peaks with mobile phase only

[juaant]

Hello I am Juan Manuel I work in a Technology Center as researcher. My group analyze carbohidrates (inulin, glucose, fructose and sacarose) with a colum PL Hi-Plex, 0.6 ml/min flow, mobile phase water, colum temperatura 80ºC and detector 55ºC RID.

Our problem is that we buyed a new colum, by error the mobile phase initial was 0,005 m H2SO4 for 1 hour. The specifications of column don't say that this is bad for column. The problem is that:

The first chromatograms of sample calibration (multistandard) was OK, but from fourth sample calibration my peaks split.

without peaks split
with peaks split

With the time the chromatograms was worse.

At last we pass only mobile phase (water) we had peaks as

¿Why, if this column is new?

If you can help us, we would be very gratefully
Thank you for your attention

[sadsal123]

One of the likely causes of your splits may be that the sulphuric acid mobile phase reacted with your sample. The other thing is have you checked your columns compatibility with acids? However, if your column did not support acidic mobile phases, you would not have been able to get decent chromatogram with water. The first is probably more likely.

Regards and good luck

[juaant]

In this moment the mobile phase is water 100% and I have the peaks as image. The peaks as you can see are continous in a interval of time. The column is compatible with sulphuric acid 0.05 M. I think that if I have signal of peaks is because something eluye.
This type of column is new for me, I am a little loose
Thanks you

[HW Mueller]

The peaks (knifes) with water appear without injection? The 0.005M acid was in the column at 80°? With the present information it´s hard to get any ideas about what could happen.
The initial "good" peaks don´t look so hot to me either (overload??), but I have to defer here as I am not familiar with this type of sugar chromatography.

[juaant]

Now readed well the specifications and you can wash the column type the form hydrogenate with 0.05 M with H2SO4 but my column is with lead and wash with water.

Do you think that I can regenerate it?

Other thing:
The peaks appear without injection.
The column was with 0,05 M with H2SO4 for a little time to 80ºC.
Why appear this peak?

I understand you. This chromatogram is very bad but is the best way for quality our process with this product.

thank you very much

[HW Mueller]

Someone else may know whether your column can take hot sulfuric.
The peaks you see without injections could be air. I have seen saw type baselines, not this time separation between peaks as in your chromatogram. If it´s really air than something must intermittantly introduce it into your system (an air bubble seems to built up in your cell, then get swept out, after that there is a rest, then it repeats.....anyway this does not appear to be created by the column, maybe modified by it....take the column out and see what you get).

[HW Mueller]

Forgot: Don´t you have some ref chromatograms to check whether your best is really the best possible? I doubt that, it seems that you are either overloading the column or it is shot?

[Mark Tracy]

These columns are a cross-linked styrene-divinylbenzene substrate that is fully sulfonated. The base resin can withstand dilute acid at 80° practically forever. The problem is that his column is charged with lead. Acid will remove the lead from the resin. You can recover from this sometimes by treating the column with lead nitrate. The real problem is that this column was exposed to sulfuric acid: lead sulfate is insoluble. I think that this column can't be saved, at least not for a reasonable amount of effort.

[Alfred88]

Dear:
I guess that you are using this system for the first time. Am I correct?

There are at least two problems here.
1. Bubble in the flow cell. Years ago, when we acquired an LC system with the RI detector, I observed the saw-tooth pattern when running just the mobile phase. We called tech support, and they did not have a clue what caused it. Now we know better about what to do: remove the column & guard, then flush the mobile phase thru the system at 2-3mL/min for 5 minutes. Also, you should degass the mobile phase (or you can bubble Helium thru the mp).

2. Peak tailing due to overload. You did not describe the dimension of the column. As HW Mueller suggested, you need to reduce the injection volume, by 50-75%, to achieve a better peak symmetry.

The column is made of a very tough polymer, so it should not be damaged with a diluted acid as you described.

Alfred.

[tom jupille]

Mark hit the nail on the head. You cannot use a lead-form column with sulfate in the mobile phase. The column is dead.

[Noser222]

If you still want to try to recover the column you could reverse the flow and flush with water at a high temperature for a while and then run 0.1M Lead Nitrate at 85 degrees, 0.2 mL/min.

[Bruce Hamilton]

Check out any information from your supplier about reconversion to the original form, but Tom's probably correct, the lead has precipitated and you're doomed .

However, if you want to try, I'd suggest using the procedure recommended by Biorad for their Aminex HPX-87P lead column, which appears to use a backflush at 60C for 16 hours of 0.1 ml/min flow of 30% acetonitrile with 0.1 molar lead nitrate that has been acidified to pH 4.0 with 0.1 molar nitric acid.

I'm not sure why that column has CH3CN in the regeneration solvent, and you might like to try first with just the lead nitrate at pH 4.0, and if that doesn't work try the CH3CN blend. You can get the Aminex column care guide from the BioRad site.

Good luck, and let us know the outcome.

Bruce Hamilton

[juaant]

Excuse me for me delayed.

We have had many problems in our equipe. We find the solution. The principal problem was to quit the noise in the detector. Clean of desgasser of vacuum, filter all the movil phase and look like the noise finished. Now there is something of noise but low intense.

The problem wasn´t the column. After run with the column was well.

Thank you very much for your attention

[JM]

well looking at your chromatograms it is clear that the peaks you are geting only in mobile phase running is due to air intake in the flow cell , this is a known problem with RI detectors. you can check by just raising the outlet tube above the detector or block your outlet tube with your finger, your spikes will go off.

Solution : use restrictor tube in the outlet of flow cell to creat some backpressure ( may be a coil of 1/2 meter HPLC steel tube).

I used ones a tube end cap ( comes with precut PEEK ) with a very fine hole and it worked.

your column is perhaps OK.

JM

[Alfred88]

Dear juaant:

I am glad that you still can use your Lead-based column!

Still there are 2 (possible) problems:

1. Peak tailing. You should reduce the injection volume, and/or reduce the conc of your 'multi standard' solution.
2. Peak split. You may have contamination of the guard and/or column. Reverse flush the column will help.

Have fun with your RID and good luck,

Alfred.