Chromatography Forum

Dear Chromatographers,

I synthesized a peptide with the sequence Leu5-Phe-Cys through solid phase peptide chemistry and I am trying to purify this compound with a HPLC-MS System using following conditions:

Column: RP18-EC (Nucleodur), Poresize: 100 A
Mobile Phase: Isocratic, MeCN/H2O (40:60) + 0,5 % AcOH
Flow: 0.5 ml/min.
Sample: 1 mg Subst in 80 % TFA, 20 % saturated Urea in H2O
Retentiontime: 10 Min.

The main side Product is the Peptide with one less Leucine, i.e: Leu4-Phe-Cys. elutes 3 to 4 minutes before the main Product.

My Problem: The sideproduct, which is chemically allmost the same as the main product, shows a clear and sharp Peak. Strangely, the main Product is extreamly broadened an elutes in a timeslot of 4 Minutes!.
My first thought would be that it might be due to size-exclution effects. Maybe one aminoacid makes the difference and the whole molecule doesnt fit into the pores anymore.
Can this be true or are there other possibilities??

My main target is to transfer this system to preparative Scale, for which a sharp peak is favourable.

Thank you very much for your advices.

Regards
Alex

Alex, could it be that you overload your column? What is the peakshape of the main product?

Thank you for your reply,

No, It can't be overloaded. I tried different loadings (5µl, 10µl, 20µl) to exclude this possibillity. All loadings showed equal peakshapes.

Impurity: Leu4-Phe-Cys: sharp and intense at around 7 min.
Main Product: Leu5-Phe-Cys: very Broad and flat from 10.0 till 14.0 min.

One big problem is the solubility (maximum of 50 mg in 10 ml solvent for preparative scale). I think if the broad peak would be as sharp as the Impuritypeak, I would be able to catch the product in the preparative run.

Regards,
Alex

To what does your %TFA, etc., refer? How do you know which peak is what?

Alex

Size-exclution effects would not occur on the Nucleodur C18 column for those peptides. With the information given I would try the following:

Make a 1:10 dilution of the peptide sample and inject followed by a blank to eliminate the possiblity of late elution analytes. If the peak shape doesn't sharpen up you may have to try a mobile phase with less viscosity or increase your temperature.

Alex,

Your peptides are too small to be exluded from a 100A stationary phase. Furthermore, if you had size exclusion phenomena for your peptide of interest, I would expect it to be eluted before and not after your impurity (sharp peak).

I can not explain such a chromatographic difference with only one more residue but in general you can experience such problems with very hydrophobic peptides and/or cystein containing peptides. The question always here is why the impurity (if that is really the impurity you think) that contain only one less residue behaves that much better...

Maybe you could try to play a little bit with the temperature as it has been shown to improve peptide peak shapes (i.e. 50 C or higher).

Kostas Petritis,

Could it be that my peptide is in equilibrium to a lot of different conformations, like alpha-helices that elute each at another retentiontime? Maybe one residue makes such conformations more likely...?

If this is so, what can I do besides rising the temperature.


Steve,

what mobile phases could I use with less viscosity?
One Problem is still the bad solubillity.


HW Mueller,

TFA is the only solvent with dissolves my peptide good enough to make a transfer on preparative scale possible.
The percentage refers to the sample preparation, i.e: 1 mg Peptide, 900 µl TFA and 100 µl Water with Urea.

Thank you for all your replies

Does your MS show the right masses (the same as if you transport the stuff into the MS without a column, after you "separate" them?)
If you really chromatograph your peptides, it would be amazing to me that they withstand such a [TFA]. If they do come through allright I could think of a whopping pH incompatibility regarding one and not the other. (I havn´t done estimations on pKa of peptides, maybe someone is more proficient here?).
Certainly conformational changes wouldn´t increase the size enough to get trouble with 100 A pores, besides, that would most too fast to "see" on a HPLC scale.
I still think that I have seen ion exclusion on silica with positive species, a possibility here of partial exclusion? Well not much sense in guessing if we don´t know that you really have only these two peptides.

Make a 1:10 dilution of the peptide sample and inject

Alex, did you try this??

regards Bert

Bert,
I get the exactly the same peakshape and the same retentiontimes. Only the intensity of the MS is much weaker.


HW Müller

TheMS shows exactly the right masses of the peptides. Of course there is little of other impurities that originate from the Solid phase peptide synthesis, but they come of the column right at the beginning.

regards
Alex

Alex- I don't understand. Are you really injecting a sample in almost pure TFA? Why- I've never heard of this before.

If so you will make a mess of your mobile phase pH as this slug of almost pure acid hits the column.

Spell out also what volume of what peptide concentration solution you are injecting.
Are you really injecting 1 mg of peptide? That is 100ul of a 10,000 mg/l solution......

Are you really injecting 1 mg of peptide? That is 100ul of a 10,000 mg/l solution......

What happens if you dilute the samples 100-1000 times??

Victor,

I had to dilute the sample in pure TFA, scince this is the only good solvent for my Peptide. Even though I still get a (weak) signal by diluting it in acidic MeCN, this would never make sense on preparative scale.
Using pure TFA (with a bit of urea in water) is the only way to get at least 50 mg in the 10 ml loop of the preparative system.
And of course I do not inject 1 mg at once. This is just the concentration of the sample in the vial.
I inject 10 µl.


bert,
I didn't try more diluted samples. As said above I am searching for a system with the highest load possible, scince the peptide is so unsoluble.


Regards
Alex

Alex:

To reduce the viscosity of this mobile phase reduce the water content to

Mobile Phase: Isocratic, MeCN/H2O (80:20) + 0,5 % AcOH.

This should also reduce your backpressure which may allow you to increase the flow rate to 1ml/min if possible.

The problem is not knowing anything about the peptides. This MP conditions will results in a must faster chomatogram with possible coelution. If that's the problem, start increasing the water concentration of the MP.

Assuming that the peak area of the first peak (the impurity) and the second peak (your desired compound) are drastically different, it might be possible that your injection is overloading the buffering capability of the 0.6% acetic acid, and that this is the reason for the good peak shape of the first peak (below the buffer concentration).

Try to increase the acetic acid concetration by a factor of 2 and see if the second peak becomes better. If it does, you know in which direction to go.