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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.
In 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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