Editing Size-exclusion chromatography (section)

== Theory and method ==
[[File:Size_Exclusion_Chromatography_Apparatus.jpg|thumb|[[Agarose]]-based SEC columns used for protein purification on an AKTA [[Fast protein liquid chromatography|FPLC]] machine]]

SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping smaller molecules in the pores of the [[adsorbent]] ("stationary phase"). This process is usually performed within a column, which typically consists of a hollow tube tightly packed with micron-scale polymer beads containing pores of different sizes. These pores may be depressions on the surface or channels through the bead. As the solution travels down the column some particles enter into the pores. Larger particles cannot enter into as many pores. The larger the particles, the faster the elution. The larger molecules simply pass by the pores because those molecules are too large to enter the pores. Larger molecules therefore flow through the column more quickly than smaller molecules, that is, the smaller the molecule, the longer the retention time.

One requirement for SEC is that the analyte does not interact with the surface of the stationary phases, with differences in elution time between analytes ideally being based solely on the solute volume the analytes can enter, rather than chemical or electrostatic interactions with the stationary phases. Thus, a small molecule that can penetrate every region of the stationary phase pore system can enter a total volume equal to the sum of the entire pore volume and the interparticle volume. This small molecule elutes late (after the molecule has penetrated all of the pore- and interparticle volume—approximately 80% of the column volume). At the other extreme, a very large molecule that cannot penetrate any the smaller pores can enter only the interparticle volume (~35% of the column volume) and elutes earlier when this volume of mobile phase has passed through the column. The underlying principle of SEC is that particles of different sizes [[Elution|elute]] (filter) through a stationary phase at different rates. This results in the separation of a solution of particles based on size. Provided that all the particles are loaded simultaneously or near-simultaneously, particles of the same size should elute together.

However, as there are various measures of the size of a macromolecule (for instance, the [[radius of gyration]] and the hydrodynamic radius), a fundamental problem in the theory of SEC has been the choice of a proper molecular size parameter by which molecules of different kinds are separated. Experimentally, Benoit and co-workers found an excellent correlation between elution volume and a dynamically based molecular size, the [[hydrodynamic volume]], for several different chain architecture and chemical compositions.<ref>{{Cite journal| vauthors = Grubisic Z, Rempp P, Benoit H |date=1967|title=A universal calibration for gel permeation chromatography|journal=[[Journal of Polymer Science Part B|J Polym Sci B]]|volume=5|issue=9|pages=753–759|doi=10.1002/pol.1967.110050903|bibcode=1967JPoSL...5..753G|issn=1542-6254}}</ref> The observed correlation based on the hydrodynamic volume became accepted as the basis of universal SEC calibration.

Still, the use of the hydrodynamic volume, a size based on dynamical properties, in the interpretation of SEC data is not fully understood.<ref>{{cite journal | vauthors = Sun T, Chance RR, Graessley WW, Lohse DJ|date=2004|title=A Study of the Separation Principle in Size Exclusion Chromatography|journal=[[Macromolecules (journal)|Macromolecules]]|volume=37|issue=11|pages=4304–4312|doi=10.1021/ma030586k|bibcode=2004MaMol..37.4304S|issn=0024-9297}}</ref> This is because SEC is typically run under low flow rate conditions where hydrodynamic factor should have little effect on the separation. In fact, both theory and computer simulations assume a thermodynamic separation principle: the separation process is determined by the equilibrium distribution (partitioning) of solute macromolecules between two phases: a dilute bulk solution phase located at the interstitial space and confined solution phases within the pores of column packing material. Based on this theory, it has been shown that the relevant size parameter to the partitioning of polymers in pores is the mean span dimension (mean maximal projection onto a line).<ref>{{Cite journal | vauthors = Wang Y, Teraoka I, Hansen FY, Peters GH, Hassager O |display-authors=3|date=2010|title=A Theoretical Study of the Separation Principle in Size Exclusion Chromatography | journal=[[Macromolecules (journal)|Macromolecules]]|volume=43|issue=3|pages=1651–1659|doi=10.1021/ma902377g|bibcode=2010MaMol..43.1651W|issn=0024-9297}}</ref> Although this issue has not been fully resolved, it is likely that the mean span dimension and the hydrodynamic volume are strongly correlated.
[[Image:Superdex 200HR gel filtration column.jpg|thumb|300 px|left|A size exclusion column]]

Each size exclusion column has a range of molecular weights that can be separated. The exclusion limit defines the molecular weight at the upper end of the column 'working' range and is where molecules are too large to get trapped in the stationary phase. The lower end of the range is defined by the permeation limit, which defines the molecular weight of a molecule that is small enough to penetrate all pores of the stationary phase. All molecules below this molecular mass are so small that they elute as a single band.<ref name=":0" />

The filtered solution that is collected at the end is known as the '''eluate'''. The '''void volume''' includes any particles too large to enter the medium, and the solvent volume is known as the '''column volume'''.

Following are the materials which are commonly used for porous gel beads in size exclusion chromatography <ref>{{Cite book|title=Fundamentals and techniques of Biophysics and Molecular biology|last=Kumar|first=Pranav|publisher=Pathfinder Publication|year=2018|isbn=978-93-80473-15-4|location=New Delhi|pages=05}}</ref>
{| class="wikitable"
|+
!Sr. No
!Material
And Trade name
!Fractionation range 
(kDa)
|-
|1
|Sephadex G-10
|0-0.7
|-
|2
|Sephadex G-25
|1-5
|-
|3
|Sephadex G-50
|1.5-30
|-
|4
|Sephadex G-75
|3-70
|-
|5
|Sephadex G-100
|4-150
|-
|6
|Sephadex G-150
|5-300
|-
|7
|Sephadex G-200
|5-8000
|-
|8
|Bio-gel P-2
|0.1-1.8
|-
|9
|Bio-gel P-6
|1-6
|-
|10
|Bio-gel P-60
|3-60
|-
|11
|Bio-gel P-150
|1.5-150
|-
|12
|Bio-gel P-300
|16-400
|-
|13
|Sepharose 2B
|2000-25000
|-
|14
|Sepharose 4B
|300-3000
|-
|15
|Sepharose 6B
|10-20000
|}