Study On HPLC Online Sample Cleanup And Inhibition Of Peak Tailing

Two-dimensional liquid chromatography(2D-LC) was developed as a very important method to analysis complicated samples. The development of 2D-LC experienced two stages, i.e. off-line and on-line mode. This kind of method has a lot of advantages such as automatic cleaning up and concentrating sample. As timesaving and saving solvent, it can be used to analysis samples with very low concentration. In this study, 2D-LC methods for the analysis of VB12 and human milk oligosaccharides(HMOs) were developed.The main contents can be expressed as follows:1. In this work, we investigated the silanol-blocking ability of cesium ion(Cs+) in reversed-phase liquid chromatographic separation with the different characterizing methods for silanol activity. The comparison of the blocking ability on ion-exchange interaction among Cs+, barium ion(Ba2+) and amine silanol blockers including triethylamine and octylamine was completed using six basic probe solutes at p H 7 on a conventional C18 phase. The effects on blocking hydrogen bonding interaction of different additives were also characterized by using toluidine isomers, p-ethylaniline, aniline and phenol as probes. The results conclude that the blocking ability to silanophilic interactions of Cs+ is better than that of Ba2+. Taking together of the blocking ability of Cs+ on ion-exchange and silanophilic interactions, Cs+ exhibits better blocking effect to tailing than Ba2+. When using Cs+ as an additive in mobile phase to analyze four alkaloids, i.e. protopine, allocryptopine, sanguinarine, and chelerythrine, better silanol-blocking effect can be obtained in slightly acidic mobile phase.2. A new chromatographic method capable of measuring vitamin B12 in adult, infant, ready-to-feed liquids, and pediatric formula powders, and liquid concentrates was developed. This method utilizes solid phase extraction to cleanup and concentrate sample extracts, using size exclusion and reverse phase chromatography to online separate vitamin B12 from interfering compounds, and visible detection at 550 nm to measure vitamin B12 concentrations. The calibration curves were ranging from 2.6 to 26 μg/L. The recoveries of spiking samples are more than 90 %.3. A sensitive Ion Chromatography with Pulsed Amprometric Detection(ICS-PAD) assay method to simultaneously determination six human milk oligosaccharides(HMOs) in human breast milk was developed and validated. The human milk was cleaned up with an online NG1 column and then separated on a PA20 column. The LOQs and LODs for six HMOs ranged from 0.02 to 0.15 μg/m L and 0.01 to 0.04 μg/m L. The recoveries of spiking samples are in the range of 90 %-105 % and most of relative standard deviations of accuracy and precision test are below 10 %.