By Chuk Foo on Tuesday, February 18, 2003 - 05:21 pm:
I am developing a method using gradient elution with the following conditions:
Mobile phase A : Octane sulphonic Acid pH 3.0
Mobile phase B : Acetonitrile
Sample Extraction Solution : Phosphate Buffer pH 2.7
Conditions:
Time A% B%
0 mins 82 18
12mins 75 25
16mins 55 45
25mins 55 45
Detection Wavelength 210, 215, 227 and 245.
The baseline drifted upwards from ~16 to 20 mins., more significantly for wavelength 210 and 215 which cause integration problems for small peaks (degradation compounds) eluting at this time. Any suggestions as to how to overcome this problem.
By Jan on Tuesday, February 18, 2003 - 11:40 pm:
Hi Chuck,
Check the quality of your acetonitrile. It should be "far UV" or "super gradient". Look for acetonitrile with the lowest possible UV cutt-off.
Good luck
Jan
By Chris Pohl on Wednesday, February 19, 2003 - 11:39 am:
Chuk,
In addition to the possibility of problems associated with contamination of your acetonitrile (even in "UV grade" acetonitrile the absorbance of the solvent is 0.1 at 205 nm and 0.01 at 225 nm), there is another strong possibility that the problem is derived from contamination of your octane sulfonate. As far as I know, Dionex is the only company selling octane sulfonic acid per se. Most other companies selling this reagent actually just acidify sodium octane sulfonate. Depending on the quality of the sodium octane sulfonate, there may well be contaminants present in the source material or in the acid used to acidify the salt. I would suggest that you try purchasing your ion pair reagent from a different source. Preferably one selling the salt as a crystalline solid. That way you have control of what additives are used to acidify the ion pair reagent.
By Tom Mizukami on Wednesday, February 19, 2003 - 04:09 pm:
Chuk,
In addition to the suggestions above, you might want to try manually mixing the 18% and 45% ACN solutions and holding the concentration of ion pairing agent constant. This usually produces better baseline in gradient ion pair methods.
It is also quite possible that the "drift" you are seeing is due to the changing refractive index of your mobile phase as it approches 45% organic. This would be consistient with seeing more drift at shorter wavelengths. The effect of changes in the refractive index is highly dependent on the detector. You might want to try a different detector.
Lastly, if the shape of your baseline is consistent from run to run and depending on your environment (regulatory wise) baseline subtraction may flatten your basline and make it easier for your datasystem to identify the begining and end of impurity peaks. Good luck.