So...I'm trying to get this old FPLC working in this lab where no one knows how to use it. The graduate student who knew how to use it got a job at a pharmaceutical company in another prefecture.

I'm running standards on a Superdex 200 column and the Dextran Blue is causing some strange things to occur. Here are some details...

Sample: Dextran Blue 1 mg/ml
Volume: 230 ul

Running Buffer:
50 mM Tris
200 mM NaCl
10% Glycerol
(pH to 7.2 and De-gas)
0.1% NP-40)

Flow Rate: 0.3 ml/min
Fractions: 1 fraction/2 min (600 ul/fraction)
UV set at 230 nm

Column cleaned with 2 column volumes of 0.1 M HCl followed by 2 column volumes of 0.5 M NaOH. 1 column volume of DIH2O was ran before switching to running buffer.

So Dextran Blue came off at around fraction 13 which was fine but then when the peak started dropping (around 3 fractions later) it dropped way below baseline (buffer only) absorbance. Following the steep drop, the absorbance shot back up and then down again nearing baseline. Unfortunately, the chromatogram is generated on paper and therefore when the drop in absorbance went off the chart I cant tell how far off from baseline the absorbance returned. I also checked the absorbance of each fraction in a seperate spectrophotometer and it matched what was seen on the chromatogram. However, the absorbance never made it back down to what the buffer is.

Please help...