Trouble with sugar profile method

[mschonert]

Hi everyone. I am having trouble with my Sugar Profile method and was hoping someone could give me some advice. My problem is that my peaks are not showing up in my chromatogram. I have been developing this method based on AOAC 980.13 and AOAC 982.14. Here are my conditions: M.P 80/20 ACN/water, HPLC with RI detector and the column is Waters Spherisorb 5.0um NH2 4.6mm x 250mm. Flow rate is 2 mL/min with an injection volume of 25uL, and column heater is at 50 C. The sugar standards I'm using are Fructose, Maltose, Glucose, Lactose, and Sucrose, and all are 50/50 reagent alcohol/water .3g/mL. Thanks! ~M

[sepscientologist]

I would try running a bit of gradient say up to 50% water and see if they come out.

[tom jupille]

I would try running a bit of gradient say up to 50% water and see if they come out.

Not with an RI detector.

But on the right track: try increasing the water content a bit (do a series of runs increasing the water content by 5% each time). This is a normal-phase separation and water is the strong solvent.

Before doing that, however, check to make sure your detector is OK (do you see anything happening at t0?).

[upamaniah]

Dear guest,
for sugar analysis there are some specific columns are there, see below, check your detector performance also.
the below gives link to Shodex polymer based NH2 (amino) column which is the best and gives reproducible results, with 100% water as mobile phase.
also some ligand exchange columns available which are also very good for sugar and sugar alcohol separations.
i used it in my college days,

http://www.shodex.com/english/dc030214.html
http://www.shodex.com/english/dc030210.html

good luck
upamaniah