CHROMATOGRAPHY, Vol. 35 (2014), No. 1, pp. -59

¹Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
²Graduate School of Pharmaceutical Sciences and GPLLI Program, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Two separated peaks were detected when a small volume (approximately less than 0.5 μL) of one compound was injected into a HPLC system using an autosampler equipped with a 6-port valve. The origin of the two peaks was found to be injection by an autosampler that sucked the sample solution twice during the injection of each sample. First, the autosampler sucked in a sample solution to fill the needle, and then, after the rotation of the 6-port valve, the autosampler sucked in the sample solution again to fill the injection loop. Because the twice suck generated two separated sample zones in a 6-port valve and these sample zones were injected into the column separately, two separated peaks were observed. The increase in the sample volume induced disappearance of second peak and only one peak was observed. And also the same phenomenon was observed with a manual injector instead of the autosampler even when small volume of sample was injected.