saccharide at the reducing end of the oligosaccharide. Because alkaline degradation produces numerous side reactions and products, the FT-ICRMS is necessary—particularly for the exact mass determinations. Lebrilla says, "An exact mass determination for each of the fragments is pretty important, so that we can fish out the real product of the reaction from the chemical noise." The size of oligosaccharides is generally not a problem for sequencing. The big problem is the branching. "If [the oligosaccharide] is too highly branched, you may get a lot of information near the reducing end, but you won't get much information at the non-reducing end." The alkaline degradation occurs at different rates, which depend on the linkages. Therefore, if a branched oligosaccharide has antennae with different linkages, it will be relatively simple to determine the linkage at the branch points. Another strength of the alkaline degradation/MALDI FT-ICRMS combination is that it can also pinpoint the location of sialic acid and fucose on oligosaccharides. In traditional oligosaccharide sequencing, these residues often fall prey to nonselective glycosidic bond cleavage. Although the method provides sequence and linkage information, it does not provide complete structural information. In particular, it does not provide information about either the stereochemistry or a and P linkages. "The bad side is that you may never get full structural information [with MS]," says Lebrilla. "For that, you still have to use something such as NMR, but those techniques require significantly more material." Lebrilla points out that obtaining sufficient quantities of material will always be a problem for oligosaccharide analysis. They have thus far used the method to sequence a range of known oligosaccharides. The next step is to use the method to sequence unknown samples. To that end, they are collaborating with Jerry Hedrick of the molecular and cell biology department at the University of California-Davis to sequence oligosaccharides involved in frog egg fertilization. "There's been some work done with NMR in which they've gotten a few full structures, but they've only looked at the more abundant components," says Lebrilla. "We want to go back and see what the less abundant components are that they couldn't do with NMR We want to do real samples and see if we can get results."
Simple, yet sophisticated How can one build a detector for CE that leaves the capillary undisturbed and therefore can be placed anywhere along its length? That does not need a window in the capillary coating that would weaken the device? That can be constructed with less than $100 worth of materials? A schematic of the contactless conductivity detector. Such a detector was prewhen an electric frequency is applied. sented in the Feb. 1 issue of Analytical Schnell works with frequencies in the Chemistry (p. 563)3 It was seveloped dnd 20- to 40-kHz range, thereby keeping tested by a team at the Institute of Anathe capacitive reactance low. lytical Chemistry and Radiochemistry at the Leopold-Franzens-University InnsIn an earlier version of the detector, bruck (Austria). The Institute has a the two electrodes were made by paintlong research tradition in isotachoing a conductive silver varnish directly phoresis, CE, HPLC, and coupled methonto the CE capillary coating, rather ods. As long ago as the 70s, Erhard than by using syringe needles. The silSchnell, a now-retired professor at Innsver varnish provided excellent sensitivbruck, had the idea of a contactless conity for the signals because no air gap ductivity detector with capacitive couexisted between the electrodes and the pling. However, it wasn't realized until capillary. The construction is more comhis retirement and the rise of CE. plicated than with cannulas, however, Giinther K Bonn the head of the Instiand it leaves the detector's position tute offered a small laboratory to fixed. In the newer cannula version, the Schnell that would allow him to bring detector can be placed anywhere along his ideas to fruition Because a retired the capillary. In fact, Zemann hopes that professor does not have an official reit will be possible to investigate the fate search group Andreas J Zemann also of CE bands during their migration a professor at Innsbruck and his group through the capillary would use and test the new device With the newer detector, conductivity The detector consists of two syringe cannulas that are mounted 2 mm apart so that the CE capillary can be pushed through both of them (see figure). The cannulas act as capacitors that, together with a homemade frequency generator and amplifying electronics, measure the electrolyte conductivity inside the gap between them. When an analyte zone passes, the conductivity increases, which will be registered as a peak. The conductivity is measured via the capacitive properties of the detection unit, which is in fact a resistor-capacitor unit
Up close with the new detector.
is measured axially along the capillary (over the length of the gap between electrodes) and not across the capillary diameter. Therefore, capillaries of a different diameter give similar signals. The length of the gap is of minor importance. The detector linearity is excellent (from less than 1 ppm to more than 1000 ppm for sodium and chloride), and the detection limits are comparable with instruments on the market. Schnell and Zemann hope to improve the detection limits even more. For this purpose the use of more sophisticated electronics will be sary to optimize the waveform of the frequency generator and to improve the grounding Zemann also plans to develop a suppressor device that could be coupled to Schnell's detector and would improve the baseline by bringing the background conductivity to almost zero Veronika R. Meyer
Celia Henry Analytical Chemistry News & Features, March 1, 1998 175 A
