Waters XSelect CSH XP 2.5 µm Columns User Manual
[ CARE AND USE MANUAL ]
XSELECT CSH XP 2.5 µm COLUMNS
CONTENTS
I. INTRODUCTION
II. GETTING STARTED
a. Column Connection
b. Column Installation
c. Minimizing Band Spread Volume
d. Measuring Band Spread Volume
e. Measuring System Dwell Volume
f. Column Equilibration
g. eCord Installation
h. Initial Column Efficiency Determination
i. VanGuard Pre-Columns
III. COLUMN USE
a. Sample Preparation
b. pH Range
c. Solvents
d. Pressure
e. Temperature
I. INTRODUCTION
Thank you for choosing a Waters XSelect™ Charged Surface Hybrid
[CSH] eXtended Performance [XP] 2.5 µm Column. The manufacture
of XSelect CSH XP 2.5 µm Columns begins with ultrapure reagents
and are manufactured in a cGMP, ISO 9001 certified facility to
control the chemical composition and purity of the final product.
Well-controlled manufacturing processes result in industry-leading
batch-to-batch reproducibility. Every column is individually tested.
A Performance Chromatogram and Certificate of Batch Analysis are
provided on the eCord™ Intelligent Chip.
XSelect CSH XP 2.5 µm Columns are based on the same base particle
technology and bonded-phase chemistry as 1.7 µm ACQUITY UPLC®
CSH Columns as well as XSelect CSH 3.5 and 5 µm HPLC Columns,
thus enabling seamless transferability between HPLC, UHPLC and
UPLC® platforms.
XSelect CSH XP 2.5 µm Columns will exhibit maximum chromatographic
performance when used on a member of the ACQUITY UPLC
System family.
IV. COLUMN CLEANING, REGENERATION AND STORAGE
a. Cleaning and Regeneration
b. Storage after Reversed-Phase Use
V. eCORD INTELLIGENT CHIP TECHNOLOGY
a. Introduction
b. Installation
c. Manufacturing
d. Column Use
VI. ADDITIONAL INFORMATION
a. Tips for Maximizing XSelect CSH XP 2.5 µm Column Lifetime
b. Troubleshooting Questions
c. Recommended Flow Rates and Anticipated Backpressures for
Reversed-Phase XSelect CSH XP 2.5 µm Columns
XSelect CSH XP 2.5 µm Columns 1
[ CARE AND USE MANUAL ]
II. GET TING STARTED
Each XSelect CSH 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 CSH 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. T hese data
should be recorded and stored for future reference or can be accessed
via the ACQUITY UPLC console.
a. Column Connection
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 leaking 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.
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).
0.005 inches
0.020 inches
0.040 inches
Diluted/Distorted Sample Band
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.
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.
XSelect CSH XP 2.5 µm Columns 2
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)
[ CARE AND USE MANUAL ]
System Volume
5
4.4 %h
Figure 2: Determination of system band spread volume using 5-Sigma Method.
Table 1: Expected System Band Spread Volumes
System Band Spread
Alliance 2695 HPLC 29 µL
Vendor A HPLC 41 µL
Vendor B UHPLC (600 bar) 28 µL
Vendor C UHPLC 21 µL
Vendor D UHPLC 17 µL
ACQUITY UPLC 12 µL
ACQUITY UPLC H-Class 9 µL
ACQUITY UPLC I-Class (FTN) 7.5 µL
ACQUITY UPLC I-Class (FL) 5.5 µL
6. At 5 minutes, program a step to 100% B, and collect data for an
additional 5 minutes.
7. Measure absorbance difference between 100% A and 100% B.
8. Measure time at 50% of that absorbance difference.
9. Calculate time difference between start of step and 50% point.
10. Multiply time difference by flow rate to calculate system volume.
Programmed time = 5 minutes
50% Absorbance = 0.35852 AU
Time = 5.6953 minutes
0.70
0.65
0.60
0.55
0.50
0.45
0.40
5.69
0.35
0.30
5.00
0.25
0.69 min
0.20
0.15
0.10
0.05
0.00
0.00 2.00
= Programmed Gradient
= Actual Gradient
4.00
6.00 8.00
System Volume
0.69 min x 1.5 mL/min = 1.04 mL
Figure 3: Measuring system band spread volume.
100% Asymptotic
Total absorbance = 0.7164 AU
10.00
12.00 14.00
16.00
18.00 20.00
e. Measuring System Dwell Volume
Dwell volume is different than system band spreading. System dwell
volume is a measurement of the volume it takes for the initial gradient
conditions to reach the head of the column. This calculation is
particularly useful when it is necessary to transfer a method between
different LC systems.
1. Disconnect the column from the system and replace with a zero dead
volume union.
2. Use acetonitrile as mobile phase A, and acetonitrile with 0.05 mg/mL
uracil as mobile-phase B.
3. Monitor UV at 254 nm.
4. Use the flow rate in the original method and the intended flow rate on
the target instrument.
5. Collect 100% A baseline for 5 minutes.
XSelect CSH XP 2.5 µm Columns 3
Table 2: Expected System Dwell Volumes
System Dwell Volume
Alliance 2695 HPLC 900 µL
ACQUITY UPLC 120 µL
ACQUITY UPLC H-Class 350 µL
ACQUITY UPLC I-Class (FTN) 100 µL
ACQUITY UPLC I-Class (FL) 95 µL
f. Column Equilibration
XSelect CSH XP 2.5 µm Columns are shipped in 100% acetonitrile.
It is important to ensure mobile-phase compatibility before changing
to a different mobile-phase system. Equilibrate the column with a
minimum of 10 column volumes of the mobile phase to be used
(refer to Table 3 for a list of column volumes). The column may
be considered fully equilibrated once a constant backpressure
is achieved.
[ CARE AND USE MANUAL ]
Table 3: Column Volumes (mL)
Column Length
(mm)
30 0.10 0.21 0.50
50 0.17 0.35 0.83
75 0.26 0.53 1.25
100 0.35 0.71 1.66
2.1 mm 3.0 mm 4.6 mm
Internal Diameter
To avoid precipitating mobile-phase buffers within the column or
system, flush the column with five column volumes of a water/organic
solvent mixture using the same, or lower, solvent content as in the
desired buffered mobile phase (i.e., flush the column and system with
60% methanol in water prior to introducing 60% methanol/40%
buffer mobile phase).
Note: If mobile-phase additives (i.e., ion-pairing reagents) are present in low
concentrations (<0.2% v/v), 100 to 200 column volumes may be required for
complete equilibration. In addition, mobile phases that contain formate (i.e.,
ammonium formate, formic acid) may require extended equilibration times.
g. eCord Installation
eCord™ Technology represents a significant advancement in column
usage tracking management which can be realized if the column is
installed on an ACQUITY UPLC System. T he eCord can be read
by connecting the yellow fob to the reader/writer located on the
right-hand side of the ACQUITY UPLC Column heater module.
Embedded information such as the column manufacturing QC data
and Certificates of Analysis may then be accessed via the ACQUITY
UPLC console.
modified to such an extent that they are not commercially viable and have
limited method flexibility other than isocratic column testing.
2. Determine the number of theoretical plates (N) and use this
value for periodic comparisons.
3. Repeat the test periodically to track column performance over
time. Slight variations may be obtained on different LC systems
due to the quality of the connections, operating environment,
system electronics, reagent quality, column condition and
operator technique.
i. VanGuard Pre-Columns
VanGuard™ Pre-Columns are 2.1 mm ID x 5 mm length guard column
devices designed specifically to protect an analytical column while
minimizing the negative dispersion impact of utilizing such a device.
VanGuard Pre-Columns are packed with the same stationary phases
as the XP 2.5 µm Column offering. VanGuard Pre-Columns
are designed to be directly attached to the inlet of a eXtended
Performance [XP] 2.5 µm Column.
Note: VanGuard Pre-Columns are shipped with a collet and ferrule that are NOT
pre-swaged. This enables the end user to mate the VanGuard Pre-Column to a
specific XP 2.5 µm Column and ensures void-free and leak-free connections.
Care must be taken when removing the O-ring that holds these two pieces on
the pre-column tubing.
2.5 µm XP Column
VanGuard Pre-Column
h. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it to track column
performance over time. This test may consist of:
a. An analyte test mixture that is commonly used in your laboratory
b. An analyte mixture as found on the “Performance Test
Chromatogram” which can be accessed via the eCord.
Note: If [b] is performed, the isocratic efficiencies measured in your laboratory
may be less than those given on the Waters Performance Test Chromatogram.
This is normal and expected. The Waters isocratic column testing systems have
been modified in order to achieve extremely low system dispersion. This presents
a more challenging test of how well the column was packed. This also guarantees
the highest quality packed column. These special testing systems have been
XSelect CSH XP 2.5 µm Columns 4
Place wrench here
Ferrule
Figure 4: Installing a VanGuard Pre-Column.
Flow
Collet
Place wrench here
VanGuard Pre-Column Installation Instructions
1. Remove the VanGuard Pre-Column from its box and shipping
tube and remove the plastic plug.
2. Orient the pre-column so that the male end is facing up and
carefully remove the black O-ring that holds the collet and
ferrule in place during shipment (collet and ferrule are not
permanently attached).
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