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to determine which approach is most appropriate in efforts to reduce cycle time.
Figure 2. Ten replicate injections in a hydrodynamic chromatography size refinement of 1.0 µm fully porous bridged-ethyl hybrid particles (peak 1) with urea (peak 2) used as a void volume marker. Adapted with permission from [20].
needle wash processes, which ensured that it could be completed prior to the finish of the preceding 20 s chromatographic run (Figure 1). Although no significant issues were observed with carryover in this example, it can become an issue depending on the specific analytes and sample solvent coming into contact with the autosampler needle and loop. In a demonstration of a more robust, qualified high-throughput OTC analgesic assay, a 40 s cycle time was achieved combining an equivalent separation time and a ~30 s autosampler sequence that included needle washes [8].
Isocratic separations were used in both of the examples described in the previous paragraph. With synchronous operation of the autosampler and chromatographic analysis, the primary concern is to ensure that no valve actuation steps are coincident with eluting peaks. Such actuations can result in pressure changes that lead to detector signal disruptions and distortions to observed peak shape. When rapid (or ‘ballistic’) gradient separations are used in low cycle time analyses, the system dwell volume is another critical consideration because it affects how long it takes the programmed gradient to reach the column [9]. Connections between the pump and injector (which may include an in-line mixer), flow paths within the injector itself, and additional connections to the column all contribute to this value. Autosampler configurations that do not flow through the needle typically provide smaller volumes that are more amenable for high- throughput gradient methods, though their design creates the potential for air bubble formation in the injector flow path [3].
A variation on synchronous injection cycle sample preparation is the multiple injections
in a single experimental run (MISER) technique [10]. In MISER, multiple injections are run in the same way that is described above, but the data is not collected as individual chromatograms and is instead collected in a single data file. This is an ideal strategy for qualitative work in which a rapid comparison of a series of runs in an injection sequence can quickly identify general trends and outliers (i.e. high and low abundance peaks) within a sample set. For example, MISER has been used to rapidly compare the amount of hop-derived components in different varieties of beer [11], caffeine concentration in beverages [12], and capsaicin content in peppers and hot sauces [13]. It is also a powerful strategy for the enantiopurity monitoring and other high-throughput screening needs within the pharmaceutical industry [1, 14]. The key drawback in MISER is the increased difficulty in peak quantification compared to collecting individual chromatograms for each unique run. Many software algorithms for quantitation use area comparisons between analyte and internal standard peaks at a specific retention time (and/or m/z value, depending on the detection mode), which can be more difficult to incorporate when a number of individual separations are all plotted on a single chromatogram. While it can still be accomplished through chromatogram splitting programs [9] or manual processing, these strategies require more user interaction to ensure all the peaks are properly matched; the overall workload increases significantly as the number of runs grows in a high- throughput screening experiment. Therefore, it is important to consider the overall purpose of the high-throughput experiments being conducted, more specifically whether they are qualitative or quantitative, in order
A modified version of the synchronous autosampler cycle approach is to actually inject the subsequent sample during a preceding run rather than to just prepare for injection. This approach is often used when performing ‘stacked injections’ for chiral purifications in preparative-scale HPLC or SFC. When isocratic elution conditions are used and the peak elution window is short relative to the total peak elution time, stacked injections reduce solvent consumption and improve production rates [15]. The approach is also effective with non- retentive chromatography modes (i.e. size exclusion chromatography, hydrodynamic chromatography) [16, 17], where the finite elution window of the peaks is known based on the volume difference between analytes at the high and low ends of the size range for a given stationary phase. Because of the initial delay between sample injection and the first eluted peak, an additional injection can be performed during this time gap so that this same first peak from the second injection elutes closely to the final peak from the initial sample. This was demonstrated for a preparative-scale hydrodynamic chromatography separation used to reduce the particle size distribution of a silica packing material [18,19]. Because multiple injections were needed to obtain the amount of purified sample that was required, this strategy eliminated any added time between runs used to deliver the sample to the injection loop [20] (Figure 2). A similar approach has also been described for aggregate characterisation of monoclonal antibodies [21, 22]. Although these examples included slightly longer runs and the added time would have been only a fraction of the overall cycle time, the technique would have an impact on high-throughput size-based separations [23]. A similar process has been developed for retentive chromatography modes with more complex mixtures, but it can be much more difficult to implement due to the optimisation required to avoid peak co-elutions [24]. In those instances, the previously described strategies may be preferred.
Considerations for Injector- Related Broadening Effects
The use of columns with smaller dimensions, and thus volumes, has made the consideration of instrument-based broadening effects a critical aspect of high- throughput LC. The most basic description of broadening due to the injection cycle is based solely on the volume of sample injected [25]:
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