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Myoglobin and Hemoglobin HPLC

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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?

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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 ...

Solution Summary

The situation is discussed and reference to published HPLC traces are provided.