Chromatography Forum

Hello,

I am simply trying to analyze samples for Triton X, but am having a heck of a time just getting my standards to work. Currently, my method is A: H20, B: ACN, 0-100 B% 30 min, 100% B 15 min, 100% A 5 min, 100% A 15 min. 1.5 mL min. Waters 2996 PDA dectector, 4.6x250 C18 5 micron column. For some reason, I see a broad peak in my standards that would seem to correspond to triton X (looks like references), however, the Uv-vis trace does not look right, and the peak has a maximum absorbance at ~215 nm. Triton is supposed to have a maxmimum absorbance near ~280 nm. I took the same sample and analyzed it on a nanodrop, and it definitely has a maximum absorbance at 280. I can not think of any reason why triton doesn't seem to be coming out of the column or why, if this peak I'm seem is indeed triton, has a lower absorbance maximum than what is published in literature and by the manufacturer. Any advice is much appreciated. Thanks!

Access the Waters manual (it is available online) and run diagnostics and wavelength calibration. It may be possible that the calibration is offset because 215 nm and 280 nm have huge difference.

Could it be that 280nm is only a "practical" absorption maximum which is usually used for analytical work while Triton has a global absorption maximum at 215nm? With quick googling I found the following paper which shows an UV-VIS spectrum of Triton X-100 (figure S-4, page S12):
http://www.rsc.org/suppdata/ay/c1/c1ay0 ... 05495c.pdf

It HAS a maximum at ~275nm, but the global maximum is clearly in the low-UV range. To be sure, do you have access to a UV-spectrophotometer to record a spectrum of your Triton reference? If not, you may directly inject the diluted reference in the PDA.

Ups, just saw the Nanodrop IS a spectrophotomerter. Never came across it before

for checking the wavelength accuracy, inject some caffein, which has a maximum at 271-273nm