Journal of Chromatography A, 1129 (2006) 41–46 Size-exclusion chromatography using deuterated mobile phases Jeanine M. Erdner a,b , Howard G. Barth a , Joe P. Foley b,∗ , William G. Payne a a Corporate Center for Analytical Sciences, DuPont Company, PO Box 80228, Wilmington, DE 19880-0228, USA b Department of Chemistry, Drexel University, 3141 Chestnut St., Philadelphia, PA 19104, USA Received 16 March 2006; received in revised form 15 June 2006; accepted 21 June 2006 Available online 11 July 2006 Abstract The effect of deuterated solvents in size-exclusion chromatography (SEC) was studied by comparing intrinsic viscosity measurements, SEC calibration curves, and column efficiency using water-soluble polymers. For aqueous SEC, the use of deuterium oxide slightly increases the SEC elution volume. To verify that adsorption onto the packing was absent, data from exclusion experiments were compared at 35 and 50 ◦ C. Our results indicate that adsorption is not occurring for pullulan or polyethylene glycol (PEG)/poly(ethylene oxide) (PEO); for the latter, however, the elution volume increased using both D 2 O and H 2 O, indicative of slight hydrodynamic volume contraction of PEG/PEO at higher temperatures. A moderate increase in band broadening (moderate decrease in column efficiency) was observed using D 2 O. Finally, the effects of chloroform versus deuterated chloroform were evaluated, but no hydrodynamic volume changes were observed. © 2006 Elsevier B.V. All rights reserved. Keywords: SEC; Size-exclusion chromatography; Deuterated solvents; Deuterium oxide; Pullulan; PEG/PEO; Proteins; Gel permeation chromatography; Gel filtration chromatography 1. Introduction Depending on the application, deuterated solvents offer an alternative to nondeuterated solvents in liquid chromatogra- phy. Deuterated solvents are useful when using hyphenated techniques like LC–NMR, LC–FTIR, etc. Owing to their different physical properties compared to their nondeuterated counterparts, deuterated solvents offer some advantages when used in conjunction with spectroscopic detectors. For example, deuterated solvents are better for LC–NMR analysis because the solvent is not protonated and thus the entire proton chemical shift range can be used; the need for solvent suppression is also eliminated since no protonated solvents are used [1]. Similarly, with on-line aqueous size-exclusion chromatography (SEC)-IR, absorption by water in the amide I region (1600–1700 cm -1 ) hinders the identification of secondary structures [2]. When deuterium oxide is used instead, the IR absorption in the amide I region is eliminated and the spectral frequencies are slightly shifted compared to water. Remsen and Freeman exploited this spectroscopic ∗ Corresponding author. Tel.: +1 215 895 6218; fax: +1 215 895 1265. E-mail address: jfoley@drexel.edu (J.P. Foley). advantage in their analysis of protein secondary structures [2]. In contrast to the well-studied spectroscopic advantages of deuterated solvents, there have been only a few studies on the effect of deuterated mobile phases on chromatographic param- eters such as retention and separation factors in HPLC. Jinno [3], for example, studied the effects of deuterated versus non- deuterated mobile phases on HPLC separation behavior of different solutes using combinations of H 2 O, D 2 O, methanol, and deutero-methanol mobile phase [3]. He reported a 30% increase in solute separation factors with deuterium oxide com- pared to water, where alkylbenzenes showed a 10% increase in separation factors when using deutero-methanol compared to methanol. The separation factors of other compounds stud- ied, aromatics and aminoacetophenones, only increased slightly, however. Jinno also observed that phthalates had lower separa- tion factors in deutero-methanol compared to methanol [3]. Online NMR detection is becoming increasingly impor- tant in size-exclusion chromatography [4], especially with the use of solvent suppression techniques that reduce or eliminate unwanted chemical shifts from the solvents [5]. As noted earlier, deuterated solvents can obviate the need to perform solvent sup- pression. In view of this, a study was undertaken to determine the effects of deuterated solvent on the SEC behavior of several 0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.06.073