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High‐resolution peptide separations using nano‐ LC at ultra‐high pressure
Author(s) -
Nováková Lucie,
Vaast Axel,
Stassen Catherine,
Broeckhoven Ken,
Pra Mauro,
Swart Remco,
Desmet Gert,
Eeltink Sebastiaan
Publication year - 2013
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201201087
Subject(s) - chromatography , chemistry , analytical chemistry (journal) , detection limit , resolution (logic) , particle size , capillary action , particle (ecology) , pressure gradient , packed bed , materials science , mechanics , oceanography , physics , artificial intelligence , computer science , composite material , geology
We report on the optimization of nano‐ LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by kinetic plots depicting the gradient time needed to achieve a certain peak capacity, while using the maximum system pressure of 80 MPa. The selection of the optimal particle size/column length combination and corresponding gradient steepness was based on scouting the performance of 75 μm id capillary columns packed with 2, 3, and 5 μm fully porous silica C 18 particles. At optimal gradient conditions, peak capacities up to 500 can be obtained within a 120 min gradient using 2 μm particle‐packed capillary columns. Separations of proteomic samples including a cytochrome c tryptic digest, a bovine serum albumin tryptic digest, a six protein mix digest, and an Escherichia coli digest are demonstrated while operating at the kinetic‐performance limit, i.e. using 2‐μm packed columns, adjusting the column length and scaling the gradient steepness according to sample complexity. Finally, good run‐to‐run retention time stability ( RSD values below 0.18%) was demonstrated applying ultra‐high pressure conditions.