Editing Size-exclusion chromatography (section)

== Analysis ==
In simple manual columns, the eluent is collected in constant volumes, known as fractions. The more similar the particles are in size the more likely they are in the same fraction and not detected separately. More advanced columns overcome this problem by constantly monitoring the eluent.
[[Image:Size exclusion standardisation.png|thumb|left|300 px|Standardization (calibration) of a size exclusion column]] [[Image:Chrom SephG-50.tif|thumb|right|300 px|Size exclusion chromatogram after bioanalytical continuous-elution gel chromatography of a plant sample<ref name=":3" />]]
The collected fractions are often examined by [[spectroscopy|spectroscopic techniques]] to determine the concentration of the particles eluted. Common spectroscopy detection techniques are [[refractive index]] (RI) and ultraviolet (UV). When eluting spectroscopically similar species (such as during biological purification), other techniques may be necessary to identify the contents of each fraction. It is also possible to analyze the eluent flow continuously with RI, [[Low-angle laser light scattering|LALLS]], [[Multi-Angle Laser Light Scattering]] MALS, UV, and/or viscosity measurements.

The elution volume (Ve) decreases roughly linear with the [[logarithm]] of the molecular [[hydrodynamic volume]]. Columns are often calibrated using 4-5 standard samples (e.g., folded proteins of known molecular weight), and a sample containing a very large molecule such as thyroglobulin to determine the [[void volume]]. (Blue dextran is not recommended for Vo determination because it is heterogeneous and may give variable results) The elution volumes of the standards are divided by the elution volume of the thyroglobulin (Ve/Vo) and plotted against the log of the standards' molecular weights.