Molecular Techniques and Methods

Degassing Water, Buffers, and Solvent

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Dissolved air is present in water, aqueous buffers, and solvents. This air will "out gas" during a gradient, leading to bubble formation which may accumulate in the flow cell. Air bubbles in the flow cell can be eliminated with a backpressure regulator set at 20 to 60 psi. Air bubbles can also be a problem for check valves.




MATERIALS

Trapped Vacuum Pump or Lyophilizer

4-liter Erlenmeyer Filtration Flasks

Rubber Stoppers (sizes 12 and 13)

Rubber Hose to connect flask to trapped vacuum pump

Borosilicate Glass Filter Holder, 47-mm dia. with removable stainless-steel support screen

Nylon-66 Membrane Filters, 0.45-um pore size, 47-mm dia.




PROCEDURES

1. Water (< 2 liters) can be degassed in a rubber-stoppered, 4-liter Erlenmeyer filtration flask by evacuation with a trapped vacuum pump. After the bubbling ceases, the water is degassed. Volatile buffers should be added after degassing. Any solution containing nonvolatile buffer salts should be filtered through a 0.45-um nylon filter into the flask and then degassed. Filter buffer concentrate (1 M solution) and then add to degassed water. Solvents should be degassed only briefly to avoid collecting solvent in vacuum pump.

2. Alternatively, the buffers can be sparged with helium (He) gas to displace air gasses.

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