I'm using an HPLC column HC75 H+ (Hamilton) for separating a mixture of mono, di and trisaccharides using water as mobile phase. This column is recomended for low MW carbohydrates, but I realized it is a cation exchange column, so my doubt is if a sugar, that have covalent and not ionic bonds, can behave as a cation in solution. Thank you.
Hi,
Carbohydrate are polar compound. So that, your hplc column separate them acording their relative polaraty. There is not (significat) ionic exange inside de the colum.
regards
cv
Hi Francesco, the Hamilton H75 column you use for your separation are generally fine for mono- and disaccharides, and are probably the best option if you dont want or can not label the carbohydrates with functional groups (i.e. fluorphores) - I used these types of columns a lot in the past which helpted me to separate various mono- and disaccharides. However, the resolution of these columns is limited and you might get difficulties of separating complex mixtures of sugars, I doubt that you can successfully separate >10 carbohydrates successfuly. There are really good works out there on separating unlabeled carbohydrates using graphite columns (ie. DOI: 10.1093/glycob/cwr138), the downside of these methods is that they are somehow high maintainance (cappilary HPLC, columns last only for a couple of samples, etc..)
I think you are on the right way, although your current approach has some limits it resolution etc...
I partially agree with John, since i think he misunderstood the question.
Francisco asked wether carbohydrates behave like cations in solution and if because of this, they can be separated using a column with a cation exchange stationary phase.
So that, I reaffirm my answer. Carbohydrates interact with the stationary phase because they are polar compound (not because they are ions). For instance, the pKa of glucose is close to 12. So, during a typical HPLC analysis, glucose has not net charge. Therefore, is impossible a ionic exchange.
There are several interaction between carbohydrates and solid phase that can take place, such as: the formation of coordination complexes, ion dipole interactions, ionic hydration and hydrogen bonding. All of these interactions are possible, since carbohydrate are polar ( I mean, a compound with an uneven electron distribution within one or more chemical bonds, owing to the difference in electronegativity of different elements). Sugar are polar due to the presence of several OH groups. On the other hand, carbohydrates with a different degree of polymerization are separated by size exclusion (they partially penetrate into solid phase). The latter can be controlled varying the cross-linking of the resin.
Francisco, I enclose a very illustrative document.
All the best
Thank you for your time and your answers, in fact all of them were very helpful!
Maybe you can try with an anion-exchange chromatographic separation, I think it would be useful if you have sugar acids or some anionic substituents like sulfate groups. In contrast, if you have neutral carbohydrates, the interaction (cations-neutral carbohydrates) is weak.
http://www.sciencedirect.com/science/article/pii/S0021967399004860
Hi Francisco, monosaccharides may have partially weak charge so you can't depened on for separation, by going larger in di and trisaccharides this partially charge disappeared. so in this case you need to use an affinity column like NH2 to separate mono,di and trisaccharides
All the best
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