Chlorophyll b and antheraxanthin separation issue

[johnnym]

Hi All

I am new to this great forum. Recently I am having difficulties in separating leaf samples chlorophyll b and antheraxanthin pigments (other pigments chlorophyll a, violaxanthin, seaxanthin and b-carotenoid are separating good) using Spherisorb® ODS-1 reversed phase column (5 µm particle size; 250 mm x 4.6 mm i.d) protected by AltimaTM C-18 guard column.

Method detail
Solvent A -acetonitrile:methanol:Tris-HCl (0.05 M, pH 7) (72:8:3, v/v/v) B, Solvent B -methanol:ethyl acetate (68:32, v/v).
Flow rate- 0.8 ml/min
Isocratic elution of 100% A for the first 2 min was followed by a linear gradient from 100% A to 100% B from 2 to 16 min. A 2 min linear gradient from 100% B to 100% A, and an isocratic elution with 100% A for the next 4 min were used to re-equilibrate the column.

Can anyone pl give insight in to what could have gone wrong with the separation of those two pigments?

Thanks in advance

Johnny

[bisnettrj2]

Four things:

1. Can you post a chromatogram showing where your difficulty lies?

2. Are you controlling the temperature of your separation. If so, what temperature?

3. What is your sample diluent? Is it the same as your A mobile phase?

4. Four minutes is a very short re-equilibration time (edit - especially with your flow rate and column combination) - I believe rule of thumb for re-equilibration is 3 times the system volume plus 5 times the column volume, so you may want to extend your re-equilibration time at the back end of your gradient just to make sure your system is fully re-equilibrated with the A mobile phase before the next injection.

[oscarBAL]

Hi Johnnym!!
I implemented a similar analysis once; like 5 years ago; it has been one of my favorites; I do not remember it very well; but as Bisnettrj2 mentioned Column temperature is important your column is fine but the mobil phase I used it was diferent, Specially because started under higher aqueous condition (MeOH:Buffer pH7.2; 80:20) I suposse the one you use it has been working before; but yes; it would be nice to see your chromatograms in order to know what the problem is.
Are you usin DAD? I could find my old procedures if you are interested.