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Size-exclusion his own dead volume and size exclusion limit.

Size-exclusion

Another example of a combination is size-exclusion
chromatography. SEC bw1 is
often used for (bio)polymers. Something that is unique about SEC LC is, that
the mobile phase should not have an effect on the separation efficiency. It
only depends on the pores in the surface
of the stationary phase. The volume of the pores typically consists of about
40% of the total volume of the column. This means that there is a need for a
large volume of the pores in the packing material. To gain a large pore volume the columns are tall. Another option to
improve separation efficiency is to connect multiple columns in series. Every
SEC column has his specific region where it’s viable. This depends on the pore
volume of the packing material.14

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The mobile phase consists of an organic modifier that’s able to dissolve the
analyte, most likely THF. With SEC, it’s possible to obtain information about
the molar weight distribution or molar mass averages. This separation mechanism
is based on the size of the molecules. The column is packed with a porous
material, because of these pores, there
is a possibility for retention. The
packing is often based on porous silica or on highly cross-linked organic gels,
that are made out of a copolymer of styrene and divinylbenzene. The larger
molecules are receiving less retention because they can’t enter the pores like
the smaller molecules can. The larger molecules can only take the shortest
route, passing the pores of the packing material. The smaller molecules enter
every pore on their way, resulting in a
longer route to the end of the column.

The SEC chromatogram is
interpreted a bit different than a
chromatogram from reversed phase chromatography. Every different SEC column has
his own dead volume and size exclusion limit. Depending on dead volume and
particle size, the size exclusion limit Vi changes. Large molecules
that have no access to the pores at all
are eluted at the size exclusion limit. The size exclusion limit shows up at
the time when the dead volume of the system is flushed with mobile phase. On
the other end of the chromatogram, there is t0. This is the point
where all the molecules that are small enough to enter every pore of the
column. This means that the molecules have travelled the longest possible route
through the column. The molecules with a size between these extreme values will
elute between Vi and t0. Because this depends on the
column, there’s a need for a correction when two different columns have to be
compared. This is fixed by dividing the retention time by the t0.

When the analytes have been
separated, there is a need for a detection of the analytes. While there are
many different techniques to detect analytes, only a handful are applicable in
SEC. Those can be distinguished into two groups. From the first group of
detectors, the response is determined by the concentration of the analyte in
the mobile phase, e.g. UV/Vis detector or
evaporating light scattering detector (ELSD). For the second group of
detectors, the response relies on the molar mass of the analyte, as well as the
concentration, e.g. mass spectrometer. Typically,
there is a need for at least one concentration detector for SEC LC.

 bw1In SEC, analyte molecules ideally do not interact with
the surface of the stationary phase, but are instead separated based on their
ability to penetrate the pores of the packing. Analytes with a smaller
hydrodynamic volume will penetrate into smaller pores than larger analyte
molecules, thus experience a larger accessible pore volume and elute later than
larger molecules. SEC is applied for the analysis and characterization of
(bio-)polymers.

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