HEEEELP! Gradient bad shape!

[hplc.chem]

Thanks Tom and Hollow for the comments and directions, and all readers for your time.

Special thanks for you Tom for the Idiom and for Sherlock Holmes quote!
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I used a brand new column today for five injections of ACN using the same method described in the first post, with no guard column. I got the below chromatogram for the last four injections. (I excluded the first one as it was not stable enough and I used it as a preparation for the new column).



The chromatograms here are almost identical to what I got after cleaning the previous column (point 8th in my first post).

I also noticed the same peaks in both set of chromatograms for the two columns. I checked the peak areas of the largest TWO peaks at 25.6 min and 26.0 min in today's set of chromatograms and got the following:

RUN #-PEAK AREA FOR 25.6 min-PEAK AREA FOR 26.0 min
#1-1967-945
#2-2296-1148
#3-2609-1274
#4-2761-1323

There is a clear increase in the peak size from an injection to the next. I am feeling that there is a build-up of a UV-absorbing material from somewhere. What do you think?

It might be possible that the problem is a combination between a detector problem and a contamination.

Before installing the new column I thought it might be better to clean the injector with some large injections, so I injected 10 replicates of 100uL injections of 100% ACN (with no column in place). The run was isocratic at 50% ACN/water, 0.5 ml/min. I got a high peak (> 0.002 V) in less than 1min. Just to have more fun, I injected 50uL and 10uL to see what I may get. The results are shown below.



I was wondering why I got a peak for only ACN! I thought the reason might be the differences in the characteristics between the injected 100%ACN and the mobile phase (50%ACN), so I injected 100uL of 50%ACN and 100%Water and got the below:



I wonder if this is a NORMAL effect, and also why to get a peak for the mobile phase injections?

After disconnecting the new column I ran an injection of ACN using the same gradient method in post 1 with NO COLUMN in place (tubing instead). I got a clear chromatogram with a high peak in front of the gradient as the chromatogram in my THIRD post.

Is there anything to worry about here?

Sorry for the LONG story but I am getting lost!

Thanks.

[hplc.chem]

Dear All,
I think we reached the end of the story!!

This is a comparison between the instrument situation during the problem and its conditions now!



I think the problem has been solved BUT I am not sure what was the cause!!

I cleaned every part in the instrument starting from the inlet filter in the solvents reservoir to the flow cell in the detector. I also replaced the inline filter which is at the outlet of the pump because it was badly dirty!! It was almost BLACK and BLOCKED! The pressure has also dropped from more than 3500psi during the problem to less than 1700psi now!!!

So, many thanks for this forum and special thanks for all contributors in this thread.

See you again in the NEXT problem

[Hollow]

you're welcome

Sorry that I "disappeared" somewhen, got busy on other things.

Nice to hear that it is working now and thanks for this update.

see you