Waters XSelect HSS XP 2.5 µm Columns User Manual
[ CARE AND USE MANUAL ]
XSELECT HSS 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. Solvent Conversion for Normal-Phase Use
h. eCord Installation
i. Initial Column Efficiency Determination
j. 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™ High Strength Silica [HSS]
eX tended Performance [XP] 2.5 µm Column. The manufacture of
XSelect HSS 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 HSS XP 2.5 µm Columns are based on the same base
particle technology and bonded phase chemistry as 1.8 µm ACQUITY
UPLC® HSS Columns as well as XSelect HSS 3.5 and 5 µm HPLC
Columns, thus enabling seamless transferability between HPLC,
UHPLC and UPLC® platforms.
XSelect HSS 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
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 XSelect HSS XP 2.5 µm Column Lifetime
b. Troubleshooting Questions
c. Recommended Flow Rates and Anticipated Backpressures for
Reversed-Phase XSelect HSS XP 2.5 µm Columns
XSelect HSS XP 2.5 µm Columns 1
[ 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. T hese 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.
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.
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.
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)
XSelect HSS XP 2.5 µm Columns 2
[ 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
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.
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
2. Use acetonitrile as mobile phase A, and acetonitrile with
3. Monitor UV at 254 nm.
4. Use the flow rate in the original method and the intended flow
5. Collect 100% A baseline for 5 minutes.
XSelect HSS XP 2.5 µm Columns 3
zero dead volume union.
0.05 mg/mL uracil as mobile phase B.
rate on the target instrument.
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 HSS 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
To avoid precipitating mobile-phase buffers within the column or
system, flush the column with five column volumes of a waters/
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.
2.1 mm 3.0 mm 4.6 mm
Internal Diameter
g. Solvent Conversion for Normal-Phase Use
XSelect HSS Cyano XP 2.5 µm Columns can be used for both reversed-
phase as well as normal-phase separations. The column is shipped in
100% acetonitrile and is ready for use in reversed-phase conditions.
If you intend to use the XSelect HSS Cyano Column for normal-
phase applications, the following solvent conversion procedure
must be followed:
1. Flush the column with a minimum of 20 column volumes of 100%
methanol using a low flow rate to avoid overpressuring the LC system.
Refer to Table 3 for column volume information.
2. Flush the column with a minimum of 20 column volumes of
100% isopropanol using a low flow rate to avoid overpressuring
the LC system. Refer to Table 3 for column volume information.
3. Flush the column with a minimum of 20 column volumes of 100%
dichloromethane using a low flow rate to avoid overpressuring the
LC system. Refer to Table 3 for column volume information.
4. Flush the column with the intended mobile-phase conditions
until a stable baseline is achieved.
h. 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.
i. 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 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.
j. 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.
XSelect HSS XP 2.5 µm Columns 4
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