I use a NUCLEOSIL C18 250mm x 4mm column and it works well for my applications but at present I need to analyse impurities in acetylocysteine and I cannot determine L-cysteine and L-cystine because they don't separate. The mobile phase is water : acetonitrile (97:3) at pH=1,5 , isocratically. Should I try a different column, a different mobil phase composition? I'd appreciate any suggestions.

have you looked at the amino acid analysis sections in the relevant column suppliers literature? Should you be using a silica based C18 at such a low pH?

A quick search of the waters application database turned up the following:

Title: Sensitive analysis of cystine/cysteine using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives

File Number: 950263

Authors: Strydom, Daniel J.; Cohen, Steven A.
Source: Tech. Protein Chem.
Year: 1993 Volume: IV Page: 299



Compounds: cystine/cysteine
Matrix:

Column: Nova-Pak
Dimensions: 15 x 0.39 cm
Waters products: 470

you could also try the following:

Title: Simultaneous determination of several amino acids, including homocysteine, cysteine and glutamic acid, in human plasma by isocratic reversed-phase high-performance liquid chromatography with fluorimetric detection

File Number: WA10463

Authors: Tcherkas, Y.V.; Denisenko, A.D.
Source: J. Chromatogr. A
Year: 2001 Volume: 913 Page: 309-313

Hi,

This separation is not very difficult. All you need is to add some ion-pairing reagents in your mobile phase (I take for granted that you speak about the separation of these compounds at their underivatized form).

You may use perfluorocarboxylic acids for that. While trifluoroacetic acid might not work I know that you can succeed the separation by using Heptafluorobutyric acid or nonafluoropentanoic acid or higher chain homologues.

Just check for your application 0.1% of ion-pairing reagent and the % of ACN you need to elute them isocratically (cysteine is eluted isocratically even with 100% water, cystine will need some ACN to be eluted (or to obtain better peak shapes) but it depends on the type of column that you will use as well).

If you go for ion-pairing reagents with higher side alkyl chains than nonafluoropentanoic acid you should carefully check the time you need for the column equilibration.

You may find relevant information about underivatized amino acid separation by using perfluorinated carboxylic acids as ion-pairing reagents and silica or carbon based columns, as well as equilibration times etc. at:

K. Petritis et al.
J. Chrom, A 833 (1999) 147-155,
J. Chrom. A 870 (2000) 245-254,
J. Chrom. A 855 (1999) 191-202.

I have seen that some chiral columns can achieve the simultaneous separation of chiral amino acids and/or separation among different amino acids, but of course that is a very expensive solution (you will though only need ACN-water as mobile phase).

You may see:
K. Petritis et al.
J. Chromatogr. A 913 (2001) 331-340.

If you wish to analyse the amino acids without derivatization and the UV does not provide the desirable sensitivity/specificity you may want to look at this publication as well:

K. Petritis et al.
A comparative study of commercial liquid chromatographic detector for the analysis of underivatized amino acids.
J. Chromatogr. A 961 (2002) 9-21.

Hope that the above helps,

Kostas