Chromatography Forum

Dear All

I am working with Hamilton SAX column (PRP-X100; 50 x 2 mm) and I have a problem: analyte does not interact with the column; it elutes from the column in the dead volumn (even using water as mobile phase). I use formic acid as mobile phase.
The analyte (alendronate) is a poliprotic acid and it has negative charges in the pH that I work. I have equilibrated the column for one hour or more with the mobile phase.
Should I make any special operation?
How can I increase the interaction between the analyte and the column?

Kindest regards.


Christian Fernandes, Msc.
Laboratório de Cromatografia
Instituto de Química de São Carlos - USP/Brazil

Instrument : Agilent series 110
Eluent : 0.2 mL HCOOH/L water adjusted to pH 3.5 with NaOH;
Flow : 1.50 ml/mim (split for the MS ok for the RI)
Column : Ion Pac AS14A, 4x250 mm, 7 microns ( Dionex) 40 celsius
Inj.vol :10 microL
Run Time: 40 Min.
UV/Vis : no absorption

MS :

Instrument : LC/MSD SL
Source type :ESI
Polarity : Negative

Substance Parent ion
Alendronate m/z 248 [M-H]-


I am using the above method without any problems. The only drawback is that the RT is about 30 min.

Boa sorte
JNF

Christian,

As suggested above by JNF, this separation can be accomplished by anion exchange but the analyte contains no UV chromaphore so if you're using a UV detector you won't be able to see the analyte. Detection requires either a conductivity detector or as in the case above, MS.

However, another point to keep in mind is that the column as you receive it will probably be an a form (p-hydroxybenzoic acid) difficult to quickly convert to the formate form. If you have an appropriate detector and so you know that your problem is not detection, you may want to try a two-step regeneration process: pass a 0.1 molar sodium chloride solution over the column for 1-2 hours and then switch to your formic acid eluent system. This should be sufficient for removing any of the p-hydroxybenzoic acid.