Hello,
To determine betaine by HPLC RP C18, the MOP is buffer with sodium hexane sulfonate (Pic B6) and sodium sulfate.
I would like to know the role of sodium sulfate.
Many thanks.
Hi Zakaria,
I guess the sodium sulfate is to increase the overall ionic strength of the solvent - you have a anionic sulfate group in your buffer system and on the sodium hexane sulfonate. Analyte molecules with e.g. positive charges would strongly stick to the sodium hexane sulfonate and not to your stationary C18 phase and elute earlier as they in theory would do just by hydrophobic interaction with the column material. The sodium sulfate "shields" that interaction, making every analyte molecule "equal" towards ionic interaction.
You can simply try that by just leaving out the addition of sodium sulfate to your buffer mixture - the HPLC elugram may change.
You could change to different solvent system, we use binary system of water/acetonitrile with 0.1% HCOOH for our RP C18 HPLC columns (solvent A: 5% AcN/water 0.1% HCOOH, solvent B: 95% AcN/water 0.1% HCOOH).
Hope that helps.
Dear Marcus ,
First of all thank you for your response.
Could you please explain to me how exactly the sulfate sodium shields the interaction between the sulfonate and cation (betaine).
I moved the sodium sulfate from the buffer and didn t affect the retention time or the tailing of my compound.
sincerely yours.
I think the purpose of adding sodium sulfate is to maintain charge balance and prevent system peaks (peaks which appear when the column equilibrium is disturbed). Assuming that the betaine were retaining by forming an ion-pair with the hexane sulfonate ion, then the hexane sulfonate ion has to give up the Na+. There must be a supply of Na+ ions to replenish the flushed out Na ions.
Hi Zakaria,
if the removal of the sodium sulfate from your buffer system does not affect peak shape, retention time etc... you probably have no interaction with the stationary C18 phase. Betain is a highly hydrophilic molecule due to its permanent positive charge and the deprotonated COOH group (that's why it will form ion pair, just as Farooq descibed) and C18 columns are best suited for more hydrophobic compounds such as drugs or peptides.
You can try other column phases such as C8, C5 or even NH2 or CN (which is much better for polar compounds).
You can get a nice overview about different HPLC column phases on the link I attached (in the menu "phasen" - you can look at the structures and can compare them in a table).
http://www.phenomenex.com/Products/HPLCDetail/luna
Dear Farooq,
As i undersatnd that the sodium sulfate increase the dielectric constant of the mobile phase, like that the interaction increase between the sulfonate and charge + on betaine then increase the retention time right?
For the Na+ from the hexane sulfonate can complexe with carboxylate of betaine and i think it is not necessary to have another Na+ right?
Dear Marcus ,
thank you for the link, you are right maybe try another column less hydrophob to have more intercation.
sincerely yours.
Dear Rachid, the dielectric decreases with electrolyte concentration in water. The sulfonate end with interact with the positively charged end of betaine. You can try other modes of chromatography. I don't like ion-pairing chromatography.
Dear Farooq ,
Sorry i mean the sodium sulfate decrease the dielectric constant (salt) comparing to pure water. maybe this help the interaction between sulfonate and betaine.
many thakns.
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