By Anonymous on Monday, October 21, 2002 - 08:48 am:
I am trying to separate a mixture of amino acids on nonpolar stationary phase. What types of solvents and buffers should be tried? The stationary phase is like a C3 or C4.
By Mel on Monday, October 21, 2002 - 09:16 am:
Is there a reason you have to use a C3 or C4 column? I think you will have a hard time separating them. Are you going to use a derivitizing reagent?
By Silvio10 on Monday, October 21, 2002 - 09:48 am:
Right now,I am using a hypersil ODS column (2,1 *200,5 microns)and in this method is very important the derivatization with OPA and FMOC.I hope this information can be useful for you.
Sincerely.
S.A.A.L
By Anonymous on Tuesday, October 22, 2002 - 10:31 am:
The amino acids are not derivatized. They are all aromatic. I am using the C3 and C4 because those are the types of columns I have available to me. Lowly graduate student (smile)
Thanks again...any buffers commonly used with amino acid separations?
By Kostas Petritis on Tuesday, October 22, 2002 - 05:41 pm:
If all of your amino acids have aromatic groups it shouldn't be a problem for their detection and if you further speak for only Tyr, Phe and Trp then these amino acids have different hydrophobicities and they should be readily separated even in the C3-C4 columns with water acetonitrile mixtures containing small concentrations of formic or trifluoroacetic acid (0.05-0.1%)(I haven't check it though).
Now if you want to separate o, m, p Tyr, things become more complicated (I have the answer for their separation) and maybe C3 columns are not enough. How complex is your sample? Is it for separation of just standards or in real world mixtures?
I recall having seen in the literature an article speaking about the simultaneous separation of more than 10 aromatic amino acids. Could you give us more information about your amino acid mixture and your goals? (if not you can try the first paragraph solution).
Kostas
By Benjamin on Wednesday, October 23, 2002 - 06:50 am:
Dear anonymus;
It seems like you do not have to worry about detecting your amino acids, if they are all aromatic it should be easy to use UV detection.
Retaining them on regular reversed-phase columns can be a problem. It all depends whether they have enough hydrophobic character. Try first a simple phosphate buffer (0.05M) at pH 3 or lower. If there is not enough retention, using 10-20% ACN in the mobile phase, then you may have to add an ion-pairing reagent. I recommend you try first, hexanesulfonic acid (0.005M)to start with. If the retention is still too short, then go to Decanesulfonic acid.
Good Luck;
Benjamin