In fact, in RP-HPLC the protein molecules are separated on the basis of their hydrophobicity. The molecule with more hydrphobicity will be attached to the column with more strength and will need more buffer for detachment so will have high retention time (late elution).

Now urea is a denaturant which can break the hydrophobic and other non-covalent interactions. It may surely be decreasing the strength of attachment of your molecule with the column, which could be a possible reason that your molecules were detached (eluted) from the column earlier.

You cannot use urea in RP-HPLC as it affects the hydrophobic interactions.

However, there is no harm of using urea in size exclusion chromatography, where the molecules are separated on the basis of their size and not of their nature.

Hope this will help 

We use urea for denaturing hydrophobic proteins before subjecting to LC-MS/MS. This method produces more membranous/surface proteins than other methods, as can be witnessed at http://www.ncbi.nlm.nih.gov/pubmed/?term=Comparison+of+five+methods+for+direct+extraction+of+surface+proteins+from+Listeria+monocytogenes+for+proteomic+analysis+by+orbitrap+mass+spectrometry

or

http://www.sciencedirect.com/science/article/pii/S0167701215000056

Dear Mohammed en Jenny

Mohammed - just a note - urea is highly polar and does not directly affect the classic hydrophobic interactions, but it does affect electrostatic/polar interactions. It is a very potent competitor for hydrogen bonds and will therefor denature proteins, in particular the secondary structures that are mostly buried in the hydrophobic interior of proteins (this is a indirect effect that will lead to loss of  the cooperative hyrdophobic interactions). It is therefore also potent in denaturing hydrophobic proteins.

Jenny, my hypothesis is that you have multiple urea molecules associating with your small peptide via H-bonds and other polar interactions, making it a more polar peptide-urea complex and sterically masking your peptide's hydrophobic character. It could also lead to hydrogen-bonded oligomers that will elute earlier in a broader peak. My suggestion is that you freeze-dry your peptide 3-5 times after diluting it to 0.2-0.5 mg/mL in water to get rid of the urea and then repeat the chromatography. Repeat if necessary. I have found the the best solvent for amphipathic peptides is 50% acetonitrile and for the more polar ones water or 20% acetonitrile. The acetonitrile helps to prevent bacterial growth and protease action. It is also best to work in glass and store your peptides dry. Refrain from adding anything to your peptide preparation that is difficult to remove via freeze-drying.

If you are interested I can supply you with a generic C18-HPLC methods that can be adapted for polar to hydrophobic peptides to limit your chromatography time to less than 30 minutes (including column wash and re-equilibration). The shorter runs will help a lot if you want to to many runs a day, and you use less solvent. Mail me at [email protected].

Good Luck!

Regards

Marina

Hi Jenny:

The best is to dissolve the sample with the initial mobile phase condition of your HPLC run.  If the solvent or solvent mixtures of your sample differs from the initial mobile phase conditions you risk:

interactions between the sample solvent and the mobile phase

interactions between the sample solvent and the stationary phase

potential insolubilities that can make your sample precipitate at the head of the column or in the frits

All of the above will decrease the performance of the column as well as decrease the life time of the column.  Sometimes these effects are reversible, which means that the column can be brought to original conditions after thorough cleaning.  Unfortunately, there are times that interactions are so strong that it is impossible to bring the column back to what it was and retention times can be compromised permanently.  If the column is permanently affected you will experience retention time shifts compared to when the column was good.  Therefore, you will need to start with a new column again. 

You may try to clean your column according to the manuafcturer's recommendations and see if the effects you experienced with urea are reversible, otherwise, you will need to get a new column.

You can dissolve peptides with a or a mixture of solvents for HPLC, such as here:

http://www.kromasil.com/applications/view.php?app=g1014

Here is an example of angiotensins and working with pH to get a better separation

http://www.scientistlive.com/content/advantage-chemically-and-mechanically-stable-reversed-chromatography-phases

You left out the most important info. What pH was the urea solution that you used? We often use urea solutions (up to pH 9.0 !) to clean protein separation RP columns which have been overloaded or abused. Some column supports can handle the higher pH without damage, but other types of silica can not and may dissolve under those high pH conditions (resulting in a plugged column, which is then placed in the trash). Before using any new compounds or solutions, first review the column manufacturers instructions regarding the care, use and compatibility of the support. Better to learn these things early.