SCIENCE
Tailing of peaks Researchers have long been trying to prevent peak tailing in reversed-phase separations of organic cations. Tailing varies from one batch of silica gel to the next, even when the same procedure is used to manufacture the gel, making the problem extremely difficult to solve. Mary J. Wirth and graduate students Melody D. Ludes and Derrick J. Swinton at the University of Delaware have turned to single-molecule spectroscopy to better understand the causes. In an earlier work, they observed strong adsorption of a cationic fluorescent dye (l,l'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, Dil) at a reversed-phase chromatographic interface. By looking at individual molecules, they were able to distinguish between strongly and weakly adsorbed molecules; however, it was unclear whether strong adsorption was caused by active silanols (Anal. Chem. 1998 70, 5264-71)) In the upcoming September 15 issue of Analytical Chemistry, the hesearchers take things one step further using fluorescence microscopy and atomic force microscopy (AFM) to examine the overall patterns of the strong adsorption of Dil at the interface between fused silica (modified with chlorodimethyloctadecylsilane) and water or ace-
tonitrile. Rather than looking at individual molecules, they now focus on the entire surface to determine whether strong adsorption sites are homogeneously distributed and whether strong adsorption is caused by silanols. "Single-molecule spectroscopy is a very powerful technique. We could see that there were two kinds of adsorbates behaving very differently—one strongly adsorbed and one weakly adsorbed, which is what has always been predicted to underlie tailing," says Wirth. "We knew we were looking at something that would indeed cause tailing, but with single molecules, you can't see if there is a heterogeneous pattern to the surface." Using the same instrumental setup as in the singlemolecule experiments, consisting of an inverted fluorescence microscope CCD corners the researchers simply increased the concentration of molecules to image more of them "Once we looked at a lot of molecules it became clear that the pattern was not at all homogeneous " says Wirth The fluorescence images showed that the dye adsorbs at particular lines and spots on the surface indicating that strong adsorotion sites are not randomly distributed The researchers also obtained AFM images of the same surface for comparison
with the fluorescence images. The AFM images revealed remarkably similar markings to those in the fluorescence images, showing that the strong adsorption sites were at nanometer indentations. "For the first time, the active silanols are shown to be associated with surface topography," says Wirth. Efforts to reduce the number of active silanols in the future manufacturing of silica may benefit from considering surface topography as a factor. No relationship between pH and strong adsorption was found, indicating that the so-called active silanols (those that are still active at low pH) were being studied. In addition, end capping, which is used in LC to reduce the number of active silanols, decreased the number of patterns observed on the surface. As in the previous work, highly pure fused silica, which is well hydroxylated to minimize isolated silanols, was used in all experiments. "The surfaces we use are polished by a chemical/mechanical process, which leaves indentations on the nanometer scale," says Wirth. "They are actually molecular scale indentations, which are difficult to get rid of. Chromatographic silica may very well have these molecular scale indentations, which would lead to tailing. Nobody really knows." Britt Erickson
(a) AFM image of dry silica surface, (b) Fluorescence image of the same region. Both images are on 50 um x 50 \sm scale. Analytical Chemistry News & Features, September 1, 1999 5 8 9 A
