Sugar Separation by UPLC/ELS

[jcastaneda]

Hello science comunity.

Im developing a method for separate and quantify Fructose, Glucose, Sacarose,, Maltose and Lactose in foods.

Im using a UPLC with an ELS, but when I run the sugars standars individually I see two peaks for each sugar, and Im not suppose to see more than one peak for each sugar standars. Im not seeing this when I run it on the HPLC. My task is to migrate from HPLC to UPLC.

Im using the Amide BEH Amide 1.7um, 1.0x100mm Column. the mobile phase I using is 90%ACN-10%H2O with a gradient to 75%ACN-25%H2O in 8 minutes. the flos is 0.3mL/min and tehe column temperature is 35C. The detector setting are: Drift tube temperature 60C, Nebulizer at 12C (cooling), Gas Pressure N2 at 50psi. Gain 200.

The sample and standars used was 60%ACN/40%H2O. The volumn inyection is 1.3uL and the concentration of the standar being tested is 3mg/mL.

As the sugars can convert from cis to trans, but the Sacarose looks good. The simple sugars are the ones thant shows two peacks. Probably contamination fo the standar....well Im using fresh standars from Merck up to 99% purity.

Thanks for your opinions.

[Vlad Orlovsky]

you might have a mismatch between diluent and your mobile phase, you might be overloading the column ( 1x100 mm is kind of small) or you formed a void in the column

[Bryan Evans]

This separation can be easily performed on Unison UK-Amino:
http://www.imtaktusa.com/site_media/fil ... TI325E.pdf

Follow the above method using Unison UK-Amino and all your problems go away...

[Uwe Neue]

What you observe is absolutely normal, and the solution is equally well known. I will explain below, but you can look at the Waters applications as well.

Sugars form anomers, which are two forms of the same molecule that can interconvert when the ring opens. This is the reason that you get two peaks for your sugars. Saccharose does not do that, this is why you get a single peak.

In order to get a single peak out of the two peaks, it is necessary to speed up the equilibrium between both forms. This can be done by adjusting the pH to alkaline and elevated temperature. For your application, we used 0.2% triethylamine (TEA) at 35 degrees, or 0.05% TEA at 90 degrees.

[Bryan Evans]

In order to get a single peak out of the two peaks, it is necessary to speed up the equilibrium between both forms. This can be done by adjusting the pH to alkaline and elevated temperature. For your application, we used 0.2% triethylamine (TEA) at 35 degrees, or 0.05% TEA at 90 degrees.

...and column lifetime under such harsh conditions???

[Uwe Neue]

Of course, if this were a silica column, if would go belly-up in a few hours. However, the BEH Amide column is based on the ethyl-bridge hybrid material, which is good to pH 12.

I would not give a presentation at the HPLC conference on stuff that has not been proven to work, and the material described in my post cam straight from my lecture.

I can send copies of the lecture to anybody interested, including you, Bryan. You might learn something...

[Bryan Evans]

Hi Uwe -

Thank you for the kind offer - but I think I've got a pretty good understanding of UPLC and BEH technology.
I do my homework