what could be the cause for the area increase?

[vliu]

After extensive cleaning of the column, 50% then 100% acetonitrile then back flask the column, the run after the extensive wash gives results much higher than previous runs, ~15-20% higher, to both samples and standards.

I have eliminated sample prep error, mobile phase prep error and I changed the blocked check valve. what could be the possible reason for the sudden increase??

mobile phase a water with 0.1%TFA
mobile phase b acetonitrile with 0.1% TFA

Please help!!

[danko]

Hi Vliu,

When you say higher results, do you mean higher peaks (i.e. peak height) or larger area?
If it’s the former, then the explanation is simple: Clearer stationary phase, resulting in higher plate count, resulting in narrower and thus higher peaks (for the same sample load).
Try to compare plate count before cleaning and after cleaning.

Best Regards

[Uwe Neue]

Let me ask a simple question first: do you quantitate by peak area of by peak height?

[vliu]

Danko:

The high result refers to both height and area.

Just to give you a clearer idea, below is the before and after result

result before cleaning:

(area; k prime; height; symmetry, plate count USP)
8180353; 4.54; 329829; 1.75; 8357

result after cleaning:
9780618;4.38;433186;1.66;10127

Uwe Neue:
Quantitate by peak area.

My results will be compare to another lab, and their results are comparable to my old result (their column is newer than ours). I am getting so confused. I have been cleaning the column (without connecting to the detector) overnight with 50% acetonitrile. My plan for today is to give the system inclu the PDA detector a good clean with;

1. warm water, both lines
2. 6M HNO3
3. warm water to rinse out all HNO3
4. reintroduce my mobile phase and do a trial injection

if still doesn't work then......I don't know what else I can do......

[danko]

Hi again,
First thing first: I don’t understand your frustration and especially the compel to obtain lower sensitivity. Usually people would be pleased to achieve higher signal. So, if you calibrate your system with known standards, you should be able to obtain the same results as the other laboratory – even better (meaning less uncertainty).
Btw, I can’t see how washing the system with HNO3 would reduce the signal – on the contrary.
Anyway, you’re seeing higher plate count which is consistent with the expected outcome.

There is a slight possibility that the current flow rate is a bit slower than it was before the column cleaning (just accidentally – not in connection with the cleaning) and that would result in longer retention time as well as larger area.
Would you please check the retention time before and after?
Another sensible action would be to try another column on the same system as well as another system with the column in question (i.e. the cleaned one).

Best Regards

[Uwe Neue]

If you have a strongly tailing peak, some peak area may get lost in the integration. Since the phenomenon is the same for standard and analyte, this is usually not a problem, or at least it is not readily visible.

It is also not clear if the observation is a column problem. Could be anywhere, actually...

[danko]

I wonder what causes the disappearance of large parts of my posts – just like in this thread

[tom jupille]

I wonder what causes the disappearance of large parts of my posts

It's a side effect of the move to a new hosting service and domain (viewtopic.php?t=9878).

Random posts got truncated at random lengths. I don't understand why (that part of the data base was exported from the old server and imported into the new one) and unfortunately, there is no "global" way to find and fix the truncated posts . I know it's painful, but as you find any of your old posts that were mutilated, you can edit them to restore the full sense.

Alternatively, if you e-mail me a list of the topics that contain your truncated posts, I can try to recover them from the old database and restore them from this end (what I need is the t=xxxx parameter from the address bar when you're reading the posts).

I do apologize for the inconvenience, but the pain of the "Temporarily Unavailable" hassles just got to be too much.

[vliu]

Danko:
Could you please re-post your last 2 posts as they got chopped off due to server problem, maybe you had the answer I've looking for

[danko]

Hi Vliu,
I don’t quite remember the exact wording, but the content of my last post was like this:

The peak height has increased following the column cleaning and that was expected due to the higher plate count.
Theoretically, the area should have remained constant i.e. the peak should have become correspondingly narrower. The reason for that inconsistency could be slight flow rate change.
So, let me ask you a question: What is the current retention time, compared to that prior to the column cleaning?
Regarding system flushing with 6 M HNO3, I don’t think it would reduce the area. In fact I’m sure it won’t. Another thing is: Why should one want to reduce the plate count etc.?

Lastly, maybe you should try the clean column on another system with the same mobile phase and see if you’d obtain the same results as usual. And if you have a new column it would be nice to try it on the system which produces the higher results.

But for a start, please tell us whether the retention times are consistent, or did you se longer retention time without changing anything else.

Best Regards

[tom jupille]

For those of you who read this thread and are puzzled by the exchange about truncated posts, the problem has been fixed.

[ColinB]

Hi, Why did you think that your column needed to be cleaned in the first place if your results are already comparable to another column on another system?
Your results indicate that everything is higher in terms of area, height, K' prime, the height increase could certainly be explained by the plate count increase due to the cleaning, higher plate count = sharper peaks as explained by Danko but the peak area should still be the same.
The most obvious answer is that you have injected a slightly more concentrated sample. How sure are you that there is absolutely nothing different about the sample preparation method?

Colin

[vliu]

Danko:

I tried the clean column on another system. I managed to get correct area but when I put a run on, jumping retention time was observed once again This time I recorded the system pressure as well. The jumping retention time is obsered when the system presure starts from 1800 psi, the usual staring pressure should be 2400psi and should be consistent till the peak of interest is eluted.

I tried extending the run time from 45 to 65 mins (the cleaning start about 36-38 min with 100%ACN and should equilibrate by 45 mins). However, increasing run time does not solve the problem.

I am trying to flush the system and column with 100% methanol, as I suspect there's a bubble in the column. Any Suggestions anyone??


ColinB:

I use calibrated eppendorff pipettes and volumetric flasks for the sample prep so my prep to prep should be fairly consistent.

Please help...I am trying out of ideas and time.......

[danko]

Hi Vliu,

Yes, flush the system. The pressure fluctuations suggest air problems.
Not in the column though, as you fear, but rather in the pump/s. So, just remove the column and flush the system with eluent B at 2 – 3 mL/min for half an hour or so.

Btw, did you check the retention times before and after the column cleaning?

Best Regards