Segmented Microfluidics-Based Packing Technology for Chromatographic Columns

Nanoflow
liquid chromatography-mass spectrometry (NanoLC-MS) has
become the method of choice for the analysis of complex biological
systems, especially when the available sample amount is limited. The
preparation of high-performance capillary columns for nanoLC use is
still a technical challenge. Here, we report a segmented microfluidic
method for the preparation of packed capillary columns, where liquid
segments were used as soft, dynamic, and well-dispersed slurry reservoirs
for carrying and delivering micrometer packing particles. Based on
this microfluidic packing technology, the column bed was assembled
layer-by-layer at a 50 μm resolution, and ultralong capillary
columns of 3, 5, and 10 m were fabricated in such a manner. The microfluidically
packed columns demonstrated excellent separation efficiencies of 116 000
plates/m. The higher efficiencies obtained at higher slurry concentrations
also indicate that a high-quality packed bed can be obtained without
sacrificing the packing speed. Kinetic performance limit analysis
shows that the microfluidic packed columns have higher peak capacity
production efficiency in the high-resolution region, presenting an
improved separation impedance of 2800, which is significantly better
than columns packed with the conventional slurry packing method. In
comparison with a commercial nanoLC column, a 5 m long microfluidic
packed column was evaluated for proteomic analysis using a standard
HeLa protein digest and presented 261% improvement in peptide identification
capability, resulting in significantly enhanced protein identification
confidence.