Chromatographic Separation Mechanism and column selectivity
Chromatographic techniques are based on four different sorption mechanisms, surface adsorption, partition, ion exchange and size exclusion.
Surface Adsorption Chromatography
The separation mechanism depends upon differences in polarity between the different feed components. The more polar a molecule, the more strongly it will be adsorbed by a polar stationary phase. Similarly, the more non-polar a molecule, the more strongly it will be adsorbed by non-polar stationary phase.
During a surface adsorption chromatography process, there is competition for stationary phase adsorption sites, between the materials to be separated and the mobile phase. Feed molecules of low polarity spend proportionally more time in the mobile phase than those molecules that are highly polar, which are retained longer. Therefore the components of a mixture are eluted in order of increasing polarity.
Almost any polar solid can be employed as a polar stationary phase. The choice of stationary phase is governed by the polarity of the feed components. If the feed components are adsorbed too strongly, they may be difficult to remove. Weakly polar mixtures should be separated on highly active absorbents, or little or no separation will occur.
The choice of mobile phase is equally important. The polarity of the mobile phase should be chosen to compliment the choice of stationary phase. In general, good separation is achieved by using fairly polar stationary phases and low polarity mobile phases such as hexane. Water, it should be noted, is a very polar solvent.
The 2 most common adsorbents used in chromatography are porous alumina and porous silica gel. Of lesser importance are carbon, magnesium oxide, and various carbonates. Alumina is a polar adsorbent and is preferred for the separation of components that are weakly or moderately polar, with the more polar components retained more selectively by the adsorbent, and therefore eluted from the column last. In addition, alumina is a basic adsorbent, thus preferentially retaining acidic compounds. Silica gel is less polar than alumina and is an acidic adsorbent, thus preferentially retaining basic compounds. Carbon is a non-polar (apolar) stationary phase with the highest attraction for larger non-polar molecules.
Adsorbent-type sorbents are better suited for the separation of a mixture on the basis of chemical type (e.g. olefins, esters, acids, aldehydes, alcohols) than for separation of individual members of a homologous series. Partition chromatography is often preferred for the latter, wherein an inert solid (often silica gel) is coated with a liquid phase
Hydrophobic interaction chromatography (HIC) is a special form of surface adsorption chromatography. The materials to be separated should be at least partially hydrophobic in nature. Separation is facilitated by differences in the relative strength of interaction between these materials and a matrix substituted with suitably hydrophobic groups. This type of process is extensively used for the preparative-scale separation of proteins.
Unique to chromatography is the liquid-supported or liquid-bonded solids, where the mechanism is absorption into the liquid, also referred to as a partition mode of separation or partition chromatography. With mobile liquid phases, there is a tendency for the stationary liquid phase to be stripped or dissolved. Therefore, the stationary liquid phase has to be chemically bonded to the solid bonding support.
In partition chromatography, the stationary liquid phase is coated onto a solid support such as silica gel, cellulose powder, or kieselguhr (hydrated silica). Assuming that there is no adsorption by the solid support, the feed components move through the system at rates determined by their relative solubilities in the stationary and mobile phases.
In general, it is not necessary for the stationary and mobile phases to be totally immiscible, but a low degree of mutual solubility is desirable. Hydrophilic stationary phase liquids are generally used in conjunction with hydrophobic mobile phases (referred to as “normal-phase chromatography”), or vice versa (referred to as a ‘”reverse- phase chromatography”).
Suitable hydrophilic mobile phases include water, aqueous buffers and alcohols. Hydrophobic mobile phases include hydrocarbons in combination with ethers, esters and chlorinated solvents.
Ion Exchange Chromatography (IEC)
In this process, the stationary phase consists of an insoluble porous resinous material containing fixed charge-carrying groups. Counter-ions of opposite charge are loosely complexed with these groups.
Passage of a liquid mobile phase, containing ionised or partially ionised molecules of the same charge as the counter-ions through the system, results in the reversible exchange of these ions.
The degree of affinity between the stationary phase and feed ions dictates the rate of migration and hence degree of separation between the different solute species.
The most widely used type of stationary phase is a synthetic copolymer of styrene and divinyl benzene (DVB), produced as very small beads in the micrometer range. Careful control over the amount of DVB added dictates the degree of cross-linking and hence the porosity of the resinous structure.
Resins with a low degree of cross-linking have large pores that allow the diffusion of large ions into the resin beads and facilitate rapid ion exchange. Highly cross- linked resins have pores of sizes similar to those of small ions.
The choice of a particular resin will very much be dependent upon a given application. Cation (+) or anion (-) exchange properties can be introduced by chemical modification of the resin.
Ion exchange chromatography has found widespread uses in industrial processes. This technique is used in the separation of transition metals, the removal of trace metals from industrial effluents and in the purification of a wide range of organic compounds and pharmaceuticals. The resin matrix is usually relatively inexpensive when compared with other types of stationary phase. Ion exchange chromatography is probably the most widely used large-scale chromatographic process, but is limited to ionisable, water soluble molecules.
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Size Exclusion Chromatography (SEC)
In this process, also known as gel permeation chromatography, molecules of a feed material are separated according to their size or molecular weight. The stationary phase consists of a porous cross-linked polymeric gel.
The pores of the gel vary in size and shape such that large molecules tend to be excluded by the smaller pores and move preferentially with the mobile phase. The smaller molecules are able to diffuse into and out of the smaller pores and will thus be retarded in the system.
The very smallest molecules will permeate the gel pores to the greatest extent and will thus be most retarded by the system.
The components of a mixture therefore elute in order of decreasing size or molecular weight.
The stationary phase gels can either be hydrophilic for separations in aqueous or polar solvents, or hydrophobic for use with non-polar or weakly-polar solvents. Sephadex, a cross-linked polysaccharide material available in bead form, is widely used with polar/hydrophilic mobile phases. The degree of cross-linking can be varied to produce beads with a range of pore sizes to fractionate samples over different molecular weight ranges. Hydrophobic gels are made by cross-linking polystyrene with DVB and are therefore similar to ion exchange resins but without the ionic groups.
SEC is used extensively in the biochemical industry to remove small molecules and inorganic salts from valuable higher molecular weight products such as peptides, proteins and enzymes.
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Knowledge Tools used in Chromatograph
THE growth driver for chromatography/adsorption technology is the creation and availability of novel molecules. An other driver are more stringent conditions as regards food safety and new production methods.
We humans suffers about 7000 different forms of diseases of which approx 1000 have been addressed by pharmaceutical and medtech products. The remaining 6000 diseases may be described as rare or orphane diseases. (Rare diseases affect approx. 1 in 2000 persons). Thus the market volume for novel pharmaceutical is smaller, the opportunity for large profits declining and the pressure for cost reduction growing. Traditional operational managers try to reduce cost by reducing choice of materials to be procured and by automating processes. This strategy works in commodity industries but not in the highly complex live science and health product sector.
In the past 10 years we experienced a decline in output of novel small molecule drugs produced by organic chemistry. In contrast molecules produced by biological means experienced a healthy growth during the same period.. However, since about two years we observe a revival in discovery of small molecules. This should lead in future to a healthy growth in the pharmaceutical sector. This will lead to new job opportunities for chromatographers and pressures to reduce development costs. The most successful way to reduce cost is to increase speed to market. We can assist by providing help to Method Developers and to supply competitive process chromatography system. We are determined to make chromatography a competitive purification technology that can compete with re-crystallisation and distillation.
There are about 4000 different brand of columns/chromatography materials in the market. They survive because everyone of them has unique properties and applications. Since many years various groups are trying to quantifying the “chromatographic properties” of stationary phases. The Tanaka* test is established worldwide as industrial standard test which assesses selectivity and performance differences between HPLC columns. These column parameters should be known for effectively choosing the appropriate HPLC column for a particulate separation and allow comparing columns easily.
A set of seven selected substances is used to describe capacity, hydrophobicity, steric selectivity, and silanophilic properties. To facilitate the illustration and to recognize the quality of a sorbent at one glance, the values of these parameters are outlined on the six axes of a hexagon. The more symmetrical the hexagon appears and the larger its area, the more balanced the stationary phase is in the sum of its chromatographic properties. (* Prof. Tanaka, Kyoto Institute of Technology, J. of Chrom. Sci. 27, 721, 1989). After Tanaka a number of other researcher have advanced tests based on new discoveries in chromatography (See N.S. Wilson, M.D. Nelson, J.W. Dolan, L.R. Snyder, R.G. Wolcott, and P.W. Carr, J. Chromatogr. A 961, 171–193 (2002). and M.R. Euerby and P. Petersson, J. Chromatogr. A 994, 13–36 (2003).) Today the following parameters are considered to be vital in sationary phase characterisation
HR = hydrophobic retention (retention based on a hydrophobic probe)
HS = hydrophobic selectivity (ability to discriminate between probes of similar hydrophobicity (hydrophobic selectivity)
SS = steric selectivity (ability to discriminate between analytes of different shape or hydrodynamic volume (shape or steric selectivity)
HBC = hydrogen bonding capacity(extent of hydrogen bonding with acids or bases (typically via the silanol surface, polar end capping reagents, or functional groups within the bonded ligand)
BA = base activity;
C = chelation;
IEX = ion-exchange capacity at pH 2.6 ( extent of ion-exchange interactions at low pH 2.8 is typical to differentiate between situations in which surface silanol species will be potentially ionized or ion suppressed)
IEX = ion-exchange capacity at pH 7.6 (extent of ion-exchange interactions at mid pH 7.0 is typical to differentiate between situations in which surface silanol species will be potentially ionized or ion suppressed)
AI = acid interaction
The problem today is that chromatography results are also dependent on application requirements (e.g. process vs analytical chromatography) and differences in equipment configurations.
seen as vital factors influencing a parameters Unfortunately, all tests were focussed on small molecules.
•ability to discriminate between probes of similar hydrophobicity (hydrophobic selectivity)
•extent of hydrogen bonding with acids or bases (typically via the silanol surface, polar end capping reagents, or functional groups within the bonded ligand)
The Tanaka Test has become the standart
The first step in Method Development is to
Characterise your sample molecules (Types, Functionalities, Solubility etc. )
Define what you aim to achieve.