Polymer HPLC complex polymers

Whereas SEC is the dominant technique for the characterisation of polymers, various nonexclusion liquid chromatographic (NELC) methods, such as GPEC and LACCC offer equally valid possibilities for deformulation of complex polymer systems. In fact, molecular characterisation of polymers in the precip-itation/adsorption mode (gradient HPLC) enables differences in chemical structure and composition to be... 

Normal-phase HPLC has been used increasingly during the past few years for the separation of PAs from grape seed and cocoa [11], PAs up to decamers were separated and resolved into equally spaced peaks in an order consistent with the degree of polymerization. However, for more complex polymers extracted from grape, the resolution becomes lower. 

Phase separation (precipitation) of a polymer strongly depends on all its molecular characteristics. On the one hand, this allows very efficient separations in polymer HPLC utilizing phase separation and re-dissolution processes [20]. On the other hand, due to complexity of phase separation phenomena, the resulting retention volumes of complex polymers may simultaneously depend on several molecular characteristics of separated macromolecules. This may complicate interpretation of the separation results. Both precipitation and redissolution of most polymers is a slow process. It may be affected by the presence of otherwise inactive surface of the column packing. Therefore, the applicability and quantitative control of the phase separation phenomena may be limited to some specific systems of polymer HPLC. 

The significant intrinsic limitation of SEC is the dependence of retention volumes of polymer species on their molecular sizes in solution and thus only indirectly on their molar masses. As known (Sections 16.2.2 and 16.3.2), the size of macromolecnles dissolved in certain solvent depends not only on their molar masses but also on their chemical structure and physical architecture. Consequently, the Vr values of polymer species directly reflect their molar masses only for linear homopolymers and this holds only in absence of side effects within SEC column (Sections 16.4.1 and 16.4.2). In other words, macromolecnles of different molar masses, compositions and architectures may co-elute and in that case the molar mass values directly calculated from the SEC chromatograms would be wrong. This is schematically depicted in Figure 16.10. The problem of simultaneous effects of two or more molecular characteristics on the retention volumes of complex polymer systems is further amplifled by the detection problems (Section 16.9.1) the detector response may not reflect the actual sample concentration. This is the reason why the molar masses of complex polymers directly determined by SEC are only semi-quantitative, reflecting the tendencies rather than the absolute values. To obtain the quantitative molar mass data of complex polymer systems, the coupled (Section 16.5) and two (or multi-) dimensional (Section 16.7) polymer HPLC techniques must be engaged. 

In conclusion, eluent gradient polymer HPLC represents a useful tool for separation of complex polymer system. It belongs to the important constituents of several two-dimensional polymer HPLC procedures. 

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. 

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... 

HPLC and the extensive complexity of ESI and MALDI spectra for multicomponent polymers with molar mass over about 10 g mol. Some applications of MALDI MS in polymer HPLC can be found for example in [300-303],... 

A useful approach to detection in polymer HPLC presents the on-line hyphenation of different measurement principles. For example, an RI detector combined with a UV photometer produces valuable additional information on the composition of some copolymers. Further progress was brought with the triple detection RI plus LALS plus VISCO [313], which is especially suitable for branched macromolecules and the tetra detection UV plus RI plus LALS plus VISCO, which enables characterization of some complex polymer systems, exhibiting a distribution not only in their molar mass and architecture, but also in their chemical composition such as long chain branched copolymers. 

The volume of effluent among particular measuring cells has to be considered in the hyphenated detection systems. In spite of recent progress, the detection remains an important challenge in polymer HPLC, especially in case of complex polymer systems. 

In the next section, the term "complex polymers" is defined, the effects of polymer complexity on conventional SEC analysis are examined, and attempts to analyze ccanplex polymers by utilizing SEC detector technology are summarized. High performance liquid chromatography (HPLC) attempts to accomplish the task are then described. This is followed by a summary of the theoretical development of OC, experimental results of OC analysis, complications which emerged, and finally a summary of the status of OC in light of recent developments. (1) and (2) provide reviews of OC. 

Attenpts to Analyze Complex Polymers by High Performance Liquid Chromatography (HPLC)... 

Some studies have successfully demonstrated separations of linear copolymers using HPLC. However, it is important to realize that unlike in the HPLC of small molecules where a peak shows the concentration of only one type of molecule, the SEC chromatogram of a complex polymer is really an envelope covering possibly thousands of different components. Even with modem detectors it is often very difficult to ascertain that the desired fractionation has really been accomplished. Universal calibration in SEC is of practical utility because the same fractionation (i.e., a fractionation according to molecular size) occurs whether monodisperse, polydisperse or complex polymer molecules are involved. The fractionation is reliable. This is a very difficult requirement for HPLC methods because of the variety of complex molecules that can be present. 

For the precise determination of the complex polymer composition including the chemical composition and MMD of the components in most cases a separation step is required. Only after obtaining fractions comprising the different polymer components, can an analysis with regard to chemical composition and MMD be conducted. The present section discusses different options which enable the use of liquid chromatography (SEC, HPLC, LC-CC) in conjunction with FTIR, mass spectrometry and NMR for the separation of complex polymers. 

Frequently, when analyzing a complex polymer, the first step must be the determination of the gross composition. Only when the chemical structures of the polymer components are known, can sophisticated separation techniques, such as gradient HPLC or LC-CC, be adapted to a specific analysis. 

Balke ST (1991) Characterization of complex polymers by SEC and HPLC. In Barth HG,Mays JM (eds) Modern methods in polymer characterization, chap 1. Wiley Interscience, New York... 

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. 

Based on a caUhration curve prepared by measuring the elution volume of a series monodispersed polymer standards with known molecular weights, the molecular weight of a polymer sample can then be determined. Based on this principle, GPC determines the distribntion of Af, while Af, Af, and Af can also be calcnlated nsing other data. As GPC only measures molar masses of polymers indirectly, some researchers regard this techniqne as senuquantitative [53]. GPC can be conpled with mnltidimensional polymer HPLC techniques to obtain the qnantitative molar mass of complex polymer systems [54]. 

On-line HPLC-NMR offers unique opportunities for analyzing complex polymer systems. Using a selective chromatographic technique, the sample can be separated according to the chemical structure. The structural analysis of the polymer can then be conducted by on-line H-NMR. Because of sensitivity enhancements and inq)roved solvent suppression techniques of the NMR, it is possible to work under experimental conditions which are common in liquid chromatography of polymers, i.e. sufficiently high flow rate, moderate sample concentration, protonated solvents, and on-flow detection. 

HPLC-NMR is a powerful tool for the analysis of complex polymer systems. Using this coupled technique, an analyst can determine the end-group structure, chain length, as well as the composition and the microstructure of the polymers. It has been shown that on-flow experiments, in particular, can be used for the structural analysis of the polymer systems. The analysis can be done with conventional HPLC-grade solvents. Quantitation of the NMR data has been facilitated through the use of multiple solvent suppression experiments (the WET pulse sequence). 

Size exclusion chromatography is undoubtedly the most important method of polymer HPLC. Therefore, it is inappropriate to speak about HPLC and SEC. The reason for above uncertainty in terminology may be the fact that the exclusion based separations are rare in the case of low-molecular substances except mainly the situation when macromolecules are to be (pre)sepaiated, or just removed from the complex sample. The terms HPLC of low-molecular substances and polymer HPLC are employed in present chapter as general designations to differerrtiate the two groups of liquid chrorrratography methods. 

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). 

A more general approach is represented by the two-dimensional procedures of polymer HPLC (see section 11.9), in which a coupled method of polymer HPLC is employed as an important (usually the first) separation step followed by SEC. This means that in the two-dimensional polymer HPLC a tandem of two distinct separation systems is needed. The three-dimensional polymer HPLC is still only in the incoming stage. The task of both coupled and two-(multi-) dimensional precedures of polymer HPLC is the comprehensive molecular characterization of complex polymers and complex polymer systems. 

Dwyer [51] used a combination of chromatography and IR spectroscopy to provide a versatile tool for characterisation of polymers. HPLC-Fourier-transform IR spectroscopy interface systems deposit the output of a chromatograph on an IR optical medium, which is then scanned to provide data as a time-ordered set of spectra of the chromatogram. Polymer analysis applications described include the identification of polymer additives, the determination of composition/molecular weight distributions in copolymers, the mapping of components of polymer alloys and blends, molecular configuration changes in polymers, and component identification in complex systems. 

The advantage of this sequence lies in the fact that SEC of complex copolymers responds to both molecular weight and chemical composition, while the influence of molecular weight on HPLC fractionation can be minimized by the selection of adequate analysis conditions. The importance of polymer cross-fractionation techniques is increasing because of the rapidly growing use of polymers with complex microstructures and would require a separate chapter for complete discussion. The interested reader is referred to the article by Pasch for a review on recent advances in the field of cross fractionation of polymers (96). 

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