Chromatography Forum

Hi,
I have a 2695 waters HPLC which has an injection issue. A very tailed peak is obtained with or without column (column was replaced by a restrictor) , as show in the picture at https://filebin.net/b4vkacbe20y89c7v

I have checked all seals in V1, V2, V3 & V4 valves and also in seal pack. A new needle is installed but the problem is still persist.
Any idea?

Sounds like you've got some crud in your system that is hanging onto your analytes.

Remove the column and the solvent frits and thoroughly flush it with some 30% H3PO4, water and IPA in turn. If that does not help, make several large injections of ~10% H3PO4 (still, no column), then purge injector several times with water and IPA in turn.

can you show us a better picture of the peaks (with and w/o column)?
The picture from the link doesn't show any baseline.

Beside this, did you check that there are no void-volumes inside the flow path?
Maybe also introduced during checking the V1-4. Also check it at the detector.
Also you mention a new seal pack and needle.

Did you pay attention on the direction of the needle opening when installing it? If not, this may be one part of the problem. (1 install the neddle to tighten the ferrule, then loose it, adjust the needle opening so it points to the outline of the seal pack, then tighten again).

Then do the seal pack adjustment for proper alignetment with the new needle.

Does your 2695 have a column selector valve? If yes, then maybe the rotor and seal there is worn out or partially pluged.

New chromatograms were shown at:
https://filebin.net/bqj9h36c0ducebu9

These chromatograms were obtained by caffeine injection (two replicates at the same concentration) without column. As could be seen, small peak is normal but big peak is not. The big peak is like an injection of air. In a worse situation, the peak tail gets more (the previous picture).

I think the big peak is related to entrance an air plug in injection volume but I still haven't found its source. Sometimes this problem is resolved by purging but comes again. I changed needle, high pressure seals in seal pack, V1-V3 seals and did leak test.
Also, autosamplere picks up accurate injection volume.

Thanks.

But I don't understand the picture.
How can you get that big differences in peak areas with replicates of the same solution and if injection volumes are correct?

what was the injection volume?
Was the caffeine dissolved in mobile phase?

The small one looks ok as you say, the bigger one could also be of too much volume injected or solvent mixing effects?
What kind of restrictor do you use? Just a piece of 0.1mm tubing or a special pressure regulator? Only youse tubing.

Maybe repeat the caffeine test witha C18 column and about 25% MeOH as mobile phase and sample diluent. (If I remember correctly).

Thanks.

I'm doing QC for my instrument according to EDQM with caffeine solutions. I installed a youse tubing restrictor (0.1 mm i.d, 10 cm L) instead of column with a back pressure of about 800 psi. Mobile phase is water (isocratic mode) and caffeine solutions were prepared in water.

I have done the QC tests many times in recent years (according to EDQM) and there is certainly no doubt about the HPLC conditions and I'm sure the cause of the problem is the malfunction of one of the modules but I still haven't found it.

I think the big difference between two injections is related to air plug. Actually, the big peak is not for caffeine only, it might be air !! My normal peak is the smaller one but after several injections with a good repeatability (for small peak) suddenly the big peak is appeared and in the worse cases, the big peak tailed more and more. Sometimes I get the large peak up to several injections (even by mobile phase injection) and then it goes suddenly (or sometime by purging) and peak shape becomes normal again.

The interesting thing is that when I inject air (when the situation is normal and I get small peak for caffeine), I see a peak similar to the big peak (with a slight difference in size). So my suspicion for injecting air plug is reinforced.

Thank you for the details. That gives a much clearer picture of the situation.

Now I can't really tell what it is but some more suggestions:

- if all the pressure and leak tests pass, then it would be weird that air should come from outside... but one never knows...
- if you immerse the out of the detector in a liquid, do you see an air-bubble when the peak tails?
- Is your degasser working properly?
If I remeber, the pressure indicator should be below 40 psi (or was it mbar)?
- do you have a pressure regulator at the out-port of the detector, so it won't siphoning or bubbles in the cell?
If you use a small piece of tubing, make sure it won't exceed the cell's pressure limit under your working conditions


I think something similar was seen in another thread here in the forum, with bubbles/weird baselines occuring once in a while after several good injections.

maybe with a real chromatogramm with several substances, one could see, if all peak are affected (real injection problem, void volume) or only the first ones ->extra column effects or timed/volumed issues.


-> LC Resources Troubleshooting Wizard:
http://www.lcresources.com/resources/TSWiz/hs40.htm