SCX Betaine

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.

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..