What are the ways to optimize peak shape and improve chromatography for nano LC separations? - WKB237325

ENVIRONMENT

• ACQUITY M-Class
• nanoACQUITY
• nanoEase M/Z
• Trap column
• Method development
• Peptide separation
• Chromatography
• nano LC-MS

ANSWER

Suggestions for optimizing peak shape and improving the chromatography include the following:

• The age and quality of the mobile phase modifier is important for peptide peak shape and solubility and should be changed out regularly when developing a new method or troubleshooting an established method.
• If the system setup allows for reverse eluting the Trap column, consider evaluating this option.
• Ensure that the analytical column is not overloaded; the Trap may have a higher loading capacity than the analytical column doing the separation.
• Evaluate different column temperatures.
• Evaluate different column chemistries.
• Ensure that the analytical flow rate is not too fast for peptide separations.
• Check gradient slope. Gradients that are too steep will also affect peptide separations.

ADDITIONAL INFORMATION

For additional information, see How often should mobile phase be replaced on a nanoACQUITY or M-Class system

and How Should I plumb my ACQUITY M-Class system for use with nanoEase M/Z and/or ACQUITY M-Class Trap and analytical columns?

and What is the suggested peptide digest loading capacity of the nanoEase M/Z Trap Symmetry C18 180um x 20mm

and What is the estimated column peptide mass loading capacity for Waters Reverse-Phase Columns when performing peptide mapping?