Chromatography Forum

Hello everybody,
I need to analyse a sugar mixture (fructose, glucose, isomaltose, sucrose) in water by using a HILIC HPLC column but I cannot separate fructose and glucose because their peaks overlap. Are there any ideas what I could try? I use the following settings:
mobile phase: acetonitrile + water (80:20)
flow: 1 mL/min (I've already tried 0.2 mL/min and 0.5 mL/min without success)
injection volume: 1 µL (samples were diluted 1:1 with mobile phase)
column: hypersil gold amino from thermo fisher
oven temperature: 35 °C
RI detector with 35 °C; 0.5 s rise time; 5 Hz data collection rate

thank you

Seems like you are running this example?

https://appslab.thermofisher.com/App/19 ... -detection

Do your standards prepared in mobile phase retain? What is your sample diluent, you might be injecting too strong a solvent.

edit: I see your sample is in water, try diluting it 1:4 sample:acetonitrile to match your mobile phase conditions for improved retention, although your injection is small that it might work in your strong diluent.

As already mentioned, try to match the mobile phase conditions with your samples. So dilute it directly with acetonitrile, I would even go to 1:5 as to give 80% ACN. If peaks go too low now, you may be able to inject more.

Also adjust the needle wash solvent to high organic (if the system is used also for RP-mode, this can be overlooked)

Another idea would be to add about 20 mM ammoinum formate or acetate to the mobile phase.

If you can also play with the mobile phase composition, try 15:85 or something in between.

Also check the peak width by injecting a single standard. Calculate the plate number and compare to the specification for the column. If the column is old/damaged, or if there is too much extra-column volume, the peaks will be wider than they should be (= low plate number) and they will overlap even if chemically separated.

Seems like you are running this example?

https://appslab.thermofisher.com/App/19 ... -detection

--> Yes, almost. In my system is isomaltose and not maltose; and I have no lactose.

Do your standards prepared in mobile phase retain? What is your sample diluent, you might be injecting too strong a solvent.

--> The sample diluent is my mobile phase, so acetonitrile and water (80:20).

edit: I see your sample is in water, try diluting it 1:4 sample:acetonitrile to match your mobile phase conditions for improved retention, although your injection is small that it might work in your strong diluent.

--> I will try diluting 1:4. I hope it will work. Thank you for your advice

As already mentioned, try to match the mobile phase conditions with your samples. So dilute it directly with acetonitrile, I would even go to 1:5 as to give 80% ACN. If peaks go too low now, you may be able to inject more.

Also adjust the needle wash solvent to high organic (if the system is used also for RP-mode, this can be overlooked)

Another idea would be to add about 20 mM ammoinum formate or acetate to the mobile phase.

If you can also play with the mobile phase composition, try 15:85 or something in between.

Thank you very much. I will try all of your tips as well

Also check the peak width by injecting a single standard. Calculate the plate number and compare to the specification for the column. If the column is old/damaged, or if there is too much extra-column volume, the peaks will be wider than they should be (= low plate number) and they will overlap even if chemically separated.

A very good hint! Thank you very much! I will check that out as well