Missing a peak in Gel Filtration Standard (SEC HPLC)?

Question

Hi Everyone. I ran GFS on Agilent 1100 HPLC, and the profile is atypical. It seems to be missing a peak and there is also a negative peak. Does anyone know what might be going on? 
 Mobile phase: 25 mM Ammonium Acetate, pH 8 (not filtered) 
 Flow rate: 0.3 mL/min 
 Wavelength: 280 nm 
 Column: AdvanceBio SEC 130&Aring;, 7.8 x 300 mm, 2.7 &micro;m (brand new) 
 GFS preparation: Reconstituted 1 vial of Bio-Rad GFS with 1 mL of HPLC Grade water, centrifuged, and injected supernatant at 5 uL.

Andy Wright · Answer

Hi A_chroma 
 The problem is with the reference wavelength. It would suggest that the third component absorbs more in the reference region (310 - 410nm) than it does in the sample region (272 - 288 nm). Try without the reference and see what you get, or try with a narrower reference bandwidth. 
 /Andy