High-resolution peptide separations using nano-LC at ultra-high pressure

• Published in: Journal of separation science Vol. 36; no. 7; pp. 1192 - 1199
• Main Authors: Nováková, Lucie, Vaast, Axel, Stassen, Catherine, Broeckhoven, Ken, De Pra, Mauro, Swart, Remco, Desmet, Gert, Eeltink, Sebastiaan
• Format: Journal Article
• Language: English
• Published: Weinheim Blackwell Publishing Ltd 01.04.2013; Wiley; Wiley Subscription Services, Inc
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Capillarity; Chromatography; Chromatography, High Pressure Liquid; Column coupling; Column packings; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Kinetic plot; Nano flow rate; Nanomaterials; Nanostructure; Nanotechnology - methods; Optimization; Packed capillary columns; Particle Size; Peptides; Peptides - chemistry; Peptides - isolation & purification; Pressure; Proteins; Proteomics; Separation; Steepness; Ultra-high pressures; Capillary column; Ultra-high pressures; Elution; Gradient; Peak resolution; Chemical analysis; Flow rate; Nano flow rate; Peptides; HPLC chromatography; Kinetic method; Kinetic plot; Packed capillary columns; Protein; Operating conditions; Column coupling; Reversed phase chromatography; Ultraviolet detector; Pressure effect; Analysis method; Plate efficiency; Peptide fragment; Proteomics; Packed column
• ISSN: 1615-9306; 1615-9314
• DOI: 10.1002/jssc.201201087

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 C18 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.