Characterization of single-chain polymer folding using size exclusion chromatography with multiple modes of detection
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- Frank, P., Prasher, A., Tuten, B. et al. Appl Petrochem Res (2015) 5: 9. doi:10.1007/s13203-014-0046-1
We highlight here recent work from our laboratory on the subject of fabricating nanostructures from single polymer chains. These so-called single-chain nanoparticles are synthesized by inducing intra-molecular cross-linking on discrete macromolecules in dilute solution. Among the biggest challenges in this rapidly expanding area of research is reliable and accurate means to characterize this process. In this paper, we review our preferred method of characterization: size exclusion chromatography featuring multiple modes of detection. Multi-angle light scattering in conjunction with a concentration detector can provide absolute molecular weight data; viscometric detection can provide information about solution size and conformation. Correlation of these data provides a simple and robust way to quantify the process by which we fold single polymer coils into architecturally defined unimolecular nanostructures.
KeywordsSingle-chain nanoparticles Polymer folding TEM SEC
Although young, this field is developing rapidly; a number of important contributions have appeared in the recent literature. [1, 12, 19, 20, 21] The Meijer group showed that the molecular weight and backbone rigidity of the parent polymer does not significantly influence the SCNP fabrication induced by supramolecular interactions. Instead, the solvent plays a key role in mediating intra-molecular folding . Meijer and coworkers also expanded the scope of possible architectures by successfully synthesizing SCNP from cylindrical brush block polymers as well as an orthogonally collapsible ABA triblock copolymer [6, 22]. Additionally, this group studied temperature and co-solvent induced changes in the secondary structures of benzene-1,3,5-tricarboxamides (BTA)-based SCNPs, and developed a compartmentalized metal ion SCNP sensor that shows impressive binding affinities [4, 23]. The Barner-Kowollik group demonstrated a photo-induced Diels–Alder ligation to create SCNP in addition to using metal–ligand complexation to induce controlled folding by linking chain ends [1, 24]. Other work has demonstrated that SCNP can be formed from a host of reactions including benzocylcobutene coupling , olefin cross-metathesis , azide–alkyne click chemistry [19, 27, 28, 29, 30], thiol-ene click chemistry [31, 32], Bergman cyclization [33, 34, 35], Curtius rearrangement , Glasser–Hay coupling , nitrene-mediated C-alkylation (sulfonyl azide) , oxidative polymerization through pendant moieties , benzosulfone chemistry , carbodiimide coupling , benzoxazine chemistry , amide bond formation [10, 31], cinnamoyl  and coumarin  photodimerization, guest–host interactions , hydrogen bonding [20, 42, 43, 44], and vulcanization .
These methods draw synthetic parallel to nature’s elegant biomacromolecules, albeit in a crude and simplistic fashion [3, 5]. Given that synthetic polymers are always subject to distributions in molecular weights and heterogeneities in microstructure [46, 47], it is important to develop convenient and consistent methods for characterizing SCNP fabrication. We discuss here the analysis of SCNPs using size exclusion chromatography (SEC), specifically the application of multi-detection SEC. We have recently shown that when applied in conjunction with microscopy and dynamic light scattering (DLS), multi-detection SEC is a powerful tool for studying the folding of individual polymer chains into architectural defined nanostructures.
Polymer design and SCNP synthesis
SEC with conventional calibration is the standard method used to characterize SCNPs formation. Comparing the retention time of the parent polymer chains to the retention time of the folded nanoparticles after intramolecular cross-linking is induced typically reveals shifts to longer retention times. This indicates a decrease in hydrodynamic volume based on the inherent principles of size exclusion chromatography [51, 52, 53]. This is clearly demonstrated in all of the polymer series discussed here [10, 31, 49].
Triple detection SEC
The application of standard SEC as described above provides useful qualitative information about changes in the solution conformation of polymer chains during SCNP fabrication. Still this method is not without ambiguities, especially with regards to actual changes in molecular weight (or lack thereof) when triggering intra-chain cross-linking. Since relative molecular weight and retention time are linked in traditional SEC, it is impossible to formally characterize SCNP molecular weight with this technique. Furthermore, while shifts in retention time certainly signify changes in hydrodynamic volume it is impossible to quantify these changes with standard SEC measurements alone. Dynamic light scattering (DLS) in conjunction with SEC can be a very useful tool for SCNP characterization, but lacks the chromatographic separation provided by SEC.
To alleviate some of these issues our group implemented SEC with multiple modes of detection for SCNP characterization. The use of multi-angle light scattering (MALS) detection with SEC allows the determination of polymer and SCNP molecular weight independent of retention time . The addition of an inline differential viscometer permits characterization of molecular conformation, including hydrodynamic radius, for each thin chromatographic slice as the sample elutes from the SEC column. With this setup, the absolute molecular weight of the parent polymers can be easily measured and used to calculate the expected SCNP molecular weight. Comparing this value with the experimental absolute SCNP molecular weight can provide useful quantitative information about the chemistry involved in this process. Changes in retention time, which are assumed to correlate with changes in solvated volume, can likewise be readily quantified by this method. Recent work from our lab demonstrating the utility of this technique is highlighted below.
SEC data for series 1b
Peak retention time (min)a
Parent polymer (1b)
SEC data for series 2 before and after complete photo-induced folding
Irradiation time (min)
Peak retention time (min)
Comparison of predicted vs. actual absolute Mw for series 3
Predicted molecular weights Mw (kg/mol)
Actual molecular weights Mw (kg/mol)
Intra-chain versus inter-chain reactions
Correlating solution measurements with other characterization tools
In summary, conventionally calibrated SEC is a satisfactory method to characterize SCNP via interpretation of comparative shifts in retention time. Evolving this tool by marrying SEC with MALS and viscometric detection affords a precise and reliable method for the qualitative and quantitative study of SCNP fabrication. This methodology facilitates the detailed investigation of structure property relationships using absolute molecular weight, intrinsic viscosity, and hydrodynamic radius of SCNP systems. During SCNP formation, the decrease in both the intrinsic viscosity and hydrodynamic radius is observed with, or in the absence of, concurrent changes in the absolute molecular weight depending on the folding technique employed. Triple detection SEC in conjunction with additional characterization tools including TEM and DLS provides a variety of useful information for studying the fascinating and rapidly expanding research area of single-chain folding processes and single-chain nanoparticles.
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