Size-exclusion Another example of a combination is size-exclusionchromatography. SEC bw1 isoften used for (bio)polymers. Something that is unique about SEC LC is, thatthe mobile phase should not have an effect on the separation efficiency.
Itonly depends on the pores in the surfaceof the stationary phase. The volume of the pores typically consists of about40% of the total volume of the column. This means that there is a need for alarge volume of the pores in the packing material. To gain a large pore volume the columns are tall. Another option toimprove separation efficiency is to connect multiple columns in series. EverySEC column has his specific region where it’s viable. This depends on the porevolume of the packing material.
14The mobile phase consists of an organic modifier that’s able to dissolve theanalyte, most likely THF. With SEC, it’s possible to obtain information aboutthe molar weight distribution or molar mass averages. This separation mechanismis based on the size of the molecules.
The column is packed with a porousmaterial, because of these pores, thereis a possibility for retention. Thepacking 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 largermolecules are receiving less retention because they can’t enter the pores likethe smaller molecules can. The larger molecules can only take the shortestroute, passing the pores of the packing material.
The smaller molecules enterevery pore on their way, resulting in alonger route to the end of the column. The SEC chromatogram isinterpreted a bit different than achromatogram from reversed phase chromatography. Every different SEC column hashis own dead volume and size exclusion limit. Depending on dead volume andparticle size, the size exclusion limit Vi changes. Large moleculesthat have no access to the pores at allare eluted at the size exclusion limit.
The size exclusion limit shows up atthe time when the dead volume of the system is flushed with mobile phase. Onthe other end of the chromatogram, there is t0. This is the pointwhere all the molecules that are small enough to enter every pore of thecolumn. This means that the molecules have travelled the longest possible routethrough the column. The molecules with a size between these extreme values willelute between Vi and t0. Because this depends on thecolumn, there’s a need for a correction when two different columns have to becompared. This is fixed by dividing the retention time by the t0.When the analytes have beenseparated, there is a need for a detection of the analytes.
While there aremany different techniques to detect analytes, only a handful are applicable inSEC. Those can be distinguished into two groups. From the first group ofdetectors, the response is determined by the concentration of the analyte inthe mobile phase, e.g. UV/Vis detector orevaporating light scattering detector (ELSD). For the second group ofdetectors, the response relies on the molar mass of the analyte, as well as theconcentration, 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 withthe surface of the stationary phase, but are instead separated based on theirability to penetrate the pores of the packing.
Analytes with a smallerhydrodynamic volume will penetrate into smaller pores than larger analytemolecules, thus experience a larger accessible pore volume and elute later thanlarger molecules. SEC is applied for the analysis and characterization of(bio-)polymers.