Chromatography Forum

I had good peaks before and then doubling of peaks occurred in my chromatograms. I washed the column and now only solvent peak elutes while no active compound elutes. I am using Agilent 1200 series hplc. Using same method only length of heat exchange capillary is changed.

Hello

More details please...
-compound details
-solvents type
-eluents
-column
-method parameters
-chromatogram

Regards

Tomasz Kubowicz

Compound is Dexamethasone and Prednisolone
solvent methanol,
mobile phase H2O and ACN ( Gradient)
Column C-18
Temp 30C , Time 8 mins,
I m trying to post chromatogram but couldnt get it done

Hello

Possible problems:
-dead volume (mixing chamber) can cause double peak. Check all connections
-sample solvent (100%methanol) is stronger than mobile phase. It can cause double peak
-you mentioned that you have no peak after column cleaning. Wash your column with methanol and with your initial mobile phase.

Regards

Tomasz Kubowicz

I had good peaks before and then doubling of peaks occurred in my chromatograms. I washed the column and now only solvent peak elutes while no active compound elutes. I am using Agilent 1200 series hplc. Using same method only length of heat exchange capillary is changed.

What do you mean by "doubling of peaks" ?
If you meant what I mean then you have voids in the column and you may say bye-bye to it.

Back flush your column with ACN, sounds like you have a blocked frit

Thank you all. Keeping in view your suggestion I have back flushed the column. But still only a small peak at 0.5 sec elutes ( I suppose it is solvent peak). No other signal appears and the baseline is almost flat. How can I know if the column needs to be replaced.

Along with all columns comes a certificate. There you will find a sample chromatogram with a standard mix. Use the compounds of this mix and run them under the same conditions, then you should get the same chromatogram. Of course not the SAME chromatogram, but you will see if your plate count is close to the one stated, and your retention times are similar.
It is a good idea to create an own test mixture you run from time to time to see if your column performance varies. Of course this only helps if this test mix is run immediately after purchasing the column.

Thankyou

The peak for solvent is smaller but the retention time is same. Only that no other compound is being detected.

But still only a small peak at 0.5 sec elutes ( I suppose it is solvent peak).

At 0.5 sec it most certainly is *not* a peak at all but rather the injection mark. You did not specify the column volume or the flow rate, but you can estimate the t0 (the time for the "solvent" peak) at
0.5 * L * dc^2 / F , where L is the column length, dc is the column internal diameter, and F is the flow rate.

If this were my problem I would do the following until I found the source of the difficulty:
1. Confirm that the flow rate is accurate (run the outlet into a 10 mL graduate cylinder and time how long it takes to fill)
2. Confirm that the detector is functioning (an Agilent 1200 should have a self-test function) and set at the correct wavelength
3. Confirm that the autosampler is functioning (observe the tray and syringe; listen for stepper motor operation; measure volume in a vial before and after injecting a reasonably large volume.
4. Confirm that the gradient system is functioning properly by running the gradient step-test (http://www.lcresources.com/resources/TSWiz/hs450.htm)
5. Prepare a fresh batch of mobile phase
6. Test with a new column

I ll surely check for all the steps today

The flow rate is faulty .When i set it to 1ml/min, the volume collected is only about 0.6 ml/min. Pump A is giving accurate flow but pump B is not

Pure ACN on pump B?

Thank you very much for your guidance. I sonicated the filter first with H2O and then with methanol and viola!!! problem is solved.