-
- Posts: 5
- Joined: Wed May 25, 2016 1:59 pm
So, first of, I am a PhD candidate in a biochemistry lab and have been appointed the HPLC "god" of our lab. Which is funny, because my previous experience with this device is limited and the responsible postdoc(s) have left without leaving proper protocols or even manuals. I have already spent a decent amount of time in figuring the software (ChemStation) out, read A LOT about the principals and such, and have managed to not mess up our standard applications which are routinely used here. Of couse, in my first month of duty we had a major device failure (FLD lamp), but now everything is back in place and I have started to work with the device more and more.
For two weeks I have been trying to get advance a new protocol to detect two compounds via derivatization. The derivatization products are fluorescent, which is good, but the elution of the relevant peaks trouble me.
I am using a C18 column with 3,5 µm, 4.6x150 mm dimensions and have initially - to figure out when my compounds elute in general - run a long gradient over 20 min, with an initial 4 min 0% B @ 1 ml/min to exclude that they do not bind to the column. After I noticed that the compounds elute early, I have run an isocratic program with which I can very nicely see my peaks elute from the column.
Buffer A: 0.5% Acetic acid, pH 3.9
Buffer B: 100% MeOH
Injection volume: 50 µl
12% B for 12 min @ 1 ml / min
100% B for 2 min @ 1 ml / min
12% B for 2 min @ 1 ml / min
Peaks elute at 5 min and 10 min, respectively. The flow-through peak - small, but noticeable - is visible at 2.4 min.
Now to my problem: in several measurements I have noticed that the peaks can only be found in the flow-through, albeit similar sample preparations and a few times even for the sample when an aliquot of it was thawed and injected.
I am absolutely clueless how to attack this program. I have performed overnight runs with methanol, have injected column-cleaning reagent, yet the problem resurfaces and I am afraid that it will screw up my measurements when the method is applied to the regular context.
I'd be grateful for any help!