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首页> 外文期刊>Journal of chromatography, A: Including electrophoresis and other separation methods >High-speed gradient separations of peptides and proteins using polymer-monolithic poly(styrene-co-divinylbenzene) capillary columns at ultra-high pressure
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High-speed gradient separations of peptides and proteins using polymer-monolithic poly(styrene-co-divinylbenzene) capillary columns at ultra-high pressure

机译：使用聚合物整体式聚苯乙烯-二乙烯基苯毛细管柱在超高压下高速梯度分离肽和蛋白质

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摘要

For the first time, polymer monolithic capillary columns have been employed at ultra-high-pressure liquid chromatographic conditions (UHPLC) to investigate their potential for high-speed separations of peptides and intact proteins. In comparison to conventional flow rates and gradient conditions, a substantial decrease in analysis time (>factor 4) can be achieved when operating monolithic columns such as ultra-high-pressure conditions while scaling the gradient volume. The effects of flow rate and column length on the peak capacity and the gradient performance limits were assessed for the separation of peptide and protein mixtures applying the maximum system pressure (80. MPa) and a fixed gradient steepness. The potential for ultra-fast gradient separations of large biomolecules was further demonstrated for very steep gradients (gradient times. ?. 1. min). A tryptic digest of cytochrome c was separated using a gradient time of only 1. min. Finally, the run-to-run repeatability and column robustness were assessed at ultra-high pressure conditions (after. >. 800 runs) with consecutive steep 1. min separations of peptides, yielding RSD values below 0.12% in retention time.
机译：聚合物整体毛细管柱首次在超高压液相色谱条件（UHPLC）上被使用，以研究其在肽和完整蛋白的高速分离中的潜力。与常规流速和梯度条件相比，当操作整体式色谱柱（例如超高压条件）并按比例缩放梯度体积时，可以大大减少分析时间（> 4）。使用最大系统压力（80. MPa）和固定的梯度陡度，评估了分离肽和蛋白质混合物时，流速和色谱柱长度对峰容量和梯度性能极限的影响。对于非常陡峭的梯度（梯度时间≤1分钟），进一步证明了大生物分子超快速梯度分离的潜力。仅用1分钟的梯度时间即可分离出细胞色素c的胰蛋白酶消化物。最后，在超高压条件下（> 800次运行），通过连续的陡峭1. min肽分离，评估了运行间的重复性和色谱柱的耐用性，得出保留时间低于0.12％的RSD值。

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