Gel permeation chromatography

The gel permeation chromatography (GPC) is a form of liquid chromatography in which solute molecules due to their size (or more precisely of their hydrodynamic volume ) can be separated. Other designations are size-exclusion chromatography, size exclusion chromatography (SEC) and is, physically wrong, even gel filtration chromatography (GFC ) or molecular sieve chromatography. The separation effect is not based on a filtration process, but on different diffusion volumes for different sized molecules. The porous polymers of the stationary phase allow smaller molecules to penetrate, thereby increasing the diffusion volume available to them and thus prolongs the retention time. Small molecules are therefore more strongly retained as large, only those spaces between the polymer granules are accessible and therefore flow more quickly through the column. Thus, large molecules in the earlier fractions of the eluate, while smaller molecules elute later. The stationary phase is usually used porous polymers in granular form. A typical application is the separation of ( polymer fractionation ) of any kind of macromolecules, such as polymers or biopolymers (such as polysaccharides, DNA, RNA and proteins). GPC determined molecular weight distribution curve, which then the average molecular weights (Mn, Mw, Mz ) and the polydispersity of the sample can be calculated. In the area of ​​separation of macromolecules, the field-flow fractionation ( FFF) has become increasingly established as an alternative method.

Building a GPC machine

The essential components of an automated GPC system are pump, injection system, columns and various detectors. The pump draws in the eluent, and generates a constant flow through the entire system. Often the eluent by a Durchlaufentgaser (inline degasser ) is drawn, which removes dissolved gases. After the pump is the injection system for sample application, either manually or by an autosampler. In the following column, the sample is fractionated according to their hydrodynamic radii. The various detectors provide then depending on certain statements. Eventually ends up the whole of the river (including sample) in a waste container. The river ( the eluate ) but can also be absorbed into individual vessels, this is called fractionation ( preparative GPC).

Separation principle

The separation properties of cross-linked dextran were discovered by Jerker Porath in 1957. The columns are filled with powdered particles of a hydrophilic, porous and cross-linked material, such as Sephadex ( a cross-linked dextran and epichlorohydrin ), or Sepharose ( a cross-linked agarose) or silica, and other silicates. The diameter of the particles is in the range of about 3-35 microns. The particles of this opaque gels possess a highly porous surface and depending on the molecule to be separated sizes varying pore sizes of about 60-2000 Å; ( 6-200 nm). Elution is now a sample of molecules of different size. Each very large and very small molecules can not be separated. All molecules that do not fit into the pores elute at the very beginning, and molecules that fit very well into all the pores, at the very end.

Separation columns

Generally there are two types of columns: the so-called single - Porosity columns and the linear -pillars, which are also called mixed- bed columns. The single- Porosity columns have pores with a very small pore size distribution. You can cut very well in a certain size range. To achieve separation of a larger molecular weight, here often three to four columns with different pore sizes are connected in series. In the mixed -bed columns, the column material was then mixed by the manufacturer, that from very small to large pores all pore sizes are represented. These columns separate a large molecular weight range and very linear with the hydrodynamic volume. The separation efficiency of a mixed- bed column is therefore limited, so that here two or three columns are combined in series for a good separation. Today, the mixed- bed columns dominate. The single- Porosity columns but have absolutely justified for special applications.

Detectors

The different variants detection (refractive index, light in the UV / VIS range, viscosity, light scattering, electrical conductivity, radioactivity, ...) must be carefully calibrated to the particular polymer type. As detectors find so-called concentration detectors such as refractive index detector ( RI detector of Engl. Refractive index) and UV detectors ( depending on the UV activity of the analyte polymer) use. In these detectors, the peak area increases in proportion to the concentration, which permits quantification of a substance. In the classical GPC alone this type of detector is used and the system is calibrated for the determination of the molecular masses of standard substances.

Under the synonym of molar mass sensitive detectors continue to find viscosity detectors and light scattering detectors use. This type of detector is used only in combination with concentration detectors because the Molmassenberechnung in each case, the concentration is required. In particular, the light scattering can be independent of polymer standards determine the molecular mass averages ( Mn, Mw, Mz ) and the radius of gyration directly. A distinction is especially multi-angle ( MALS ), small-angle ( LALS ) and right -angle light scattering detector ( RALS ). With the viscosity detector, the parameters K and α of the Mark -Houwink equation, a universal calibration and statements about the conformation of the polymer can be made.

In addition, look for infrared detectors and fluorescence detectors can be installed, but are limited to specific applications.

Conventional calibration

The conventional calibration takes place only when using a concentration detector (RI or UV) use. For calibration usually several different sizes available polymer standards with low polydispersities. From the indicated molecular weights of the standards and the retention time obtained after analysis, you can create the calibration curve. Using the calibration curve, the molecular weights of the unknown sample can now be determined. The result is relative molecular weights, based on the standard substance. Since narrowly distributed standards are not available for each polymer, the Molmassenberechnung can be problematic when using different standards. Although used in such cases as possible "similar" standards that Molmassenberechnung may differ dramatically from the real. A much more secure method would be to use a direct method, here, such as light scattering.

Universal Calibration

GPC using a concentration detector (RI and UV) in conjunction with a Viskositätsdetekor. For calibration available polymer standards with low polydispersities and a calibration log set (molar mass × Intrinsic viscosity). As the product of is proportional to the hydrodynamic radius (molar mass × Intrinsic viscosity), the real or absolute molar masses can thus be calculated.

Light Scattering

By using a light scattering detector setting up a calibration curve is not necessary. A light scattering detector indirectly measures the absolute molecular mass. For evaluation a concentration detector is also required. Of John William Strutt, 3 Baron Rayleigh established equation describes the relationship between the scattered light intensity, which is expressed by the so-called Rayleigh ratio R ( θ ), the polymer concentration, C, and the weight average molecular weight Mw. Where K is an optical constant and A2 the second virial coefficient.

Alternative methods

Occurs in particular in macromolecules, nanoparticles, and agglomeration in the gel permeation chromatography are often problems as a result of interaction with the stationary phase. Clogging of the column, the absorption and removal of agglomerates of the molecules by shearing in the column, problems frequently described. By separating the open river channel without static phase, these effects can be avoided in the field-flow fractionation ( FFF).