Waters Advanced Purification Glass Columns - Manual (Page 8)

[ Care and Use ManUal ]
Waters Advanced Purification Columns 8
Replacing an inlet connector O-ring
To replace the inlet connector O-ring:
1. Deactivate the inlet O-ring by turning the seal adjustment knob.
Remove the inlet connector assembly from the column by unscrewing
the plunger.
2. Use a small blunt tool, such as a push pin, to gently pry up the O-ring
so that you may roll it off the connector with your fingers. Carefully
push a new O-ring onto the connector tip. Be sure to select the proper
inlet connector O-ring.
Do not scratch the O-ring groove. If the groove is scratched, the column
will leak.
3. Return the inlet connector assembly to the column. Reactivate the
O-ring.
Replacing a filter O-ring
This small O-ring is located at the edge of the filter skirt. With the filter off,
roll the O-ring off the connector tip and replace it with a new one.
f. Storing the Column
Storage considerations
Store a packed glass column according to the recommendations of the
manufacturer of the packing material. Note the following:
•
Store the column with end plugs in place to prevent column drying.
• Use bacteriostatic solutions in appropriate concentrations, for example,
0.2% sodium azide or 30% ethanol.
• Remove all salts from the column before adding an organic solvent.
An organic solvent added to a salt can cause precipitation, resulting in
increased backpressure.
• Extended storage in high salt buffers may result in the formation of
microcrystals which can lead to high backpressure. (Dissolve microcrys-
tals by slowly introducing a low ionic strength buffer).
• Do not allow the mobile phase to freeze. Storage at 4 °C is acceptable.
IV. sCalIng up separatIons
a. Scaling a Separation
When scaling an analytical separation to a preparative separation, use the
following isolation approach:
1. Define the objective by determining the:
• Complexity of the sample mixture
• Component to be isolated
• Required quantity
• Degree of purity
• Properties of the components in the mixture
2. Perform crude separations to remove large quantities of extraneous
material or to isolate groups or classes of compounds. Simplify a com-
plex sample through coarse separation techniques, such as liquid-liquid
or solid-liquid extraction, crystallization, or precipitation. Centrifuge or
filter the sample to remove particulates.
3. Develop the separation method. To optimize the separation for com-
pounds of interest, make small-scale injections of the sample mixture,
varying the gradient.
4. Perform a loading study. Make progressively larger injections of the
sample mixture on a small scale column, such as the AP-Minicolumn,
to determine the effect of overload on resolution. This study defines the
amount of sample that can be separated in a column of a given volume.
5. Scale up the separation. Translate the small scale parameters to the
large scale system values. Adjust the:
• Sample load
• Flow rate
• Gradient volume
b. Adjusting Sample Load and Flow Rate
When scaling the separation, use analytical and preparative
columns that are:
•
The same length
• Packed with the same particle size material