Hello All,
Currently, I am developing a method for one of the drug substance that has 8 impurities. I would like to come up with an iso-cratic method that can elute all of the compounds within 40 mins or so. Out of the eight impurites, three of them are structurally similar and are more hydrophobic. The other five impurities are less hydrophobic (structurally simliar, slightly more polar than the other three impurities) and one of the impurity is an isomer of the drug substance. Haven said this, with 65% phosphate buffer (pH=7)and 35% CH3CN, the first five impurities elute within 10 minutes or so and the other three impurities doesn't come out of the column until 50 to 80 minutes. This behavior remains the same with C8, C18, phenyl and fluro columns. I even tried PEG and cyano columns where all peaks elute early and isomer peak co-elutes with drug substance peak. I have tried all kinds of tricks, like peak reagent, pH, THF and perchlorates, etc.
I know gradient elution will solve the problem, but we want can't have a method that is gradient since our pumps are old:-)
My question is, how good are these rocket columns (Alltech) in reducing the retention times? How much of resolution loss I might encounter for my first five impurity peaks? I hope I made my question clear:-)

Sounds like you need a reverse-phase column with an embedded polar group such as Supelco Discovery, Waters Symmetry-Shield. That would increase retention of your more polar cpds while allowing you to increase the organic concentration to reduce retention of your more hydrophobic cpds. Whether you'll get enough selectivity to separate the isomers is a guess.

Hi Ravi,
sometimes the other way round can bring you the solution. What jclark said you should also have a close look. Another option you have with HILIC - Hydrophilic Interaction Chromatography. Just check the publication of Mark A. Strege from Lilly Research Laboratories, published in Anal. Chem. 1998, 70, 2439 - 2445: Hydrophilic Interaction Chromatography-Electrospray Mass Spectrometry Analysis of Polar Compounds for Natural Product Drug Discovery. I know it is not what you have, but it can give you an idea about the effect of this HILIC technology you can perhaps use to solve your problem. Good luck. Gerhard

Another possibility is a manipuation of the pH. Of course, this depends on your analytes. We have had good luck compressing chromatograms for a wide range of compounds by switching from acidic pH (around 3) to basic pH (around 10).

Ravi,
All the suggestions should work and I would through in an ion pair reagent also as a try to hold up the polars and allow stronger eluant.

Another approach is to switch columns. For example if your first group eludes together. You could switch a short column a minute or so after the inject to the end of the long column. Sometimes you get lucky and the late peaks are sufficently separated. If the separation occurs you can adjust the switching time and column length ratios to get good separation in a short time.
If the gradient is not just a cost problem, a three way valve could create a solve strength change. The usual problems of an fast change might not affect you because of the separation of the two groups of peaks. You may need a mixer, sometimes just any empty precolumn will do the mixing (put after the valve before the pump.) Let us know what you try and how it works.
Jim

What about two injections, each with optimum conditions for the two groups of analytes? (Two mobile phases)

Hello all
Thanks for all the suggestions. I have tried Discovery RP amide, ProntoSIL EPS C18 and other polar embedded columns (Check out the recent paper in LCGC Jan 2002 issue, page 62, "The Benefits of Reversed phases with EPS in analyzing wide polarity range samples"). All of these columns work for the first five impurities, but the last three elute really late. Only columns that could elute both within say 50 minutes is Waters XTerra RP 8 (Hybrid silica), but the resolution is not great between the isomer and the drug substance peak. I have ordered Xterra C18 and also longer Xterra RP C8. ALso, rocket Alltech column is on its way to my lab:-) I will keep you posted guys. Thanks again for all your suggestions.

I have also encountered this problem once. "Rocket" column did not help me in that instance but Synergi RP-Polar worked miracles. Also, if your compounds do not require much of organic in the mobile phase, you could try IPA or THF. I assume that this compound is N-containing. I would stick with lower pH, AcN and/or IPA, Synergi RP-Polar, and slightly elevated temperature (~35-40C). You may also conceder using ammonium acetate-TEA-acetic acid buffer instead of phosphate.