Chromatography Forum

hi

I am working with "mecobalamine" I am looking for HPLC Related substance method that elute the " hydroxocobalamin" after the mecobalamine since i found severals method but all of them gives me rapid elution of hydroxocobalmine (approx. 2min). Can any one gives me some ideas about it?

thank you

It is normal for a reversed-phase method that a compound with a hydroxy group will elute before the same compound without such a group. To inverse the elution order, the best bet is to do HILIC = hydrophilic interaction chromatography.

what do you mean by HILIC = hydrophilic interaction chromatography , do you have a method, and if anyone have please help me.

I do not have a method, but if you want to invert the elution order seen in RP between a compound and its hydroxylated metabolite, you will not be successfull with straight reversed-phase chromatography. However, if you can run your compound in HILIC, the principle is the inverse of RP, and your hydroxylated metabolite will elute last.

but still i dont know what is the HILIC = hydrophilic interaction chromatography ? and how can i use it

You use a silica column, equilibrate it in 95% acetonitrile, 5% water, then run a gradient to 50% acetonitrile, 50% water. Further refinements are possible by using a buffer.

It is in principle a partitioning of the analyte into the water-rich layer on the surface of a very hydrophilic packing.

More in my book on "HPLC Columns".

how can i get your book? where can i found it?

If you're interested in learning more about HILIC, you should get the SeQuant "Practical Guide to HILIC". It is a comprehensive booklet that gives a thorough introduction to HILIC as well as it expemplifies areas of use via a selection of applications. Please visit www.sequant.com, leave your contact details, and it will be sent to you for free.

My book on "HPLC Columns" can be purchased through Amazon.com, or from Waters, part number WAT038216.