Stationary phase modification

To obtain a simple interpretation of the experimental findings in IIC, theoretical chromatographers first adopted a stoichiometric strategy that pioneered this separation mode. Unfortunately, the reaction schemes of stoichiometric models in both the mobile phase (ion pair model) and stationary phase (dynamic ion exchange model) lack a firm foundation in physical chemistry because they are not able to account for the stationary-phase modification that results from the addition of the HR to the eluent, and they fail to properly describe experimental results, as pointed out by Bidlingmeyer et al. " Key insights on these retention models were also provided by... 

Rapid separations are clearly desirable where sufficient selectivity exists for the particular component(s) of interest. The low pressure drop across capillary SFC columns allows the solvating properties of the fluid to be controlled by changing the pressure on a nearly instantaneous basis ( ). Additional advantages are related to the ease of pressure control and the absence of stationary phase modification which decreases reproducibility in HPLC. 

Stationary Phase Modification to Improve the Separation of Naproxen Enantiomer... 

Borgerding MF, Hinze WL, Stafford LD, Fulp GW Jr, and Hamlin WC Jr (1989) Investigation of stationary phase modification by the mobile phase surfactant in micellar liquid chromatography. Analytical Chemistry 61 1353-1358. 

The last modification in the Rayleigh integral concerns the delay of propagation between the two points which is simply the time taken by the energy to propagate along the path of stationary phase. It is denoted T and given by,... 

However, the same changes in the mobile phase will also modify the interactions of the solute with the mobile phase, and the effect on solute retention can be as great or even greater than the modification of the stationary phase. Changes in the interactive character of the stationary phase usually occur at relatively low concentrations of... 

In the first version with a mobile phase of constant composition and with single developments of the bilayer in both dimensions, a 2-D TLC separation might be achieved which is the opposite of classical 2-D TLC on the same monolayer stationary phase with two mobile phases of different composition. Unfortunately, the use of RP-18 and silica as the bilayer is rather complicated, because the solvent used in the first development modifies the stationary phase, and unless it can be easily and quantitatively removed during the intermediate drying step or, alternatively, the modification can be performed reproducibly, this can result in inadequate reproducibility of the separation system from sample to sample. It is therefore suggested instead that two single plates be used. After the reversed-phase (RP) separation and drying of the plate, the second, normal-phase, plate can be coupled to the first (see Section 8.10 below). 

The most widely used columns contain a chemically modified silica stationary phase, with the chemical modification determining the polarity of the column. A... 

Mobile phases are of a greater variety than the restricted number of stationary phases. Many solvents and their mixtures are used as a mobile phase. The possibility of slight modification of solvent proportions in a mixmre permits the increase of mobile phase number and, thus, different results in the component separation of the analyzed sample. That is why the optimum mobile phase selection becomes one of the basic operations for the success of the analysis. 

B. Waiczak, L. Morin-Allory, M. Chrdtien, M. Lafosse and M. Dreux, Factor analysis and experiment design in high-performance liquid chromatography. III. Influence of mobile phase modifications on the selectivity of chalcones on a diol stationary phase. Chemom. Intell. Lab. Syst., I (1986) 79-90. 

Solutions to the above problea are required if efficient open tubular colunns are to be prepared. The energy of the saooth glass surface can Sse Increased by roughening or chemical Modification, or the surface tension of the stationary phase can be lowered by the addition of a surfactant. Roughening and/or cheMical modification etre the most widely used techniques for column preparation the addition of a surfactant, although effective, modifies the separation properties of the stationary phase and may also limit the thermal sted>ility of columns prepared with high temperature stable phases. 

Surface modification reactions are used to improve the wettability of glass surfaces by polar stationary phases and to Improve the extent of deactivation by sllylation" [138-146,166]. Miaaiuua procedures have been investigated but only a few are in use. Of these, the most important reactions are etching by hydrogen chloride, leaching with aqueous hydrochloric acid, formation of whiskers and solution deposition of a layer of solid particles. Because of the high purity and thinness of the... 

Chemical surface modifications The first surface modification for the purpose of eliminating EOF and protein adsorption was recommended by Hjerten.28 The attachment of vinyl silanes allowed the polymerization of a variety of molecules to the surface. Most of the chemical modifications used for preparing capillaries for electrophoresis originated from the experience acquired over the years preparing GC and LC stationary phases. Chemical modification should conform to certain requirements, including the prevention of adsorption, the provision of stable and constant EOF over a wide pH range, chemical stability, ease of preparation, and reproduciblity of preparation. The effects of silanization of the inner surface of capillaries on electrophoretic separations have been extensively studied.26-29... 

In gc there is only one phase (the stationary liquid or solid phase) that is available for interaction with the sample molecules. Because the mobile phase is a gas, all sample vapours are soluble in it in all proportions. In hplc both the stationary phase and the mobile phase can interact selectively with the sample. Interactions such as complexation or hydrogen bonding that are absent in the gc mobile phase may occur in the hplc mobile phase. The variety of these selective interactions can also be increased by suitable chemical modification of the silica surface, hence hplc is a more versatile technique than gc, and can often achieve more difficult separations. 

Reverse-phase HPLC (RP-HPLC) separates proteins on the basis of differences in their surface hydophobicity. The stationary phase in the HPLC column normally consists of silica or a polymeric support to which hydrophobic arms (usually alkyl chains, such as butyl, octyl or octadecyl groups) have been attached. Reverse-phase systems have proven themselves to be a particularly powerful analytical technique, capable of separating very similar molecules displaying only minor differences in hydrophobicity. In some instances a single amino acid substitution or the removal of a single amino acid from the end of a polypeptide chain can be detected by RP-HPLC. In most instances, modifications such as deamidation will also cause peak shifts. Such systems, therefore, may be used to detect impurities, be they related or unrelated to the protein product. RP-HPLC finds extensive application in, for example, the analysis of insulin preparations. Modified forms, or insulin polymers, are easily distinguishable from native insulin on reverse-phase columns. 

In order to avoid tedious procedures required to prepare packed CEC columns, some groups are studying the use of empty capillaries. Since solute-stationary phase interactions are key to the CEC process, appropriate moieties must be bound to the capillary wall. However, the wall surface available for reaction is severely limited. For example, a 100 pm i.d. capillary only has a surface area of 3xl0 4m2 per meter of length, with a density of functional sites of approximately 3.1 xlO18 sites/m2, which equals 0.5 pmol sites/m2. Moreover, surface modification cannot involve all of the accessible silanol groups, since some must remain to support the EOF. As a result, the use of bare capillaries in CEC has been less successful. 

Interestingly, in HPLC the stationary phase and the mobile-phase is able to interact with the sample selectively. Besides, such interactions as hydrogen bonding or complexation which are absolutely not possible in the GC-mobile phase may be accomplished with much ease in the HPLC-mobile phase. Furthermore, the spectrum of these selective interactions may also be enhanced by an appropriate chemical modification of the silica surface the stationary phase. Therefore, HPLC is regarded as a more versatile technique than GC and capable of achieving more difficult separations. 

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