I conducted HPLC analysis of myoglobin and hemoglobin using a C18 and C4 column. Both proteins were in a buffer solution only. On the C4 column the order of the peaks was a reverse of the C18 column.
The chromatographic output showed two peaks. Looking at the UV absorbance, I can attribute one of the peaks to the heme group (400 nm) and the other protein (280 nm), but I have no clue why this would have happened and can find nothing in the literature to further verify that this happens regularly or why it happens.
Can anyone help?
My first question to you would be, why would you not expect them to have a different retention times and thus be separated by reverse phase HPLC? They are indeed two different proteins, yet I agree with you they are very similar proteins. However, from personal experience I can tell you that I can routinely tell apart small proteins where only 1 or 2 amino acids are different by HPLC. It is a matter of setting the elution gradient up just right. Now for your case, I am absolutely not surprised that you can observe hemoglobin and myoglobin as separated species and a brief ...
The situation is discussed and reference to published HPLC traces are provided.