Chromatography Forum

I am using C18 RP silica in Flash LC for separatin of tetracycline and its oxidation products at prep scale (~200 mg) but some of the products react with the RP silica and stay their permanently as visible by the brown color...I feel the acidic compounds are responsible for that. Also TC is highly tailing compound. One more problem is that I start gradient with water(amm. formate):ACN 10:90 at which sample is not very soluble and increasing ACN to dissolve the sample causes some compound to elute at solvent front.

RP is suitable for this or should I change to HILIC or Ion chromatography?Are the stationary phase of HILIC or Ion C available in market for flash LC?

Or simple normal phase silica is sufficient?

Here is HPLC method for tetracycline and degradation products. Mixed-mode separation performed on Primesep 200 column:

http://www.sielc.com/compound_285.html

You can use this method with prep friendly conditions (by using TFA instead of phosphoric acid)

Contact me if you have any questions.

Regards,

Vlad

Thanx for reply

For analytical estimation I m u sing HPLC/MS/MS so peak resolution is not a big factor during LC/MS/MS.

But I wish to separate ~200 mg of TC+products...It contain ~5 major compounds and ~10 minor compounds as I can see in LC/MS/MS. I wish to confirm the molecular structures by NMR and for that I need pure compounds. I tried with RP silica in flash column (10mm ID 150 mm lenght) but there is no clear separation. I do it with gradient and as I increase solvent strenght the conc. of compounds increases but all of compounds elute at the same time. so I could not get a nice separation though I added amm. formate in it. Many of the TC and product retaion on the silica strongly and never elute.

Plz guide me precisely.........

Thnx