Chromatography Forum

I run Glucosamine testing by USP method. The following information is my method.
Column: Phenomonex NH2 4.6x150mm, 5um P/N 00F-4378-E0
Mobile phase: Phosphate Buffer at pH 7.5 with Acetonitrile mixture(250/750 mL).
Wavelength: 195 nm.
My problem is the baseline drift. Normally, it takes me to do a few sample injections to achieve a good baseline. I don't understand why Buffer solution causes the baseline drift. I run a different UV method which use Phosphate Buffer to test Thiamine with C18 column at 254 nm. I have no baseline drift. Can anyone explain why the baseline-drifting only happens in some circumstance? Is there any good paper about baseline-drifting? Thanks

What you are seeing is no surprise to anyone who has ever run chromatograms at low-wavelength UV. In general, a conjugated double-bond system of some type is required for absorbance at "mid" wavelengths like 254 nm. Anything that has a double bond (e.g., a carbonyl group, or even O2) will have at least some absorbance at 200 nm and below.

If I had to guess, I would suspect that the dissolved air level in your mobile phase is changing until everything equilibrates.

Dear Xiaiopang2

I have recenly worked with this API, and the stabilization time was very long! I think two hours is a good waiting time.

I sligthly modified the USP method: These are within USP allowances.

Injection: 50 µl of an 50 mg of Glucosamine SO4.2KCl dissolved in 5 ml of water, then to 50 ml in mobile phase.

Mobile Phase: (30:70) Buffer-MeCN

All the rest, the same. My LC is a 20 year old HP1100, I used reference wavelength. After a good stabilization the RSD was 0.76 % for standard and 0.38 % for samples.

Good luick!

Fernando

Hi Xiaopang2

I forgot to mention that as always Tom is correct! DEGAS thoroughly you moble phase.

Good luck!

Fernando

If you want to consider a different method, you can look for methods employing derivatisation of glucosamine using Fmoc. We use this for routine analysis of glucosamine on supplements, and it circumvents the issues that Tom described; detection at ~265nm

ELSD/CAD/MS/RI might be a better choice. Any very UV active impurity will affect your chromatogram.