The message of this tutorial is that reverse phase HPLC is simple. Compounds stick to reverse phase HPLC columns in high aqueous mobile phase and are eluted from RP HPLC columns with high organic mobile phase. In RP HPLC compounds are separated based on their hydrophobic character. Peptides can be separated by running a linear gradient of the organic solvent. I often tell my fellow researchers to run the 60/60 gradient when chromatographing an unknown. The 60/60 gradient means that the gradient starts at near 100% aqueous and ramps to 60% organic solvent in 60 minutes. The majority of peptides (10 to 30 amino acid residues in length) will elute by the time the gradient reaches 30% organic. To learn some of the simple principles of RP HPLC please read on.
Our Preferred Solvent System for ESI LC/MS
A = HPLC grade Water, 0.1 % formic acid
B = HPLC grade Acetonitrile, 0.1% formic acid
It is important to use the correct flow rate for your HPLC column. Below is a table with standard flow rates for easy reference. If you are running a column with a different diameter than those shown in the table please review the maintaining linear velocity page to learn how to calculate the appropriate flow rate for your column.
The sample is normally reconstituted in the A solvent to maximize binding to the column. The sample should not be dissolved in an organic solvent or it may not stick to the stationary phase. The sample should not be dissolved in detergent containing solutions. Some detergents may bind to reverse phase columns and modify them irreversibly. In addition detergents preferentially ionize in electrospray mass spectrometry and can obscure the detection or suppress the ionization of the analyte.