Enhanced-Fluidity Liquid Chromatography–Mass Spectrometry for Intact Protein Separation and Characterization

Abstract

Recent advances in the analysis of proteins have increased the demand for more efficient techniques to separate intact proteins. Enhanced-fluidity liquid chromatography (EFLC) involves the addition of liquefied CO2 to conventional liquid mobile phases. The addition of liquefied CO2 increases diffusivity and decreases viscosity, which inherently leads to a more efficient separation. Herein, EFLC is applied to hydrophobic interaction chromatography (HIC) stationary phases for the first time to study the impact of liquefied CO2 to the chromatographic behavior of proteins. The effects of liquefied CO2 on chromatographic properties, charge state distributions (CSDs), and ionization efficiencies were evaluated. EFLC offered improved chromatographic performance compared to conventional liquid chromatography (LC) methods including a shorter analysis time, better peak shapes, and higher plate numbers. The addition of liquefied CO2 to the mobile phase provided an electrospray ionization (ESI)-friendly and “supercharging” reagent without sacrificing chromatographic performance, which can be used to improve peptide and protein identification in large-scale application.

Analytical challenges encountered and the potential of supercritical fluid chromatography: A perspective of five experts

“…EFLC starts with the equivalent of 100% cosolvent and adds CO 2 . Most recently our group illustrated that ELFC interfaced with electrospray ionization (ESI) sources, provides marked improvements in detection limits for proteins and provides the capability to shift the charge distribution for proteins to higher charge states 4 …”

Zero electrical power pump for portable high-performance liquid chromatography

“…Techniques such as enhanced-fluidity LC involves the addition of liquefied CO 2 to conventional liquid mobile phases and has been shown to provide enhanced diffusivity, faster solute mass transfer, and reduced system backpressure. 12,13 We have not, as yet, explored how this effect might be exploited using our system. We have found the low solubility of nitrogen permits the driving of mobile phase without necessitating a physical barrier or membrane to isolate the phases.…”

Automated Programmable Generation of Broad pH Range Volatile Ionic Eluents for Liquid Chromatography

“…However, these were often not optimal, a recent paper provides statistics on the dramatic decrease of the use of IPC over the intervening period. 24 For mostly aqueous eluents used in LC-ESI-MS, which are usually based on ammonium acetate or formate, the maximum recommended concentration is <25 mM, preferably <10 mM with an eluent pH within 2 units of analyte pK a , 25 with the vast majority of reported applications using ≤10 mM salt; 26 albeit higher salt concentrations are possible under high organic content EFLC conditions 27 or in nanospray MS applications. 28 Higher salt concentrations may be possible under other conditions, but significant ionization suppression occurs, especially with purely aqueous eluents.…”