Chromatography Forum

Hi,

Whenever the gard cartriage is installed, I got split peak for the standard. I have replaced a new cartriage and related fittings, still the same problem. . can anybody give some suggestions? Thanks a lot.

I assume from your post that you don't see this behavior when operating without the guard. Make sure the guard is the same phase type as the column (i.e. don't use a C18 guard with a C8 column). Make sure your fittings are connected properly. If you have steel fittings between your guard and main column, or are using a "direct connect" style guard, make sure you aren't trying to connect a standard fitting to a column with a Waters style fitting.

In fact, just in order to find out the what cause the problem. I didn't use any colum in the line (Normally I use C18 and the gard column is also C18). Without any column(both gard and analysis column), Flow injection give perfect peak, when gard column itself it installed, a peak followed by a very broad peak is obtained. In other word, it is not only splitting, it become two peaks, one is sharp, the other is broad. The gard cartriage and column are from Phenomenex (P/N: Kj0-4282), all other connection are standard. Hope this can help you understand my problem more.

By the way, I do consider useing waters column, what kind of gard column cartriage should I use for water column? What is the difference for the waters style fitting?

Thanks a lot for your valuable time

Waters style fittings have a deeper bore than non-Waters fittings. If you do not pay attention to this, you may get inferior performance from a Waters column due to the increased dead volume.

I recommend Sentry Guard columns. They are packed using the same techniques as used for analytical columns, and thus they have a high performance and good stability.

meillid, you don't say what happens if you inject a standard onto your column without a guard. Doing this once for testing purposes, with a pure solvent standard, shouldn't hurt your column. This will tell you whether the problem is really with your guard, or with your column or method.

Just in case it's something else, let me ask: What solvent is your standard dissolved in? What is your injection volume? Is your separation isocratic or gradient? What is your mobile phase (or starting mobile phase if gradient)?

My standard is a very basic compound with amine group on it(tobramycin). I disolve it in water. the mobile phase is gradient. A: H2o, 0.1% FA, 1.2 mM HFBA, 2mM ammonium acetate; B: methanol, 0.1% FA,2mM ammonium acetate. The gradient begin with 80% A. I used to use the Phenomenex c18 gard column and Xterra C18 Column. obtained very good peak shape under this experiment condition. Only recently, I got this problem, my test to find the problem is indicated in the earlier reply. I also did one experiment as your suggested, the peak do split (still two peaks) when use only Xterra column without gard. But when I try to run the second one, the connection into the column break(? so maybe still connection probelem? high pressure make the connection break?) It seems the problem become even more complexed. I appreciated your kind reply.

It sounds like the problem may be with your column.

Also, I don't have experience with HFBA, but I can tell you that TFA containing mobile phases will "go bad" after about a week. If your "A" mobile phase is more than a week old, it might be worth re-prepping it. Without HFBA, I would not expect that you would get any retention of that compound.

Confusing thread

Here are the points I've picked up in order going through the posts.

* Guard + column = split peak
* New guard + column = split peak
* No guard or column = sharp peak
* Guard only = split peak
info: Guard and column are from Phenomenex, C18 type unspecified (p/n KJ0-4282 is the cartridge holder only)
info: Used to use Phenomenex guard + XTerra column.
* XTerra column only = split peak

I'm not sure what type of fitting is being employed; PEEK fingertight on PEEK tubing, swaged SS on SS, etc, but IMO when different types of column endfitting are to be accomodated PEEK-on-PEEK or PEEK-on-SS make it easy to ensure your tubing is properly seated. If the poster has a swaged SS fitting, I might of expected sharp peaks with either the Guard or XTerra column alone. If it's fingertight fittings, I wonder if it's possible for one to repeatedly incorrectly connect the tubing..

The following short article gives some info on potential connection problems: http://www.mac-mod.com/pdf/08_PlumbingTBS.pdf

If a simple 4 mm guard cartridge can chromatographically resolve sample components then maybe there is an incompatibility in the methodology giving a broadened peak.

Looking at the structure below, I would of thought aminoglycoside sugars like this are easily retained on a C18. Is there the possiblity of epimerisation in this structure and hence some sensitivity to temperature?