SEPARATION PROBLEM

[oalamu]

Hi everyone,
I am using RP-C18 column from Waters and analysing carotenoids.But I am experiencing problem in having consistent peaks i.e.If inject a sample there might be peaks ,the next injection of the same sample gave no peaks. Please note , i was operating uder the same conditions .
What could cause this?please advice.
Thanks.

[Rafael Chust]

Without more info on flow rate, sample concentration, detector, etc. is quite difficult to provide an input.

Anyway, it looks like low absorbance for the sample, caused by low concentration of analytes.

[oalamu]

Thanks Rafael,
Flow rate ;0.7ml/min ,conc injected 10microlitres,Detector used;Photodiodearray detector.
The absorbance used is that of carotenoids,450nm

[Rafael Chust]

Are you using standards or the sample itself.

I keep my original guess (low concentration), although you might also have high retention.

Try to use a stronger eluent (more organic solvent) and see if there are analytes retained in the column.

The problem might also resides at detector. Deuterium lamps are quite trick over 400 nm and sometimes there are also a Tungsten lamp for high nm range - if it is not "hot" enough you get low absorbance.

[Mark Tracy]

Have you checked your injector?

[Uwe Neue]

Do you see the peaks come and go? Does the retention of the peaks change? From what you have described ubtil now, I agree with Mark. Do you get peaks from different sample vials? What injector are you using?

[oalamu]

Thanks to all, am looking at the injector as well as checking if the analytes are retained on the column.Whatever that happens i will keep you posted.

[oalamu]

Hi,
I have checked the injector it was okay, and I decided to try a new column which yielded an appreciable result but I have a change in the retention time.And sometimes a drifted baseline.The problem is not yet over.

[unmgvar]

oalamu do you have a thermostated column oven?

what is the height of the peaks you are looking for?

could it be that the signal you are getting is too close to LOQ or LOD, hence the many "peaks"/?noise?, and drift?

can you say by how much AU you see the baseline drift?

what is the composition of your mobile phase?
it's possible that one of the component has a major influence on the retention of your peaks. do you adjust your mobile phase to a certain PH?

how do you inhect? manually or with an autosampler?
and if possible give us a chomatogram. that will save a lot of words

[oalamu]

Thanks,
Am not using thermostated column oven ,operating at ambient temperature.The peak height expected varies depends on the conc.of analyte in the sample.While ,am running standards that fall between the expected concentration in the sample.The drift is about minus 0.03AU and at a time it is positive , say .05AU.My mobile is ternary, ratio 80:10:10(ACN,MeOH,EthAC).No PH adjustment.
Finally,I used autosampler.

[oalamu]

Sorry, the drift is in three decimal places instead of two mentioned earlier on.That is.003AU and minus..005AU.

[Mattias]

We have seen this behaviour on our Waters Alliance systems. No peak in one chromatogram and two in the next..

It is usually the check valves that don't work properly. You can get the wrong flow, or the wrong composition. Especially acetonitrile seems to be a problem, we have to sonicate our check valves every month...

[oalamu]

I agree, this may be the source of our problem,because we have sonicated the check valve once.To keep you posted as it goes.Any other suggestions are highly welcome.
Thanks to all.