Sudden change in peak shape

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

24 posts Page 2 of 2
thank you for the additional info.

So, in simple terms; (1) the original method used may not have been rugged enough for the actual application and is now showing its limitations or (2) some type of sample degradation is taking place (which you should be able to test for, separately).
Multidimensional wrote:
thank you for the additional info.

So, in simple terms; (1) the original method used may not have been rugged enough for the actual application and is now showing its limitations or (2) some type of sample degradation is taking place (which you should be able to test for, separately).



Correct and to follow up with the possibility of degradation:

We are seeing the same peak shape within different final product formulations (using different lot numbers of the peak of interest) as well as in the analytical standard. I believe it could be more method based than material based if I had to lean one way or another. I can always run a GC/MS analysis to get a better picture.

I'll do some trouble shooting early next week looking at solvents and see if that alleviates any problems.
We see a lot of methods which are "Validated" and show limitations in actual use. "Validating" a method is not an indication that the method is specific or selective for the sample types. That may be the intent of validation, but the real life truth of the matter is that many methods are poorly developed by people with little or no training so the methods flaws are often only revealed later on, after use. A good example of this can be seen in the peer reviewed journals. Perhaps 10-20% of the published methods show poor design and a general lack of fundamental understanding of the technique. I am not saying that is 100% the cause, but many people seem to get stuck using poor quality methods that just do not work.
Multidimensional wrote:
We see a lot of methods which are "Validated" and show limitations in actual use. "Validating" a method is not an indication that the method is specific or selective for the sample types. That may be the intent of validation, but the real life truth of the matter is that many methods are poorly developed by people with little or no training so the methods flaws are often only revealed later on, after use. A good example of this can be seen in the peer reviewed journals. Perhaps 10-20% of the published methods show poor design and a general lack of fundamental understanding of the technique. I am not saying that is 100% the cause, but many people seem to get stuck using poor quality methods that just do not work.



I agree and I didn't imply that because our method was validated it was flawless. Also, to update the situation, I prepared the formulation using methanol/water in the same ratio as the mobile phase and we still have the same peak shape. Even when I lower the concentration, we see a similar phenomena occurring.

I also changed the needle wash to be less strong of a solvent as well. It may be an issue with the rotor seal as someone suggested, I just find it odd that it's only this one molecule that has a significantly different peak shape that occurs in the analytical standard as well as among the multiple formulations we have that contain this molecule.

If I am able to solve the problem I'll be sure to update but at this point I'm not sure what my next step will be.
I think it was worth the try. You're ruled out quite a bit already:

- Column issue
- Instrument issue
- Standard/sample degradation
- Mismatch between injection solvent/mobile phase
- Temperature should be stable

In my opinion, the next thing worth taking a look at is the mobile phase. Is the MeOH/water mixture prepared in a single bottle or do you use the pump to mix? Is it prepared freshly? Did you by any chance change vendors for MeOH/water recently? Could you take a different source of MeOH and water to prepare the mobile phase and try?

If prepared in a single bottle, note that making a MeOH/water mixture is trickier than it sounds. Taking 100mL MeOH and adding to the 500mL mark with water will NOT give the same mixture as taking 400mL water and adding to the 500mL mark with MeOH. The way you prepare it should be written down in an SOP and always be the same.
Rndirk wrote:
I think it was worth the try. You're ruled out quite a bit already:

- Column issue
- Instrument issue
- Standard/sample degradation
- Mismatch between injection solvent/mobile phase
- Temperature should be stable

In my opinion, the next thing worth taking a look at is the mobile phase. Is the MeOH/water mixture prepared in a single bottle or do you use the pump to mix? Is it prepared freshly? Did you by any chance change vendors for MeOH/water recently? Could you take a different source of MeOH and water to prepare the mobile phase and try?

If prepared in a single bottle, note that making a MeOH/water mixture is trickier than it sounds. Taking 100mL MeOH and adding to the 500mL mark with water will NOT give the same mixture as taking 400mL water and adding to the 500mL mark with MeOH. The way you prepare it should be written down in an SOP and always be the same.


Vendor for methanol has not changed, we use Thermofisher. Our mobile phase is mixed online, with each individual component being degassed as well as possible prior to placing the solvents on the HPLC.

I would have to double check the actual lot number of the HPLC water and Methanol I am using, it could be an issue with the lot itself - great thinking.

I have changed the methanol and water out, but as I just mentioned earlier, I may have replaced it with the same lot - just different physical bottle.

Unfortunately my predecessors in this position were not very thorough with writing SOP's or spelling out how they prepared their mobile phase. I am very familiar with solvent expansion and contraction when mixing organics and aqueous liquids together and I usually practice measuring them separately in separate graduated cylinders prior to mixing together but unfortunately the way it was prepared for this method specifically was never written down (whether the method was developed by online mixing or mixing together prior to putting on the instrument).

Because it was never written down I always just mixed online and had results as expected.

I'll be having a PM performed on the instrument soon and I will be sure to have a very thorough maintenance replacing rotor seals, pump seals, etc...

Until then I will look at the solvents and see if I can find out anything more. As I had mentioned earlier, even though the peak looks to be poorly shaped - the phenomena occurs in both standard and formulations and isn't adversely impacting my quantification. Even so, I want to figure out what's going on.
It's mixed online so 1 of the lines has 100% water. Do you often pump that particular line with organic? It's good practice to do that since stuff can start to grow or get stuck in there, and impact the chromatography. Our lab performs a weekly flush of the aqueous lines with methanol for around 10min - it doesn't take time.

Also, 100% aqueous mobile phases shouldn't be left more than a couple of days on the instrument before being replaced. Rinse with methanol, rinse with water, and replace. Avoid placing them on the dishwasher.

Sorry if i'm stating obvious stuff, just hoping that something will help in fixing the problem.
Rndirk wrote:
It's mixed online so 1 of the lines has 100% water. Do you often pump that particular line with organic? It's good practice to do that since stuff can start to grow or get stuck in there, and impact the chromatography. Our lab performs a weekly flush of the aqueous lines with methanol for around 10min - it doesn't take time.

Also, 100% aqueous mobile phases shouldn't be left more than a couple of days on the instrument before being replaced. Rinse with methanol, rinse with water, and replace. Avoid placing them on the dishwasher.

Sorry if i'm stating obvious stuff, just hoping that something will help in fixing the problem.


Yes, I do make sure be quite careful with HPLC water sitting for too long. I usually keep just enough on the system for each analysis prior to replacing and I do a methanol wash maybe twice a month.

Sometimes it's the obvious stuff that's overlooked.
When you say you tried different columns, were they "new"? I've had this problem, and it just happened to be a reduction in ability of the column to separate properly. If the columns you tried were fresh out of the box, I'd go another route, but if they were about the same age/same amount of usage as the initial column, I'm inclined to say it's a column issue.
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