Chromatography Forum

I am using a C-18 column with a mobile phase of 50m M phosphate buffer, pH7.4 and ACN. the gradient employed is:
1-3 min 95:5
3-4 min 80:20
15-16 min 95:5
20 min 95:5


The compound is an oligonucleotide and the peak elutes at 13 min. however, when the drug is eluting the baseline starts to drift and comes back after 30 min.

Any suggestions?

A couple of additional questions:

1. What wavelength?
2. What sensivity (range) on the detector?
3. Is the drift linear? curved?random? increasing? decreasing?
4. Do you see the same drift when you run a blank and when you run a "dummy" gradient (no injection at all)?
5. How bad is the drift (any chance you can post a chromatogram)?

Are you sure you gave us the correct information? If I understand you correctly, you are running under isocratic conditions between 4 minutes and 15 minutes. This is usually a problem for large-molecular weight compounds. Your peak elutes at 13 minutes, and the drift after the peak could be still your compound (actually in which direction is the drift going?).

The way I understand your gradient conditions is that from 4 minutes you start going back to the initial conditions which does not make a lot of sense and in this case you might get the kind of problems that you describe.

As Uwe mentioned, can you be more clear on your gradient conditions?

Here are the details of the HPLC conditions for the oligonucleotide on C-18

A: phosphate buffer, B: ACN

0-3 mins = 95:5 A:B
3-5 min = 80:20 A:B
5-18 min= 80:20 A:B
18-21 min= 95:5 A:B
21-26 min= 95:5 A:B

The range is from 1 mcg/ml to 50 mcg/ml. Wavelength of detection is 254 nm.

When I injected a blank I did see the drift in the baseline which was after 14 mins , it increased for 2 mins and then was constant. It returned to baseline after 26 mins. Once the baseline drifts upward it stays there (straight line, linear) and then comes back to the original after 26 mins. ( I am using a HP printer for the chromatograph, will scan the chromatograph and post it asap)

Thanks

Is the base line drift the same when you inject your sample or during your blanck injection? If yes then the drift is due to absorbance difference between your two mobile phases (although you shouldn't observe such differences if your wavelenght is 254 nm).

Some suggestions:

1) The drift might be due to contaminated ACN. If you have any doubts on the ACN quality I would reprapare the mobile phases with brand new acetonitrile and repeat the experiment.

2) If you still see the drift, I would try to make the mobile phases as similar as possible by changing the mobile phase B to 50:50 water:acetonitrile and add 50 mM of phosphate buffer (check for insolubility issues). I would then change the gradient from 10%B to about 40-50% B. Having said that, I would expect you to have a negative and not positive drift if there were contaminations in your phosphate buffer.

3) If you still see the drift and you can not live with it, I would try to change my mobile phase composition all together...

The other possibility is refractive index change. That tends to be more pronounced at longer wavelength (e.g., worse at 254 nm than at 210 nm). Often the baseline takes on a pronounced "hump", because RI is a non-linear function of composition.

sonal, seeing the baseline will help a lot.

http://tinypic.com/view.php?pic=6ewrzmq

Here is the link for the baseline. The baseline starts to rise at 13 mins. and keeps rising for 2 mins with a small hump at 16 mins and stays constant till 26 mins. After this it starts to come down to the original baseline.

Thanks

Probably RI. The duration of the baseline shift roughly matches the duration of your "gradient" (actually closer to a step change).

What was the flow rate? What column dimensions? and what is the dwell volume (gradient delay volume) of your system?