Chromatography Forum

I have had some unusual problems associated with a gradient qualification that I was attempting to run on a Dionex U3000. The method I was attempting to run was laid out in a LC/GC supplement entitled "The Application Notebook." The method is described in the section "Performance Qualification of HPLC instrumentation in Regulated Laboratories" and appears on page 70 of the supplement, there is also a link to the article below.

The article suggests injecting a uracil spiked sample and running one of the mobile phases (in my case B) with a uracil spike. A gradient from 0 to 100% B is then started over ten minutes and held at 100% B for 5 minutes before going back to 10% B at 15 minutes and holding for 3 minutes. A set of plateaus with 10 to 90% B is then set up with intervals of 3 minutes. The article uses a column, but I used a restrictor coil in its place, I monitored at 273nm. I had water for mobile phases A and C and I had B as a solution of 0.035mg/mL Uracil in Acetonitrile. The article had a sample chromatogram, which I also have attached below.

My concern is the fact that my chromatogram (also attached below) has a few features that give me cause for concern. The first is the drop-off in absorbance once the % B reaches 100. There is then a spike in absorbance at 15 minutes when the %B changes from 100 to 10%, and similar but smaller spikes with the %B changing from 90 to 10%. I would welcome any suggestions on why the absorbances are behaving this way.

http://chromatographyonline.findpharma. ... &sk=&date=

Sample Chromatograph of the Gradient Qualification



My Chromatograph of the Gradient Qualification

can you be more specific regarding the exact modules type that you have.

was this your first gradient run, or do you have several more trials?

have you tried doing the test with a column as well?

The chromatogram I attached was the third run on the instrument.

What I believe is happening is the uracil is not as soluble in acetonitrile as I thought it would be. When I prepared the mobile phase solution I added 35mg of uracil to 1L of acetonitirle and had to sonicate the solution for a few minutes in order to dissolve everything, and even then I thought I could see one or two specks of white material floating around. If solubility was the problem this would explain why when 100% of the mobile phase of ACN w/ uracil was used that the absorbance would decrease, and then spike when the mobile phase was switched to water, in which the uracil is more soluble in. When I switched the mobile phase to uracil in water everything turned out perfectly.

jbw