Chromatography Forum

Hi! I need guidance. I've started to use a waters spherisorb amino column, with the aim to analyze sugars. I activated the column in the conditions especified in the care manual, and then I started to flush several mobile phases through the column to conditioning it for sugar analysis. I used isopropanol/pentane, isopropanol, isopropanol/acetonitrile and finally acetonitrile. Each 20 min at 1mL/min. After equilibrating the system with the mobile phase I needed to determine sugars (ACN/H2O 80:20), I injected a sucrose standard and an inulin standard but both had the same retention time. It seems like the column is not interacting with the analytes. What can I do? How can I solve this?

Hi

"What can I do?"
Give us more details, like:

Column dimension?
Injection volume?
Sample/standard solvent?
Concentration of standard?
HPLC model?
Detector?
Was it a new or used column and how old was it (even if new)?

But in general, be aware that you're doing HILIC mode with this application.
Therefore water is the strong solvent.

So if you don't have enough retention, decrease the water content in steps of ca. 5% and test again until you get enough retention (always equlibrate with about 20 column volumes).

if possible, reduce the water content in your injection solution by adding acetonitril. Best would be same composition as your mobile phase.
If that is not possible, reduce injection volume.
You may also need to reduce the water content in your Needle-Wash solvents if peak form is bad.

https://books.google.de/books?id=Sx8VNI ... in&f=false

Check out this paper please.
What pH has your sample solution?

Thaks for your help, here are the answers to your questions:

Column dimension? "250x4.6 mm"
Injection volume? "10uL"
Sample/standard solvent? "standard asolutions were made in miliQ water"
Concentration of standard? "between 50-150 mg/L"
HPLC model? "KONIK 500 A serie"
Detector? "RI detector waters 2414"
Was it a new or used column and how old was it (even if new)? "it is a new column, I made only 5 injections"

But in general, be aware that you're doing HILIC mode with this application.
Therefore water is the strong solvent.

So if you don't have enough retention, decrease the water content in steps of ca. 5% and test again until you get enough retention (always equlibrate with about 20 column volumes). "I already did that, and retention times increased but the analytes all had the same retention time"

if possible, reduce the water content in your injection solution by adding acetonitril. Best would be same composition as your mobile phase.
If that is not possible, reduce injection volume.
You may also need to reduce the water content in your Needle-Wash solvents if peak form is bad. "This I will try next, after I clean the column and equilibrate it again"