The experimental effects of sampling time on the resolving power of on-line LC×LC were investigated. The first dimension gradient time (1tg) and sampling time (ts) were systematically varied (1tg=5, 12, 24 and 49min; ts=6, 12, 21 and 40s). The resolving power of on-line LC×LC was evaluated in terms of two metrics namely the numbers of observed peaks and the effective 2D peak capacities obtained in separations of extracts of maize seeds. The maximum effective peak capacity and number of observed peaks of LC×LC were achieved at sampling times between 12 and 21s, at all first dimension gradient times. In addition, both metrics showed that the " crossover" time at which fully optimized 1DLC and LC×LC have equal resolving power varied somewhat with sampling time but is only about 5min for sampling times of 12 and 21s. The longest crossover time was obtained when the sampling time was 6s. Furthermore, increasing the first dimension gradient time gave large improvements in the resolving power of LC×LC relative to 1DLC. Finally, comparisons of the corrected and effective 2D peak capacities as well as the number of peaks observed showed that the impact of the coverage factor is quite significant.
Bibliographical noteFunding Information:
This work was supported by a grant from NIH ( GM 054585 ). We also wish to acknowledge funding from the Agilent Foundation and the gifts of columns from Supelco Inc. and ZirChrom Separations Inc. The authors wish to thank Dr. Wenzhe Fan and especially Prof. Dwight Stoll for many helpful conversations during the course of this work. The authors also wish to thank Klaus Witt of Agilent Technologies for his many valuable suggestions.
- Peak capacity
- Resolving power
- Sampling time
- Two dimensional liquid chromatography