36 February / March 2021


used in high-throughput LC. Thus, further optimisation of channel structure and overall injector design are still needed to ensure high chromatographic performance in rapid LC separations.


Potential Directions for High-Throughput Sample Injection


Figure 3. Comparison of the exponential decay ‘tau’ broadening for full loop (full squares) and timed pinch (open squares) injections when the injector is coupled to a ~1 m segment of 50 µm i.d. capillary. Adapted


with permission from [27]. (1)


However, this is a simplification that assumes a rectangular injection band profile and is not a true estimation of the actual broadening that occurs due to the injection process [26]. A number of studies have identified additional causes of broadening in the injection process, many of which are flow-dependent and can grow as the separation speed is increased. In one experiment, a miniaturised electrochemical detector was used to track injector band profiling in a nano-volume internal loop design coupled a variety of tubing diameters and lengths. It was determined that the eluted peaks contained both a Gaussian and exponential component, the exponential component of which grew with increasing flow rate (Figure 3) [27]. The likely source of this broadening was the abrupt changes in channel diameter that existed between the stator, rotor, and connected tubing, where each channel mismatch created a ‘mixing chamber’. This experimental observation was further supported through fluid dynamic modelling of a computational reconstruction of the injector flow path. To reduce this broadening contribution, the tailed portion of the peak can be removed from the injection profile by performing a partial loop ‘timed pinch injection’ with rapid actuation of the valve back to the ‘load’ position after injection (Figure 3), although this can reduce injection repeatability compared to traditional full loop injection [27].


Commercial autosamplers in analytical- scale systems have also been studied to determine the broadening effects due to injection. In systems that contain a capillary connection between the needle seat and the injector (common for flow-through needle designs), smaller-diameter tubing is critical to minimise broadening [25, 28]. There was also an observable increase in the injector-based broadening with increasing flow rates, which levelled off around 0.8 mL/ min [28]. For loop-based injectors that are more similar to those that were investigated in the preceding paragraph, the impact of both injection volume and flow rate on the observed broadening were both lower than with the flow-through needle injector. However, with larger inner diameter loops, added contributions at high flow rates were also observed in this format [28]. Fluid dynamic modelling was also used to further explore these observations, and the presence of two regimes within the injector were identified: the convective regime and the diffusion regime [29]. The dominant regime depends on a number of variables, including the injection volume, needle diameter (specifically for flow- through needle injectors), analyte diffusion coefficient, and chromatographic flow rate. Under typical LC operating conditions that would be used for chromatographic analysis, loop-based injectors produce narrower injection bands than flow-through designs [29]. These various studies all indicate that band broadening contributions due to injection are usually underestimated and that there is typically an increase in this broadening at the higher flow rates


As described above, the modern injection process has been improved significantly, with the fastest autosampler cycle times now down to 7 s [17]. However, as ultrafast subsecond separations become more feasible [30], 7 s will still be far too slow to achieve high-throughput screening rates that compare to MS-based techniques which can now exceed 30 samples per second [31]. One approach is to use parallel sampling that introduces multiple samples into the flow stream simultaneously [32]. To some degree, this has already been incorporated into modern autosamplers that contain dual needle designs [33, 34], although this multiplex approach would need to be increased significantly to accommodate more rapid separations. The potential to use segmented flow droplets in a multiplex array to further increase sample throughput was described in a presentation at Pittcon 2020 [35]. The highest analysis rates that have been achieved when coupling droplets to a separation technique thus far rely upon microchip electrophoresis [36, 37, 38], as there are many successful approaches to injecting droplet streams into an electrophoretic separation channel [39]. This process can be more challenging with pressure-driven LC separations. A swan probe approach has been used to collect sample from spatial droplet arrays and deliver it to an in-line monolithic column [40], although it relies upon a channel sealing technique that limits operating pressure to 25 bar and is no faster than state-of-the-art commercial LC autosamplers. In a reaction optimisation platform that included on-line LC-MS analysis, droplet streams segmented with an inert gas were coupled directly to an injection loop with an additional vacuum port used to clear remaining liquid in the loop and reduce carryover [41]. This latter technique may have the best capacity for increased throughput, but it is critically important to precisely time the valve actuation so that only sample is injected and not the carrier phase. This may only cause minor issues if the droplets are segmented with inert gas, but could be much more detrimental when a fluorinated oil that could


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