Protein separations using enhanced-fluidity liquid chromatography.

Area of Science:

• Analytical Chemistry
• Chromatography
• Biochemistry

Background:

• Conventional liquid chromatography methods face challenges in achieving rapid and efficient separation of complex protein and peptide mixtures.
• Hydrophilic interaction liquid chromatography (HILIC) is a common technique, but often requires longer analysis times.
• The incorporation of carbon dioxide (CO2) into mobile phases presents an opportunity to enhance chromatographic performance.

Purpose of the Study:

• To explore the efficacy of enhanced-fluidity liquid chromatography (EFLC) using methanol/H2O/CO2 mobile phases for protein and peptide separation.
• To evaluate the impact of CO2 addition on chromatographic parameters such as peak asymmetry and analysis time.
• To demonstrate the feasibility of EFLC for separating hydrophilic proteins within a specific molecular weight range.

Main Methods:

• Development and application of enhanced-fluidity liquid chromatography (EFLC) with methanol/H2O/CO2 mobile phases.
• Comparison with traditional acetonitrile/H2O based hydrophilic interaction liquid chromatography (HILIC).
• Utilized trifluoroacetic acid (TFA) additive and elevated temperatures to optimize separation conditions.
• Analyzed a 13-analyte intact protein mixture and hydrophilic proteins up to 80kDa.

Main Results:

• EFLC methods using CO2-containing mobile phases enabled the separation of a 13-analyte protein mixture in under 5 minutes.
• Modest improvements in peak asymmetry and analysis time were observed with EFLC compared to conventional ACN/H2O separations.
• Electrospray ionization - quadrupole time of flight detection confirmed that protein analytes were unaffected by the presence of CO2.
• Successfully demonstrated the separation of hydrophilic proteins up to 80kDa, including transferrin.

Conclusions:

• Enhanced-fluidity liquid chromatography (EFLC) with CO2 is a viable technique for rapid protein and peptide separations.
• The addition of CO2 enhances mobile phase properties, leading to reduced analysis times without compromising analyte integrity.
• EFLC shows promise for the analysis of hydrophilic proteins, expanding the capabilities of liquid chromatography.