Chromatography Forum

Good afternoon,

I am working on a new HPLC method and I seem to be having injector problems. If I make multiple injections from the same vial I will get some chromatograms that are normal and some chromatograms that are flat lines all the way across. I tried setting up a run with a methanol blank in between all of my samples thinking that this would flush out any particulates, but I still had this intermittent problem. My samples look clear and most of the ones with this problem have been filtered through 0.2 um syringe filters. Any ideas of what is causing this and what I can do to fix it? I wondered about the vial septum plugging the needle, but we haven't noticed this with other methods.

Thanks,

KarenJ

Hi Karen

A host of questions spring to mind. Is the solvent you are dissolving your sample in the same as your starting eluent? Do you see the problem intermittently or once it happens does that put paid to all of your other injections subsequently? I assume your are using an autosampler? What do you do to rectify the problem? If its an autosampler have you watched to make sure its always seating properly? Think what I'm saying is I need a bit more info to be able to help at a distance.

What model of injector or autosampler do you use? There are several different designs, and they can fail in different ways.

I am using an Aglient HP1100 system with a G1367A autosampler. I haven't seen any obvious malfunction of the autosampler operation.

I have two differnt solvents for my samples. The first sample is dissolved in neat methanol. The second sample is dissolved in a mixture of 5% ethanol, 25% propylene glycol, 70% phosphate buffer saline. My mobile phase is a gradient of 0.1% acetic acid and acetonitrile.

One thing that I tried was to run a methanol blank between each sample. In that run the problem came and went. When I made duplicate injections of the ethanol/PG/PBS sample (both injected following the methanol blank) the first injection was a flat line and the second injection was normal.

Please let me know if you need any additional information. Thanks very much for the help.

I'm guessing, but since the second shot always works, I suspect the vials. One guess is a partial vacuum inside that is relieved after the first puncture of the septum. The other is that the septa are too thick, and the needle can't penetrate on the first try; this actually happened to me on an old HP1050.

A plugged needle would cause the system pressure to exceed limits as soon as the injection valve moves. A poor seating would cause leaks. You don't report either of those symptoms.

I have some additional information. In my previous runs, when the injection was 'bad' the chromatogram was just a noisy baseline - no peaks at all.

I suspected the vials so this morning I changed from glass screwcap vials (which could have been sealed tightly enough to create the vacuum problem mentioned) to plastic low volume snapcap vials. The methanol samples yielded nice chromatograms with normal height peaks on the first injection. The ethanol:PG:PBS samples yielded chromatograms with peaks, but the peaks were small. After several injections, the peak size returned to normal.

Right now I have programmed my injector to pickup a small amount of methanol before the sample aliquot so that the methanol will flush out the sample when it injects. So far I have been getting good chromatograms.

You know, 25% glycol is pretty viscous. You can try a slower syringe draw, or a longer wait before lifting the needle from the vial.

The design of this A/S is such that the needle is actually part of the sample loop. Therefore everything that gets into the needle is injected and you don't need to worry about flushing it out. If it is not injecting your sample, it never got into the needle.