Kubán Pavel, Hauser Peter C
Department of Chemistry, University of Basel, Spitalstrasse 51, 4004 Basel, Switzerland.
J Chromatogr A. 2007 Dec 28;1176(1-2):185-91. doi: 10.1016/j.chroma.2007.10.109. Epub 2007 Nov 9.
Gradient programs were applied to the determination of peptides and proteins in HPLC with contactless conductivity detection. A monolithic capillary column was used for the fast and sensitive determination of the biochemical species in acidic mobile phases consisting of acetic acid or trifluoroacetic acid in various concentrations of acetonitrile in water. The drift in baseline, which is caused by conductivity changes during the elution program, was minimized by careful optimization of the composition of the mobile phase and remaining drift was removed by computational baseline normalization. The flow rate from a conventional HPLC pump was reduced to a flow rate suitable for capillary systems using a pre-column flow splitter and a final total flow rate of 1.65 microl/min was used for all capillary HPLC separations. The contactless conductivity detector was positioned directly on the outlet capillary of the separation column and positively charged peptides and proteins were determined as sharp and symmetrical peaks. Detection limits in a concentration range from 3.7 x 10(-8) to 5.1 x 10(-7)M and a reproducibility of peak areas and peak heights between 2.3% and 7.3% were achieved for all biochemical species tested.
梯度程序被应用于高效液相色谱法（HPLC）中采用非接触式电导检测法测定肽和蛋白质。使用整体式毛细管柱在由不同浓度乙腈水溶液中的乙酸或三氟乙酸组成的酸性流动相中快速且灵敏地测定生化物质。通过仔细优化流动相组成，将洗脱程序期间由电导率变化引起的基线漂移降至最低，剩余漂移通过计算基线归一化去除。使用柱前分流器将常规HPLC泵的流速降低至适合毛细管系统的流速，所有毛细管HPLC分离均使用1.65微升/分钟的最终总流速。非接触式电导检测器直接置于分离柱的出口毛细管上，带正电荷的肽和蛋白质被测定为尖锐且对称的峰。对于所有测试的生化物质，实现了3.7×10⁻⁸至5.1×10⁻⁷M浓度范围内的检测限以及2.3%至7.3%的峰面积和峰高重现性。
