Experiencing poor Sucrose elution

[SeanAustin]

Hi

With your existing equipment and your separation needs, I would recommend to use an amine column - some similar suggestions have alreay been made. The new Sugar-D column looks quite interesting but I have no experience with it. I normally use the Asahipak NH2P-504E (from Shodex), but they have just released a new version of this column with smaller particles which looks interesting. The only possible issue you might have with these types of columns are the trisaccharides and larger. You need to use isocratic conditions with the RI typically a mixture somewhere in the region of 80/20 to 70/30 AcN /H2O will resolve the mono and disaccharides. I think the longer oligos are eluted with a bit more water. The amide columns (e.g. Tosoh Amide-80) should also be able to perform the separation under similar conditions - but again I don't know about the situation with the trisaccharides.

If you had an ELSD, or CAD then the above solutions would be fine since you could introduce a gradient. Another option that would allow you to use a gradient would be to introduce a post-column derivatisation that would give you something detectable in UV/VIS - there are several options described in the literature for this, but you would need an additional pump for introducung the reactants.

Pre-column derivatisation would also be a good solution. But most of these derivatives exploit the reducing end of the sugars for labelling - so it won't work for sucrose. I don't know if a suitable label exists that you could use for sucrose.

Hope some of this is helpful.

Sean

[mwoodman]

Hi; I saw this in my morning mail.
One quick RI comment. You do not have good stability at 25C and I would recommend 35C or higher with any RID that does not have a Peltier controller. There is little relationship between column and RI temperature and I usually run 35-40C.
Do you need to separate sucrose and maltose in the same run? If you really do, this limits your ion exchange choices and it is the main reason why people switch to amino phases with ACN/water mobile phase. Because you have RID you do not need to worry about silica or resin based amino, while with ELSD you would need resin based amino.

With great looking maltose/glucose/fructose we can assume your column is fine mechanically. My experience with many non-Hydrogen form columns is that with some use they lose the named ligand (Ag, Ca, Pb, etc.) and can convert to H or be contaminated by Iron. If they convert slightly to Hydrogen you will indeed invert the sucrose and get a messy peak that is due to dynamic inversion on the column. Reducing column temperature will help but the lower limit is probably 70C or so. Historically, I like to add a little of the Cation to the mobile phase. CalciumDisodiumEDTA is a great additive (50mg/L) for Calcium columns. Lead has the obvious toxicity issues but a low level of Lead Acetate might be beneficial for those columns. No experience on Silver. Perhaps others here...

Tom, I believe, noted the possible high molecular weight material in your sucrose c'gram. This peak is also possible from ion exclusion effects, and the RI has no way to help you distinguish that problem. I wonder if you have prepared the sucrose with any possible "salt" contamination or if your sucrose standard might somehow contain "salt"?

Finally, I believe you should change the column. Unless you can restore Ag to this column (vs. Hydrogen) you either need a replacement column or a different cation column. If sucrose/maltose separation is required your choices for ion exchange will be very limited. Otherwise any non-Hydrogen IEX should be acceptable if you can keep the column in its' designated cation form. There is also amino phase, eluting Fru/Glu/Suc/Malt/Lac in that order. Be aware that sensitivity with the ACN/water mobile phase is much poorer than water -- I'd say around 10X.

[Kees Olieman]

Cation exchange columns in the Ag mode are not suitable for sucrose analysis, because there are some remaining H+ ions, which will invert the sucrose. This results in detoriated peak shape, because the inversion takes more less contineous place.
A Pb column is capable of giving a nice separation; however, this column can also lose some Pb ions and then you have the same problem. Moreover, a precolumn ("desalting") is necessary; however, you should place it outside the column oven, as the strong acid ion exchange resin shall invert your sucrose at 80°C.
If sucrose starts to show multiple peaks, we regenerate the Pb column by pumping a solution of Pb nitrate (or acetate).