Waters Oligonucleotide Separation Technology XBridge OST C18 Columns - Manual (Page 6)

[ CARE AND USE MANUAL ]
6
III. COLUMN CLEANING, REGENERATING AND STORAGE
a. Cleaning and Regeneration
Changes in peak shape, peak splitting, shoulders on the peak, shifts
in retention, change in resolution or increasing backpressure may
indicate contamination of the column. Flushing with approximately
20 column volumes of filtered, neat organic solvent (e.g., acetonitrile
with the T EAA method of methanol with the T EA/HFIP protocol)
is usually sufficient to remove the contaminant. If the neat organic
solvent flushing procedure does not solve the problem, purge the
column with 20 column volumes of OST Mobile Phase A followed
by 20 column volumes of either 7M guanidine hydrochloride or 7M
urea. Be sure to flush column with an additional 20 column volumes
of filtered, LC Grade Waters prior to reuse OST Mobile Phases. If the
column performance is poor after regenerating and cleaning, call your
local Waters office for additional support.
b. Storage
For periods longer than four days at room temperature, store the
column in 100% acetonitrile. Immediately after use at elevated
temperature and/or pH, store column in 100% acetonitrile for
the best column lifetime. Do not store column in highly aqueous
(<20% organic) mobile phase, as this may promote bacterial growth.
Completely seal column to avoid evaporation and drying out of the
packed bed.
VI. ADDITIONAL INFORMATION
a. Band Spreading Minimization
Detritylated synthetic oligonucleotide separations are almost
exclusively performed using gradient elution techniques. As such,
the effect of pre-column sample band broadening can be minimized
by allowing the sample to bind to the column before beginning the
actual separation gradient. However, proper connection from the
XBridge OST C
18
column outlet to the detector is critical in
order to minimize the deleterious effect of post-column sample
band spreading. Use of appropriate internal diameter tubing
(e.g., 0.005 inch PEEK
™ tubing for UV detector applications) is
recommended for reasons illustrated in Figure 7.
Figure 7: Impact of column connecting tubing on band spreading
b. Measuring System Bandspreading Volume and System Variance
1. Disconnect column from system and replace with a zero dead
volume union.
2. Set flow rate to 0.5 mL/min.
3. Dilute a test mix in mobile phase to give a detector sensitivity
of 0.5 - 1.0 AUFS (system start up test mix can be used which
contains uracil, ethyl and propyl parabens; Waters part number
WAT034544).
4. Inject 2 to 5 µL of this solution.
5. Measure the peak width at 4.4% of peak height (5-sigma method):
5-sigma Bandspreading (µL) = Peak Width (min) x Flow Rate
(mL/min) x (1000 µL/1 mL)
System Variance (µL2) = (5-sigma bandspreading)2 / 25
Figure 8: Determination of System Bandspreading Volume Using
5-Sigma Method
Diluted/Distorted Sample Band
0.005 inches
0.020 inches
0.040 inches
System Volume
4.4 %h
5