Swartz:Protocols/HPLC
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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