12 Size-Exclusion Chromatography of Dextrans and Pullulans with Light-
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Scattering, Viscosity, and Refractive Index Detection William S. B a h a r y , M i c h a e l P. H o g a n , M a h m o o d J i l a n i , and Michael P. A r o n s o n Unilever Research, 45 River Road, Edgewater, NJ 07020
The objective of this work was to characterize pullulans and dex trans in order to assess the performance of the size-exclusion chro matography (SEC) system with multi-angle laser light-scattering (MALLS), viscosity, and refractive index (RI) detectors using a buffered aqueousmedium as the mobile phase. In 0.2 Μ NaNO , with standard pullulans that had low polydispersity, the weight -average molecular weights()obtained by light scattering and by universal calibration agreed well with the expected values from the supplier with a mean deviation of about 5%. With standard dextrans that had higher polydispersities (1.6), theM sexhibited greater mean deviation, about 12%. Mark-Houwink exponents of 0.64 and 0.39 for pullulans and dextrans, respectively, obtained from plots were reasonable. The scaling law of radius of gyration versus gave exponents of 0.48 and 0.38 for the polymers after adequate filtration. These were lower than the slopes calcu lated theoretically using the Ptitsyn-Eisner equation, but similar values have been reported elsewhere. Overall, triple detection SEC proved to be a powerful and time-saving technique for character izing water-soluble polymers absolutely and confirming their values. 3
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/YLTHOUGH SIZE-EXCLUSION CHROMATOGRAPHY (SEC) w i t h the con ventional or universal calibration technique i n combination with viscosity detection is w e l l established i n nonaqueous media ( J , 2), its application in aqueous media is more complicated because of unusual difficulties 0065-2393/95/0247-0151$12.00/0
© 1995 American Chemical Society
Provder et al.; Chromatographic Characterization of Polymers Advances in Chemistry; American Chemical Society: Washington, DC, 1995.
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CHROMATOGRAPHIC CHARACTERIZATION OF POLYMERS
associated w i t h water-soluble polymers and biopolymers i n aqueous systems (3, 4). These complications include hydrogen bonding, h y d r o phobic interactions, polar interactions, and i o n - i o n interactions that can make the separation by size nonideal. T h e first three can lead to ad sorption or delay of sample elution times. These effects can be suppressed through the proper selection of column and mobile phase. I o n - i o n i n teractions of the polymer w i t h column support can lead to ion-exclusion and early elution relative to noninteracting polymers. Intraionic inter actions of polyelectrolytes can lead to different polyion sizes and shapes w i t h different elution times. These ionic effects can be m i n i m i z e d by the addition of salt to the mobile phase. F o r samples for w h i c h no standards exist, low-angle laser light scat tering ( L A L L S ) is a possible solution, but has the drawback of excessive scattering by dust at low angles and it does not provide the macromolecular radius (5). Multi-angle laser light-scattering ( M A L L S ) photom eters are a potentially more powerful detection technique (6, 7). T h e r e fore the objective of this work was to characterize pullulans and dextrans to assess the performance of the size-exclusion chromatograph w i t h M A L L S and viscosity and refractive index (RI) detectors. It was antici pated that w i t h the combination of detectors, the performance of one detector could be effectively checked against that of the other. This type of checking becomes especially important i n aqueous systems be cause i n addition to the other issues, aqueous mobile phases are more difficult to clarify than nonaqueous ones.
Experimental Procedures Materials. Standard pullulans having weight-average molecular weights « M > s ) of 23.7, 48.0, 100, 186, and 380 k D a with a polydispersity ( M / M ) of about 1.1 were obtained from J. M . Science (Buffalo, N Y ) . Stan dard dextrans having
