HPLC separation of cross links from collagen

[rajeev]

HPLC experts,

This post is the continuation of the previous question about ODS reversed phase column.

I have been trying separate the collagen cross links such as hydroxy pyridinium, dehydroxy pyridinium and pentosidine cross links from collagen. (There is no option here to upload the file that contain these structures)

In the published paper, they used ODS reversed-phase column (8mm x 10cm), 10um, C18 column. 20 mM phosphate buffer pH 3.5 (Analytical biochemistry 278 99-105 (2000).

Presently i have been trying to use the above buffer system to separate all the cross links in single experiment using C18 (4.6mm x 25cm, 5u) column but, with no success.

I got the suggestions that i should work out by changing the pH of the buffer etc. But the questions are

1.Do i need to try different ranges of pH or

2. Different molarity of the buffer

3. Should i try to retain all the cross links first on the column and then try to resolve it

With all the questions in my mind, i thought if i post a specific question, then i may get the specific answers from HPLC people .

The above explained cross links can be obtained from Google search.

Thanks in advance for the inputs

Rajeev

[Uwe Neue]

An 8 mm x 10 cm 10um ODS column sounds pretty much as if they have used a radially compressed column. Contact me, and I should be able to get you the correct column or at least the correct packing that will have the selectivity that you want.

[SIELC_Tech]

Rajeev,

I am not sure if this will work for you but here is reference on analytical and prep separations of compounds in collagen. The research utilizes mixed mode Primesep A column:

Analysis of amino acid 13C abundance from human and faunal bone collagen using liquid chromatography/isotope ratio mass spectrometry
James S. O. McCullagh, Dieter Juchelka, Robert E. M. Hedges
Rapid Communications in Mass Spectrometry
Volume 20, Issue 18, 2006. Pages 2761-2768

Regards,

Vlad