Chromatography Forum

Hi

I am trying to follow this method https://www.agilent.com/cs/library/appl ... 5169EN.pdf

I am only doing the HPLC part and was hoping that this would give the separation needed, unfortunately I am not having much luck


HPLC is set up as method states except for a few changes

https://ibb.co/t4ghwWj

- column: Poroshell 120 EC-C18 4.6 x 100mm 2.7 Micron (this is one we already have onsite and thought it would be similar enough but clearly i was wrong)
- mobile phase A 0.1% formic acid in water
- std prep: 100mg/l AAT std in Acetonitrile
- bx prep: red sample spiked with 100mg/l AAT std in acetonitrile

1st run
std:
https://ibb.co/j6YMLJm

bx:
https://ibb.co/jzZYR7w

std gave either a 2nd peak immediately after or 1 badly shaped peak with an unexplained drop to zero mid peak

bx gave a peak that dropped below zero, not sure why this may be as the internet said this is usually caused by a mismatch of your mobile phase and dilutant solvent, but both are acetonitrile.

I then ran repeats of my std to see if the peak shapes were consistent, which the shape and retention time were but the peak heights varied between injections, no pattern ie increasing peak height suggesting sample being retained.


https://ibb.co/bbcg4Sz


I then made another dilution of the std at 5mg/l and changed the injection volume to 10μl in case the hplc was struggling with the 0.5μl injection volume, and again ran 4 repeats but this has just resulted in more confusion. only the 2nd injection looks like the previous ones and the remaining 3 still go negative but have a peak later in the sequence not present in previous runs.


https://ibb.co/yBvjdxQ

please could I get some advice of what i need to change to get the method to work or is it just a fruitless endeavour and i should stop wasting my time. or if any of you have a working method for detecting low levels (<0.1%) of AAT (o-aminoazotoluene) from liquid fuel dyes youd be willing the share.

Thank you

- column: Poroshell 120 EC-C18 4.6 x 100mm 2.7 Micron

First of all, you changed the column i.d. from 3.0 mm to 4.6 mm. This requires the increase in the flow rate by a factor of (4.6/3.0)^2 = 2.351. Read this:
https://www.phenomenex.com/documents/20 ... ents-guide
https://www.usp.org/sites/default/files ... m99380.pdf

Edit: I believe that you will likely need help beyond the forum to answer this question, as this will become very convoluted.

Hi Khopper,

To piggy-back off what vmu was saying, this looks like an inexperience issue.
Problems such as the column i.d, negative peaks, "no pattern ie increasing peak height". These issues are relatively obviously and explain, even at surface level, why your analysis will not work with this method. Even when I was going over the method you are imitating, I could not find the correct retention time OR in your case, factor. I would ask around for someone that knows how to set up a method from a USP before coming here to ask questions, there are just far too many to answer here.

TS

This an incredibly rude response, don't you think? A forum is a place you go for guidance and advice, regardless of experience. Get off your high horse because it's giving boomer...

While I did not initially mean to come off as rude, I believe you are right. However, to answer a question such as this one, one would have to teach the user basic fundamentals about LC to even build up to the question. I agree with you about the forum being a place of guidance and advice, we are not here to teach users an introductory course in chromatography. I don't think many users/commenters would revel in the paragraphs needed to explain much of this, and that's why I recommended asking someone with expertise around them. While I believe you can learn quite a lot from the forum and other users, you would not receive the level of instruction required to run an LC from just asking and answering questions. A large amount of posts on the site that go unanswered or have merely one or two responses are, in my opinion, mainly due to the poster's lack of detail/insight into what they are attempting (thus making it difficult to answer without further knowledge from the poster), or general ignorance about LC that would require a class/seminar to fully illustrate.

I do fundamentally agree that I was rude within my response, but it was after I spent time examining the USP method, analyzing the results of their images, and re-reading the prompted question. This was also after I had typed a few paragraphs regarding their analysis and basic questions I had for the poster, such as the fact that listed method is a LC/MS method that does not describe retention times/factors for hit analyte (AAT), and only discusses the MS results. If they made it through the process of actually getting their analyte to run, could they even quantify the result as accurate without an MS? Will they simply pick a peak from the chromatogram that lines up with theirs, perhaps verify by UV, and say that's good? While I don't think it is impossible to do this work, I find the method a relatively poor choice. Obviously, that is not up to me, but it just makes things harder. For example, with one google search I was able to discover an article that describes AAT by retention using a column that is also a C18 (mind you, not the exact same column, but a good place to start) at the correct ID but different length. You could argue his initial method is more apt to his set-up, except for the crucial part of actually detecting and confirming the peak that you want. It is located here: https://doi.org/10.1016/S0021-9673(02)01162-7

From the article, and what I have gathered and in the initial discussion of this, I suspect that due to his flow-rate issue and his sample diluent, much of his sample is eluted from the column immediately in his injection solvent due to a mismatch with the mobile phase. While OP is correct about wanting the solvent to match your running buffers, you actually would like to inject in a solvent mixture that is as close to your running buffer as possible. Not completely on one side of the spectrum. This can cause, essentially, a bubble of your analyte to run right through your column without interacting with your phase. What he may be seeing in the later chromatograms (due to the flow-rate issue) is his analyte that actually did retain (from prior runs) and is finally eluting off the column in a subsequent run. However, AAT is pretty stable and I assume this peak is AAT, but it should be confirmed (hopefully with the help of an established method with comparable results). At first, I was going to ask if there is any washing step between runs, or if these two-peaks that we're seeing (early, immediate elution vs longer elution) are the same, but I am assuming that they are due to a lack of detail. It is simply in my opinion that questions like these, while valuable to many inexperienced users, could be easily remedied by a class on LC, a quick 30-minute youtube video, or a professional/higher-up quickly inspecting their method.
In my opinion, OP should not be wasting his time on this method if he would like concrete confirmation of his product because the USP method he is using does not fit his parameters for detection. While I would not call it a waste of time, I would simply ask someone with more experience to set up the method for them or ask them for help. I do not believe it is the forum's place to teach LC to new users. However, that does not mean that there are simple things that cannot be discussed and argued about, and I should not be so condescending and rude in my responses. Curiously, what would you advise OP do in this situation? Should they continue with their MS methods and running standards with modified settings? Or should they find a better method more applicable to their analysis and detection methods?

One thing you can experiment with would be your detector. From what I can see, the Agilent method gives no guidance on detector types or associated parameters. In the one chromatogram chart they show, it says MRM[254.0 -> 183.0]. I think MRM is in association with mass spec. Since I have little to no experience with either MRM or MS, I am a bit ignorant of what they are referring too there. I see that you are using a DAD for your detector. Perhaps you can try some other wavelengths (254?) or refer to a UV chart of AAT and see where it is most absorbent. These would at least be simple items to experiment with and see if it improves your chromatograms. Good luck to you.