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?© BrainMass Inc. brainmass.com October 4, 2022, 4:38 pm ad1c9bdddf
SOLUTION This solution is FREE courtesy of BrainMass!
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 check into the literature confirms this. Look for example here, were hemoglobin variants were screened and it was indeed possible to distinguish them by reverse phase HPLC:
HPLC Retention Time as a Diagnostic Tool for Hemoglobin Variants and Hemoglobinopathies: A Study of 60000 Samples in a Clinical Diagnostic Laboratory Alla Joutovsky, Joan Hadzi-Nesic and Michael A. Nardi Clinical Chemistry 50: 1736-1747, 2004
Or see here for an actual chromatogram displaying both hemoglobin and myoglobin separated on a hydrophobic interaction column: http://books.google.com/books?id=aWY1FwtlYZkC&pg=PA866&lpg=PA866&dq=HPLC+analysis+of+myoglobin+and+hemoglobin&source=bl&ots=DdKkmMR_pP&sig=VHGNRt_kjJhOOMid4e4rREW5y1s&hl=en&ei=PDu8S5ffAo6E8wSi59z2Bw&sa=X&oi=book_result&ct=result&resnum=5&ved=0CB4Q6AEwBA#v=onepage&q=HPLC%20analysis%20of%20myoglobin%20and%20hemoglobin&f=false
As for why do they behave different on C4 versus C18 column, I agree with you in that I would not necessarily expect them to reverse the elution order, but it can all be explained. The more hydrophobic compound eluted later in the C18 column, but earlier in the C4, as the C4 column does not provide a hydrophobic enough environment, smaller concentrations of polar solvent are sufficient to elute the compound of the C4 column. The elution profile for the other compound very likely changed as well, but let?s just assume that compound has a more balance surface composition of hydrophobic and hydrophilic residues, it would still bind to the C4 reasonable tight, but would elute in appropriate polar solvent concentrations. Given a feasible amino acid distribution for hemoglobin and myoglobin it could certainly be explained why the switched elution behavior.© BrainMass Inc. brainmass.com October 4, 2022, 4:38 pm ad1c9bdddf>