Chromatography Forum

I use the Phenomenex Phenosphere Amino (4.6 x 250, 5 micron) with a mobile phase of 19% H20, 80% Acetonitrile, and 1% Chloroform. (the chloroform is only there for adduct formation in MS detection).

My problem is that they ship the column in Hexane + 3% Acetonitrile. Before I started my analysis I flushed as follows:

Hexane/THF
THF
THF/Acetonitrile
Acetonitrile
Mobile phase listed above.

I know the performance of these silica amino columns deteriorates because of the pH problem, but I have used this column once before a couple of years ago and got good separation between rhanmose, fucose, xylose, arabinose, mannose, glucose, and galactose. However, the last two times I have purchased this column, the retention factor and resolution on mannose, glucose, and galactose were much worse on my initial injections.

I also have the Shodex Asahipak, and Phenomenex Luna amino which are pH stable, but neither of those separates all of the sugars that have the same MW under MS friendly conditions.

So, my question is, how would the flush procedure damage the column? I thought water would be the only solvent to damage it.

OK, you know already that these amino columns selfdestruct slowly when you put them into water. Your mobile phase contains water.

You will get significant changes in retention over at least a day or so, and then slow changes later. You will be better off with a column that is stabilized, such as the Waters Carbohydrate Analysis column. Also, for the separation of your carbohydrates, you may be better off with a bit less water in the mobile phase.

We use a Phenomenex Amino column for our fructose, glucose and sucrose separation. The mobile phase is 20/80 H2O/ACN. I'm surprise your mobile phase contains such a high proportion of H2O.

In any case, these columns don't seem to last very long, compared to common C18 phases. In our case, peak shapes started good and gradually broadens and then splits. I try to extend it's life by increasing the flow gradually, thereby reducing pressure shocks....this I actually try to do for any column, but more so here.

Thanks for the input guys.

The mobile phase worked for me in the past and was able to separate them all, but with the Phenosphere Amino I may be operating near the edge of a cliff, so to speak, since they degrade rapidly at first. I may have gotten lucky with my first column and not so lucky with the next two.

Uwe, I've considered getting the Waters Carbohydrate column since I saw in your book, HPLC Columns, that it is prestabilized. I noticed in the Waters catalog that they did not list a retention time for rhamnose. Do you know if it is separated from fucose? That's the issue I have with the Shodex Asahipak, that it does not separate rhamnose and fucose and I can't distinguish them in the MS.

As sfe-co2 mentioned, I also suspected the sugars were retained by a HILIC mechanism consequently requiring a lower % of water than the O.P's conditions.

If you have an amino column in relatively low aqueous HILIC mode with the water layer held close to stat. phase could the column degrage more rapidly by hydrolysis as compared to a higher % of water where the surface layer isn't set up?

I assumed that the use of 80% water with an amino column for the separation of carbohydrates was just a typo. In 80% water, all these things are unretained.

I do not know, if rhamnose is separated from fucose. However, from the standpoint of retention, an amino column is an amino column. I'll see if I can get an answer from somebody who knows everything that you ever wanted to know about sugar separations, especially on amino columns, but I may not get an answer for a while...

That was a typo. 20% H2O is correct...I fixed it in my initial post.

I've never run them at 80% H2O since I know that would kill the column and the sugars would fly through there. So, in summary, since I caused some confusion, my first Phenosphere Amino worked well with 80% Acetonitrile and the next two I bought were problematic from the start.

I had a little success yesterday with the Shodex Asahipak NH2. When I've run it with 80% acetonitrile it separated everything except rhamnose and fucose. Going up to 90% acetonitrile did not help.
I found that if I use a mobile phase of 45% Acetonitrile, 45% THF, and 10% H2O that I get separation of rhamnose and fucose, but it is at the expense of separating the other sugars (xylose, arabinose and mannose, glucose, galactose).
I tried a gradient from 100% Solvent A (45% ACN, 45% THF, 10% H2O) to 100% Solvent B (80% ACN, 20% H2O) and it worked out in separating all of them. I get a chromatogram like the one from Shodex's website, but I don't have to use the phosphoric acid, so now I have the MS friendly method that I needed.

Good job!!!