Waters XBridge XP 2.5 µm Columns User Manual
[ CARE AND USE MANUAL ]
XBRIDGE 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 XBridge® Ethylene Bridged Hybrid
[BEH] eXtended Performance [XP] 2.5 µm Column. The manufacture
of XBridge 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.
XP 2.5 µm Columns are based on the same base particle technology
and bonded-phase chemistry as 1.7 µm ACQUITY UPLC® Columns as
well as XBridge 3.5, 5 and 10 µm HPLC Columns, thus enabling
seamless transferability between HPLC, UHPLC and UPLC® platforms.
XBridge XP 2.5 µmColumns 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
c. Storage after HILIC Use
V. eCORD INTELLIGENT CHIP TECHNOLOGY
a. Introduction
b. Installation
c. Manufacturing
d. Column Use
VI. ADDITIONAL INFORMATION
a. Tips for Maximizing XBridge XP 2.5 µm Column Lifetime
b. Troubleshooting Questions
c. Recommended Flow Rates and Anticipated Backpressures for
Reversed-Phase XBridge XP 2.5 µm Columns
d. Getting Started with XBridge HILIC XP 2.5 µm Columns
e. Getting Started with XBridge Amide XP 2.5 µm Columns
XBridge XP 2.5 µm Columns 1
[ CARE AND USE MANUAL ]
II. GET TING STARTED
Each XBridge 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 XBridge 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 leak-
ing can occur, if the style of the column endfitting does not properly
match that of the compression screw/ferrule tubing depth setting.
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 spread-
ing 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.
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.
XBridge XP 2.5 µm Columns 2
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)
[ CARE AND USE MANUAL ]
Programmed time = 5 minutes
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
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 gradi-
ent 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.
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 5% 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.
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.
Table 2: Expected System Dwell Volumes
100% Asymptotic
Total absorbance = 0.7164 AU
10.00
12.00 14.00
16.00
18.00 20.00
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
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.
XBridge XP 2.5 µm Columns 3
f. Column Equilibration
XBridge 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.
For XBridge HILIC XP 2.5 µm Columns, flush with 50 column volumes
of 50:50 acetonitrile:water with 10 mM final buffer concentration.
For XBridge Amide XP 2.5 µm Columns, flush with 50 column
volumes of 60:40 acetonitrile:water with 10 mM final buffer
concentration. Prior to the first injection, equilibrate with 20 column
volumes of initial mobile-phase conditions (refer to Table 3 for a
list of column volumes). See “Getting Started with XBridge HILIC
Columns” or “Getting Started with XBridge Amide Columns” sections
for additional information.
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
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 eX tended
Performance 2.5 µm Column.
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 device 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.
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:
XBridge XP 2.5 µm Columns 4
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.
XP 2.5 µm Column
Place wrench here
Ferrule
Flow
Figure 4: Installing a VanGuard Pre-Column.
Collet
VanGuard Pre-Column
Place wrench here
[ CARE AND USE MANUAL ]
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).
3. Orient the XP 2.5 µm Column perpendicular to the work surface so that the column inlet is on the bottom.
4. From below, insert the VanGuard Pre-Column into the column inlet; turn the assembled column and pre-column 180° so that the pre-column is now on top.
5. Tighten with two 5/16” wrenches placed onto the XP 2.5 µm Column flats and VanGuard Pre-Column hex nut (male end) as shown in Figure 4.
6. While keeping pressure on the VanGuard Pre-Column against the XP 2.5 µm Column, tighten turn to set the collet and ferrule.
7. Check that the ferrule is set by loosening the connection and inspecting the ferrule depth.
8. Reattach the pre-column to the XP 2.5 µm Column, apply flow and inspect for leaks.
III. COLUMN USE
To ensure the continued high performance of XBridge XP 2.5 µm Columns, follow these guidelines:
a. Sample Preparation
1. Sample impurities and/or particulates often contribute to column contamination. One option to avoid column contamination is to use Waters Oasis® or
Sep-Pak Solid-Phase Extraction (SPE) devices. To select the appropriate sorbent for a specific sample type, visit www.waters.com/sampleprep
2. It is preferable to prepare the sample in the initial mobile-phase conditions or a weaker solvent for the best peak shape and sensitivity.
3. If the sample is not prepared in the mobile-phase, ensure that the sample, solvent and mobile-phases are miscible in order to avoid sample and/or buffer
precipitation.
4. Filter sample with a 0.2 µm membrane to remove particulates. If the sample is dissolved in a solvent that contains an organic modifier (e.g., acetonitrile,
methanol, etc.) ensure that the membrane/filter material is compatible with the solvents in use. Alternatively, centrifuge the sample for 20 minutes at
8000 rpm, followed by the transfer of the supernatant to an appropriate vial could be considered.
b. pH Range
Table 4: Recommended pH Range
Chemistry pH Range
XBridge BEH C
XBridge BEH C
XBridge BEH Phenyl 1 - 12
XBridge BEH Shield RP18 2 - 11
XBridge BEH HILIC 1 - 9
XBridge BEH Amide 2 - 11
18
8
1 - 12
1 - 12
Column lifetime will vary depending on the combination of temperature, mobile-phase pH and
type of buffer/additive used. Table 5 lists the recommended buffers and additives for XBridge
XP 2.5 µm Columns.
Note: Working in combinations of extreme pH, temperature and pressure may result in reduced
column lifetime.
XBridge XP 2.5 µm Columns 5
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