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SEC-GPC Columns
SEC columns and GPC columns for size exclusion chromatography and gel permeation chromatography are used for the high-performance separation and characterisation of macromolecules such as polymers and biomolecules. At MZ-Analysentechnik GmbH, we offer an extensive range of GPC columns and SEC columns designed to help you perform precise and reliable analyses and gain deeper insights into the structure and properties of your samples.
Our in-house MZ-Gel SDplus GPC columns have been used in analytical laboratories worldwide for more than 30 years and are characterised by the highest quality at a moderate price. The bulk media and chromatography columns are manufactured in our own production facility under strict quality controls to ensure that they meet the highest standards. We also offer GPC and SEC columns from renowned manufacturers such as Sepax, Tosoh, Shodex and others.
Technical Data
Which parameters influence my SEC separation?
Different parameters have varying degrees of influence on SEC separation. The most important parameters are the pore size (defines the molecular weight range) and the type of stationary phase (defines the solvents that can be used). The first choice in an SEC separation is the choice of the solvent that should or must be used. In the following, the most important parameters of an SEC column will be described in more detail and their influence on the separation will be considered.
Influence of the column dimension
How does the length of the column influence the separation?
As with HPLC separations, a longer column extends the retention time but also increases the number of plates. With increasing length, the loadability of the column also increases, as more material is available. In general, the longer a column is ...
- the longer the retention time/running time.
- the higher the soil number.
- the higher the dissolution.
- the higher the loadability.
- the higher the back pressure.
How does the inner diameter of the column influence the separation?
The standard diameter of an SEC column is approx. 8 mm and is larger compared to HPLC columns. This has become established as separation using SEC is based solely on diffusion into the available intramolecular volumes (particle volumes) and columns with larger internal diameters therefore also provide better separation. With larger internal diameters, larger volumes are available for diffusion. For narrower columns, 4.6 mm is usually used, whereby the sample volume should also be reduced accordingly. In general, the larger the inner diameter of an SEC column ...
- the higher the resolution tends to be.
- the higher the loadability.
How do I choose the right combination of length and inner diameter?
The above explanations of the column dimensions in the SEC indicate a preferred dimension. The longer the column, the higher the resolution and the larger the internal diameter, the greater the resolution tends to be. A standard dimension of 300x8.0 mm has therefore become established (some manufacturers also offer an internal diameter of 7.5 mm, 7.7 mm or 7.8 mm). However, columns with a length of 600 mm are also available from some manufacturers, which can further improve the resolution.
Since the selectivity or the interaction in the SEC does not play a role, the resolution can only be improved by either changing other parameters such as temperature or flow rate or by lengthening the column or achieving a higher plate number. It is therefore also common to connect several columns in series in order to obtain a longer column.
How do I choose the right packaging material?
Firstly, SEC must be divided into its two sub-areas. If aqueous solvents are used, this is generally referred to as GFC (gel filtration chromatography). If organic solvents such as THF are used, the term GPC (gel permeation chromatography) is used. The choice of method depends entirely on the solubility of the analytes. Depending on which solvent is chosen, there is a different selection of possible materials.
Packaging materials for the GFC
A typical application of the GFC is the separation of proteins. Silica columns are favoured for proteins as they have a narrower pore size distribution than polymers and are also more stable under pressure and do not swell or shrink in different solvents. Due to the narrower pore size distribution, a higher resolution can be achieved in a certain molecular weight range, but at the same time this molecular weight range is smaller compared to polymer columns. Polymer columns can have larger pores than silica materials, so polymer materials are used for extremely large water-soluble molecules or polymers. However, these are not as pressure-stable and can swell and shrink in various solvents and thus also change the pore size. Due to the wider pore size distribution, the resolutions are also poorer but a larger molecular range is covered.
| Silica | polymer |
| + Pressure stable | - no high pressure stability |
| + Narrow pore size distribution for high resolution | - wide pore size distribution for lower resolution |
| + Dimensionally stable | - Can swell or shrink (depending on the solvent) |
| - narrow pore size distribution restricts molecular weight range | + wide pore size distribution offers larger molecular weight range |
| - Pore size restricted | + very large pores available for large polymers |
Packing materials for GPC
Hydrophobic packing materials such as polystyrene-divinylbenzene (PS/DVB) polymers are used in GPC. The most common solvent is THF. However, other solvents such as chloroform or DMF can also be used. The compatibility of the polymer with the desired solvent is important when choosing the right column in relation to the material. Not every column of every pore size can be operated with every solvent. Therefore, with more specialised solvents, always check whether the selected column can also be used with this solvent. If in doubt please contact us! We will be happy to help you.
How does the pore size influence my separation?
The pore size is the most important parameter for SEC columns. The pore size determines the molecular weight range of the column. As a rule, each column has a specific linear range in which it can be used. The larger the pore, the larger the mass range and the higher the molecular weight that can be separated. The choice of the appropriate pore size depends on the expected masses of the analytes and should be selected accordingly.
When making the selection, it is recommended that the molecular weight range of the column is as small as possible for the analytes in order to increase the resolution. However, if very small and very large molecules are to be analysed in one run, there are two options:
- The use of so-called linear columns. Here, particles with different pore sizes are packed into one column in order to increase the linear range of a column (however, the resolution is quite low and should only be used for very different masses).
- The connection of several columns of different pore sizes in series. Here, analytes of a large mass range can be separated from each other with high resolution. This is the standard method used in many laboratories. The columns are run through from small to large pores. One disadvantage of this method is the long run time.
How does the particle size influence my separation?
As with HPLC columns, the smaller the particles, the higher the plate number and the higher the back pressure. The pore size also plays a role in the choice of particle size. Above a certain point, it is no longer possible to produce smaller particles with larger pores. Therefore, materials with large pores often have larger particles. From an analyte or polymer weight of more than 1,000,000 Da, shear forces also play a role. The analytes can be torn into smaller pieces by the forces.
It is therefore advisable to analyse large analytes with large pores and large particles. Small analytes can be safely separated with small pores and small particles.
Other parameters that can influence the separation
Although other parameters primarily have nothing to do with the column, they can still influence the SEC separation. However, the selected column defines the framework within which these parameters can be varied.
Temperature
In general, temperature does not play a major role in the selectivity of SEC columns. The peaks shift only slightly at higher temperatures. However, the peaks become narrower, which means that a higher resolution can be achieved. In addition, the viscosity of the mobile phase is also reduced, which can lead to a lower back pressure and therefore longer column lifetimes.
Flow rate
As separation mainly occurs by diffusion into the pores, a lower flow rate can lead to narrower peaks. In addition, a lower flow rate also reduces the shear forces, which can lead to better analyses for very large analytes. As the diffusion paths for larger particles are longer than for smaller particles, the effect of a lower flow rate is particularly noticeable for larger particles.
Organic modifiers
Organic modifiers in the mobile phase are mainly used with biological samples to reduce the hydrophobic interactions between proteins and the stationary phase. This minimises secondary interactions and the peaks become narrower. The maximum concentration of organic modifier in the mobile phase depends on the stability of the analytes and the stationary phase. Therefore, the permissible concentration of organic solvents on a column should be looked up beforehand
In general, the selectivity of an SEC column is difficult to influence. The choice of the right column is therefore extremely important. We will be happy to help you select the best column for your separation. Please contact us! Further information on separation technology can be found either at Aqueous size exclusion chromatography (GFC) or Organic Size Exclusion Chromatography (GPC) can be found.
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