Swartz:Protocols/HPLC

https://docs.google.com/document/d/1u-pF8-jbFt8zLzd3oW01XVPn4mkQ400ldB_muA2qObs/edit

HPLC guide

The HPLC on the right uses the older Windows interface; left uses newer Windows

.M files are methods; .S files are sequences

Use the tan filter in front of the column; the Discovery columns don’t require an actual guard column

Set up method by clicking on: method:edit entire method

-be sure end point times are in agreement

Pump:

Be sure buffer and speed are correct (VLP buffer is 10mM Tris pH 7.4 100mM NaCl and typical speed is 0.2ml/min

Set max pressure to 100bar; should run at around 20bar

Detector:

Remove reference index (RID)

-click on instrument: configure: RID: remove

-restart program

Use DAD (diode array) with UV measurement (not visible) at 210 and 280nm

-to collect samples manually, remove inlet to RID

Injection samples:

Be sure to leave 10ul or so extra in small sample adaptors

Needle wash before injection

Sequence:

Sequence Table: setup a wash step at the start and end of the run (and every 3 or 4 samples in between)

-should be a different location (91 and 92) than vial for washing the needle

Sequence parameters: put on standby after running

-can leave overnight; turn off otherwise

-be sure to change either sequence name or counter starting position if rerun same samples to prevent overwriting