Column Chromatography Protocol
Prestegard Lab, 11/20/03 aik
Equipment:
Peristaltic pump
UV detector
Chart recorder
Column
Tubing (see cabinet above chromatography set up) and flow adaptors
Flask (buffer) – Peristaltic Pump (inlet, then outlet) – column inlet/outlet – UV detector
inlet/outlet – waste collection flask
1. To set up pump: run tubing through inlet/outlet wheel, check to make sure liquid
flows smoothly. Salt deposits often build up in tubing and may need to be
massaged out before liquid will flow.
2. Connect outlet of pump (or column when column setup) to inlet of UV detector.
Change reference solution to buffer solution or water and screw back in place
carefully so you don’t lose liquid or create air bubbles.
3. UV detector electronic connections: optical cable (several pins) between lamp
(optical unit) and flow cell. Also connect power cables: red to red, black to black,
green to green (ground). Keep setting at 0.2 Absorbance. Check to make sure
correct wavelength filter is in place (254nm for nucleotides, 280nm for proteins).
Turn switch on (flow cell unit) and make sure blue UV lamp in optical unit turns
on. (faint blue)
4. Chart recorder: Use 50-100mV (Pres 50 or 100) and use zero line dial to adjust to
a zero baseline. 1-2mm/min setting is OK (Press 1 or 2 and press min). Make
sure nothing else is pressed!
Troubleshooting:
1. Is the pump working? Check for salt blocks in the tubing, leaky tubing.
2. Are the tubes and fittings OK, tight?
3. Is the column well packed, no air bubbles?
4. Is there too much head space above the column for sample to get diluted before
adhering to column?
5. Is buffer flowing through column? Is the tap open? Is the pressure on the column
acceptable (for the resin, the sample, for the desired flow rate)?
6. Is buffer getting to the UV cell? Is the lamp on?
7. Is there solution in the reference tubing?
8. Is the detector sensing ANYTHING? Do you see noise? Does the chart recorder
respond when the lamp is on/off, cables plugged/unplugged? Are the detector
settings OK?