Waters XSelect HSS XP 2.5 µm Columns - Manual (Page 2)

XSelect HSS XP 2.5 µm Columns 2
[ CARE AND USE MANUAL ]
II. GET TING STARTED
Each XSelect HSS XP 2.5 µm Column comes with a Certificate of
Analysis and a Performance Test Chromatogram embedded within
the eCord intelligent chip. The Certificate of Analysis is specific to
each batch of packing material contained in the XSelect HSS XP 2.5 µm
Column and includes the gel batch number, analysis of unbonded
particles, analysis of bonded particles and chromatographic results
and conditions. The Performance Test Chromatogram is specific to
each individual column and contains such information as: gel 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 or can be accessed
via the ACQUITY UPLC console.
XP 2.5 µm Columns are designed to operate on any HPLC, UHPLC or
UPLC System. Due to the absence of an industry standard, please be
aware that the type of fittings and connections on each system will
vary by manufacturer and should be mated specifically to a column
when it is installed.
The chromatographic performance can be negatively impacted, or leak-
ing can occur, if the style of the column endfitting does not properly
match that of the compression screw/ferrule tubing depth setting.
b. Column Installation
Note: The flow rates given in the procedure below are described for a 2.1 mm ID
column. Scale the flow rate according to the flow rate and pressure guidelines
described in Section VI (Additional Information).
1. Purge the pumping system of any buffer-containing mobile
phases and connect the inlet of the column.
2. Flush the column with 100% organic mobile phase (methanol or
acetonitrile) by setting the pump flow rate to 0.1 mL/min and
increase the flow rate to 0.5 mL/min over 5 minutes.
3. When the mobile phase is flowing freely from the column outlet,
stop the flow and attach the column outlet to the detector. This
prevents air entering the detection system and provides a more
rapid baseline equilibration.
4. Gradually increase the flow rate as described in step 2.
5. Monitor until a steady backpressure and baseline have
been achieved.
c. Minimizing Band Spread Volume
Band Spreading is a measurement of the system dispersion that
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
Note: This test should be performed on an LC 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 to 1 mL/min.
3. Use a test mixture (dissolved in the mobile-phase conditions)
that delivers a maximum peak height of 0.5 – 1.0 AU (System
Start Up Test Mixture can be used, Part No. WAT034544).
4. Inject 2 – 5 µL of this solution.
5. Using the 5-Sigma method, measure the peak width at 4.4% of
peak height:
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)