Chromatography Forum

Hello,

I am in the process of using a simple hot water and acid extraction procedure to hydrolyse and extract soil carbohydrates. The solution I am left with will be a 0.5M sulphuric acid solution. We have a Prevail Carb ES HPLC column set up on our HPLC at the moment and have an ELSD. I have read in the literature that this column operates between pH2 and 14, but have also noticed that nearly everyone neutralizes their solutions. So, my first question is whether the neutralizing step is necessary?

If I were to neutralize my solution, I could do so with sodium hydroxide, but a lot of recent literature uses a barium base such as barium hydroxide, so that a precipitate can subsequently be centrifuged off. I was struggling to find in the literature any tests of this columns specificity, though the manufacturers make it appear that the specificity is very good. So, second question is, does anyone have any idea as to whether a Na2SO4 salt, or even a buffer salt such as PBS, would affect the monosaccharide peaks detected by the ELSD?

Finally, the examples of successful elution and detection of monosaccharide peaks within simple sugar solutions on the manufacturer's website are very pretty and perfect, showing the column in a very good light. Is there anyone with experience of detecting carbohydrates within more complex solutions that could give me any tips of particularly troublesome things to look out for?

Thank you,

Harry

There was a discussion about this topic a while ago that will to understand the NH2 phase:

http://tinyurl.com/properuseofaminocolumn

Not sure about the sample prep. But no matter which you decide, you should use a guard cartridge before the column.

Hi there

I’m currently running wine and plant root extract samples on the Prevail ES 250*4.6mm with ELSD, using a 1mL/min 25:75, MilliQ:MeCN. (ELSD set at 80C with gas flow of 2.35). Oh column is held at 30C

As all samples are 100% aqueous, there are limits to how much i can inject due to the high % organic in the mobile phase. Seems as though 35-40uL is that limit, after which you start to see partially split peaks. Well over this volume im sure you would see full split peaks, but i have not done that so far. Im looking specifically at fructose, glucose and sucrose. For nearly all these samples, i get a reasonable baseline where these sugars elute, and runtimes of 15 minutes. The tissue extracts are from a 5% perchloric acid, neutralised to pH 4 with K2CO3 and some of the wine samples are diluted 10 fold with 25mM KH2PO4 pH 2.5. Although there is a large “spreadingâ€