Waters ACQUITY UPLC BEH Columns User Manual
[ CARE AND USE MANUAL ]
ACQUITY UPLC BEH Columns
CONTENTS
I. INTRODUCTION
II. GETTING STARTED
a. Column Connectors
b. Column Installation
c. Column Equilibration
d. eCord Installation
e. Initial Column Efficiency Determination
f. VanGuard™ Pre-Columns
III. COLUMN USE
a. Sample Preparation
b. pH Range
c. Solvents
d. Pressure
e. Temperature
IV. COLUMN CLEANING, REGENERATING
AND STORAGE
a. Cleaning and Regeneration
b. Storage
I. INTRODUCTION
Thank you for choosing a Waters ACQUITY UPLC® BEH Column.
The ACQUITY UPLC BEH packing materials were designed
specifically for use with the ACQUITY UPLC System and are
manufactured in a cGMP, ISO 9001 certified plant using ultra pure
reagents. Each batch of ACQUITY UPLC BEH material is tested
chromatographically with acidic, basic and neutral analytes and the
results are held to narrow specification ranges to assure excellent,
reproducible performance. Every column is individually tested and
a Performance Chromatogram and Certificate of Batch Analysis are
provided on the eCord™ intelligent chip.
ACQUITY UPLC Columns were designed and tested specifically for
use on the ACQUITY UPLC System. ACQUITY UPLC Columns will
exhibit maximum chromatographic performance and benefits ONLY
when used on the holistically-designed ACQUITY UPLC System since
the ACQUITY UPLC System and column were created and designed to
operate together. For these reasons, Waters cannot support the use
of ACQUITY UPLC Columns on any system other than an ACQUITY
UPLC System.
V. INTRODUCING ECORD INTELLIGENT
CHIP TECHNOLOGY
a. Introduction
b. Installation
c. Manufacturing Information
d. Column Use Information
VI. ADDITIONAL INFORMATION
a. Tips for Maximizing ACQUITY UPLC BEH
Column Lifetimes
b. Recommended Flow Rates and Backpressures for
Reversed-Phase ACQUITY UPLC BEH Columns
c. Getting Started with ACQUITY UPLC BEH
HILIC Columns
d. Getting Started with ACQUITY UPLC BEH
Amide Columns
[ CARE AND USE MANUAL ]
II. GETTING STARTED
Each ACQUITY UPLC BEH Column comes with a Certificate
of Analysis and Performance Test Chromatogram embedded
within the eCord intelligent chip. The Certificate of Analysis
is specific to each batch of packing material contained in the
ACQUITY UPLC BEH Columns 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, capacity factor, and
chromatographic conditions. These data should be stored for
future reference.
a. Column Connectors
The ACQUITY UPLC System utilizes tubing and gold plated
compression screws which have been designed to meet stringent
tolerance levels and to minimize extra column volumes.
Optimized column inlet tubing (Part Number 430001084) is
supplied with the ACQUITY UPLC System. The inject valve end of
the tubing is clearly marked with a blue shrink tube marker. Insert
the opposite end of the tubing into the ACQUITY UPLC Column and
tighten the compression fitting.
For information on the correct column outlet tubing, please refer
to the relevant detector section in the ACQUITY UPLC System
Operator’s Guide (Part Number 71500082502).
4. Gradually increase the flow rate as described in step 2.
5. Once a steady backpressure and baseline have been achieved,
proceed to the next section.
Note: If mobile-phase additives are present in low concentrations
(e.g., ion-pairing reagents), 100 to 200 column volumes may be
required for complete equilibration. In addition, mobile phases that
contain formate (e.g., ammonium formate, formic acid, etc.) may
also require longer initial column equilibration times.
c. Column Equilibration
ACQUITY UPLC BEH 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 1 for a list of column volumes).
The column may be considered thermally equilibrated once a
constant backpressure is achieved.
Table 1. Empty Column Volumes in mL (multiply by 10 for flush
solvent volumes)
Column Length
(mm)
30 — 0.1 0.2
50 0.04 0.2 0.4
100 0.08 0.4 0.8
150 0.12 0.5 1.0
1.0 mm 2.1 mm 3.0 mm
Internal Diameter
b. Column Installation
Note: The flow rates given in the procedure below are for a typical
2.1 mm i.d. by 50 mm length 1.7 µm column. Scale the flow rate up
or down accordingly based upon the flow rate and pressure guide
provided in Section V (Additional Information).
1. Purge the pumping system of any buffer-containing mobile
phases and connect the inlet end of the column to the injector
outlet.
2. Flush 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 entry of air into the detection system
and gives more rapid equilibration.
To avoid precipitating mobile-phase buffers on your column or
in your 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. (For example,
flush the column and system with 60% methanol in water prior to
introducing 60% methanol/40% buffer mobile-phase.)
For ACQUITY UPLC BEH HILIC Columns, flush with 50 column
volumes of 50:50 acetonitrile:water with 10 mM final buffer
concentration. For ACQUITY UPLC BEH Amide Columns, flush
with 50 column volumes of 60:40 acetonitrile:aqueous. Prior
to the first injection, equilibrate with 20 column volumes of
initial mobile phase conditions (refer to Table 1 for a list of
column volumes). See “Getting Started with ACQUITY UPLC BEH
HILIC Columns” or “Getting Started with ACQUITY UPLC Amide
Columns” for additional information.
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[ CARE AND USE MANUAL ]
d. eCord Installation
The eCord button should be attached to the side of the column
heater module. The eCord button is magnetized and does not
require specific orientation.
e. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it. This
test may consist of:
a. An analyte test mixture that is commonly used in your
laboratory, and/or
b. An analyte mixture as found on the “Performance Test
Chromatogram” which accompanied your column.
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. The Waters
isocratic column testing systems have been modified in order
to achieve extremely low system volumes. This presents a
more challenging test of how well the column was packed. This
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.
ACQUITY UPLC Column VanGuard Pre-Column
Place wrench here
Ferrule
Collet
Place wrench here
Flow
Installation Instructions
1. Remove VanGuard Pre-column from box and shipping tube and
remove plastic plug.
2. Orient pre-column so that male end is facing up and carefully
remove rubber O-ring that holds collet and ferrule in place
during shipping (collet and ferrule are not yet permanently
attached).
3. Orient ACQUITY UPLC Column perpendicular to work surface
so that column inlet is on the bottom (column outlet on top).
4. From below, insert VanGuard Pre-column into ACQUITY UPLC
Column inlet and hand-tighten (collet and ferrule are not yet
permanently attached).
2. Determine the number of theoretical plates (N) and use this
value for periodic comparisons.
3. Repeat the test at predetermined intervals to track column
performance over time. Slight variations may be obtained
on two different UPLC® Systems due to the quality of the
connections, operating environment, system electronics,
reagent quality, column condition and operator technique.
f. VanGuard Pre-Columns
VanGuard Pre-columns are 2.1 mm ID x 5 mm length guard column
devices designed specifically for use in the ACQUITY UPLC
System. VanGuard Pre-columns are packed with the same UPLC
chemistries and frits as our 2.1 mm ID UPLC columns. VanGuard
Pre-columns are designed to be attached directly to the inlet side
of an ACQUITY UPLC Column.
Note: In order to ensure void-free and leak-free connections, the
VanGuard Pre-column is shipped with the collet and ferrule NOT
permanently attached. Care must be taken when removing the
O-ring that holds these two pieces on the pre-column tubing.
5. While pushing the VanGuard Pre-Column into the column inlet,
turn assembled column and pre-column 180˚ so that precolumn is now on top.
6. Tighten with two 5/16” wrenches placed onto ACQUITY UPLC
Column flats and VanGuard Pre-column hex nut (male end) as
shown above.
7. Tighten 1/4 turn to set collet and ferrule.
8. Check that ferrule is set by loosening connection and
inspecting the ferrule depth. A properly set ferrule depth will
resemble other connections in the ACQUITY UPLC System.
9. Reattach pre-column, apply mobile-phase flow and inspect
for leaks.
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[ CARE AND USE MANUAL ]
II. COLUMN USE
To ensure the continued high performance of ACQUITY UPLC BEH
Columns, follow these guidelines:
a. Sample Preparation
1. Sample impurities often contribute to column contamination.
One option to avoid this is to use Oasis® Solid-phase
Extraction Cartridges/columns or Sep-Pak® Cartridges of the
appropriate chemistry to clean up the sample before analysis.
For more information, visit www.waters.com/sampleprep
2. It is preferable to prepare the sample in the operating mobile
phase or a mobile phase that is weaker than the mobile phase
for the best peak shape and sensitivity. Acetone should
not be used as a sample solvent/diluent unless a Hexane
Tetrahydrofuran Compatibility Kit (P/N: 205000464) has
been installed.
3. If the sample is not dissolved 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 0.2 µm membranes to remove particulates.
If the sample is dissolved in a solvent that contains an organic
modifier (e.g., acetonitrile, methanol, etc.) ensure that the
5. membrane material does not dissolve in the solvent. Contact
the membrane manufacturer with solvent compatibility
questions. Alternatively, centrifugation for 20 minutes at
8000 rpm, followed by the transfer of the supernatant liquid
to an appropriate vial, could be considered.
6. For Hydrophilic Interaction Chromatography (HILIC)
separations, the samples must be prepared in a high
percentage of organic solvents (e.g., 95% acetonitrile). See
“Getting Started with ACQUITY UPLC BEH HILIC Columns” or
“Getting Started with ACQUITY UPLC Amide Columns” for
additional information.
b. pH Range
The recommended operating pH range for ACQUITY UPLC BEH
Columns is 1 to 12 for the C18, C8 and Phenyl chemistries; 2 to 11
for the Shield RP18 and BEH Amide chemistries; and 1 to 9 for
the BEH HILIC chemistry. A listing of commonly used buffers and
additives is given in Table 2. Additionally, the column lifetime
will vary depending upon the operating temperature, the type and
concentration of buffer used. For example, the use of phosphate
buffer at pH 8 in combination with elevated temperatures will lead
to shorter column lifetimes.
c. Solvents
To maintain maximum column performance, use high quality
chromatography grade solvents. Filter all aqueous buffers prior
to use through a 0.2 µm filter. Pall Gelman Laboratory Acrodisc®
filters are recommended. Solvents containing suspended
particulate materials will generally clog the outside surface of
the inlet distribution frit of the column. This will result in higher
operating pressure and poorer performance. See Section V for
more information.
d. Pressure
ACQUITY UPLC BEH Columns can tolerate operating pressures up
to 18000 psi (1241 bar or 124 MPa). Note: Working at the extremes
of pressure, pH and/or temperature will result in shorter column lifetimes.
e. Temperature
Temperatures between 20 ˚C – 90 ˚C are recommended for
operating ACQUITY UPLC BEH Columns in order to enhance
selectivity, lower solvent viscosity and increase mass transfer
rates. When operating at high pH, lower operating temperatures
are recommended for longer column lifetime. Working at high
temperatures (e.g. > 70 °C) may also result in shorter column
lifetimes. Under HILIC conditions, ACQUITY UPLC BEH Amide
Columns can be used at high pH and at high temperatures without
issue (see recommended conditions in Getting Started with BEH
Amide section).
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