Chromatography Forum

I am running a purity assay on a number of fractions from SMB. I am failing spec for an unknown impurity at 3.5 minutes. (%AUC =0.1%) I have eliminated the sources from the diluent and the system. It dos not appear to be a solvent. How else can I troubleshoot the identity of this unknown impurity? (No access to LCMS)
I can include chromatogram if someone will tell me how to attach it.

thanks.

Diluent: Milli Q water
Column: SymmetryShield RP18, 4.6 X 150mm, 3.5m
HPLC: 1100 w/ UV

Chromatographic Conditions:

Flow rate: 0.75 ml/min
Detection wavelength: 242 nm, BW 4 nm, Ref off.
Autosampler: 4oC
Injection volume: 5 uL (adjustable for column fractions)
Column Temperature: 45oC
Run Time: 25 minutes
Post Time: 7 minutes

Method Gradient:
Time (min) %A %B
0 70 30
5 63 37
13 63 37
22 15 85
25 15 85

What are your solvents?

If you want to post a chromatogram, make an image and use a photo upload site like flickr, and then use the Img button to display it in your post.