Reichmuth David S, Shepodd Timothy J, Kirby Brian J
Sandia National Laboratories, P.O. Box 969 MS 9291, Livermore, California 94551, USA.
Anal Chem. 2005 May 1;77(9):2997-3000. doi: 10.1021/ac048358r.
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.
利用一种集成了亚纳升芯片上进样和分离以及小型化荧光检测器的微型设备，在12巴/厘米（180磅力/平方英寸/厘米）的压力梯度下对肽和蛋白质进行了快速微芯片反相高效液相色谱分析。蛋白质和肽在通过接触光刻法定义的C18侧链多孔聚合物整体柱上进行分离，并通过使用投影光刻法定义的压力可切换含氟聚合物阀实现进样。肽标准品和蛋白质混合物的初步分离在40 - 200秒内完成，并且在无可检测样品残留的情况下进行了样品间切换。进样和分离具有可重复性；保留时间的相对标准偏差（RSD）为0.03%，峰面积RSD为3.8%。通过使用传统的定时和阀控来控制压力进样脉冲持续时间，可以对220至800皮升范围内的样品体积进行线性计量。当前的原型系统显示了以不同模式进行快速自主高效液相色谱分离的潜力，以及在纳喷流速下直接连接质谱仪的潜力。
