Chromatography Forum

Asking for your advise on quantitative HPLC analysis of this compound. (We can't use GC).

I would try HILIC, with low UV or ELSD. Use a silica HILIC column, with a gradient from 90% acetonitrile 10% water to 50/50. If the peak does not show up, you need to consider pH control with something that is compatible with your detection method.

You can use Primesep for direct analysis of your amino acids.

For UV use sulphuric or phsophoric acid
For ELSD use TFA:

http://www.sielc.com/compound_002.html

I agree with Uwe, HILIC will be the best alternative for you and most likely an isocratic run will work fine. Although it depends on the matrix, also. If you look for real low concentrations an MS could be needed.

You may find more advice at
http://www.sequant.com/sn/p_notes.php?id=7

for HILIC it will be hard to dissolve aminoacid in 90% of ACN. I am wondering if somebody can provide a reference to retention of aminoacids in HILIC mode-how people are dissolving aminoacids in ACN?

Just wonder whether the two methyls don´t make it possible to use even RP? I know that proline, which has the N embedded in a 5-membered ring is too polar for RP.
On solubility: If you must use UV detection above 200nm you may get to the limit, but for that you need enourmous concentrations (like a mg/mL).

Forgot, with UV detection, a bigger problem than the solubility is likely to be overloading of the HILIC column.
(Strangely, or maybe not?, the zwitter ionic variety of HILIC columns has proven to have a higher loading capacity than silica)

I don't think 90% acetonitrile is needed, the range 70-80% is more likely for an acceptable k' value.

Send me an e-mail and I can provide you with graphs on k' vs %ACN for various amino acids (I don't have these available on this computer).

In this case I believe that mobile phase pH may be the most important parameter to control.

I agree with Einar, I do not think that 90% organic is needed. For your analyte, I expect elution in a much more polar solvent. For this reason, and for the reason that sample solubility is rarely an issue in analytical chromatography, I do not subscribe to the argument by SIELTECH that there is a solubility problem with HILIC.
If we would be talking about preparative chromatography, the story would be different...

The method that I've used is 0.1%methanesulfonic acid in 98%H2O/2%MeOH and an aqueous C-18 column with UV detection. Sarcosine comes out first, then dimethylglycine, then betaine, with other exotic glycines being found at various time points.

Thomas,
some more details on column packing, dimensions, on retention time/flow rate and injection amount/concentration could be interesting.

Alltech Prevail 4.6 x 150mm, 3um, 0.8mL/minute flow, 205nm wavelength, 2mg/mL sample/H2O. Because the chromophores of some of the relateds are stronger absorbers than the dimethylglycine (DMG), run a trusted standard. The dimethylglycine tails, but the other items of interest are separated, including some unknowns relative to the supplier of DMG. It's a less than 10 minute run, quick and cheap. You can try TFA in place of MSA if you wanted mass spec.

Did you have proline or hydroxyproline in this soup (wonder where they would be relative to DMG)?

I had unknowns appear at about 6 and 7 minutes (did not line up with any of the other off-the-shelf gylcines that I purchased), the DMG appearing at 3 minutes. The unknowns had a strong chromophore relative to the DMG. But, I was only proving the best DMG lot by assay, and did not get into unknown determinations. Area% ratios were irrelevant due to the chromophoric differences.