Waters CORTECS 2.7 um Columns - Manual (Page 2)

2 CORTECS 2.7 µm Columns
II. GET TING START ED
Each CORTECS 2.7 µm Column comes with a Certificate of
Analysis, which includes the bonded phase batch number and
the analytical test results for the unbonded and bonded particle.
The included Performance Test Chromatogram summarizes
the performance of each individual column and provides batch
number, column serial number, USP plate count, USP tailing
factor, retention factor, and chromatographic test conditions.
These data should be recorded and stored for future reference.
When available, the information can be accessed via the ACQUITY
UPLC Console using the attached eCord.
a. Column Connection
CORTECS 2.7 µm Columns are designed to operate on any HPLC,
UHPLC, or UPLC system. Due to the absence of an industry
standard, be aware that the type of fittings and connection on
each system will vary by manufacturer. All tubing connections
must be reseated to match the new column when it is installed.
If the style of the column endfitting does not properly match the
system connections, the chromatographic performance of your
new column will be negatively impacted, or leaking can occur.
b. Column Installation
Note. The flow rates given in the procedure below are described for
a 2.1 mm i.d. column. Adjust the flow rate up or down based on the
column pressure and system limits.
1. Purge the pumping system of any buffer-containing mobile
phases using 100 % HPLC-grade water.
2. Purge the pumping system to 100% organic mobile phase
(methanol or acetonitrile).
3. Connect the inlet of the column to the chromatographic system.
4. Flush the column with 100% organic mobile phase (methanol
or acetonitrile) by setting the pump flow rate to 0.1 mL/min.
Increase the pump flow rate to 0.5 mL/min over 5 minutes.
5. Once the mobile phase is flowing from the column outlet, stop
the flow.
6. Attach the column outlet to the detector. This prevents air
entering the detector flow cell.
7. Increase the flow rate as described in step 4.
8. Monitor until a steady backpressure and baseline have
been achieved.
c. Minimizing Band Spread Volume
Band spreading is the measurement of the system dispersion,
which impacts the chromatographic performance. Internal tubing
diameter and fluidic connections can significantly impact system
band spreading and chromatographic performance. Larger
tubing diameters cause excessive peak broadening and reduced
sensitivity (Figure 1).
Diluted/Distorted Sample Band
0.005 inches
0.020 inches
0.040 inches
Figure 1. Impact of Tubing Diameter on Band Spread.
d. Measuring Band Spread Volume
This test should be performed on a liquid chromatographic system
equipped with a UV detector.
1. Disconnect the column from the system and replace with a zero
dead volume union.
2. Set the flow rate on the pumping system to 1 mL/min.
3. Use a test mixture (dissolved in the mobile phase conditions)
that delivers a maximum peak height of less than 0.5 AU.
4. Inject 2–5 µL of this solution.
5. Using the 5-Sigma method, measure the peak width in minutes
at 4.4% of peak height:
System Volume
4.4% h
Figure 2. Determination of System Band Spread Volume using 5-Sigma method.
Band Spreading (µL) = Peak Width (min) x Flow Rate (µL/min)
(For example, If peak width = 0.1 min and flow rate = 1000 µL/min,
band spread = 100 µL)