Chromatography Forum

I am trying to separate underivitized
DL Homocysteine
L Cysteine
D-Methionine
L Homocystine

with C18 column, gradient of 0.1% TFA(aq) to 0.1% TFA(Acetonitrile)

but they coelute. Any suggestions for underivitized Amino Acids?

Do they co-elute at the beginning of the run or at the end of the gradient?
Also, please describe your gradient (% B per min, gradient duration) and of course flow rate, temperature and column dimensions.

Best Regards

Yes you can separate them by higher chain perfluorocarboxylic acids such as Tridecafluoroheptanoic acid.

Have a look at Figure one and Table 2 of one of my previous publications (Rapid Communications in Mass Spectrometry 2005; 19: 1587–1602). There are more publications on the subject of underivatized amino acids analysis, make a search by using my name if you are interested....

at the beginning.
I have since found the reference of Kostas', HPLC-CLND paper.
That is what I will go to next.
Thanks much.

The LC-CLND method is an isocratic method which uses pentadecafluoro octanoic acid (PDFOA) and will not be suitable for your needs. You will only see cystein and maybe cystine, while all other amino acids will be retain in the column and won't be eluted unless you perform a gradient elution. As both ion-pairing reagents allow you to separate your compounds under gradient elution, I will go with the one which has a smaller side chain thus is faster to equilibrate (PDFOA can take up to 3 hours for 15 cm columns). Furthermore, PDFOA is more difficult to solubilize in water.

So my advice would be to try either tridecafluoro heptanoic acid (TDFHA) or nonafluoro pentanoic acid (NFPA). There is also an intermediate one which was not available when I was making the studies but became available later (however, I think that the purity is not very good).

Good luck...

I ordered tridecafluoroheptanoic acid. Thanks for the headsup.
I read something about monitoring the fermentation process by analyzing for the amino acids. I have a few researchers that routinely stress microbial populations. Might you have a good introductory reference for this?

What are your detection options?

Here are methods for analysis of underivatized amino (including ones from your list) acids without ion-pairing reagent. You have two mechanisms to adjust you selectivity-amount of ACN and amount of ions in the mobile phase. You can also control ionization state of amino acids by playing with pH. With Primesep 100 column you can achieve almost any retention. You can use isocratic conditions if you don’t have amino acids with two amino groups.
http://www.sielc.com/compound_025.html
http://www.sielc.com/compound_283.html
Here is another approach to analyse amino acids, sugars and carboxylic acids in one run (fermentation):

http://www.sielc.com/application_183.html

You can adjust retention by ACN, buffer pH and buffer concentration.

In case of sulfur containing amino acids you will have pretty good UV activity. You can always use ELSD for amino acids

Just browse our applications and you will find a lot of cool examples and ways to control retention (and we never use IP reagents )

http://www.sielc.com/Applications_By_Compound.html

Regards,

Vlad

Below are examples of underivatized amino acids using Unison UK-Amino:
http://www.imtakt.com/TecInfo/TI311E.pdf
http://www.imtakt.com/TecInfo/TI329E.pdf