Microchip HPLC of peptides and proteins
Microchip HPLC of peptides and proteins
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DOI：
10.1021/ac048358r
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发表时间：
2005-05-01
影响因子：
7.4
通讯作者：
Kirby, BJ
中科院分区：
文献类型：
--
作者：
Reichmuth, DS;Shepodd, TJ;Kirby, BJ
快速微芯片反相HPLC的肽和蛋白质在12巴/厘米（180 psi/cm）的压力梯度已进行使用微型装置，集成了亚纳升芯片上的注射和分离与小型化的荧光检测器。蛋白质和肽分离的C18侧链多孔聚合物整料定义的接触光刻，并通过使用投影光刻定义的压力可切换的含氟聚合物阀实现注射。肽标准品和蛋白质混合物的初步分离在40-200秒内进行，并在样品之间进行切换，没有可检测的样品残留。进样和分离具有重现性;保留时间的相对标准偏差（RSD）为0.03%，峰面积RSD为3.8%。样品体积范围从220到800 pL可以线性计量，通过控制压力注射脉冲持续时间与传统的定时和阀门。目前的原型系统显示了快速和自主HPLC分离的潜力，具有不同的模式和直接连接到质谱仪在纳米喷雾流速的潜力。
Rapid microchip reversed-phase HPLC of peptides and proteins at pressure gradients of 12 bar/cm (180 psi/cm) has been performed using a microdevice that integrates subnanoliter on-chip injection and separation with a miniaturized fluorescence detector. Proteins and peptides were separated on a C18 side-chain porous polymer monolith defined by contact lithography, and injection was achieved via a pressure-switchable fluoropolymer valve defined using projection lithography. Preliminary separations of peptide standards and protein mixtures were performed in 40-200 s, and switching between samples with no detectible sample carryover has been performed. The injections and separations were reproducible; the relative standard deviation (RSD) for retention time was 0.03%, and peak area RSD was 3.8%. Sample volumes ranging from 220 to 800 pL could be linearly metered by controlling the pressure injection pulse duration with conventional timing and valving. The current prototype system shows the potential for rapid and autonomous HPLC separations with varying modalities and the potential for direct connection to mass spectrometers at nanospray flow rates.