Chromatography Forum

Hello,

we just evaluated a sequence of the following type:

Standard 80%
Standard 100%
Standard 120%
Samples (about 30 vials)
Standard 80%
Standard 100%
Standard 120%
Samples (about 30 vials)
Standard 80%
Standard 100%
Standard 120%
Samples (about 30 vials)
Standard 80%
Standard 100%
Standard 120%
Samples (about 30 vials)
Standard 80%
Standard 100%
Standard 120%

For the Standard 100%, the area of the first injection is about 350'000 AUs and it increases continuously up to a value of 420'000 for the fifth injection.

All other vials lead to a area set with the usual low variance.

Standard 100% was filled in two additional vials that have been injected 7 times each directly after the injections described above. The injections of the first vial lead to a mean area of 380'000 (low variance) and the injections of the second vial lead to a mean area of 340'000 (low variance).

What might be happen with the standard 100%? It is prepared by dissolving the working standard in mobile phase and is the further diluted twice (down to the estimated sample concentration).

This type of sequences have been ran in our lab for years and this is the first time that we have a problem.

Thank you very much in advance for eny ideas.

Florian

More information is needed like solvents, chromatography conditions etc., is the standard solution viscose for example. What injection system do you use? Can be a carry over! Temperature of column and auto sampler is increased?

Since the repeatability of each vial is good , check your dilution procedure and equipments.

Hello,

Temperature: slight T-wave across the entire sequence (+/- 2°C for room, samples and column); Target T = 25°C
Mobile phase = sample solvent = acetonitrile : 0.05% Tris(hydroxymethyl)aminomethane 35:65
Flow = 1.0 mL/min
InjVol = 20 µL
Column: LiChrospher 100, RP-18, 125x4 mm, 5 µm
Injector: Merck Hitachi AS-2000 (6-port injection valve)

Regards

Florian

If I understand you correctly, you only have repeatability issues with one single vial. If so, that's probably where your problem is located. I'm thinking of the vial septum not closing properly (due to it being over tightened for example), allowing some of your solvent to evaporate over time (gradually increasing your concentration).

LCFan, Experiment performed only once is not a genuine experiment.
Fill up new vials and perform the experiment, at least once more.
If you see the same tendency, then you’ll have something to discus.

Best Regards

If I understand you correctly, you only have repeatability issues with one single vial. If so, that's probably where your problem is located. I'm thinking of the vial septum not closing properly (due to it being over tightened for example), allowing some of your solvent to evaporate over time (gradually increasing your concentration).

I agree here. It could be as simple as a crip cap not being tight enough if you use crimp top vials as we do. Sometimes there is a chip in the rim of the vial or the septa is a little thinner than normal and the crimp cap will actually turn with little force instead of being completely tight. It is easy for even acetonitrile to evaporate under that condition, even worse when you are using methanol. Since it was confined to that one vial that is probably what is happening.

You could always try weighing the standard vials before and after the run and see if the mass loss is equal across the vials as it should be if you remove the same volume from each.