Reversed stationary phase, adsorption

However, Kazakevich and co-workers demonstrated the importance of the adsorption of chaotropic ions onto the reversed stationary phase [106]. The rank of an ion in the Hofmeister series is another measure of its tendency to accumulate at the stationary phase in RP-HPLC and be quantified via its adsorption isotherm [88]. Clearly a specific surface excess of the chaotropic reagent results in the development of a potential difference between the stationary phase and the bulk eluent, modulating retention of analytes [107]. We discussed in Chapter 3 the way a comprehensive theory can take this experimental evidence into account. 

The stationary phase can be inside a tube as a packing or a thin layer (column chromatography) or on a flat surface. The interactions of the components of the mixture depend on the nature of the stationary phase. Adsorption on surfaces, solvation in a liquid layer or solid layer (membrane), and reversible chemical reaction between ionic parts of molecules are examples of interaction mechanisms. 

It is seen, from equation (5), that a graph relating the reciprocal of the corrected retention volume to the concentration of the moderator can provide values for the adsorption/desorption coefficient and the surface area of the stationary phase. Scott and Simpson [1] used this technique to measure the surface area of a reversed phase and the curves relating the reciprocal of the corrected retention volume to moderator concentration are those shown in Figure 2. 

Adsorption of macromolecules has been widely investigated both theoretically [9—12] and experimentally [13 -17]. In this paper our purpose was to analyze the probable structures of polymeric stationary phases, so we shall not go into complicated mathematical models but instead consider the main features of the phenomenon. The current state of the art was comprehensively summarized by Fleer and Lyklema [18]. According to them, the reversible adsorption of macromolecules and the structure of adsorbed layers is governed by a subtle balance between energetic and entropic factors. For neutral polymers, the simplest situation, already four contributor factors must be distinguished ... 

Owing to the weak hydrophobicity of the PEO stationary phases and reversibility of the protein adsorption, some advantages of these columns could be expected for the isolation of labile and high-molecular weight biopolymers. Miller et al. [61] found that labile mitochondrial matrix enzymes — ornitine trans-carbomoylase and carbomoyl phosphate synthetase (M = 165 kDa) could be efficiently isolated by means of hydrophobic interaction chromatography from the crude extract. 

The more dispersive solvent from an aqueous solvent mixture is adsorbed onto the surface of a reverse phase according to Langmuir equation and an example of the adsorption isotherms of the lower series of aliphatic alcohols onto the surface of a reverse phase (9) is shown in figure 9. It is seen that the alcohol with the longest chain, and thus the most dispersive in character, is avidly adsorbed onto the highly dispersive stationary phase, much like the polar ethyl acetate is adsorbed onto the highly polar surface of silica gel. It is also seen that... 

As the solvent concentration increases, the PIC reagents will interact more strongly with the mobile phase and will be less strongly adsorbed on the reverse phase surface. As a consequence, there will be less ion exchange material on the stationary phase surface. This is clearly demonstrated by the adsorption isotherm of octane sulfonate shown in figure 10. 

The form of the effective mobility tensor remains unchanged as in Eq. (125), which imphes that the fluid flow does not affect the mobility terms. This is reasonable for an uncharged medium, where there is no interaction between the electric field and the convective flow field. However, the hydrodynamic term, Eq. (128), is affected by the electric field, since electroconvective flux at the boundary between the two phases causes solute to transport from one phase to the other, which can change the mean effective velocity through the system. One can also note that even if no electric field is applied, the mean velocity is affected by the diffusive transport into the stationary phase. Paine et al. [285] developed expressions to show that reversible adsorption and heterogeneous reaction affected the effective dispersion terms for flow in a capillary tube the present problem shows how partitioning, driven both by electrophoresis and diffusion, into the second phase will affect the overall dispersion and mean velocity terms. 

Tswett s initial column liquid chromatography method was developed, tested, and applied in two parallel modes, liquid-solid adsorption and liquid-liquid partition. Adsorption ehromatography, based on a purely physical principle of adsorption, eonsiderably outperformed its partition counterpart with mechanically coated stationary phases to become the most important liquid chromatographic method. This remains true today in thin-layer chromatography (TLC), for which silica gel is by far the major stationary phase. In column chromatography, however, reversed-phase liquid ehromatography using chemically bonded stationary phases is the most popular method. 

Prus and Kowalska [75] dealt with the optimization of separation quality in adsorption TLC with binary mobile phases of alcohol and hydrocarbons. They used the window diagrams to show the relationships between separation selectivity a and the mobile phase eomposition (volume fraction Xj of 2-propanol) that were caleulated on the basis of equations derived using Soezewiriski and Kowalska approaehes for three solute pairs. At the same time, they eompared the efficiency of the three different approaehes for the optimization of separation selectivity in reversed-phase TLC systems, using RP-2 stationary phase and methanol and water as the binary mobile phase. The window diagrams were performed presenting plots of a vs. volume fraetion Xj derived from the retention models of Snyder, Schoen-makers, and Kowalska [76]. 

Restricted access phases are another approach to exploiting the differences in characteristics of analytes. Large analytes are excluded from an internal surface on which an adsorptive stationary phase is present. A herbicide analysis for Metsulfuron methyl, Bentazone, Bromoxynil, methylchlorophenoxy acid, and Mecoprop in the presence of humic acid was performed on restricted access reversed phase media.52 The cytostatic compound epirubicin and its metabolites were separated from plasma using a Pinkerton GFF II column.53 Gradient separations of polymers on reversed phase and on normal phase represent an alternative to gel permeation chromatography. Polyesters of noncrystalline materials were separated on a variety of such phases.54... 

Membranes offer a format for interaction of an analyte with a stationary phase alternative to the familiar column. For certain kinds of separations, particularly preparative separations involving strong adsorption, the membrane format is extremely useful. A 5 x 4 mm hollow-fiber membrane layered with the protein bovine serum albumin was used for the chiral separation of the amino acid tryptophan, with a separation factor of up to 6.6.62 Diethey-laminoethyl-derivatized membrane disks were used for high-speed ion exchange separations of oligonucleotides.63 Sulfonated membranes were used for peptide separations, and reversed-phase separations of peptides, steroids, and aromatic hydrocarbons were accomplished on C18-derivatized membranes. 

The stationary phases available for HPLC are as numerous as those available for GC. As mentioned previously, however, adsorption, partition, ion exchange, and size exclusion are all liquid chromatography methods. We can therefore classify the stationary phases according to which of these four types of chromatography they represent. Additionally, partition HPLC, which is the most common, is further classified as normal phase HPLC or reverse phase HPLC. Both of these are bonded phase chromatography, which was described in Chapter 11. Let us begin with these. 

Answer the following with normal phase or reverse phase. For which type of liquid chromatography is a C18 column used Which is similar to adsorption chromatography in terms of the polarity of the stationary phase ... 

The most popular and versatile bonded phase is octadecylsilane (ODS), n-C18H37, a grouping that is non-polar and used for reverse phase separations. Octylsilane, with its shorter chain length, permits faster diffusion of solutes and this results in improved peak symmetry. Other groups are attached to provide polar phases and hence perform normal phase separations. These include cyano, ether, amine and diol groups, which offer a wide range of polarities. When bonded stationary phases are used, the clear distinction between adsorption and partition chromatography is lost and the principles of separation are far more complex. 

Two types of system are used for ion-pair liquid chromatography. When polar stationary phase materials, such as silica gel, are used an ion-pair partition mechanism is applied. When non-polar stationary phase materials, such as octadecyl-bonded silica gel and polystyrene gel, are employed a paired-ion adsorption mechanism is involved. The former is called normal-phase ion-pair partition liquid chromatography, and the latter is called reversed-phase ion-pair liquid chromatography. 

Two mechanisms for retention in reversed-phase ion-pair liquid chromatography have been considered. One is the adsorption of the hydrophobic paired ion on the hydrophobic surface of stationary phase material. In the second mechanism, the hydrophobic counter-ion is held on the surface of the hydro-phobic stationary phase, and the analyte ion is retained by ion-ion interactions, as shown in Figure 4.16. In the latter case, of a dynamic ion-exchange... 

Gritti, R and Guiochon, G, Adsorption mechanism in reversed-phase liquid chromatography—effect of the surface coverage of a monomeric C-18-sihca stationary-phase, 7. Chromatogr. A, 1115, 142, 2006. 

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