Capillary Flow LC–MS Using Micro Pillar Array Columns: Combining Nano Flow Sensitivity with Analytical Flow Robustness and Throughput

Register free: http://www.chromatographyonline.com/lcgc_w/capillary_flow

Event Overview:

Mass spectrometry based proteomics has become an essential tool in biological, biomedical and biopharmaceutical research. Due to the increased sensitivity that can be achieved as compared to analytical flow LC–MS, the majority of mass spectrometry based proteomics research is performed using nanoflow LC–MS, with flow rates typically below 1 µL/min and total run times exceeding 60 minutes. Compared to the LC–MS methods employed in other ‘omics’ fields, the need for high separation power often prevails over sample throughput, making relatively long separations the gold standard in LC–MS based proteomics. However, capillary and microflow LC–MS solutions are recently gaining interest as the demand for large quantitative proteomics studies that require increased throughput and robustness is growing, and as the sensitivity of MS instrumentation is increasing.  By working at higher flow rates, the impact of gradient delay and sample loading volumes on the total analysis time can be reduced and low flow rate or column dimension related technical errors (such as electrospray instability, column clogging or the presence of void volumes in the analytical flow path) can also be minimized. This robustness in measurements is especially relevant in the context of the growing interest in data-independent acquisition (DIA) methodologies. These DIA LC–MS techniques have been developed in the last decade in order to remove the stochastic precursor selection of data-dependent acquisition (DDA). Thus, DIA techniques acquire a reproducible periodic signal for each individual fragment for all eluting analytes. Maximizing the robustness and reproducibility of the LC benefits the efficiency of DIA analysis algorithms, and also liberates label-free MS from batch size restrictions by maximizing what is arguably the most valuable asset of DIA-its reproducibility.

A micro pillar array column based solution for capillary flow LC–MS is presented. In contrast to conventional LC columns that contain randomly packed beads as their stationary phase, micro-chip based pillar array chromatography columns have a separation bed of perfectly ordered and freestanding pillars obtained by lithographic etching of a silicon wafer. The regular mobile phase flow pattern through these micro-chip pillar array columns adds very little dispersion to the overall separation, resulting in better peak resolution, sharper elution and increased sensitivity. The freestanding nature of the pillars also leads to much lower back pressure buildup, and makes it possible to operate longer columns.

In this webcast, Dr. Jeff Op de Beeck of PharmaFluidics will explain the principles of the micro-chip based pillar array columns and demonstrate how the PharmaFluidics µPAC capLC column can contribute to LC–MS workflow robustness. Dr. Maarten Dhaenens of ProGenTomics will show high resolution DIA data of 500 ng of human proteome analyzed using either packed bed or micro-chip based pillar array columns operated at different flow rates and gradients.

Key Learning Objectives:

• Learn the principles of micro-chip based pillar array chromatography columns

• Understand the benefits and separation performance that can be achieved with the µPAC capLC column (1–15 µL/min)

• Be convinced about the robustness and sensitivity of modern day capLC and its applicability in proteomics

• Get a glimpse of an alternative future in proteomics: highly reproducible label-free quantification of thousands of peptides over large sample batches using capLC-DIA

Speakers: Dr Jeff Op de Beeck, Application Development Manager, PharmaFluidics

Dr Maarten Dhaenens, Principal Investigator, ProGenTomics

Time and date: Friday, Dec. 13, 2019 at 10am EST | 9am CST | 3pm GMT | 4pm CET

On demand available after airing until Dec. 13, 2020.

Sponsor: PharmaFluidics and ProGenTomics

Register free: http://www.chromatographyonline.com/lcgc_w/capillary_flow