Stationary phase testing different types

The HcReynolds abroach, which was based on earlier theoretical considerations proposed by Rohrschneider, is formulated on the assumption that intermolecular forces are additive and their Individual contributions to retention can be evaluated from differences between the retention index values for a series of test solutes measured on the liquid phase to be characterized and squalane at a fixed temperature of 120 C. The test solutes. Table 2.12, were selected to express dominant Intermolecular interactions. HcReynolds suggested that ten solutes were needed for this purpose. This included the original five test solutes proposed by Rohrschneider or higher molecular weight homologs of those test solutes to improve the accuracy of the retention index measurements. The number of test solutes required to adequately characterize the solvent properties of a stationary phase has remained controversial but in conventional practice the first five solutes in Table 2.12, identified by symbols x through s have been the most widely used [6). It was further assumed that for each type of intermolecular interaction, the interaction energy is proportional to a value a, b, c, d, or e, etc., characteristic of each test solute and proportional to its susceptibility for a particular interaction, and to a value x, X, Z, U, s, etc., characteristic of the capacity of the liquid phase... 

Shape Selectivity Molecular recognition of the solute by the stationary phase with respect to its geometrical dimension is called shape selectivity. For this test, one can employ aromatic components with identical hydrophobicity that differ only in their three-dimensional shape. The chromatographic selectivity of o-terphe-nyl/triphenylene or tetra-benzonaphthalene/benzo[a]pyrene are commonly used and show dependencies on several features of the phase, for example, pore structure, ligand type, and density. Figure 3.6 shows a chromatogram of a test mixture of uracil (to marker), n-butylbenzene, and w-pentylbenzene (to assess hydrophobic properties and efficiency), and o-terphenyl and triphenylene (to assess shape selectivity). The test mixture was chosen to provide a short analysis time and to facilitate calculation of parameters from baseline-separated peaks. 

The different types of cross-axis coil planet centrifuges (cross-axis CPCs) were tested for the retention of the stationary phase of aqueous-aqueous pol3fmer-phase systems such as poly(ethylene glycol) (PEG) l(X)0-potassium phosphate and PEG SOOCf iextran T500. The XL and XLL CPC are suitable for the PEG 1000-potassium phosphate, which has a relatively large difference in density between the two phases. On the other hand, the XLLL and L CPC are required to acheive satisfactory phase retention phase retention of the PEG-dextran system. These cross-axis CPCs were utilized for the separation of several proteins. 

These facts, together with the hRy values of the test dye mixture, can be used to characterize the different types of chamber and, simultaneously, indirectly characterize the vapor phase conditions used for a given separation (40). These results can be used for comparison of separations with given stationary and mobile phases, thus enabling prediction of the separation under other vapor phase conditions, e.g., the different forced-flow planar chromatographic techniques. The technique also provides guidelines for the transfer of mobile phases between the different planar chromatographic methods. 

When one develops new reversed-phase (RP)-HPLC methods, one usually uses the selectivity of the mobile phase as the primary method development tool. The chromatographic separation can be influenced by the choice of the organic solvent (mainly methanol and acetonitrile), or by variation of pH or buffer type. Schemes for method development using these parameters have been described in the literature [1,2]. Most important are the selectivity changes caused by pH changes, which are well-understood and easily predictable (3). It is well known that the stationary phase influences the selectivity as well, but this effect is often not very well understood. The primary reason for this is the fact that reliable methods for the description of the stationary phase selectivity have only become available fairly recently. In the last few years, several papers have been published that deal with the subject of selectivity in a fimdamental way [4—9] or represent a data collection based on older methods [10-15]. In this chapter, we describe in detail the method used in our laboratory. We then look at our selectivity charts and discuss our results. It needs to be pointed out in advance that selectivity charts only accurately represent the properties of a stationary phase under the conditions of the measurement. If we depart from the mobile phase composition of the test, the relationships between different columns will change, since selectivity arises from a combined effect of the mobile phase and the stationary phase. 

Afterwards, one lowers the temperature, perhaps from ambient to 15 °C, in order to improve separation. If one does not hit the mark, a further stationary phase is tested in the same manner. In this way, the influence of the factors type of stationary phase , solvents , and column temperature are tested, but without being able to recognize the influences of the three factors on each other. We do not learn whether the individual factors mutually negate themselves, strengthen one another, or jointly have some completely different unimaginable effects on the chromatographic separation. 

The exploratory practical work in the laboratory begins during method development after conclusion of the preliminary theoretical work on a separation problem. First, one specifies the possible influencing factors that are to be tested, e.g. six columns with different types of stationary phases, ideally with identical dimensions, the solvents acetonitrile and methanol, the column temperatures (15 °C and 45 °C), and if necessary, the influence of the pH (e.g., 2,7, and 9). Handbooks that have already been published on the strategic changes of chromatographic parameters can afford valuable assistance [3, 4] and should always be consulted to save time. 

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