Peaks only above a certain concentration?

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

8 posts Page 1 of 1
Hi,

I am developing an LC method (DAD detector) for Lonzabac 12 (Bis(3-aminopropyl)dodecylamine).

All I've done is make standards (Lonzabac 12) in 50/50 H2O/MeOH at 200, 400, 500 & 600 ppm.

I've tried a simply H2O/MeCN 5-95% gradient with a range of columns, most give no peak.

A Waters SCX column gives a well-resolved peak, but only at 600ppm, i.e. no peak AT ALL at 200/400/500, obviously you'd expect a linear increase of response with concentration. We should also note that I've tried 400ppm at 2x inj volume, with no peak.

*Note the 600ppm peak is NOT contamination.

Any ideas? I'm puzzled as to why I only see a peak at the biggest concentration. Is there like some kind of critical concentration?

Many Thanks
We can only help you if you give us more information! Thus, post the types of columns, chromatographic conditions, and an example chromatogram...

However, I am surprised you see anything considering Lonzabac 12 has no C=C double bonds! You may have to use another type of detector (like MS, ELSD...)
I agree with the previous poster. It sounds like you have not had any formal training in liquid chromatography yet and may be looking at the solvent peak. You injected your sample in a different liquid than your mobile phase so you should see a nice peak at Tzero. You provided none of the normally accepted metrics needed to evaluate and troubleshoot your issue and appear to be using UV/VIS for a compound that is not normally detected by that method.
If you don't have any ions in the mobile phase you are not going to elute your compound from RP column even if it is well end-capped. Your compound will bind to residual silanols on the surface by ion-exchange mechanism. Even if you find ways to elute it by adding ions, most likely peak shape will be ugly due to the overloading of these residual silanols. In addition to this, DAD might not be the best detector for this compound. It has very poor UV activity and you will be forced to inject more and look at this compound at 200 nm. This will cause further overloading and ugly peak shape. Also any highly UV active impurity will throw you off.

These problems don't exist in mixed-mode columns where polar groups shield residual silanols. so you get something like this:
https://helixchrom.com/compounds/cetylpyridinium/

It is always a good idea to inject compounds without the column and compare peak areas with and without the column. If peak areas do not match, then you are not eluting your compound. You can read these FAQ to learn more:
https://helixchrom.com/chromatography-faq/
Vlad Orlovsky
HELIX Chromatography
My opinions might be bias, but I have about 1000 examples to support them. Check our website for new science and applications
www.helixchrom.com
Multidimensional wrote:
I agree with the previous poster. It sounds like you have not had any formal training in liquid chromatography yet and may be looking at the solvent peak. You injected your sample in a different liquid than your mobile phase so you should see a nice peak at Tzero. You provided none of the normally accepted metrics needed to evaluate and troubleshoot your issue and appear to be using UV/VIS for a compound that is not normally detected by that method.


No need for the remark about training, that's offensive as oppose to constructive. Not sure why you'd feel the need to add that comment at all.

I've had plenty of training and have been given a project to specifically develop a UV-VIS method *if* possible, obviously ELSD/CAD etc. being easier and more favorable.

I've already said the peak obtained is not from the solvent, over-laying a solvent blank and standard can easily dis-credit this.

I provided little and asked a general question to see if people had ever seen the phenomena of a sudden, tall, well re-solved peak above a critical concentration or not.
We can best help those who help us. I think your initial request was naive and there is no possible way to assist you without more info. I stand by my earlier comment and if you had received training in this technique, you would have known what type of basic info should be shared to receive free advice. Without such basic info we can not constructively advise you.

"You provided none of the normally accepted metrics needed to evaluate and troubleshoot your issue and appear to be using UV/VIS for a compound that is not normally detected by that method"

BTW: I agree that you may want to start w/o a column to try and create a method which detects the concentration you wish to analyze for FIRST. If you can not reliably "see" it w/o a column, then you do not need to try it with a column. Concentration is critical here so use a value that represents what your actual method needs to be specific for. Injecting lower values is not the answer. Injecting higher concentrations is, until you can "see" it. Is there someone at your lab who can help you?
Dear Gdowsett19

I will start injecting a concentration of 20 µg/ml, of the drug, for example, examine the chromatograph and if it is all OK then go down to the ppm scale.

Good luck

Fernando
No need for the remark about training, that's offensive as oppose to constructive. Not sure why you'd feel the need to add that comment at all.

I've had plenty of training and have been given a project to specifically develop a UV-VIS method *if* possible, obviously ELSD/CAD etc. being easier and more favorable.


First, a general comment: from a single post, it can be difficult to gauge the experience level of the poster in order to frame an appropriate reply (adequate detail without being patronizing). You didn't provide a lot of detail in the original post and, to be honest, it wasn't obvious to me that you were experienced and were trying the DAD on purpose instead of from naivete.

As to whether I have ever seen a "nice" peak only above a certain concentration, the answer is "yes". The first time was a data-processing artifact decades ago. The data system (actually, in those days, "digital integrator") would only show a signal above a certain threshold level. Apparently, the idea was not to show noise. Hopefully, not something you would see today, but you might check to see if what you're plotting is the "raw" signal or processed data.

The second is a more likely scenario if you are using a cation exchange column without any buffer in the mobile phase (your initial post *did* say "water", not "buffer"). Without any competing ions in the mobile phase, it's possible that any sample below a reasonable level is simply fully bound to the column. I've run into this with proteins on GFC columns that had residual active sites. At low levels, all of the protein was strongly bound. We had to "prime" the column with several high-level injections of BSA before we could use them.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
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