Chromatography Forum

I am facing a challenge to develop an impurity method which requires a weak mobile phase strength (water/ACN 70:30 or lower) at initial condition to separate a pair of impurities. However, at this condition one of the actives will precipitate out and cause problem. Any suggestions would be appreciated.

A little more information may help get a better focused answer. What column are you using? (it may be that a change of column could allow for separation with a stronger mobile phase.) Can you describe the compounds - or better yet tell us what they are? pH could be part of the solution - or not.

Are you trying to quantitate the active also, or can that be eliminated (like on an SPE column) before chromatography?

The application requires quantitation of the related compounds and impurities of steroid neutral compounds, so pH would not affect much. I have tried on C18 and C12 columns so far and it is time consuming and costly. I am looking for other approaches than trying on different columns and mobile phase type/ratio.

it is time consuming and costly

If it was quick, easy, and cheap, they could hire anyone off the street to do it, and we would all be unemployed

One possibility is HILIC, which typically uses high ACN percentages and generally has the opposite elution order from reversed-phase. From the sound of your RP mobile phase, you may not get good retention in HILIC ( but you won't know until you try).

Another possibility is to go to standard normal phase with something like a cyano bonded phase column and organic solvent mobile phases. Again, there are no guarantees, and you will have to determine the feasibility empirically.

At what concentrations do your impurities precipitate out? Is there any chance of diluting your impurities to the point that they won't precipitate out with your starting gradient conditions but still be able to detect them adequately? I'm guessing this is standard practice for most impurity analyses when using gradients. To prove you are not getting precipiatation you could always conduct a recovery experiment (assuming you have pure impurities at your disposal

I believe that the precipitation was caused by one of the active not the impurities. Actually I also have sensitivity issue as well. I need to quantify at 1/100000 level, so I cannot afford to go any lower on concentration. Normal phase with cyano column could be a solution, but I try to avoid it unless it is absolutely necessary. Thank you all for your help!!

How are you qunatifying your impurity profile by % w/w or % area/area? If it is % area/area, then you may be able to carry on and disregard the precipitation (insoluble active) as you could assume all components (active and impurities) are soluble to the same degree.

Rob, why should anybody assume the solubilities to be the same? And even if they were, why should one assume that the precipitation is proprtional at vastly different concentrations of these? And why should there be a diff. between %w/w and %a/a in this regard?

If they are related steroids imps you might assume solubilities to be approximately the same.

However i guess you are right about your other two points...

Are you really sure it's precipitating on the column?
Maybe if you dissolve your sample in 50% ACN, but only inject 20µl on a 25cmx4.6mm column?

Ace

Rob, I am quite timid, old age?

I would like to second aceto on this one: The concentrations are generally so low that solubility is not a problem during chromatography. Overloading the column might be more likely.

To Rob - we have to quantify impurities by %w/w because the response of the active already out of linear range.

To aceto - we have fixed sensitivity to meet, so need as much as we can to inject into the column, however, we can not exceed the maximum mass load under fixed initial organic ratio in mobile phase, which back to my original question.

It looks like that trying on different columns which allows a higher organic ratio in mobile phase without resolution suffering is the most effect way to solve this kind of problems.

Thank you all for your inputs!

Believe it or not, there is a simpler way of doing this:

Dissolve the sample at 1/10th of the current concentration in a solvent composition that has 10% MORE water, i.e. in 60/40 MeCN/water, and then inject a 10 times larger volume. (or 1/20th and 20x etc.)