:

# '''Gel permeation chromatography (GPC)'''—separation of synthetic polymers (aqueous or organic soluble). GPC is a powerful technique for polymer characterization using primarily organic solvents.
# '''Gel filtration chromatography (GFC)'''—separation of water-soluble biopolymers. GFC uses primarily aqueous solvents (typically for aqueous soluble biopolymers, such as proteins, etc.).

The separation principle in SEC is based on the fully, or partially penetrating of the high molecular weight substances of the sample into the porous stationary-phase particles during their transport through column. The mobile-phase eluent is selected in such a way that it totally prevents interactions with the stationary phase's surface. Under these conditions, the smaller the size of the molecule, the more it is able to penetrate inside the pore space and the movement through the column takes longer. On the other hand, the bigger the molecular size, the higher the probability the molecule will not fully penetrate the pores of the stationary phase, and even travel around them, thus, will be eluted earlier. The molecules are separated in order of decreasing molecular weight, with the largest molecules eluting from the column first and smaller molecules eluting later. Molecules larger than the pore size do not enter the pores at all, and elute together as the first peak in the chromatogram and this is called total exclusion volume which defines the exclusion limit for a particular column. Small molecules will permeate fully through the pores of the stationary phase particles and will be eluted last, marking the end of the chromatogram, and may appear as a total penetration marker.

In biomedical sciences it is generally considered as a low resolution chromatography and thus it is often reserved for the final, "polishing" step of the purification. It is also useful for determining the [[tertiary structure]] and [[quaternary structure]] of purified proteins. SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works also in a preparative way by trapping the smaller molecules in the pores of a particles. The larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules: that is, the smaller the molecule, the longer the retention time.

This technique is widely used for the molecular weight determination of polysaccharides. SEC is the official technique (suggested by European pharmacopeia) for the molecular weight comparison of different commercially available low-molecular weight [[heparin]]s.<ref>{{Cite journal |last1=Mulloy |first1=Barbara |last2=Heath |first2=Alan |last3=Shriver |first3=Zachary |last4=Jameison |first4=Fabian |last5=Al Hakim |first5=Ali |last6=Morris |first6=Tina S. |last7=Szajek |first7=Anita Y. |date=2014-08-01 |title=USP compendial methods for analysis of heparin: chromatographic determination of molecular weight distributions for heparin sodium |url=https://doi.org/10.1007/s00216-014-7940-3 |journal=Analytical and Bioanalytical Chemistry |language=en |volume=406 |issue=20 |pages=4815–4823 |doi=10.1007/s00216-014-7940-3 |pmid=24958344 |hdl=1721.1/104914 |s2cid=492085 |issn=1618-2650}}</ref>