HI

We need to create a method to control our ordered fmoc amino acids against Fmoc amino acid of another company (because standards of Fmoc amino acids can't be found)

We actually use a gradient method using TFA 0.1% as A and MeOH/ACN/H2O 2/2/1 as B. We use an Agilent ZORBAX 300SB-C8 5 microns 4.6*150 mm column with a guard column of the same packing.

Is there somebody who can suggest me a method please ??

Dear Yoann;

I have done such analyses in the past. I can recommend you the following;

1.- You are using the right mobile phase components, but I suggest you try just ACN/H2O gradients with 0.1% TFA. Depending on the AA you may need different ACN gradients, my suggestion is to try from 20% to 60-70% in 15min. Using isocratic conditions may not elute all the impurities in the samples.

2.- Using UV detection at 264nm quite probably you can use Area % as quantitation.

3.- The column you use is adequate. But it does not have to be a Zorbax 300 type. I would use a Zorbax SB-C8 5um 150x4.6mm. The one you are using may not have enough retention to separate all the components because of the reduced surface area of the 300 A packing.

4.- I remember that many samples were relatively pure (>80%), but quite a few small signals were usually detected.

5.- Some double protected amino acids can be quite hydrophobic and require high % ACN to elute in a reasonable time.

I hope all these help;

Benjamin

Why not try the Agilent Eclipse AAA column and conditions?

See

http://www.chem.agilent.com/temp/radF9E00/00023867.pdf

Skip the derivatization steps. The 3.0 mm x 150 mm Eclipse column can be used at 0.85 mL/min. successfully, if you take dwell volumes into consideration.

Tom Waeghe
Agilent Technologies

Why not try the Agilent Eclipse AAA column and conditions?

See

http://www.chem.agilent.com/temp/radF9E00/00023867.pdf

Skip the derivatization steps. The 3.0 mm x 150 mm Eclipse column can be used at 0.85 mL/min. successfully, if you take dwell volumes into consideration.

Tom Waeghe
Agilent Technologies