I have NH2 columns: Сarbohydrate Analysis 3.9x300mm (Waters), Supelcosil LC-NH2 5um 4,6 mm x 25 cm, 5 mkm and YMC Pack NH2 300x6.0mm S-5 mkm, 12nm. I have to detect oligossaccharides from my glycoprotein after enzymatic digestion by PNGase F.
Can I see monosaccharides on these columns? Is it possible to see oligosaccharides using this columns?
ELSD has a non linear respons, so quantification requires standards at different levels. Moreover, the response of the ELSD decreases strongly when analyte concentrations are low. You should use ELSD only when you need to run a gradient, otherwise use RI detection.
There are two disadvantages of the NH2-column:
1) Reducing sugars react with the amino-group, resulting in partial loss of these sugars and modification of your stationnary phase.
2) Injection volume is severely restricted if the sample is dissolved in water, because water is a strong eluent (sugars display bad solubility in the eluent).
I wonder why the NH2-column is getting so much attention nowadays, because its use was ca. 25 years ago abandonned. The cationic ion-exchange columns using just water and RI-detection were suberb for simple mixtures of mono- and disaccharides and the anionic-exchange columns (Dionex) were the right choice for complicated mixtures and/or high sensitivity.
Dionex has several types of columns suited for (oligo)saccharide analysis, in combination with pulsed amperometric detection (PAD) you get very good separations and high sensitivity (ca. 100x more sensitive than RI or ELSD).
Yes definetly. You can use HPLC-ELSD for oligosacchrides. ACN:WATER(80:20), drift tube temp=80C, nebuliser temp=70C, Gas pressure=2.8slm. ELSD give good results with sugars by NH2 column.
Gud luck
:)
Yes, the separation ought to be possible. We separated glucose oligosaccharides using an -NH2 column in this work: Côté, G.L. and Biely, P. 1994. Enzymically produced cyclic (1,3) and (1,6) linked oligosaccharides of D-glucose. European Journal of Biochemistry 226: 641-648.
The answers below are for simple mono- and disaccharides. You will never achieve resolution on an amine phase column with oligosaccharides. Dionex makes a series of columns called Carbopac and they will separate oligosaccharides (cellular glycans from glycoproteins) and will separate the oligo by chain differentiation of 1 sugar residue. Go to their website and look at the applications. The problem with the system that Dionex uses versus ELSD is that Dionex uses electrochemical detection (PAD), which is probably a magnitude more sensitive than ELSD. This is great if you are looking at cellular oligosaccharides. I have never seen anyone use this column on any type of ELSD or RI. The other difference in this separation is the use of NaOH (10 mM to about 0.5 M gradients) as opposed to ACN:Water for amine phase. When you look at literature of people who separate these oligosaccharides, they use traditional ion exchange and are usually isolating and then doing TLC. Another alternative would be to look into CE and you can look at Beckmans applications for CE. Sorry to complicate your life but I did not want you to be disappointed when you were not able to do this intricate and complicated separation on a method designed for mono-, di- and DP sugars separations.
It means we cant separate mono sacchride, di-sachrides on HPLC-ELSD on amino column.???
Yes it is possible to have a good sepration on amino coloum of oligosaccharides. We have developed the method of analysis of 2 ingredients on HPLC on RI detector in AC:H2O as a mobile phase and the results are good. Just go on exercising for several development steps and you will surely get it done ultimately.
Thank you all. Now I'm almost cleary understand that it will be not so easy to separate oligosaccharides on NH2 columns and waiting for a new column SUPELCOGEL™ Ag2. But I litlle bit worry about sensitivity. If i have 4 sites of glycosilations on my protein how much should I deglycosilate to see oligosacch. on ELSD?
Exactly, we can separate oligisachhrides on NH2 column using HPLC-ELSD as said by Mr Quershi. We have also developed a method on oligisacchrides on HPLC-ELSD.
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