Method development

[vienna]

Dear all

I try do develop method so separate Thiamine, Pyrydoxine and diclofenac sodium with simultaneous method.
I use RP18 Column and mobile phase = Methanol : Sodium acetat 0.05 M (30 :70). I also use PIC reagent Heptane sulfonic acid sodium salt.
When I use that condition the vitamins separated well, but diclofenac give too long retention time (almost 35 min). but if I switch the comparation of mobile phase (70 :30), the retention of diclofenac is 5 min but the vitamins didnt separate. What should i do to reduce the retention of diclofenac but the vitamins still separated well... ??
I really need more information.
Thanks

[Alexandre]

Have you tried 50:50? Or try (30 :70), but use ACN, not MeOH.

Other tricks, after good separation of thiamine from pyridoxine that you have already achieved rump gradient. If needed try faster, sharper and more organics.

Try your last 70:30 conditions on more powerful column. There are new sub-3um columns on the market, if you do not have UPLC. Use MS if can. LCMS can do it practically without chromatographie.

[vienna]

Yes I've try 50:50 and 30 : 70. The thiamine and pyridoxine separated well, but the diclofenac give retention too long (30 min). If I use more organic , thiamine and pyridoxine didnt separated but diclofenac give a faster retention ( 5 min).

I want to get good separation for thiamine and pyridoxine but also get faster retention for diclofenac.

[Alexandre]

Can you use gradient?

[vienna]

Can you use gradient?

Yes I still use gradient, because i haven't found the right comparation of mobile phase

[Alexandre]

What happens if you ramp to 100% MeOH immediately after elution of two separated peaks, say ramp to 100% in 1-2 min and stay at 100%.

[vienna]

What happens if you ramp to 100% MeOH immediately after elution of two separated peaks, say ramp to 100% in 1-2 min and stay at 100%.

I've try that, thiamine and pyridoxine didnt separated. Do you have any sugestion, what PIC reagent that I should use to separated thiamine and pyridoxine and have faster retention for diclofenac?

[mobunvar]

@ Vienna

What is pH of the Buffer and which column ur using (length and i.d of the column)

Try to change the pH. I hope it ll give u major effect. Try pH below 3. whats is flow rate ?

Did u try Scout Gradient run. Better run for 20 mins from 0 - 100% MeOH.

[oscarBAL]

Hello; to know the pH of your Movil phase is very important; you can also try a shroter column. just remember that acid compuonds have a higher retencion in acidic MP and increasing concentration of PIC you increase retention of Ionic molecules (vithamines) and viceversa.

[HPLCaddict]

There have been some good tips here already. If you want it quick and dirty without too much development work involved, just do what Alexandre suggested: Use a step gradient.
Start with Buffer/MeOH 70:30 (this should give you good separation of pyridoxine and thiamine, as you said) and after pyridoxine and thiamine have eluted, ramp to 30:70 (or even 20:80) to quickly elute the diclofenac. Don't forget to reequillibrate back to 70:30 before the next injection. I woulnd't go to 100% MeOH since reequillibration with the PIC reagent will take longer in this case...