I am atempting to analyse mono and disaccharides according to the AOAC method 982.14. However the column I have is an old Alltech CHO amino column (300x4.6mm, 10um). When running the standards, I keep getting a split peak for glucose and lactose. Also, sucrose, maltose and lactose do not separate very well. I have tried changing the acetonitrile:water ratio, changing flow rate and decreasing sample load but nothing seems to make much of a difference. Any ideas. Could it be that the particle size of the packing is just too large (ie method specifies 5um) and not suitable for what I want to do?

Give the column a decent burial and buy a new one.

You can try to wash your column in reversed way, this may help, but you have to buy a new one because this is just a temporary solution.

Amino columns in general are notoriously short-lived in the presence of water.

Hi Cathryn,
all Anons are right,but this give you no help. NH2 columns sometimes give not very reproducible results (could be the column), and column live time could be very short. You are right, 5µm materials will provide you with a better resolution. In my experience with NH2 columns I found that also storage of this column is not so easy. What was the mobile phase in which the column was stored. Was the column stored in a cool place? When was the last time you used the column? Also quality of the Acetonitrile is important. NH2 columns causes a lot of problems. But there are some alternatives available, which will lead to better results. NH2 columns are only one example for a HILIC phase (hydrophilic interaction), and stationary phases modified with Carbamoyl (Amide) group show better stability, better resolution and better reproducibility. Please have a look on such columns as well. One example you can find on our application data base, just click on Tosohbiosep.de and you have free access. Last week at Analytica one of my customers told me, that such a column works also well for Oligosaccharides, and she can run about 4000 injections on this column. Good luck.
Gerhard

Let me start off by saying that the simplest solution is to buy a new column. However, there could be several things that are going on. It is possible that the column is just plainly dead, meaning that it has formed a void. It is also possible that somebody has used it for another separation and has acidified it. If this is the case, you get double peaks for sugars due to anomer separation. If this is the problem, you got to make the column basic again by washing it with an amine. Triethylamine might do. You need a basic phase to avoid the formation of the double peaks. Therefore not just any HILIC column will work.
To Gerhardt: is your carbamoyl column basic, i.e with amino functions?

Hi Uwe, yes there are amino functions on that column material. The polar functional groups (hydroxy groups) of the sample form hydrogen bonds with the polar groups (amino groups) of the stationary phase. The number of hydroxy groups, conformation and solubility in the mobile phase determine the order of elution. Cathryn should also check column temperature. Below certain temperatures some carbohydrates will elute as split peaks (also on the NH2-column). Below 65°C Glucose will elute as split peak. Our column material can be used from 4-80°C, and has an excellent chemical and physical stability (compared with NH2-columns). Gerhard