Mobile phase characteristics

Mobile-Phase Characteristics in the Reversed-Phase Chromatography of... 

Fortunately, the effects of most mobile-phase characteristics such as the nature and concentration of organic solvent or ionic additives the temperature, the pH, or the bioactivity and the relative retentiveness of a particular polypeptide or protein can be ascertained very readily from very small-scale batch test tube pilot experiments. Similarly, the influence of some sorbent variables, such as the effect of ligand composition, particle sizes, or pore diameter distribution can be ascertained from small-scale batch experiments. However, it is clear that the isothermal binding behavior of many polypeptides or proteins in static batch systems can vary significantly from what is observed in dynamic systems as usually practiced in a packed or expanded bed in column chromatographic systems. This behavior is not only related to issues of different accessibility of the polypeptides or proteins to the stationary phase surface area and hence different loading capacities, but also involves the complex relationships between diffusion kinetics and adsorption kinetics in the overall mass transport phenomenon. Thus, the more subtle effects associated with the influence of feedstock loading concentration on the... 

The development of a chromatographic procedure for an unknown sample (mixture) requires the selection of a variety of experimental conditions (type and composition of the mobile phase, characteristics of the column and the stationary phase, temperature, flow-rate, pressure, type of gradient, etc.). This problem was traditionally solved in an empirical way and with the aid of the vast literature on similar situations already dealt with. The last few years have seen attempts at the rationalization and automation of the optimization of chromatographic processes which have resulted In Interesting systematic approaches of great use. The monographs by Berridge [56], devoted to HPLC, and that by Shoenmakers [57], which deals with both HPLC and GC, represent the most systematic and complete compilations in this field at present. 

Two variations of the technique exists isocratic elution, when the mobile phase composition is kept constant, and gradient elution, when the mobile phase composition is varied during the separation. Isocratic elution is often the method of choice for analysis and in process apphcations when the retention characteristics of the solutes to be separated are similar and not dramaticallv sensitive to vei y small changes in operating conditions. Isocratic elution is also generally practical for systems where the equilibrium isotherm is linear or nearly hnear. In all cases, isocratic elution results in a dilution of the separated produces. 

SERS has also been applied as a sensitive, molecule-specific detection method in chromatography, e.g. thin layer, liquid, and gas chromatography. SERS-active colloids were deposited on the thin layer plates or mixed continuously with the liquid mobile phases. After adsorption of the analytes, characteristic spectra of the fractions were obtained and enabled unambiguous identification of very small amounts of substance. 

The theoretical treatment given above assumes that the presence of a relatively low concentration of solute in the mobile phase does not influence the retentive characteristics of the stationary phase. That is, the presence of a small concentration of solute does not influence either the nature or the magnitude of the solute/phase interactions that determine the extent of retention. The concentration of solute in the eluted peak does not fall to zero until the sample volume is in excess of 100 plate volumes and, at this sample volume, the peak width has become about five times the standard deviation of the normally loaded peak. 

The hydrophilic surface characteristics and the chemical nature of the polymer backbone in Toyopearl HW resins are the same as for packings in TSK-GEL PW HPLC columns. Consequently, Toyopearl HW packings are ideal scaleup resins for analytical separation methods developed with TSK-GEL HPLC columns. Eigure 4.44 shows a protein mixture first analyzed on TSK-GEL G3000 SWxl and TSK-GEL G3000 PWxl columns, then purified with the same mobile-phase conditions in a preparative Toyopearl HW-55 column. The elution profile and resolution remained similar from the analytical separation on the TSK-GEL G3000 PWxl column to the process-scale Toyopearl column. Scaleup from TSK-GEL PW columns can be direct and more predictable with Toyopearl HW resins. 

The characteristics of the sorbent in the precolumn may lead to problems when coupling the two systems. Therefore, when the analytes are more retained in the precolumn than in the analytical column, peak broadening may appear, even when the analytes are eluted in the backflush mode (40). This has been solved with a special design in which the analytes retained in the precolumn are eluted with only the organic solvent of the mobile phase and the corresponding mobile phase is subsequently formed (40, 41). 

Returning now to the subject of the chapter, in addition to appropriate retentive characteristics, a potential stationary phase must have other key physical characteristics before it can be considered suitable for use in LC. It is extremely important that the stationary phase is completely insoluble (or virtually so) in all solvents that are likely to be used as a mobile phase. Furthermore, it must be insensitive to changes in pH and be capable of assuming the range of interactive characteristics that are necessary for the retention of all types of solutes. In addition, the material must be available as solid particles a few microns in diameter, so that it can be packed into a column and at the same time be mechanically strong enough to sustain bed pressures of 6,000 p.s.i. or more. It is clear that the need for versatile interactive characteristics, virtually universal solvent insolubility together with other critical physical characteristics severely restricts the choice of materials suitable for LC stationary phases. 

As a result of its highly polar character, silica gel is particularly useful in the separation of polarizable materials such as the aromatic hydrocarbons and polynuclear aromatics. It is also useful in the separation of weakly polar solute mixtures such as ethers, esters and in some cases, ketones. The mobile phases that are commonly employed with silica gel are the n-paraffins and mixtures of the n-paraffins with methylene dichloride or chloroform. It should be borne in mind that chloroform is opaque to UV light at 254 nm and thus, if a fixed wavelength UV detector is being used, methylene dichloride might be a better choice. Furthermore, chloroform is considered toxic and requires special methods of waste disposal. Silica gel is strongly deactivated with water and thus, to ensure stable retentive characteristics, the solvent used for the mobile phase should either be completely dry or have a controlled amount of water present. The level of water in the solvent that will have significant effect on solute retention is extremely small. The solubility of water in n-heptane is... 

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