Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.
Waters Corporation, 34 Maple Street, Milford, MA 01757, USA.
J Chromatogr A. 2019 Apr 26;1591:110-119. doi: 10.1016/j.chroma.2019.01.021. Epub 2019 Jan 11.
The impact of porous frits (2.1 mm i.d. × 1 mm thick, 0.2 μm porosity, 20% void, 0.69 μL) in short 0.5-5 cm long × 2.1 mm i.d. columns packed with sub-2 μm superficially porous particles on gradient performance was investigated using a low dispersive i-class ACQUITY UPLC system (25 cm × 75 μm outlet tube + 250 nL optical cell). In order to maximize data accuracy, the sample dispersion through a single frit was measured from four independent methods: (1) plate height subtraction, (2) peak capacity versus efficiency plot, (3) flow reversal, and (4) direct measurement. Frit dispersion increases non-linearly with increasing flow rate. The corresponding volume variances of small molecules were measured at 0.16 ± 0.04, 0.24 ± 0.05, and 0.31 ± 0.06 μL at flow rates of 0.1, 0.2, and 0.3 mL/min, respectively. These observed variances are lower than and consistent with the maximum volume variance of 0 . 69 ∼ 0.5 μL expected for a mixer-like behavior. The peak capacity of short columns were then calculated for mixtures of peptides using a general model of gradient elution by considering (or not) the actual analyte dispersion taking place in the outlet frit, in the post-column connecting tube, and in the detection cell. The results show that the sole presence of the outlet frit is responsible for about 50% loss in peak capacity (relative to the expected gradient performance in absence of frit and post-column tube) for a 1 cm long column operated under standard gradient steepness. The actual structure and volume of column frits needs adjustment if one wants to take full advantage of the true performance of short high-throughput columns packed with sub-2 μm particles.
研究了在短（0.5-5cm 长×2.1mmID）、填充亚 2μm 表面多孔颗粒的柱子中使用 2.1mmID×1mm 厚、0.2μm 孔径、20%空隙率和 0.69μL 的多孔 frit（2.1mmID×1mm 厚、0.2μm 孔径、20%空隙率和 0.69μL）对梯度性能的影响。使用低分散性 i 级 ACQUITY UPLC 系统（25cm×75μm 出口管+250nL 光学池）进行了研究。为了最大限度地提高数据准确性，通过四种独立的方法测量了单个 frit 样品的分散度：（1）板高减法，（2）峰容量与效率图，（3）流量反转和（4）直接测量。 frit 分散度随流速的增加呈非线性增加。在流速分别为 0.1、0.2 和 0.3mL/min 时，小分子的相应体积方差分别测量为 0.16±0.04、0.24±0.05 和 0.31±0.06μL。这些观察到的方差低于并与混合器样行为下预期的最大体积方差 0.69∼0.5μL 一致。然后，使用考虑（或不考虑）在出口 frit、在柱后连接管中和在检测池中的实际分析物分散的梯度洗脱的一般模型，计算了短柱中肽混合物的峰容量。结果表明，在标准梯度陡度下操作的 1cm 长柱中，仅出口 frit 的存在就导致峰容量损失约 50%（相对于无 frit 和柱后管的预期梯度性能）。如果要充分利用填充亚 2μm 颗粒的短高通量柱的真实性能，则需要对柱 frit 的实际结构和体积进行调整。
