SCX Betaine

I’ve got a little issue that I need help with.I’m trying to test for Betaine HCl in a finished good (there’s a bunch of other stuff in there including Creatine, which is probably the issue.) Our current Betaine method on the C18 column has basically no retention time on it. The customer gave us their HPLC method, which uses a Luna SCX column. Is there a UPLC column similar to that, for easy HPLC-UPLC method transfer?If not, is there another way that I can test for Betaine HCl using the equipment I have that will separate it from the other stuff in the finished product. We currently have:UPLC / PDA / ELSDBEH C18/C8/Amide/HILIC columnsAccQTag Amino columnsThanks for your help.

Answers

  • Hi Don,

    Betaine is a great candidate for Hydrophilic interaction chromatography [HILIC]. In HILIC, compounds are retained by an intricate combination of different retention mechanisms, one of which being ion-exchange. In HILIC, the most retention for various analytes will occur when they are in their charged/ionized form. Since you already have a BEH HILIC column, you can try this out quite easily.

    On the BEH HILIC column, I recommend running a gradient from 95 - 50% acetonitrile (containing 10 mM ammonium acetate and 0.05% acetic acid) over the course of 5 minutes as a starting point. A buffered solution is absolutely essential towards gaining retention in HILIC. On the ACQUITY UPLC system, you can premix the solvents so you can get a constant ionic strength of the buffer.

    For example,

    Mobile phase A: 10 mM ammonium acetate and 0.05% acetic acid in 95:5 acetonitrile water

    Mobile phase B: 10 mM ammonium acetate and 0.05% acetic acid in 50:50 acetonitrile water

    Run a gradient from 1 - 99% B over the course of 5 minutes.

    It is also important to remember to switch the composition of the weak needle wash to 95% acetonitrile to match the initial mobile phase conditions.

    Waters actually has quite a lot of information with regards to how to develop HILIC methods which can be found at:

    www.waters.com/hilic

    Also, I've attached a quick reference wall chart on how to best develop HILIC methods.

    You may also want to order a new HILIC technology primer that we recently released that explains: retention mechanisms, selecting the mobile phase and column chemistry, practical considerations with regards to sample diluent, wash solvents and column equilibration as well as method development strategies.

  • I appreciate the information and as I read, I saw a lot of potential in using the HILIC column to get better separation in the finished good samples I receive to recieve more accurate results on other "problem compounds" that I've been having issues with.

    However, I'm not seeing a Betaine HCl peak appear. I'm diluting it in Solvent A, and it appears to gone into solution, but no peak is appearing. I have 95% ACN as the weak needle/seal wash, a 9:1 solution of ACN/Water as the strong needle wash. I'm thinking it might be a diluent problem, but I'm not sure. I'm including a chromatograph of 1.1mg/ml of the Betaine standard at  210 nm (where we see Betaine normally). Once I get the peak, I believe I can optionalize the run using the principles at the website reference that was given. Thanks a lot for your help Grumbach

  • Hi Don,

    I believe you are facing a few different issues. For one, without seeing a blank injection, the very large peak at 1.287 minutes is most likely betaine. If this is the case, you may be loading too much on column to get a good peak shape. I suggest cutting the injection volume or concentration in half and monitor the absorbance you get, If it significantly reduces, this is in fact betaine.

    Alternatively, the ion exchange interaction may be too strong at this pH resulting in the betaine being stuck on the column.

    Additionally, these buffered ammonium formate/acetate mobile phase necessary for HILIC retention do have a high UV cutoff. Which means the buffer has a high absorbance at 210 nm which may reduce the sensitivity you need to achieve for this assay. The ELS detector may be more suitable. However this would only be useful if this is a semi-quantitative assay.

    It may be worthwhile to try a lower pH to minimize the ionic interaction that you are achieving.

    One option is to use 10 mM ammonium formate with 0.2 % formic acid instead of the ammonium acetate that I suggested in the previous post. However, this will still suffer from the high UV cut off as the acetate buffer.

    Alternatively, if you must use UV, the only transparent buffer at this wavelength is phosphate. The caveat, however, is that phosphate is not terribly soluble in high organic mobile phases. Therefore, if potassium phosphate buffer is used, a low concentration of 2 - 5 mM must be implemented to mitigate precipitation.

    One other thing to try is to prepare the analyte in a different diluent rather than solvent A. I have found that a mixture of 75% acetonitrile, 25% methanol with 0.2% formic acid, provides a good balance between solubility and peak shape..

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