Unexpected Peaks for Water Sample and other Issues

Hello. I've been trying to run some samples for different concentrations of a 1-Hydroxypyrene with Methanol solution. However, the water sample that I've also been using as a reference / control of sorts has been giving undesired peaks which makes it hard to determine the appropriate data for our 1-OHP samples. The water, which I'm doing three injections of, is showing small peaks at later retention times when I would expect it to be flat the whole way through. I do have to scale a bit to see them clearly but they are noticeable even without scaling. Could this be due to A.) bad water, B.) bad solvent, or C.) bad column? Also, I was wondering if someone could answer which factors (peak width, threshold, etc.) affect integrated peak area. For the samples that are twice as concentrated as the previous, wouldn't we expect the area to be twice as large? Because that is not what we're getting. They're either the same size or slightly larger. Basically, could someone clue me in as to what the retention peaks for both water and a 1-OHP solution are supposed to look like, or is it different for each run based on things like temperature, flow rate, etc.


  • Hi AliK.  Thanks for the question and for the details.  It sounds like you're working toward developing a new method and experiencing some challenges in designing a robust solution.  Waters' website has a lot of resources you can access to help you as you work toward the development of your method.  Some of them can be found here: https://www.waters.com/waters/promotionDetail.htm?id=10048879&locale=en_USu0026cid=10073244

    Recently, we have had a focus on Analytical Quality by Design for separation methods, and managing a method through it's lifecycle.  If you're interested in these approaches, you can access a compendium on the topic here: https://www.waters.com/waters/library.htm?cid=511436&lid=135022271&locale=en_US

    To answer your direct question about what could be causing the peaks you are seeing in your water sample, I would say all are possibilities, and a systematic approach of testing each may help to elucidate which is the largest contributor.  It may be helpful to collect some mass spectral data on the small peaks you are seeing, as that may help identify not only what the compounds are, but where they may be coming from.

    To address your other question, data processing is a complex subject.  I don;t think it would be possible to answer your question in sufficient detail here, but, please have a look at our Data Acquisition and Processing Theory Guide.  https://www.waters.com/webassets/cms/support/docs/empower_3_data_acquisition_processing_theory_guide_rev_a.pdf

    Finally, our professional services organization offers method development services.  If you're interested in having a Waters engineer design your method, please reach out to your local service representative.

    Thank you for being a Waters customer.  We appreciate your business and look forward to the next opportunity to support your science.
  • If I assume lots of things that were left out of the original post, I can suspect that your water would benefit from a little pretreatment.

    Assumptions: you're running a RP gradient on a C18 (or C8) column and water is a major fraction of your A phase.

    Vacuum filtering your water through a MeOH dampened C18 extraction disk prior to making your MP A with it may be all you need to clean up those unwanted peaks eluting when the concentration of MP B gets to a certain point. This has been discussed several times over the years on chromforum.org.