Waters Protein-Pak SEC Columns - Manual (Page 2)

[ CARE AND USE MANUAL ]
Protein-Pak SEC Columns 2
II. PREPARATION FOR OPERATION
a. Steel Column Installation
Remove the end plugs from your colum with a 5/18” wrench and
save them for storage when the column is removed from the system.
The column outlet is indicated by an arrow on the label (showing the
direction solvent should flow). Tighten the fittings 1/4-to-1/2 turn.
DO NOT OVERTIGHTEN – THIS WILL DAMAGE THE FITTING SEAT.
A properly prepared and assembled compression fitting in good
conditions is all that is required.
Follow the next four steps of this procedure if tubing cutting is required
to connect a new steel column or to improve the end connections on your
existing fittings.
1. Using a file with a cutting edg, escribe the circumference of the tubing at
the desired break.
2. Grasp the tubing on both sides of the scribe mark with cloth covered pliers
(to prevent marring the tube surface), and gently work the tube back and
forth until it separates.
3. File the ends smooth and assemble as shown.
Note: Proper positioning of the fetrrule in the fitting seat will prevent
formation of unwanted dead volume which could result in sample mixing.
4. Slide the compression fitting, followed by the ferrule (large end of the
taper first) over the tube. Be certain to bottom the tube in the fitting seat
for which its use is intended to assure a leak-free connection.
Note: Attach a union in place of the column and flush the lines free of
microparticulates before attaching the column.
Figure 1: Ferrule and Compression Screw Assembly
b. Glass Column Installation
Waters Protein-Pak 200 or 300 SW glass columns can be connected to
any HPLC or medium-pressure system remembering not to exceed 300 psi
backpressure with the installed glass SEC columns. The following parts can
be used to adapt these columns to any of our standard Waters systems:
WAZ TO 1/4 UNF Adapter WAT089472
Compression Screw 1/8 o.d. 1/4 - 28 WAT088467
Reverse Ferrule 1/8 o.d. WAT088466
Tube Cut 0.125 x 0.009 x 120 WAT088431
c. Solvent Requirements
Protein-Pak 60 and 125 columns are shipped containing an organic
solvent compatible with water-miscible solvents (such as methanol,
acetonitrile, etc.) The Protein-Pak 200 and 300 SW columns are shipped
in water containing 0.05% sodium azide, added as a bacteriostat. If
an immiscible solvent is to be used for analysis, make the changeover
gradually using at least one itermediate solvent (similar to changing over
solvents during analysis). When changing the organic concentration in
the mobile phase to reduce the flow rate. When adding organic solvents
to aqueous buffer solutions avoid salt precipitation.
d. Buffers
Protein-Pak columns are stable in normal salt buffer solutions such as
sodium sulfate, ammonium acetate, ammonium formate, phosphate
buffers, tris acetate and acetate buffers. Ionic strength equivalent to 0.1 M
minimizes the ionic interaction between the protein sample and the silica
stationary phase. Generally the salt concentrations in aqueous solutions
should be maintained below 0.5 M.
e. Protein Denaturants
Aqueous solutions of SDS, guanidine hydrochloride and urea are
compatible with the Protein-Pak columns although there is a tendency
to display shorter column life when compared to non-detergent
aqeuous solutions. We recommend dedicating columns used with these
solutions rather than changing back to other aqueous solutions with the
same column.
Compression screw or nut
Tube
Ferrule
End must be straight
and smooth to achieve
maximum column efficiency Critical Distance to be determined by each
application (union, column fitting, etc.)