Chromatography Forum

Hello everybody,

We currently analyzed vitamin c with a kromasil-nh2 column with a mobile phase 60:40 phosphate: acetonitrile, however we´re having many problems in the preparation of the column, lasting for more than 48 hours . Is this very common for this kind of column?

Thanks a lot

Yes, this is common. Silica based NH2 phases are notoriously unstable.
I recommend Unison UK-Amino. It is durable under highly aqueous mobile phase conditions.

Below is an ascorbic acid appliction - you
can use this column under both normal phase and anion exchange mode:

http://www.silvertonesciences.com/files/TI317E.pdf

Another alternative is the Acclaim Mixed-Mode WAX-1 column. Look at Fig. 7 in the brochure: http://www1.dionex.com/en-us/webdocs/48 ... 021407.pdf
I have found this column to be quite rugged in a variety of applications.

Amino columns have problems, but in a buffered mobile phase, these problems shoudl go away. However, it may take a while until the column is equilibrated. A 4.6 x 25 column contains about 3 mmol of amine. If you are working with a 10 mM phosphate buffer, you will need to equilibrate the column for over 500 mL until you get stable results.

It may very well be that the problem goes away, if you take this into account. For example, you can convert the amino column in water to the desired pH at a high buffer concentration, and then return to your HPLC mobile phase.

I have done C-vit analyses on a normal C18.. It involved derivatising the sample so as to get be able to detect it by FLD and it was possible to see both ascorbic and iso-ascorbic acid. If this has interest I will try to dig up my old notes..

For example, you can convert the amino column in water to the desired pH at a high buffer concentration, and then return to your HPLC mobile phase.

Uwe -

Forgive me for being a bit dense, but could you clarify this just a bit please?

Would this be equivalent to running ca 50 mL of a 100mM phosphate buffer through the column, then returning to the 10 mM MP to run the analysis?

What exactly does this do?

Many thanks!

Chris

or 5 mL of a 1 M phosphate buffer...

I am talking about some ball-park figures, with sufficient excess to get to the pH that you need. The 3 mM of amine in your column are also the same kind of a ball-park figure.

Anyway, once you got your column to the pH that you want, you can reduce the buffer concentration. You can do this procedure also in water (better solubility of buffers) and then go to the organic composition that you need with the reduced buffer concetration.

hmm.....thought there is a AOAC method, where no derivatization is needed. However, TBAH is use in the mobile phase. The analyte is stabilized using a mixture of metaphosphoric acid and glacial acetic acid. The column is a C18 type, 250 x 4.6 mm.

The above is from memory only....you will need to check it out.

Good luck.