Polymer HPLC coupled techniques

Although the OTHdC has several unique applications in polymer analysis, this technique has several limitations. First, it requires the instrumentation of capillary HPLC, especially the injector and detector, which is not as popular as packed column chromatography at this time. Second, as discussed previously, the separation range of a uniform capillary column is rather narrow. Third, it is difficult to couple capillary columns with different sizes together as SEC columns. 

Depending on the particular method of polymer HPLC, is defined in different ways. It is the total volume of pores, Vp, in a porous packing but it can be also related to the total surface of packing (mostly to the surface situated within the pores) or to the effective volume of bonded phase. The volume of pores is relatively well defined in the case of many packings applied in polymer HPLC and plays an especially important role in the exclusion-based separations (Sections 16.3.3, 16.3.4, and 16.4.1). The exclusion processes, however, play an important role in the coupled techniques of polymer HPLC (Section 16.5). In the latter cases, the surface of packings and the effective volume of bonded phase are to be taken into account. In some theoretical approaches also, surface exclusion is considered. 

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. 

All above homopolymers are used also for the identification of suitable conditions for the coupled polymer HPLC techniques. Typical examples are liquid chromatography under critical (LC CC) and limiting (LC LC) conditions, and eluent gradient liquid chromatography (EG LC). For the development of latter methods, several defined statistical and block copolymers are available. 

The specific constraints and requirements of continuous-flow NMR will be explained in the first chapter, whereas specific applications, such as biomedical and natural product analysis, LC-NMR-MS and LC-NMR in an industrial environment, together with polymer analysis, will be discussed separately. Thus, the reader will obtain a broad overview of the application power of LC-NMR and the benefits of its use. He/She will also be introduced to the pitfalls of this technique. Special attention will be given to the exciting newer coupled techniques such as SFC-NMR and capillary HPLC-NMR. However, new emerging future developments will also be discussed thoroughly. 

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). 

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). 

Anionic polymerization has been used to its fullest advantage for preparation of polymers with functional groups at the chain ends. All previous work has been on butadiene and styrene polymers because the living ends are sturdier and there are fewer side reactions than with the methacrylates. Our early work on prolonging the life of the living ends and elimination of side reactions allowed us to bring the methacrylates under control. This synthetic control, coupled with HPLC analytical techniques, has enabled us to prepare and characterize a new series of functionalized methacrylate polymers. 

The combination of XRF with electrophoresis can constitute a quasi-on-line coupling technique. Great efforts have been made to develop on-line analysis techniques with XRF. Mann et reported a technique of on-line SRXRF detection of metal ions, such as Fe, Co, Cu, and Zn, in their high binding-constant complexes for capillary electrophoresis (CE) separation. An X-ray transparent polymer coupling is used to create a window for the on-line X-ray detection. In contrast to ICP-MS, this detection technique is not limited by sample or the buffer volatility or atomization efficiency. Simultaneous XRF and UV absorbance detection can be used to provide on-line determination of metal/chelate ratios. A bench-top energy-dispersive micro X-ray fluorescence system was also combined with the CE apparatus constructed by a thin-walled fused-silica capillary for elemental analysis of the species containing Fe, Co, and Cu, for example. This coupled technique used for metalloprotein speciation analysis can avoid the compromise between optimal separation and sensitive detection, which must be taken into account in the HPLC-ICP-MS procedure. In addition, this detection scheme is non-destructive, so the separated material can be recovered for additional characterization. 

For the analysis of nonvolatile compounds, on-line coupled microcolumn SEC-PyGC has been described [979]. Alternatively, on-line p,SEC coupled to a conventional-size LC system can be used for separation and quantitative determination of compounds, in which volatility may not allow analysis via capillary GC [976]. An automated SEC-gradient HPLC flow system for polymer analysis has been developed [980]. The high sample loading capacity available in SEC makes it an attractive technique for intermediate sample cleanup [981] prior to a more sensitive RPLC technique. Hence, this intermediate step is especially interesting for experimental purposes whenever polymer matrix interference cannot be separated from the peak of interest. Coupling of SEC to RPLC is expected to benefit from the miniaturised approach in the first dimension (no broadening). Development of the first separation step in SEC-HPLC is usually quite short, unless problems are encountered with sample/column compatibility. 

Integration of sample preparation and chromatography by on-line coupling aims at reduction of analysis time. It is apparent from Section 7.1 that these hyphenated techniques are not yet contributing heavily to the overall efficiency of polymer/additive analysis in industry. On-line SFE-SFC requires considerable method development, and MAE-HPLC is off-line. Enhancement of sensitivity for trace analysis requires appropriate sample preparation and preconcentration schemes, as well as improved detection systems. 

Liquid chromatography (LC) encompasses several techniques, which includes HPLC and SEC (sometimes referred to as GPC). Many variants of and developments from these techniques exist, and are used in the study and analysis of polymer degradation/oxidation. As discussed earlier, SEC is often coupled with MALDI-ToF-MS to facilitate the identification of the products of polymer degradation. SEC has also been coupled with mid-infrared detection and similarly used for studies of polymer degradation. SEC/GPC is discussed further below. 

Selection of the analytical instrumentation for the analysis of the pyrolysate is a very important step for obtaining the appropriate results on a certain practical problem. However, not only technical factors are involved in this selection the availability of a certain instrumentation is most commonly the limiting factor. Gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS) are, however, the most common techniques utilized for the on-line or off-line analysis of pyrolysates. The clear advantages of these techniques such as sensitivity and capability to identify unknown compounds explain their use. However, the limitations of GC to process non-volatile samples and the fact that larger molecules in a pyrolysate commonly retain more structural information on a polymer would make HPLC or other techniques more appropriate for pyrolysate analysis. However, not many results on HPLC analysis of pyrolysates are reported (see section 5.6). This is probably explained by the limitations in the capability of compound identification of HPLC, even when it is coupled with a mass spectrometric system. Other techniques such as FTIR or NMR can also be utilized for the analysis of pyrolysates, but their lower sensitivity relative to mass spectrometry explains their limited usage. 

Solid phase micro extraction (SPME) is a widely used extraction technique that was developed by Pawlistyn and co-workers in 1990 (74). SPME uses a fused silica fiber that is coated on the outside with an appropriate stationary phase (a S to 100 pm thick coating of different polymers, e.g. polydimethylsiloxane, PDMS). The small size of the SPME flber and its cylindrical shape enables it to fit inside the needle of a syringe-like device. Target molecules from a gaseous or a liquid sample are extracted and concentrated to the polymeric fiber coating. SPME has been used coupled to GC and GC-MS (75), as well as to HPLC and LC-MS 7ll>). Figure 7 shows a conunercially available SPME device. 

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