HPLC retention mechanism

Adsorption model of HPLC retention mechanism allows clear definition of the column void volume as the total volume of the liquid phase in the column, but this model requires the use of the surface-specific retention and the correlation of the HPLC retention with the thermodynamic (and thus energetic) parameters, which is not well-developed. This model requires the selection of the standard state of given chromatographic system and relation of all parameters to that state. 

The basis of all these theories is the assumption of the energetic additivity of interactions of analyte structural fragments with the mobile phase and the stationary phase, and the assumption of a single-process partitioning-type HPLC retention mechanism. These assumptions allow mathematical representation of the logarithm of retention factor as a linear function of most continuous parameters (see Chapter 2). Unfortunately, these coefficients are mainly empirical, and usually proper description of the analyte retention behavior is acceptable only if the coefficients are obtained for structurally similar components on the same column and employing the same mobile phase. 

PROCESSES IN COLUMNS FOR POLYMER HPLC -RETENTION MECHANISMS... 

A number of different retention mechanisms operate in HPLC and interested readers may find further details elsewhere [2-4]. It is sufficient to say here that the interaction may be considered in terms of the relative polarities of the species involved. As indicated in Section 2.1 above, there are two extremes of interaction, neither of which is desirable if separation is to be achieved. 

P. A., Testa, B. Solvatochromic analysis of the retention mechanism of two novel stationary phases used for measuring lipophilicity by RP-HPLC./. Liquid Chromatogr. 1992, 35, 2133-2151. 

Buszewski, B., Gadza-la-Kopciuch, R. M., Markuszewski, M. L, Kaliszan, R. Chemically bonded silica stationary phases synthesis, physicochemical characterization, and molecular mechanism of reversed-phase HPLC retention. Anal. Chem. 1997, 69, 3277-3284. 

M., La Rotonda, M. L, Testa, B. Structural properties governing retention mechanisms on immobilized artificial membrane (lAM) HPLC columns. Helv. Chim. Acta 2002, 85, 519-532. 

Scheme 4.4 Classification of liquid chromatography according to the retention mechanism. After Weston and Brown [365], Reprinted from A. Weston and P.R. Brown, HPLC and CE. Principles and Practice, Academic Press, Copyright (1997), with permission from Elsevier...

Selection of columns and mobile phases is determined after consideration of the chemistry of the analytes. In HPLC, the mobile phase is a liquid, while the stationary phase can be a solid or a liquid immobilised on a solid. A stationary phase may have chemical functional groups or compounds physically or chemically bonded to its surface. Resolution and efficiency of HPLC are closely associated with the active surface area of the materials used as stationary phase. Generally, the efficiency of a column increases with decreasing particle size, but back-pressure and mobile phase viscosity increase simultaneously. Selection of the stationary phase material is generally not difficult when the retention mechanism of the intended separation is understood. The fundamental behaviour of stationary phase materials is related to their solubility-interaction... 

Enthalpy-entropy compensation has been investigated in reversed-phase HPLC with octylsilica stationary phase [77]. The compensation temperatures were determined for this system, and the results show that their change with the composition of the mobile phase is almost similar to that with octadecylsilica stationary phase. It can be concluded that the retention mechanisms of the separation of alkyl benzenes is the same in both systems with the mobile phase exceeding 20% water content. 

The data prove that the retention order of anthocyanins deviates from each other in HPLC and TLC suggesting the involvement of a different retention mechanism. It was stated that the preseparation of anthocyanins by size-exclusion chromatography is a prerequisite of the successful preparative separation by RP-HPLC [244],... 

The PO mode is a specific elution condition in HPLC enantiomer separation, which has received remarkable popularity especially for macrocyclic antibiotics CSPs and cyclodextrin-based CSPs. It is also applicable and often preferred over RP and NP modes for the separation of chiral acids on the cinchonan carbamate-type CSPs. The beneficial characteristics of the PO mode may arise from (i) the offset of nonspecific hydrophobic interactions, (ii) the faster elution speed, (iii) sometimes enhanced enan-tioselectivities, (iv) favorable peak shapes due to improved diffusive mass transfer in the intraparticulate pores, and last but not least, (v) less stress to the column, which may extend the column lifetime. Hence, it is rational to start separation attempts with such elution conditions. Typical eluents are composed of methanol, acetonitrile (ACN), or methanol-acetonitrile mixtures and to account for the ion-exchange retention mechanism the addition of a competitor acid that acts also as counterion (e.g., 0.5-2% glacial acetic acid or 0.1% formic acid) is required. A good choice for initial tests turned out to be a mobile phase being composed of methanol-glacial acetic acid-ammonium acetate (98 2 0.5 v/v/w). 

Pagliara, A., Khamis, E., Trinh, A., Carrupt, P.A., Tsai, R.S. and Testa, B. (1995) Structural properties governing retention mechanisms on RP-HPLC stationary phase used for lipophilicity measurements. Journal of Liquid Chromatography, 18, 1721-1745. 

RETENTION MECHANISMS IN POLYMER HPLC 16.3.1 General Considerations... 

Similar to other coupled methods of polymer HPLC, for example, LC CC (Section 16.5.2), the choice of the column packing and the mobile phase components for EG-LC depends on the retention mechanism to be used. Adsorption is preferred for polar polymers applying polar column packings, usually bare silica or silica bonded with the polar groups. The eluent strength controls polymer retention (Sections 16.3.2 and 16.3.5). The enthalpic partition is the retention mechanism of choice for the non polar polymers or polymers of low polarity. In this case, similar to the phase separation mechanism, mainly the solvent quality governs the extent of retention (Sections 16.2.2, 16.3.3, and 16.3.7). It is to be reminded that even the nonpolar polymers such as poly(butadiene) may adsorb on the surface of bare silica gel from the very weak mobile phases and vice versa, the polymers of medium polarity such as poly(methyl methacrylate) can be retained from their poor solvents (eluents) due to enthalpic partition within the nonpolar alkyl-bonded phases. 

As explained in Sections 16.4 and 16.5, the comprehensive characterization of complex polymer systems is hardly possible by the SEC alone. SEC employs only one retention mechanism which simnltaneonsly responds to all molecular characteristics of sample. Similarly, also the coupling of the different retention mechanisms within one single column only exceptionally allows fulfilling this task. Evidently several retention mechanisms should be applied in a tandem approach that is within at least two different on-line chromatographic systems. This is the basic idea of the two- and multidimensional polymer HPLC. In the present section, the principles of two-dimensional polymer HPLC, 2D polymer HPLC or (2D-LC) will be briefly elucidated. There are several reviews available [23-31,249,250] dealing with the 2D polymers. It is anticipated that also the three- and multidimensional polymer HPLC will be developed in future. 

The silica gel-based column packings are the active materials of choice for polymer HPLC employing both exclusion and interaction retention mechanisms. These are either bare or bonded with various groups. C-18 alkyls and -CH2-CH2-CH2-NH2 groups are most popular for reversed-phase and normal-phase procedures of polymer HPLC employing the nonpolar and polar interactions, respectively. 

Mixed retention mechanisms are most evident in the separation of polyelectrolytes. These are large, multivalent molecules, which possess polar and non-polar groups (or sites ) on the surface of the molecule in solution, that may interact physically with the backbone of the ion-exchanger. The most important examples of polyelectrolytes are proteins. 1EC has long been the major tool for the separation of proteins by HPLC, but it is being replaced more and more by RPLC [344]. One of the reasons for this is that due to mixed retention mechanisms broad and non-symmetrical peaks are common for the 1EC separation of proteins. 

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