GPC-NMR Coupling

Figure 7.1.11 Study of the copolymerization course of the butyl acrylate-styrene system, using GPC-NMR coupling, in the first two time-intervals (- -) 15 min (- -) 30 min...

In conclusion, on-line GPC-NMR coupling provides a fast and efficient method for the characterization of polymers in general and, in particular, for copolymers of different chemical compositions and stereoregularity. 

GPC/NMR coupling opens new opportunities mainly for the characterization of polymers. SEC is very often considered as a niche separation technique mainly for nonpolar analytes. Coupled to NMR, however, SEC/NMR demonstrates the inherent advantage that no solvent suppression is needed and the whole proton chemical shift range can be used... 

Figure 7.1.9 Contour plot obtained for a typical GPC-NMR on-line coupling analysis of a styrene-butyl acrylate copolymer...

Besides coupling with HPLC, NMR can also be hyphenated with other separation techniques such as gel permeation chromatography (GPC/NMR), supercritical fluid chromatography (SEC/NMR), capillary HPLC (CapLC/NMR), and elecrodriven separation techniques such as capillary electrophoresis (CE/ NMR). 

In early work, while compositional heterogeneity was recognized and could be predicted, it was difficult to measure. Now, methods such as GPC combined with NMR and/or MALDI,238 239 GPC coupled with FTIR240 and two dimensional HPLC or GPC241"245 can provide a direct measure of the composition distribution. 

An important question that needs to be addressed in any screening study is the determination of whether or not the ligand is non-covalently bound to the active site of the target protein. A number of simple GPC spin column ESI-MS screening methods have been developed to answer this question. These methods include the use of mutated proteins where the active site has been modified, GPC spin column/ESI-MS coupled with NMR (GPC spin column/MS/NMR) and displacement of known binders. Titration experiments with molar excesses of ligand to protein (described below in Section 2.3.3.2.4) can also be used to determine whether single or multiple binding sites are available in the protein. 

The coupling of the GPC spin column/ESI-MS screening results with NMR (2D HSQC) is a powerful method for confirming that the non-covalent binders identified by the MS experiments truly bind at the predicted active site by observing NMR chemical shift perturbations in the vicinity of the protein active site [1, 15]. In contrast, the absence of chemical shift perturbations or a random distribution of chemical shift changes on the protein surface would imply a lack of an interaction of the compound with the protein or potentially the existence of non-specific binding. The development of the GPC spin column/MS/NMR assay... 

The site of non-covalent binding of the ligand to the protein is not directly measurable by GPC spin column/ESI-MS. To directly obtain the binding site, X-ray and NMR techniques are used. Site directed mutagenesis and displacement of known binders coupled with GPC spin column/ESI-MS can be used to identify non-covalent binding sites. 

The kinetics of the different condensation steps till (7) (and equivalent structures in the TBA system) are similar for TPA and TBA. This is illustrated using GPC data in Fig.2. The GP chromatograms of extracts after 5, 10, 15 and 45 minutes of hydrolysis illustrate the consecutive formation of species identified as dimers of tetracyclic undecamer (4) and (4 ), precursor (5), dimers of precursors (6) and trimer of precursors (7). The assignment is based on the retention volumes and previous identification with 29Si liquid NMR for the TPA system [2]. The GPC traces show that the transformation of species of type (4), (4 ) and (5) into (6) and (7) is slower with TBA than with TPA. This coupling of precursors seems to be hindered by the long butyl chains of occluded TBA. 

A cyclic polydimethylsiloxane was also prepared by the end-to-end reaction, i.e., a,ft)-dianion-functionalized polymers, which are then cyclized simply by reaction with a difunctional electrophile to give a cyclic polymer. The cyclic polydimethylsiloxane was synthesized from a commercially available o. tw-dihydroxy-polydimethylsiloxane (M = 2.460 g/mol). The Unear precursor was deprotonated using sodium hydride in dilute THF (< 10 2 M) and then end-coupled using a dichlorosilane coupUng agent (Fig. 53). The uncy-clized anionic linear precursors are then removed by a macroporous anion exchange resin. The successful cyclization and purification is monitored by IR and 29Si NMR, GPC, and MALDI-TOF mass [176],... 

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