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Size-exclusion

Another example of a
combination is size-exclusion chromatography. SEC bw1 is
often used for (bio)polymers. This separation technique is based on the size of
the analytes. The separation only depends on the stationary phase, unlike other
chromatographic techniques. To be more precise, the separation only occurs in
the pores in the packaging material. This
is typically the reason why SEC columns are tall, allowing for a higher total
pore volume. To improve the separation efficiency, even more, there is the option to put multiple columns in a series. The mobile phase has no influence at all
on the separation efficiency. The stationary phase is often based on porous
silica or on a polymer made out of styrene and divinylbenzene.
The mobile phase consists of an organic
modifier that’s able to dissolve the analyte, most likely THF. 14

With SEC, it is possible to
obtain information about the molar weight distribution or molar mass averages. 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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