Chromatography Forum

I have been working on a heterogenous mixture of a compound on an C18 column. Usually it give 6 distinct peaks. But now it is giving none. All I get is baseline noise.
What could be happening? Is the compound accumulating in the column?
I tried revese flusing to the column with methanol, IPA, ACN, acetone at a flow rate of 1ml/min, but to no avail.

Check the detector sættings i.e. wavelegth.
Potentially run the calibration procedure (usually by powering down and the up the detector)
If the above OK, remove/short-cut the column and perform an injection of your sample solution or better yet the standard. Do you see a huge front peak?

Best Regards

Inject compounds without column and measure peak area. This will give you indication of concentration of the sample is suitable and if you detector and injector are working.

I have already tried these trouble shooting techniques.
Can the compound be accumulating? Because immediately after running the compound I injected 3ppm pyrene and it did not give me a peak like it had when injected prior to the compund, at 333nm. In fact, the peak disappeared and the peak obtained at 254nm for pyrene was not acceptable.
Any other suggestions?
Is it possible to revive a column by removal of clogging factors, if so, how? Can anyone forward any references, where instead of replacing the column, it has been revived?

I have already tried these trouble shooting techniques.
Can the compound be accumulating? Because immediately after running the compound I injected 3ppm pyrene and it did not give me a peak like it had when injected prior to the compund, at 333nm. In fact, the peak disappeared and the peak obtained at 254nm for pyrene was not acceptable.
Any other suggestions?
Is it possible to revive a column by removal of clogging factors, if so, how? Can anyone forward any references, where instead of replacing the column, it has been revived?

Is there any marked back pressure difference now and before when you used to get all the peaks? If the pressure is on the higher side then the column maybe clogged. You can try regenerating the column by flushing the column from Polar to non polar solvents like flushing with water followed by Methanol then Isopropanol and finally with Dichloromethane and then follow the sequence in reverse order till water.
With each solvent flush the column for about 10 mins at 1.0 ml/min.
Good Luck !

SChautray, You keep talking about the column, but exactly which “trouble shooting techniques” did you try?
Did you run an injection without a column and if so, what did you observe?
If you didn’t see a peak, then it’s the detector you are having trouble with.

Best Regards

This will go much quicker if you provide details of the conditions and what compounds are involved, and what troubleshooting you have done so far.

Are you sure that you have flow through the detector ?

Peter

If you didn’t see a peak, then it’s the detector you are having trouble with.

Best Regards

It can be injector as well.