I am trying to seperate Methionine from Cysteine on the same gradient, in under an hour, any ideas.

I've seen a column on the market from Thermo Hypersil-Keystone called Hypersil Amino Acid. The application separates 18 o-phthaladehyde derivatised amino acids in just over 20 mins. Unfortunately the application only includes methionine, but I'm sure it would do the job. The conditions were as follows:

Column: Hypersil Amino Acid 5µm, 200x4.6mm
Eluent: A=Water + 0.03M NaAc + 0.25% THF (pH 7.2)
B=MeCN/0.1M NaAC 4:1
Gradient:
Time %B
0 3.3
20 40
24 100
30 100
31 3.3
Flow: 1ml/min

Hope this is of some use to you.

Hello Dave,

I've seen many columns on the market where you get nice chromatograms, also for amino acids. Most problem is the matrix, and if your amino acids are derivatized, yes or no, and with which method derivatized. What concentration you will have, and which detector is available. Do you whant to have a method for LC-MS?
Speed up a separation without loosing resolution is possible, and we have many options to optimize a method, with small particle sizes and so on. So please give us some more informations.

Hope I can give you some more help before christmas.
Any way, to you and all out there, merry christmas and a happy new year, with peace, health and a lot of fun.
Regards
Gerhard

Is there a way to detect amino acids directly without derivatization by using LC/MS? Thanks.

Yes!

Well here are some more informations than the one provided by the anonymus above (the "yes!" one).
Indeed, you can do underivatized amino acid analysis by LC-ESI-MS.
Please look at: J. Chromatogr. A 855, 1999, 191-202. We have also succeed the LC-ESI-MS-MS analysis of underivatized amino acids: J. Chromatogr. A 896 (2000) 253-263. LC-MS-MS provides several advantages for chiral analysis of underivatized amino acids as well: J. Chromatogr. A 913 (2001) 331-340.

Other detectors have been also used for the underivatized amino acid analysis such as:
the amperometric one: Anal. Chem. 71 (1999) 2774
the chemiluminescence nitrogen detector: LC-GC Europe: 14 (2001) 389-395,
indirect conductivity detection: J. Sep. Sci. 24 (2001) 397, etc.

Finally, in a forthcoming paper we compare 7 different chromatographic detectors which can detect amino acids in their native form.

Now about Cysteine and methionine as mentioned by Gerhard, all depends from what exactly you want to do and what instumentation is it available in your laboratory (is there any other amino acids that might interfere?). Solely for cysteine and methionine, these amino acids are sufficiently different so in my opinion only TFA and a higly hydrophobic C18 column can do the job. If it doesn't work (which will be surprisingly) the higher to TFA homologues could do the job.

For the use of long perflurocarboxylic acids as volatile ion-pairing reagents for the separation of underivatized amino acids please see:
J. Chromatogr. A 833 (1999) 147-155 for silica based columns and
J. Chromatogr. A 870 (2000) 245-254 for carbon based columns.

Hope the above helps,

Regards
Kostas

Thanks for the info. I wonder if there is a column avaliabe for underivatized amino acid separation without adding any ion-pairing reagents. Thanks for the help.

Dionex sells an anion exchange column specificaly designed for separation of underivatized amino acids (AminoPac PA10). The column is designed to be used with electrochemical detection but other detection modes are possible. More info is available at www.dionex.com or you can email me directly.