Polymer HPLC retention mechanisms

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

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. 

Gradient high-performance liquid chromatography (HPLC) has been useful for the characterization of copolymers (14-19). In such experiments, careful choice of separation conditions is a conditio sine qua non. Otherwise, low resolution for the polymeric sample will obstruct the separation. However, the separation in HPLC, dominated by enthalpic interactions, perfectly complements the entropic nature of the SEC retention mechanism in the characterization of complex polymer formulations. 

It seems that the recent investigations made on the AED have resulted in the development of an important new tool for the investigation of mildly heterogeneous surfaces such as modern packing materials used in HPLC. This method has already allowed the derivation of important new results regarding retention mechanisms in reversed phase liquid chromatography [61,117] and on molec-ularly imprinted polymers [56,118]. Its application in preparative liquid chromatography should be fruitful. 

A cursory review of the macromolecular HPLC literature immediately rewals sharp disagreement concerning even the basis of these separations. For the common case of protein or polymer gradient-elution, a number of different retention mechanisms" or processes have been pro x>sed as relevant ... 

The solubility of macromolecules as a rule improves with the rising temperature. Solvent - polymer mixtures usually exhibit the upper consolute temperature or upper critical solution temperature, UCST, with a maximum on the plot of system concentration versus temperature. Above the critical solution temperature, polymer is fully soluble at any concentration. For practical work, the systems with UCST below ambient temperature are welcome. There are, however numerous polymer - solvent systems, in which the solvent quality decreases with increasing temperature. The plot of system concentration versus temperature exhibits a minimum. The phenomenon is called lower consolute temperature or lower critical solution temperature, LCST Polymer is only partially soluble or even insoluble above lower critical solution temperature. This unexpected behavior can be explained by the dominating effect of entropy in case of the stiff polymer chains or by the strong solvent - solvent interactions. The possible adverse effect of rising temperature on polymer solubility must be kept in mind when woiking with low solubility polymers and with multicomponent mobile phases. It may lead to the unforeseen results especially in the polymer HPLC techniques that combine exclusion and interaction retention mechanisms, in coupled methods of polymer HPLC (see section 11.8, Coupled Methods of Polymer HPLC). 

Separation of distinct rrracromolecules in polymer HPLC results from then-different retention within colirmn Retention mechanism of analyte is the general term recommended by lUPAC. It denotes the mutrral difference of elution rate of distinct macromolecirles. Similar to HPLC of low-molecirlar substances, elution rate of macromolecirles and molecules of mobile phase differ also in polymer HPLC. As a rule, in HPLC of low-molecular substances, the separated species elute with lower velocity than molecules of their original solvent, they are retained, decelerated. With few exceptions, elution rate of macromolecules is the same or slower than elution rate of eluent molecules also in various the coupled methods of polymer HPLC. 

For practical reason, however it is useful to retain the term eluent strength also in polymer HPLC with the nonpolar column packings and for the enthalpic partition retention mechanism. This means that a thermodynamically good mobile phase is termed strong and a poor mobile phase is denominated weak. 

Practical liquid chromatography methods based on the coupling of entropic and ethalpic retention mechanisms in the same polymer HPLC column will be briefly elucidated in present section. The two-dimensional polymer HPLC will be discussed in Section 11.9. 

HPLC-CSPs are based on molecules of known stereochemical composition immobilized on liquid chromatographic supports. Single enantiomorphs, diastereomers, diastereomeric mixtures, and chiral polymers (such as proteins) have been used as the chiral selector. The chiral recognition mechanisms operating on these phases are the result of the formation of temporary diastereomeric complexes between the enantiomeric solute molecules and immobilized chiral selector. The difference in energy between the resulting diastereomeric solute/CSP complexes determines the magnitude of the observed stereoselectivity, whereas the sum total of the interactions between the solute and CSP chiral and achiral, determines the observed retention and efficiency. 

Pores (mean diameter usually from 6 to 100 nm) play a cmcial role in hplc, as they provide the surface where retention and hence separation occur. The chemical stmcture of the surface determines the thermodynamic environment for the analyte in thin surface layer (stationary phase) and in such a way determines the mechanism of retention. Flexible macromolecules approaching the internal surface of a solid particle change their spatial conformations because of steric interaction, which plays an important role in any mode of polymer chromatography. This interaction restricts the fluctuation motion of a macromolecule, decreases its... 

Proteins are isolated and characterized with different chromatographic techniques. Depending on the protein, ion-exchange (lEX), size-exclusion (SEC), affinity (IMAC), hydrophobic interaction (HIC), and reversed-phase chromatography (RPLC) may be applied. Traditionally, separation materials with low mechanical stability and limited resolution were used. In recent years, HPLC materials were developed that offered the separation power required by the protein chemist. Now, pellicular ion-exchange resins as well as organic polymer monoliths exhibit high resolution at relatively short retention times, while... 

Since most readers will be experienced in general HPLC applications, major differences in the practice of GPC are outlined in Table 1. The separation in GPC is governed by the well-known size-exclusion mechanism its principles can be found in the relevant literature [1]. In the ideal case, only the conformational entropy change causes retention. Secondary enthalpic interactions should be avoided by appropriate selection of the phase system. Since GPC does not measure molar masses directly, the retention axis has to be calibrated with so-called narrow polymer standards in order to transform peak position to molar mass. The absolute position of the peak(s) and its shape(s) are evaluated to determine the molar mass average and molar mass distribution, respectively. 

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