Highly Reproducible MW Measurement of Polypeptoids Using an EcoSEC Semi-Micro GPC System Reduces Anlysis Time and Solvent Consumption

There is considerable attention placed on the synthesis and characterization of polypeptoids, a new class of synthetic polypeptide analogues. Because of intense hydrogen-bonding among chains of these polymers, aggressive solvents such as hexafluoroisopropanol (HFIP) must be used as the mobile phase to support solubility. Furthermore, highly reproducible data is needed to obtain subtle molecular weight distribution trends. To save considerable amounts of harmful and expensive solvents we applied mixed bed GPC columns and the EcoSEC semi-micro GPC system saving both, analysis time and solvent.

Introduction

Polypeptoids are similar to peptides. While the side chain of the amino acid residue in a peptide is attached to the alpha carbon, the side chain of a polypeptoid is attached to the nitrogen. The resulting structure imparts proteolytic stability, may mimic polypeptide behaviour and could have novel polymer properties for commercial use, as they are close in structure to nylon-type polyamides. One of the groups most active in this area is Dr Li Jia and co-workers at the University of Akron who are investigating different synthetic routes for the formation of polypeptoids with alternating block structures.^1–4

A series of poly- β-alkylalanoids, obtained by using living alternating copolymerization of N-alkylaziridines and carbon monoxide,⁵ were characterized using an EcoSEC GPC system with TSK-GEL mixed-bed columns and hexafluoroisopropanol (HFIP) as the mobile phase. The EcoSEC GPC system is ideally suited for the detailed study of polymerizations owing to its superior instrument performance. The unprecedented reproducibility, accuracy and RI sensitivity of this system is due to its design: accurate temperature control, dual-compartment oven control, newly engineered pumping system, low RI dead volume and low injection volume.

Experimental Conditions

A series of block poly-β -alkylalanoids, provided by Dr Li Jia, were characterized by SEC analysis. Polymers were dissolved as received in HFIP at a level of 0.5 mg/mL, and passed through a 0.5 µm membrane filter. The column set used was a series of two TSKgel GMH_HR-M* columns (4.6 mm i.d. x 15 cm, Tosoh Bioscience) packed in HFIP. These mixed-bed columns have a separation range of about 10² to 4 x 10⁶ . The mobile phase consisted of HFIP containing 5 mmol/L sodium trifluoroacetate to help prevent sample adsorption. An EcoSEC GPC system was used at a flow rate of 0.35 mL/min at a column and system temperature of 40 °C. Injection volume was 10 µL. A refractive index monitor was used for detection.

Calibration was based on a series of nine poly(methylmethacrylate) (PMMA) standards (American Polymer Standard) ranging in molecular weight from 2825 to 2100000. A cubic fit was used for the calibration curve (correlation -0.967) (Figure 1).

Results and Discussion

Samples of block poly-β -alkylalanoids were chosen that encompassed a wide selection of different block lengths and compositions. SEC chromatograms of three representative samples are shown in Figures 2 to 4 and molecular weights summarized in Table 1. As indicated, all samples had narrow polydispersity values of 1.2, as expected from living polymerization reactions. Because PMMA was used for calibration, which has a different composition than the blocks, reported molecular weight (MW) data are relative or apparent MW, and should only be used for comparisons among samples. In fact, these values are significantly higher than calculated MW of samples computed from experimentally determined block lengths and block composition. Overestimation of MW implies that these blocks have highly extended conformations in HFIP as compared to PMMA. However, to obtain more accurate MW data, standards that are compositionally similar to polypeptoids, such as polyamide standards (e.g., nylon 66) or an on-line viscometer with PMMA universal calibration or a light scattering detector should be used.

For this column set, the exclusion limit is about 1.75 mL (5 min), while the total permeation volume is close to 3.5 mL (10 min). As indicated by the chromatograms, these samples contain almost symmetrical, narrow polymer profiles eluting in the range of about 6 min, while residual solvents, water and dissolved air elute in the 10 to 12 min range. There is very little tailing, and in all cases, the peak tail returns to the initial baseline with no baseline drift. This feature allows for highly reproducible data not available with conventional GPC systems.

The MW data in Table 1 are averaged values from three consecutive injections of the three representative samples, along with the percent relative standard deviations of each set of three injections. The average percent standard deviations range from about 0.04 to 0.5%, with grand average of 0.3% for Mn and 0.2% for Mw. These percent standard deviations are more than 10x lower than the values reported for polyamides in HFIP mobile phase.⁶ Lastly, the percent relative standard deviation of polydispersities (PD) ranges from 0.1 to 0.5%. The high accuracy allows for the detailed study of polymerization reactions.

Conclusion

The EcoSEC GPC System and a set of TSK-GEL mixed-bed columns were used successfully for obtaining high quality MWD data of a series of block poly- -alkylalanoids with HFIP as the mobile phase in under 15 minutes. The apparent MW averages based of PMMA calibration ranged from 27000 to 49000 for Mn and from 30000 to 61000 for Mw with an average polydispersity of 1.20. Because of the EcoSEC GPC system's excellent flow rate and temperature control and baseline stability, average MW values ranged from 0.2 to 0.3% relative standard deviation, a 10-fold reproducibility improvement as compared to SEC literature data of polyamides using a similar mobile phase system.

References

1. L. Jia et al., Chem. Commun., 1436–1437 (2001).

2. L. Jia et al., JACS, 124, 7282–7283 (2002).

3. J. Zhao et.al., J. Polym. Sci., Part A: Polymer Chemistry, 41, 376–385 (2003).

4. G. Liu and L. Jia, Angew. Chem. Int. Ed., 45, 129–131 (2006).

5. L.A. Roper et al., FASEB Journal, 22, 1061.8. (2008).

6. E.C. Robert et al., Pure Appl. Chem., 76, 2009–2025 (2004).

*TSK-GEL GMHHR-M, 5 µm, 4.6 mm i.d. x 15 cm columns are available from Tosoh Bioscience as custom columns.

Tosoh Bioscience GmbH

Zettachring 6, 70567 Stuttgart, Germany

tel. +49 711 13257 0 fax +49 711 13257 89

E-mail: info.sep.eu@tosoh.com Website: www.tosohbioscience.com