Sample Preparation - Sep Columns

<p>I am preparing methods for the analysis of some very complex matrixes (Dietary supplements).  Because my company is a contract manufacturer the number of products we have is immense and so are the matrixes.  So far my sample prep has been very basic, weight in dilution flask and then filtered with 0.2um filter and then injected.  In terms of getting accurate results, this has worked in most situations.  However I really don't know what is happening with the contents of the matrix because they are so complex.  My question is how do I know when more involved sample preparation is necessary either with regards to poor results or system care (sample crash out, clogging column etc etc)?</p>


  • I am tempted to say "If it ain't broke, don't fix it".  In other words, if that sample prep works keep using it.  On the other hand, in my field the analytes we look for are often bound to the matrix and it takes some pretty intensive extraction to get a clear idea of the total amount.  So I wonder if that could be the case in your analytes and if you are missing some of your response because you aren't extracting it from the matrix.

    In terms of column care I think if you keep an eye on peak shape, retention times and pressure you should know if the column is starting to get clogged.  Schedule frequent cleans following the column brochure.  Waters has a webinar scheduled for this Friday on UPLC-SEC for Biopharmaceutical Characterization which you might want to sign up for if you haven't already.

  • For the times when a simple filtration works well for your samples -- do not change your method.  If you start to see column issues (high backpressure, peak shape degrading, etc.) or if you start to see poor recoveries, failed QC's, etc., there are 2 main troubleshooting areas. One, check the LC method and see if you have a good cleaning method for the column in place.  If gradient, ramping to high organic and if isocratic, implementing a high organic wash after so many injections.  The other area is the sample prep.  Start with understanding how soluable your analytes and excipients are -- are you using the right diluent?  Is anything crashing out of solution?  Is your diluent the same or close to your mobile phase composition?  Then, you can start to better assess if the right solvent(s) ae being used and if filtration will work, or, if you need to move to other techniques such as SPE.  Then, there are many options for sorbents and methods -- will depend on the nature of your analytes and excipients.  Take look at some of the Application notes and guides on the Waters website under Products -- Sample Preparation for some help getting started.