Stationary Phase Systems

The large number of CSP s developed, tested and marketed present somewhat of a problem for how best to categorize them. Wainer has suggested a classification scheme for HPLC CSP s based on the mode of formation of the solute-CSP complex [16]. There are five categories, labeled Type l-V, and molecular modeling has been done on most of these. The categories and modes of association are  

Type I. Where solute-CSP complexes are formed by attractive interactions like hydrogen bonding, pi-pi interactions and dipole stacking as represented by Pirkle-like CSPs. 

Type II. Where the solute-CSP complexes are formed by attractive interactions and through the inclusion into a chiral cavity or ravine as represented by some cellulose based CSPs. 

Type III. Where the primary mechanism involves the formation of inclusion complexes as represented by cyclodextrins. 

The major difference between these three categories, irrespective of the type of intermolecular attractions, is the extent of inclusion. Type I has no inclusion complexation, Type II has partial inclusion and Type III uses inclusion complexation as the primary mechanism. For this review I shall create a greater line of demarcation than Wainer between Type II and Type III phases. Here Type III shall be considered to be exclusively guest-host complexes as found in crown ethers, cyclodextrins and related systems, whereas Type II uses only partial guest-host complexation. 

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The injection of a sample in a chromatographic column may result in more peaks than there are components in the mixture if the mobile phase contains one or several additives. These additional peaks result from the perturbation of the additive equilibrium between the two phases caused by the injection of a sample. It may be assumed that there is a competitive equilibrium of the sample and the modifier. Solutes enter the column, moving with the velocity of the mobile phase and not with the equilibrium velocities dictated by the equilibrium between mobile and stationary phases. System peaks are visualized with an appropriate detector, particularly a refractive index detector. This may cause trouble for the analyst, since the system peak may exhibit k values more than 1 (37). 

Hence, resolution, a key criterion for HPLC column users, is maximized for a given mobile phase/stationary phase system by maximizing N. Therefore, even if researchers are inclined to measure h as their column efficiency parameter, manufacturers... 

Columns For most analyses, separation is achieved by partition of compounds in the test solution between the mobile and stationary phases. Systems consisting of polar stationary phases and nonpolar mobile phases are described as normal phase, while the opposite arrangement, polar mobile phases and nonpolar stationary phases, is called reversed-phase chromatography. Partition chromatography is almost always used for hydrocarbon-soluble compounds of a molecular weight that is less than 1000. The affinity of a compound for the stationary phase, and thus its retention time on the column, is controlled by making the mobile phase more or less polar. Mobile phase polarity can be varied by the addition of a second, and sometimes a third or even a fourth, component. 

For characterization and exploitation of the diamide-phase system, a chiral diamide, e,g., (Ill) was examined as a modifier in the mobile phase (solvent) in conjunction with a non-bonded (bare) silica. Such a chiral carrier separated enantiomeric N-acyl-d-amino acid esters and amides with separation factors comparable to those for bonded stationary phase systems. The resolution can be as cribed to diastereomeric complexation through amide-amide hydrogen bonding between the amide additive and enantiomeric solute molecules in the carrier solvent, followed by separation of the diastereomeric complexes by the (achiral) silica phase. This process should be applicable as widely as that involving chiral diamide-bonded stationary phase systems. 

The suitability of a stationary phase for a particular application depends on the selectivity and the degree to which polar compounds are retarded relative to what their retardation would be on a completely non-polar stationary phase. Since retention time is a function of temperature, flow-rate, stationary phase type and loading or film thickness it cannot be used to relate the retention characteristics of one column to another. Various retention index methods have been described such as evaluating the partition and separation properties of solute-stationary phase systems. Kovats (1958) devised a system of indexing chromatographic retention properties of a stationary phase with respect to the retention characteristics of n-alkanes, alkanes being used as reference materials since they are non-polar, chemically inert and soluble in most common stationary phases [8-10]. The retention index (RI) for the n-alkanes is defined as... 

The relatively slow kinetics of complexatlon has been proposed as a cause of the generally low efficiencies of boronate stationary phase systems (19). The plate numbers of interacting solutes in these studies are about 90% of otherwise observed and assyraetry is unchanged. The latter is unusual since previous work showed tailing ascribed to the presence of a "non-linear Isotherm" (20). Perhaps the combination of surface buffering and mixed bonded phase effect creates a more favorable, more uniform sorption environment. 

Since a(CH2) is the ratio of the retention factors of two compounds differing only in a methylene group, it should be independent of the series type for a given mobile and stationary phase system, such is the case for the aqueous-organic mobile phases. In contrast, as shown in Table 9.1, for micelles the a(CH2 ) values are dependent on the type of series, as the methylene selectivity for alkylphenones are consistently greater than for alkylbenzenes [7]. 

Small particle size material requires a high-pressure slurry packing procedure. It is preferable to work with spherical particles since they have better mechanical stabiUty and thus longer life than irregular ones. Axial compression columns allow for efficient in-house packing of columns with bulk stationary phases. Systems utilizing radially compressed, preloaded disposable cartridges are commercially available. 

Displacement chromatography has an enormous potential as a preparative bioseparation technique. In many situations from the mg to the kg scale and beyond the displacement chromatography may theoretically be the most practical, the most economic and the most efficient approach to a given separation problem. However, in order to exploit the full potential of displacement chromatography, suitable displacer/stationary phase systems must become available. This chapter is intended as an introduction to our current understanding of the requirements for systematic displacer design. 

Fig. 13. Schematic representation of the proposed mechanism for the phospho-choline stationary phase system, (a) Establishment of the Dorman membrane, (b) use of CeCls and (c) use of a NaC104 mobile phase. (After Cook et alP)...

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