Recently I'm developing a HPLC method for the impurity detection of a drug.I've tried several mobile phase on three C18 and one C8 columns,and the common point is that the main peak begins to split when I inject (manaully£© the same sample for several times and the split increases with the number of injection. It seems that if it is the first time the column is used to seperate this drug the splitting appears slowly. I've tried to destroy the drug by heat/acid/alkali/oxidation and could not not get the splitting-out substance.The drug is an aminoguanidine-indole derivative,and there lies a bi-bonds.Is it possible that the splitting peak are isomers? And how could I know it?

Are you using a photodiode array detector? Have you collected fractions from the split peak and injected them separately?

Neither.We have no photodiode array detector in our lab.The split peak is so near to the main one that I'm afraid I cann't get the split fraction purely.Do you mean that if I inject the fractions separately I can know whether there are two substance? I'll try.

Dear WHQ

I have seen many cases like the one you describe. I suggest the following;

1.- Make sure the sample solvent and the mobile phase are not too different. If they have to be because of solubility problems, inyect a very small volume of sample of a concentrated solution.
Then the split may disappear.

2.- If the two peaks look very much the same intensity, then it is possible you are separating an isomer. Changes in mobile phase ( pH, % ACN, etc) or temperature usually are enough to coelute the two isomers.

3.- If the peaks are indeed isomers and you want to separate them, then change to another type of column that is likely to resolve them. Perhaps a phenyl column would be a good one to try.

Good Luck;

Benjamin

I encountered a similar problem in the past with a Proline derivative. the supposed reason was an equilibrium between two conformers whose rate was slower than the exchange rate between mobile and stationary phases; we solved it by doing the chromatography at a rather high temperature (70°C).
I don't know if your drug is prone to this equilibrium, anyway you can try increasing the temperature.

The change of splitting with number of injections is strange. Seems that part of your sample stays on the column, changing its characteristics. How is the total area behaving, is it constant or going up, down?
Right now I am having a problem with peak form changes with number of injections. It appears that the mobile phase is not rigidly compatible with the analyte in our case (apparently the mobile phase is borderline to giving "non-chromatography"). Slight changes in anything will then cause large changes in the chromatography. It may well be that part of the analyte is staying on our column, but must be too low to be obviously evident.

i am facing a problem and i was wondering if you people could help me. we are asked for a project at uni. it's about "chromatography for non- chromatographic applications" i've been surfing the net for info or data enough to build a project upon but i can't find any... could anyone help me?

Hi Abby,

Your project title confuses me. Can you clarify the theme more? Is it about chromatography used beyond the research laboratory? ie. for forensics, drug testing, purifying water to make it taste better?

The only non-chromatographic application I can do with my gas chromatograph, is bake cup-cakes in it :)

We can also provide better links and leads for you if you give more details as to what your project criteria are (is it a demonstration at a science fair, or a term paper). As well as giving us a due date (so we're not giving advice after the project is done).

thanks,