Polymer HPLC separation selectivity

In this chapter, high-performance liquid chromatography of oligomers and (high) polymers (polymer HPLC) will be briefly presented. As mentioned in Section 16.1, there exist several monographs, chapter in books, and review papers on this subject, for example [1-33], Most of them contain numerous examples of the HPLC separation and molecular characterization of particular macromolecular substances. Therefore, this chapter discusses almost exclusively the general principles of polymer HPLC and only few selected examples of practical applications will be mentioned for illustration. 

To do so, however, a tandem of several columns would be needed. In the case of a two-component polymer system, the molar mass effect can be suppressed selectively for one component and the molar mass of the second component can be assessed by means of one single SEC measurement. A more general approach is represented by the two- and multidimensional procedures in polymer HPLC (Section 16.7), in which the coupled methods of polymer HPLC are included as an important (usually the first) separation steps. 

In any 2D HPLC, it is important to attain certain degree of both the complementarity and the orthogonality between the two separation dimensions [255-257]. The so far most universal approach to 2D polymer HPLC assumes the partial or possibly full suppression of the molar mass effect in the first dimension of the separation so that the complex polymer is separated mainly or even exclusively according to its chemical structure. Selected coupled methods of polymer HPLC are to be applied to this purpose. In the second dimension of separation—it is usually SEC—the fractions from the first dimension are further discriminated according to their molecular size. Exceptionally, SEC can be used as the first dimension to separate complex polymer system according to the molecular size. This approach is applicable when the size of polymer species does not depend or only little depends on their second molecular characteristic, as it is the case of the stereoregular polymers... 

The details regarding preparation of clenbuterol imprinted polymers, HPLC columns and detection have been described previously (Crescenzi et al., 1998). A typical chromatogram showing the resolution of clenbuterol and timolol from a mixture at pH values 2.0 and 3.4 is shown in Fig. 4.2. In terms of the selectivity of the stationary phase, expressed as separation factor a, the values at pH 2.0 and pH 3.4 were 3.1 and 14.4, respectively. For control particles, the a value was 1. 

HPLC separation of polymers requires the removal of polymer prior to the analysis, low molecular weight SEC can operate without such need, as the polymer will Just be exeluded from the system. SEC presents a quick and easy method development strategy the process involves the selection of a suitable solvent and the use of any small pore size eolumns 50 A and 100 A. All eomponents will elute within a predetermined interval, amounting to one eolumn volume, and the elution volume of any material ean be predicted for a given eolumn provided a ealibration curve is available. 

From its principle, SEC suffers from the limited separation selectivity because the retention volumes of samples are restricted by F and F values (Figure 3(a) and 12). This drawback prevents application of SEC to quantitative characterization of numerons complex polymer systems that contain macromolecules of distinct chemical stmcture or physical architecture possessing similar molecular sizes (compare Figure 16). In turn, low sample capacity and often insufficient sensitivity of detection makes it impossible to identify and characterize the minor components of complex polymer systems that are present in a matrix of a major constituent in the amonnt below abont 10% - even if molar masses of minor and major constitnent differ substantially. Still, SEC is applied in many laboratories for just the above- mentioned pnrposes. To solve the latter analytical challenges, coupled and two-dimensional methods of polymer HPLC are to be employed (see sections 11.8 and 11.9). 

In this case, the enthalpic interactions within the HPLC system exceed the exclusion effects (see Figure 3(e)). The retention volumes of polymer species as a rale exponentially increase with their molar masses. The important limitation of the resulting procedures was presented in section 11.5.2.3. The retention of (high) polymers is usually so intense that the latter do not elute from the column any more. Therefore, the majority of enthalpy controlled HPLC procedures is applicable only to ohgomers - up to molar mass of few thousands g.mol. Still, the reduced sample recovery may affect results of separation even in case of oligomers. The selectivity of enthalpy driven HPLC separation is much higher than in the case of SEC but, naturally, the sequence of molar masses eluted from the column is reversed. If the effect of enthalpy is reduced, problems with sample recovery are mitigated - but at the same time the separation selectivity is reduced. 

The application areas for LC-MS, as will be illustrated later, are diverse, encompassing both qualitative and quantitative determinations of both high-and low-molecular-weight materials, including synthetic polymers, biopolymers, environmental pollutants, pharmaceutical compounds (drugs and their metabolites) and natural products. In essence, it is used for any compounds which are found in complex matrices for which HPLC is the separation method of choice and where the mass spectrometer provides the necessary selectivity and sensitivity to provide quantitative information and/or it provides structural information that cannot be obtained by using other detectors. 

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