Chromatography Forum

Has anyone experienced this before? What suggestions do you have?

My goal:
Develop an assay for EGCG, a green tea compound. It is rich in hydroxyl groups (do a google images search for EGCG to see the structure).

My assay:
water/acetonitrile/0.04% formic acid mobile phase
Luna (or ACE or hypersil) C18 column
alliance/Quattro Micro HPLC/MS system

My problem:
Tailing (Tf ~ 2). I might expect from a basic compound but I did not expect to see it with this compound. Since I'm not sure why the compound is tailing so severely I'm not sure how to solve the problem. Luna is an ultrapure silica and it is end capped so I'm hesitant to think it is being caused by free silanol interactions.

What I've tried:
-I spent some time on the phone with the Phenomenex folks who are surprised by the excessive tailing.
-I have eliminated system problems as the cause. Other compounds on the system exhibit acceptable tailing (Tf ~ 1.2). The IS, also a hydroxide rich compound tails slightly less (Tf ~ 1.8).

What I am going to try:
-A more basic mobile phase
-Heating up the column (had been running at RT)
-Injecting the sample at a high aqueous mobile phase to pre-concentrate the sample at the head of the column.

After those three things, I'm out of ideas. As mentioned above, we see this tailing on a variety of columns (all silica based because those are readily available in the lab).

Any advice would be helpful. Thanks!!

Below are applications for catechins in green tea using
Cadenza CD-C18, Unison UK-C8, and Unison UK-Phenyl:


http://www.silvertonesciences.com/files/TI144E.pdf
http://www.silvertonesciences.com/files/TI145E.pdf
http://www.silvertonesciences.com/files/TI146E.pdf

First, I would make sure the fluidics are good(good fittings, good flow path, and etc). Testing a known well-behaved compound can dignose id it is a problem.

Have you tried to use UV detection (which can be placed between the column and the MS)? This way you can check if the tailing is from the MS part.

I checked its structure and suspected this compound might be a metal chelator. If it is the case, replace the column bed supports with nonmetallic ones migh help.

Ascentis RP-Amide, 565324-U

1% formic acid in (water/acetonitrile) gradient can vary to vary separation. Start at 0% up to 20% acetonitrile.



1 – GC 2 – EGC 3 – C 4 – EC 5 – EGCG 6 – GCG 7 – ECG 8 - CG

best wishes,

Rod

The following links to EGCG on a Luna column with UV detection. You may have to join the WWW link to get the abstract, which conveniently has a chromatogram. I'm sure there will be plenty of other similar papers out there with method details.

If your system doesn't match it's performance, look at the significance of the chromatographic differences, starting with your MS. I suppoort the suggestion of putting a UV detector on your system, or running on another system with UV, to eliminate the column and other front end issues.

http://www.aapspharmsci.org/abstracts/A ... 002711.PDF

Bruce Hamilton