Waters Protein-Pak HR Ion-Exchange Glass Columns - Manual (Page 6)

[ Care and Use ManUal ]
Protein-Pak HR Ion-Exchanged Glass Columns 6
strength solutions may give irreproducible or ambiguous results. In
such cases, desalt or dilute the sample to approximately 50 mM prior
to injection.
c. Equilibrating the Column
Note: Pre-packed columns are shipped in a 50% aqueous ethanol
solution and must be flushed out with at least 10 column volumes
of water prior to equilibration with buffer.
Once the shipping solution is removed, gradually increase the flow
until the desired rate is reached.
Note: To avoid compression of the packed bed, the differential
pressure between column inlet and outlet should not exceed
3.5 MPa (500 psi or 35 atm).
While equilibrating the column, check for voids. Although small
voids in large-diameter columns should have minimal effect on your
results, large voids in small-diameter columns can be detrimental.
Use the following steps to eliminate voids if present in pre-packed
AP-1, AP-2, or AP-5 columns. Refer, to the Waters Advanced
Purification Glass Column Care and Use Manual for additional
information.
1. Reduce the flow to 0 mL/min.
2. Deactivate the O-ring seal.
3. Disconnect the inlet column tubing from the system to allow displace-
ment of fluid while adjusting the plunger.
4. Use the fine adjustment knob to eliminate the void.
Note: To eliminate the voids present in AP Minicolumns, use the following
steps. Refer to the Advanced Purification Minicolumn Care and Use Manual
for additional information.
1. Reduce the flow to 0 mL/min.
2. Disconnect the inlet column tubing from the system to allow
displacement of fluid while adjusting the plunger.
3. Use the inlet connector adjustment to eliminate to void.
Note: Equilibrate the column with 4 column volumes of elution
buffer followed by 10 column volumes of initial mobile phase before
performing a test run.
d. Column Efficiency
Columns are thoroughly tested before shipment for adherence to
Waters specifications. Slight variations in your results will occur
depending on the equipment used, test sample makeup, and
equipment settings and condition.
Although efficiency is not a dominant factor in ion-exchange
separations, it is useful in monitoring uniformity of the packed bed.
Measure the efficiency of the Protein-Pak HR ion exchange columns
by using the Half Height Peak Method. Plate count, as an expression
of efficiency, is determined as shown in Figure 1.
Figure 1: Half-Height Peak Method Test Calculations
Injec
t
n
2 w
N= 5.54
1/2 wh
= Column efficiency (theoretical plates)
= Elution distance from injection to peak apex on chart paper
= Void volume
= Height of peak measured from baseline to apex
= Peak width measured at half height N
n
w