I want to separate glycine and its oilogomer with hplc having uv-vis detector at wavelength of 195 nm but i failed to do that. Can anyone suggest some suitable method to do that?
If your detector is variable wavelength, try making small adjustments to see if signal-to-noise improves. I recall a separation of a- and b- lysines being performed at ~ 210 nm (lit search for D. John Aberhart as author). Be sure to use highest purity water and other modifiers (salts, organic cosolvents) and degas thoroughly because O2 absorbs strongly at these wavelengths.
In the 1980s a group studying clay-promoted glycine oligomerization compared ion exchange separation with post-column derivatization (separation similar to what you wish to do) vs pre-column derivatization and reversed-phase separation of the o-phthalaldehyde derivatives. The pre-column approach worked better, in case you can use longer wavelengths and/or fluorescence detection. .
J Chromatogr. 1987;386:243-9.
We use waters hplc with 2487 uv vis dectector
chain length is upto 4 i.e gly,gly2,gly3 and gly 4
it is detected but one peak shows with same retention time irrespective of the sample injected.
we use 100% sodium hexanesulphonate buffer with 0.5 ml/min rate as per the literature but i failed more than 20 times .we use waters C-18 column
kindly provide proper silution if you can it will be highly apprecitaed
Yes.use TFA : ACN gradients elusion @215 nm.
Glycine will not bound to column. While your peptide will.
Try
If your mystery peak is near the void volume (usually around 1.5 min at your flow rate) it may be oxygen or a refractive index artefact. Have you tried injecting initial mobile phase and using mobile phase to dissolve samples and standards? If the peak is still observed, you can ignore it.so long as it doesn't overlap any of your (gly)n standards. If it does overlap (shown by increasing size with increasing concentration of standard injected) , you'll have to alter chromatographic conditions - possibly your column or theirs is more aged, has different degree of end-capping or whatever.
The C6H13SO3Na implies this is ion pairing reversed phase separation. Is there an organic solvent in the mobile phase (most likely methanol)? If so, try small changes - 5% instead of 10% for example. Most likely you will want to make the solvent weaker (less organic solvent) or else make the columnn "stickier" by increasing the hexanesulfonate. SNK's recommendation of TFA as ion-pairing agent is fine if you can work with unretained glycine, but if you need to observe and quantitate monomer, it must be retained to get it away from the injection peak, and the hexanesulfonate is a more effective additive for that. (At short wavelengths there is often a negative peak.)
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