The use of flow splitters between the two dimensions in on-line comprehensive two dimensional liquid chromatography (LC×LC) has not received very much attention in comparison to their use in GC×GC where they are quite common. In principle, splitting the flow after the first dimension column and performing on-line LC×LC on this constant fraction of the first dimension effluent should allow the two dimensions to be optimized almost independently. When there is no flow splitting any change in the first dimension flow rate has an immediate impact on the second dimension. With a flow splitter one could for example double the flow rate into the first dimension column and do a 1:1 flow split without changing the sample loop size or the sampler’s collection time. Of course, the sensitivity would be diminished but this can be partially compensated by use of a larger injection; this will likely only amount to a small price to pay for this increased resolving power and system flexibility. Among other benefits, we found a 2-fold increase in the corrected 2D peak capacity and the number of observed peaks for a 15 min analysis time by using a post first dimension flow splitter. At a fixed analysis time this improvement results primarily from an increase in the gradient time resulting from the reduced system re-equilibration time and to a smaller extent it is due to the increased peak capacity achieved by full optimization of the first dimension.

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.

We recently introduced a new method ([1]) to deposit carbon on fully porous silicas (5 μm) to address some of the shortcomings of carbon clad zirconia (C/ZrO2), which has rather low retention due to its low surface area (20–30 m2/g). The method enables the introduction of a thin, homogeneous layer of Al (III) on silica to serve as catalytic sites for carbon deposition without damaging the silica’s native pore structure. Subsequent carbon deposition by chemical vapor deposition resulted in chromatographically useful carbon phases as shown by good efficiencies and higher retentivity relative to C/ZrO2. Herein, we use the above method to develop a novel carbon phase on superficially porous silica (2.7 μm). This small, new form of silica offers better mass transfer properties and higher efficiency with lower column back pressures as compared to sub 2 μm silica packings, which should make it attractive for use as the second dimension in fast two-dimensional LC (LC x LC). After carbon deposition, several studies were conducted to compare the new packing with C/ZrO2. Consistent with work on 5 μm fully porous silica, the metal cladding did not cause pore blockage. Subsequent carbon deposition maintained the good mass transfer properties as shown by the effect of velocity on HETP. The new packing exhibits efficiencies up to ~ 5.6 fold higher than C/ZrO2 for polar compounds. We observed similar chromatographic selectivity for all carbon phases tested. Consequently, the use of the new packing as the second dimension in fast LC x LC improved the peak capacity of fast LC x LC. The new material gave loading capacities similar to C/ZrO2, which is rather expected based on the surface areas of the two phases.