I have a peculiar problem with a gradient HPLC method. I am using a Lichrospher RP18 column - 250mmx4. Mobile phase is water and acetonitrile (both acidified with acetic acid). Sample is dissolved in acetonitrile. A known standard at times when I inject start giving a number of extra peaks. At this point of time even if I change the column and do the analysis this phenomenon is seen in the new column as well. I use Merck HPLC water and acetonitrile. The intriguing factor is that when I inject a blank acetonitrile and run the same gradient the blank is clean. Any such expereince for anybody. If water is the culprit why is that the extra peaks are seen only when sample is injected and not in blanks. The standard is known to be pure and clean and has not deteriorated.

Is there a possibility of carryover? How do you know your standard is pure and not deteriorated? What is the times of your peak and the extra peaks? How do they compare in size to main peak?

Could be the mismatch of your sample solvent and mobile phase, or a mismatch of the pH of these two. (Usually, one gets smeared out peaks though)

How many peaks are you getting? Just one extra peak or more?

Thanks for the responses. Let me make the clarifications.
1. Carry over is not suspected since the blank injections after sample injection is clean.
2. The standard is pure and not deteriorated as shown by analysis subsequently after long column flushing, changing mobile phase etc. HOwever after a set of injections the problem reoccured and since the in between improvement seen was not due to any explainable intervantion, the concern remains.
3. My main peak gives a peak purity of ~99.7%. When this problem starts the purity drops down to ~99.2%. The impurity peaks are of the order of 0.1-.2% by area. The main peak RT = 19min. There are 4-5 small peaks (previously not seen) coming after the main peak which is giving the problem
4. There is ofcourse a mismatch of mobile phase and sample diluent. The mobile phase is acetonitrile and water acidified with acetic acid. The sample diluent is acetonitrile alone. But i cant see why this should be a problem. Still I will try by acidifying the sample diluent.

Hope the clarifications are useful

Just check the stability of your standard in diluting solvent ( ACN ) with respect to time. Most provably u would find diffrence in freshly prepared standard and after some times injection.

din

If this is all the problem you have..........
Wish I had problems like that.

I think it's possible that your problem is caused by diluting the sample in acetonitrile. Try diluting the sample in eluent.