Chromatography Forum

We are trying to run the USP monograph method for Folic Acid Assay, on the raw material. I am having trouble with creeping retention times (creeping up). I think that we have tried everything, temperature control, column, new mobile phase, etc. I am wondering if anyone has had similar problems with this method, or are we just overlooking something real simple that will make us hang our heads in embarrassment when we figure it out?

Thanks in advance for your help

What kind of column have you used? The mobile phase contains large amount of aqueous solution (over 90%). We used Waters Atlantis dC18 and Phenomenex Synergy 4u Fusion. Using both columns we found no problem in retention.

We are using a zorbax SB-C18. However, I just realized, when reviewing this, that we are using a 4.6 mm diameter column and the USP calls for a 4.0 mm diameter, although I don't think that this is the cause of the problem. You are right, there is almost NO organic in this mobile phase, so it may be just that the column has been well beaten up by now, although it is relatively new and we have not used it very often.

SYX:
I am new in this facility and trying to figure out what the people here do. Can you tell me how you wash these columns after they have seen so much inorganic mobile phase?

Thanks

Same as other RP-columns: mobile phase of a similar composition but without buffer (water instead buffer) for 5 - 10 column volumes. Then with 20 column volumes of pure organic solvent.

Dear Sir,
You can use totally hydrophillic columns like CHEMSIL AQUA/ YMC ODS-AQ/ Hydrosphere to use with 100 % water as mobile phase. Please note that hydrophilic columns have advatange for aqueous mobile phaes. General ODS columns will have "matting effect" of C18 ligands on the surface which minimises the surface area when interacted with water in mobile phase. To over come this particular problem, above columns are treated with hydrophilic bonding .
Hope usuage of above columns will sort out your problem.
regards,
A.L.N. MURTHY
CHIEF EXECUTIVE
CHEMINDIA , HYDERABAD, INDIA

TEL: +91-40-55904780/55904781

jcstrides,

You're on the right track with the Atlantis dC18 column.

Vitamin peaks will migrates for several reasons that i've found.

1. Light and heat sensitive. Don't ignore your solution stability and make sure you keep solutions dark and cool. degraded solutions of folic acid will split and migrate.

2. the solubility of folic acid is low. i think usp calls for ammonium hydroxide. don't forget it.

3. don't forget to add the sodium perchlorate to buffer the solution.

good luck...