Adsorption and Exclusion Chromatography

The dissolved solute molecules X compete with mobile phase molecules S for a place on the adsorbent surface  

The strength of the interaction between the adsorbent and the solute molecules increases as the polarity of the solute increases. Thus we can increase retention of our solute molecules (X) by decreasing the polarity of the mobile phase (S), which will shift the equilibrium above to the right. Polar solute molecules are strongly held on unmodified silica and tail badly, so the method is useful only for solutes having low or medium polarity. 

II (a) In what order would you expect the other solutes to elute  

Column Mobile phase Flow rate Detector Sample  

Exclusion chromatography is a technique for separating molecules based on their effective size and shape in solution. The technique is often called gel permeation chromatography if used with organic solvents or gel filtration if used with aqueous solvents. 

The stationary phases used in exclusion chromatography are porous particles with a closely controlled pore size. Unlike other chromatographic modes, in exclusion chromatography there should be no interaction between the solute and the surface of the stationary phase. 

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Adsorption Chromatography 25-4 Liquid-Liquid Chromatography 25-5 Gel and Exclusion Chromatography 25-6 Thin-Layer and Paper Chromatography 25-7 Gas Chromatography 25-8 Identification and Measurement References Problems... 

Interest in countercurrent chromatography was revived in an effort to overcome problems frequently encountered in adsorption or exclusion chromatography, which uses a solid support. Most prominent among these problems was the difficulty in resolving mixtures of sensitive or highly polar compounds. Irreversible adsorption onto the solid phase, solid phase catalyzed decomposition, especially with silica gel, and severe tailing are obstacles to the purification of such molecules. 

Another example of vims clearance is for IgM human antibodies derived from human B lymphocyte cell lines where the steps are precipitation, size exclusion using nucleases, and anion-exchange chromatography (24). A second sequence consists of cation-exchange, hydroxylapatite, and immunoaffinity chromatographies. Each three-step sequence utilizes steps based on different properties. The first sequence employs solubiUty, size, and anion selectivity the second sequence is based on cation selectivity, adsorption, and selective recognition based on an anti-u chain IgG (24). 

The total stationary-phase volume required to process a given feed stream is proportional to the inlet concentration and volume of the feed. For example, for a typical inlet concentration of protein of 10 g/L, in a 100 L volume of feed, a column volume of at least 100 L is needed for size-exclusion chromatography. In comparison, an ion-exchange column having an adsorption capacity of 50 g/L would only require 20 L of column volume for the same feed. 

The stationary phase in gel permeation (also called size exclusion) chromatography contains cavities of a defined size distribution, called pores. Analytes larger than the pores are excluded from the pores and pass through the column more rapidly than smaller analytes. There may be secondary effects due to hydrophobic adsorption, ionic interaction, or other interactions between the stationary phase and analyte. Gel permeation and non-ideal interactions in gel permeation are described more fully in Chapter 6. 

Ng, K.L., Pauli, B., Haddad, P.R., and Tanaka, K., Retention modeling of electrostatic and adsorption effects of aliphatic and aromatic carboxylic acids in ion-exclusion chromatography, /. Chromatogr. A, 850, 17, 1999. 

In exclusion chromatography, the total volume of mobile phase in the column is the sum of the volume external to the stationary phase particles (the void volume, V0) and the volume within the pores of the particles (the interstitial volume, Vj). Large molecules that are excluded from the pores must have a retention volume VQ, small molecules that can completely permeate the porous network will have a retention volume of (Vo + Fj). Molecules of intermediate size that can enter some, but not all of the pore space will have a retention volume between VQ and (V0 + Fj). Provided that exclusion is the only separation mechanism (ie no adsorption, partition or ion-exchange), the entire sample must elute between these two volume limits. 

In each chromatographic technique, one of the four mechanisms predominates, but it should be emphasized that two or more may be involved simultaneously. Partition and adsorption frequently occur together and in paper chromatography, for example, ion-exchange and exclusion certainly play minor roles also. 

Although the overwhelming majority of theoretical papers in liquid chromatography are dealing with the various aspects of RP-HPLC separation, theoretical advances have also been achieved in some other separation modes. Thus, a theoretical study on the relation between the kinetic and equilibrium quantities in size-exclusion chromatography has been published, hi adsorption chromatography the probability of adsorbing an analyte molecule in the mobile phase exactly r-times is described by... 

A variety of procedures were utilized to analyze this reaction mixture and to characterize a,10-diaminopolystyrene. Thin layer chromatographic analysis using toluene as eluent exhibited three spots with Rf values of 0.85, 0.09, and 0.05 which corresponded to polystyrene, poly(styryl)amine and a,w-diaminopolystyrene (see Figure 1). Pure samples of each of these products were obtained by silica gel column Chromatography of the crude reaction mixture initially using toluene as eluent [for polystyrene and poly(styryl)amine] followed by a methanol/toluene mixture (5/100 v/v) for the diamine. Size-exclusion chromatography could not be used to characterize the diamine since no peak was observed for this material, apparently because of the complication of physical adsorption to the column packing material. Therefore, the dibenzoyl derivative (eq. 5) was prepared and used for most of the analytical characterizations. 

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