amino acid and products analysis

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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hi

i recently started a new project that requires the analysis of (single) amino acid substrates and its reaction products.
a typical sample will for example contain serine and ethanol amine or in another sample lysine, caprolactam and amino-caprolactam.
i have been working with isocratic solvent compositions in order to be able to use the RID detector for analysis....
I have tried seperating these compounds using a varierty of different columns including, an agilent C18 Zorbax Eclipse Plus as well as different HILIC columns...additionally i have tried eluiting compounds with various different solvent compositions including 100 H2O to 85/15 Acn-H2O mixtures with Ammonium acetate buffes...the best results sofar I obtained using a 85/15 AcN-H2O-0.1 M NH4Ac mixture adjusted to ph = 8...however the chromatograms are still not suitable for analysis because many of the peaks, i.e. lysine and amino-aprolactam have identical retention times.

im not sure where my mistake is but i feel like it should be releatively simple to sepearte these compounds due to there vastly different polarity (amino acid vs. reaction products)...does anyone have any advice on where i could be going wrong?

thanks alot in advance

best regards

david
Can you be more specific about your column parameters? What particle size are you using 3 um? 5 um?
hey

sorry for the late response...but i was testing a new HILIC column that i got on tuesday...
so i was working primarily with columns having 3 micrometer particle sizes...as suggested by the manufacturer i operated this new HILIC column (triazodole anchored silica) at 30 °C with a flow of 1.0 mL/min...the initial results where by far better than those achieved with the other columns...however i was still having trouble with multiple peaks appearing when ever i injected pure substances (10 different samples all prepared from representative samples in millipore)....specifically there was a peak that reappeared over all 10 samples (in both RID and UV @ 210 nm)...however as i then saw at the same retention time and intensity for all 10 samples. I figured that the solvent used to prepare the samples was contaminated...so i injected pure fresh solvent and saw the same peak...the reason is simply the vast difference in composition between the eluent (70-30 AcN-H2O) and sample solvent (h2o)...when you use 50/50 mixtures and inject millipore the peak is virtually gone. not 100% sure why this peak I UV active (not very but still)…
also there was a huge negataive peak that appeared near where all substances eluted...i was unsure of the reason for this...but with alot of trying I found out that it is related to the salt concentration (10 mM NH4Ac almost no negative peak compared tp 50 mM NH4Ac - aqueous) as well as the injected substance (large negative peak when injected amino acids...small to almost now negative peak when injected less polar/ionic substances)...

with tailoring of the eluent composition (70/30-85/15) as well as the salt concentration (35 mM NH4Ac) i finally managed to seperate the compounds...
sorry that I didn’t get back to you earlier but I was more than puzzled by the results I was obtaining and wanted to test different conditions before I got back to you with a more detailed description of the problem…

have a good weekend

david
When a mobile phase is used that's predominantly organic, then injecting a sample in pure water is analogous to injecting a sample onto a reversed-phase column in 80% acetonitrile. Why did you think that the peak shape and retention would be any better here? Specifically: When there's a gross disparity in composition between the mobile phase and sample solvent, then they will differ significantly in viscosity. Remember that the Gulf Stream maintains its integrity all the way across the Atlantic Ocean on the basis of a comparatively minor difference in density between it and the surrounding water. Your sample plug won't mix readily with the surrounding mobile phase either. Instead, the mobile phase will tear off parts of the sample plug at its edges. These will stream down the column faster than the rest of the sample, resulting in the multiple peaks you observed for a pure sample. In a less extreme case, you will get a badly skewed single peak for each analyte.

The lesson here is: Match the composition of the sample solvent as closely as possible to that of the mobile phase. This goes as well for the salts, per your negative peaks.
PolyLC Inc.
(410) 992-5400
aalpert@polylc.com
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