Chromatography Forum

We have had a request from a client to directly inject egg white onto reverse phase HPLC with a 50/50 acetonitrile/water mobile phase. Has anyone had any experience of this as it doesn't sound like the best idea to me.

Thanks

Umm...not that I have ever tried this, but my first thought is that you'd have significant protein precipitation. What are they interested in?

You need to tell that client to: 1) purchase/provide the analytical column and guard column and 2) purchase and provide tubing; and 3) purchase and provide a flow cell. These are precautions b/c by doing so you will shorely ruin your own column.

Assuming they want you to analyze the proteins in the egg white...
you can try it using reverse phase on Intrada WP-RP, 300A pore size
(but you will need to run a TFA reversed-phase gradient, not 50:50 ACN:Water).

Below are proteins found in egg white (albumin and lysozyme):
http://www.imtakt.com/TecInfo/TI289E.pdf

First thing to do: make sure that your client is willing to pay for the column.
Second thing to do: do it!

If the injection volume is low enough, you may get away with it - for a while. But don't expect to get a lot of injections out of the column in 50/50 MeCN/water.

If your injection is large enough, my bet is that you get a boiled egg on the column inlet.

It is difficult to pump through a boiled egg...

Still not sure what useful information they hope to obtain by
injecting egg white directly into an HPLC column but...

Below is a chromatogram for bovine serum (no protein precipitation)
injected directly on to the Intrada WP-RP column. For this example -
Imtakt used a 0.4 uL injection volume onto a 250x4.6mm, 3um column.

The chromatogram shows that the proteins elute off the column -
you'll want to use a very small injection volume
for the egg white as well (or larger i.d. column for a larger inj volume):

http://www.imtakt.com/TecInfo/TI274E.pdf

My experience with ovalbumin was that the MeCN concentration that will precipitate it is only slightly different than the concentration that will elute it from an RP column.