An experimental study of sampling time effects on the resolving power of on-line two-dimensional high performance liquid chromatography

Abstract

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 49 min; ts = 6, 12, 21 and 40 s). 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 was always achieved at sampling times between 12 and 21 s, 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 5 minutes for sampling times of 12 and 21 s. The longest crossover time was obtained when the sampling time was 6 s. 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.

Improving Peak Capacity in Fast Online Comprehensive Two-Dimensional Liquid Chromatography with Post-First-Dimension Flow Splitting

“…The procedure is based on a painstaking visual inspection of each individual second dimension chromatogram and the manual merging of peaks that correspond to the same first dimensional peak. This procedure has been described in detail 4 .…”

“…Previously we and others have shown that in this form of LC×LC there is necessarily an optimum sample acquisition time 4, 5 . In on-line LC×LC this sampling time ( t s ) must be equal to the second dimension cycle time ( 2 t c ).…”

“…More recently the various benefits of flow splitting were discussed by Tranchida et al 2 . However, we have only seen a few references to the use of post first dimension flow-splitting in on-line LC×LC 3, 4 ; in none of them was the flow-splitting used for independently optimizing the first dimension. Block diagrams of on-line LC×LC systems without and with post first dimension flow-splitting as implemented in this work are shown in Figs.…”

Two dimensional assisted liquid chromatography – a chemometric approach to improve accuracy and precision of quantitation in liquid chromatography using 2D separation, dual detectors, and multivariate curve resolution

“…In practice, the effective peak capacity is lower for several reasons, including the difficulty in choosing a pair of columns which maximize the use of the 2D separation space [3], partially due to the lack of orthogonality between commercially available columns [4], and the undersampling of the first dimension separation [5]. Despite these limitations Stoll, Wang, and Carr determined through both theoretical and experimental studies that when separation times are greater than 10 min and the 2 D separation is conducted sufficiently rapidly, LC Â LC has superior effective peak capacity as compared to 1D-LC [5][6][7][8]. While multi-dimensional chromatography has definite advantages in terms of peak capacity and peak capacity per unit time, the precision and accuracy of these methods compared to 1D chromatography often are not considered.…”

Effect of first dimension phase selectivity in online comprehensive two dimensional liquid chromatography (LC×LC)

“…The concept of LC × LC was introduced three decades ago; the enormous improvements since 1990 have confirmed the huge potential of LC × LC for increasing peak capacity; this mainly results from the “multiplicative advantage” of multi-dimensional methods under ideal conditions [12-17]. Recently, Carr and coworkers performed a series of LC × LC studies on metabolomics samples with total analysis times in the range of 15–60 min [18-20] but with the second dimension run on the time scale of 6–40 s by doing the second dimension separations at high temperatures (100–120 °C). The decreased eluent viscosity at higher temperatures makes it possible to use high linear velocities, high flow rates (~3 mL/min) and short cycle times on the second dimension.…”