Chromatographic separation of a mixture of different molecular weights and its applications


Chromatographic separation of a mixture of different molecular weights, also referred to as Size-exclusion chromatography is a method in which molecules in solution are separated by their sizes or molecular weight in some cases. Usually, it is applied to large molecules or macromolecular complexes such as proteins and industrial polymers.
This technique is regarded as gel-filtration chromatography when the medium of transportation of the sample through the column is an aqueous solution, while gel permeation chromatography is used when an organic solvent is used as the mobile phase. It is a widely used polymer characterization process because of its ability to provide good mass distribution results for polymers. 

Theory of Chromatography

Size-exclusion chromatography works by trapping smaller molecules in the pores of the adsorbent materials. This process is usually performed with a chromatography column consisting of a hollow tube tightly  packed with fine, porous beads which are composed of dextran polymers (Sephadex), agarose (Sepharose), or polyacrylamide (Sephacryl or BioGel P) and designed to have pores of different sizes. The pore sizes of these beads are used to estimate the dimensions of macromolecules. They may be depressions on the surface or channels through the bead
Larger particles cannot enter into as many pores. The larger the particles, the faster the elution. The larger molecules simply pass by the pores because those molecules are too large to enter the pores.
Larger molecules therefore flow through the column more quickly than smaller molecules. That is, the smaller the molecule, the longer the retention time.
One requirement for SEC is that the analyte does not interact with the surface of the stationary phases, with differences in elution time between analytes ideally being based solely on the solute volumes the analyte can enter, rather than the chemical or electrostatic interactions with the stationary phases. Thus, a small molecule that can penetrate any region of the stationary phase pore system can enter a total volume equal to the sum of the entire pore volume and the interparticle volume. This small particle elutes late (after the particle has penetrated all of the pore and interparticle volume-approximately 80% of the column volume). At the other extreme, a very large molecule that cannot penetrate any of the smaller pores can enter only the interparticle volume (-35% of the column volume) and elutes earlier when this volume of mobile phase has passed through the column.
The underlying principle of SEC is that particles of different sizes elute through a stationary phase at different rates. This results in the separation of particles based on size. Provided that all the particles are loaded simultaneously or near-simultaneously, particles of the same size should elute together.
Each size exclusion column has a range of molecular weights that can be separated. The exclusion limits defines the molecular weight at the upper end of the column ‘working’ range and is where molecules are too large to get trapped in the stationary phase. The lower end of the range is defined by the permeation limit, which defines the molecular weight of a molecule that is small enough to penetrate all pores of the stationary phase. All molecules below this molecular mass are so small that they elute as a single band.
The filtered solution that is collected at the end is known as the eluate. The void volume includes any particles too large to enter the medium, and solvent volume is known as the column volume.

Application of Chromatography

Gel-filtration is mainly applied in the fractionation of proteins and other water-soluble polymers, while gel permeation chromatography is used to analyze the molecular weight distribution of organic soluble polymers.
Another application is to examine the stability and characteristics of natural organic matter in water.

Advantages of Chromatography

There is good separation of large molecules from the small molecules with a minimal volume of eluate, and that various solutions can be applied without interfering with the filtration process, all while preserving the biological activity of the particles to separate. The technique is generally combined with others that further separate molecules by other characteristics, such as acidity, basicity, charge and affinity for certain compounds. With SEC, there are short and well-defined separation times and narrow bands, which lead to good sensitivity. There is also no sample loss because solutes do not interact with the stationary phase.
Also in certain cases, it is feasible to determine the approximate molecular weight of a compound. The shape and size of the compound (eluent) determine how the compound interacts with the gel(stationary phase).
In separating mixture of different polarities, a thin-layer chromatography method or column chromatography method is employed. In those methods, two phases are involved;

  1. stationary phase
  2. mobile phase

Silica gel or kiesel gel is commonly used in stationary phase, which can be either a normal phase chromatography or a reverse phase chromatography. In normal phase, the silica gel is polar and therefore the mobile phase used must be non polar or semi-polar whereas in reverse phase chromatography, the silica gel is changed into a phenyl-n-hexane that is adding an alkyl group e.g. Si-C18.

Thin layer chromatography

Using A Normal Phase chromatography:
Thin layer chromatography is an analytical technique for easy identification of compounds in a compound mixture. The TLC plates are prepared with the stationary phase i.e. applying the silica gel on the surface of the plate. This is then allowed to dry and stabilize. With a pencil, a thin mark is made at the bottom of the plates to apply the sample spots. The sample is applied in the spots. The mobile phase is then poured in the TLC chamber and the TLC plates immersed into it too. The chamber is then closed with a lid. Sufficient time is given to dry and viewed in a suitable UV light chamber. Since it’s a normal phase chromatography, the most non polar component comes out first while the most polar comes out last. This means that the compound with high affinity to the stationary phase travels slowly while others travel faster. It also means the larger the retention factor, the less polar (most non polar) the component is.

Using A Reverse Phase Chromatography:

It follows the same principle as the normal phase chromatography just that the silica gel is reversed. This is done by dipping the silica gel in phenyl-n-hexane. This makes the silica gel to be non polar and therefore the first component is a polar component while the last is non-polar

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