[ 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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[ CARE AND USE MANUAL ]
Table 2. Buffer Recommendations for Using ACQUITY UPLC BEH Columns from pH 2 to 12
Additive/Buffer pKa Buffer range
Volatility
(±1 pH unit)
TFA 0.3 — Volatile Yes
Acetic Acid 4.76 — Volatile Yes
Formic Acid 3.75 — Volatile Yes
Acetate (NH
Formate (NH
COOH) 4.76 3.76 – 5.76 Volatile Yes
4CH2
COOH) 3.75 2.75 – 4.75 Volatile Yes
4
Used for
Mass Spec
Comments
Ion pair additive, can suppress MS signal, used in the 0.02-
0.1% range.
Maximum buffering obtained when used with ammonium acetate
salt. Used in 0.1-1.0% range.
Maximum buffering obtained when used with ammonium
formate salt. Used in 0.1-1.0% range.
Used in the 1-10 mM range. Note that sodium or potassium salts
are not volatile.
Used in the 1-10 mM range. Note that sodium or potassium salts
are not volatile.
Phosphate 1 2.15 1.15 – 3.15 Non-volatile No Traditional low pH buffer, good UV transparency.
Phosphate 2 7.2 6.20 – 8.20 Non-volatile No
Phosphate 3 12.3 11.3 - 13.3 Non-volatile No
Above pH 7, reduce temperature/concentration and use a guard
column to maximize lifetime.
Above pH 7, reduce temperature/concentration and use a guard
column to maximize lifetime.
4-Methylmorpholine ~8.4 7.4 – 9.4 Volatile Yes Generally used at 10 mM or less.
Used in the 5-10 mM range (for MS work keep source >150 ˚C
). Adjust pH with ammonium hydroxide or acetic acid. Good
buffering capacity at pH 10
Note: use ammonium bicarbonate (NH4HCO3), not ammonium
carbonate ((NH4)2CO3)
Ammonia (NH
OH)
4
Ammonium Bicarbonate
9.2
10.3 ( HCO
9.2 (NH
4
-
3
+
)
)
8.2 – 10.2
8.2 – 11.3
Volatile
Volatile
Yes
Yes
Ammonium (Acetate) 9.2 8.2 – 10.2 Volatile Yes Used in the 1-10 mM range.
Ammonium (Formate) 9.2 8.2 – 10.2 Volatile Yes Used in the 1-10 mM range.
Borate 9.2 8.2 – 10.2 Non-volatile No
CAPSO 9.7 8.7 – 10.7 Non-volatile No
Reduce temperature/concentration and use a guard column to
maximize lifetime.
Zwitterionic buffer, compatible with acetonitrile, used in the
1-10 mM range. Low odor.
Glycine 2.4, 9.8 8.8 – 10.8 Non-volatile No Zwitterionic buffer, can give longer lifetimes than borate buffer.
1-Methylpiperidine 10.2 9.3 – 11.3 Volatile Yes Used in the 1-10 mM range.
CAPS 10.4 9.5 – 11.5 Non-volatile No
Triethylamine
(as acetate salt)
10.7 9.7 – 11.7 Volatile Yes
Zwitterionic buffer, compatible with acetonitrile, used in the
1-10 mM range. Low odor.
Used in the 0.1-1.0% range. Volatile only when titrated with ac etic acid
(not hydrochloric or phosphoric). Used as ion-pair for DNA analysis
at pH 7-9.
Pyrrolidine 11.3 10.3 – 12.3 Volatile Yes Mild buffer, gives long lifetime.
Note: Working at the extremes of pH, temperature and/or pressure will result in shorter column lifetimes.
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[ CARE AND USE MANUAL ]
III. COLUMN CLEANING, REGENERATING
AND STORAGE
Increasing column temperature increases cleaning efficiency. If
the column performance is poor after regenerating and cleaning,
call your local Waters office for additional support.
a. Cleaning and Regeneration
Changes in peak shape, peak splitting, shoulders on the
peak, shifts in retention, change in resolution or increasing
backpressure may indicate contamination of the column. Flushing
with a neat organic solvent, taking care not to precipitate buffers,
is usually sufficient to remove the contaminant. If the flushing
procedure does not solve the problem, purge the column using the
following cleaning and regeneration procedures.
Use the cleaning routine that matches the properties of the
samples and/or what you believe is contaminating the column (see
Table 3 below). Flush columns with 20 column volumes of solvent.
Table 3: Recommended pH and Temperature Limits for ACQUITY UPLC BEH Columns
Name of Column Particle Size Pore Diameter Surface Area pH Limits
BEH C
18
BEH C
8
BEH Phenyl 1.7 µ m 130Å 185 m
BEH Shield RP
18
BEH HILIC 1.7 µ m 130Å 185 m
BEH Amide 1.7 µm 130Å 185 m
1.7 µ m 130Å 185 m2/g 1-12 80 °C 60 °C 3.1 µmol /m
1.7 µ m 130Å 185 m2/g 1-12 60 °C 60 °C 3.1 µ mol /m
2
/g 1-12 80 °C 60 °C 3.0 µmol/m
1.7 µ m 130Å 185 m2/g 2-11 50 °C 45 °C 3.2 µmol/m
2
/g 1-9 60 °C 45 °C — —
2
/g 2-11 90 °C 90 °C 7.5 µmol /m
Flush ACQUITY UPLC BEH HILIC columns with 50:50
acetonitrile:water to remove polar contaminants. If this flushing
procedure does not solve the problem, purge the column with 5:95
acetonitrile:water.
To clean polar contaminants from ACQUITY UPLC BEH Amide
Columns, run a 10 minute gradient from 0-100% water. Please
note that as aqueous concentration increases, backpressure will
rapidly increase as well. Reduce flow rate when operating at
greater than 60% aqueous. Repeat if necessary.
Temperature Limits
Low pH High pH
Surface Carbon %
2
2
2
2
2
17.7
12.8
14.5
16.6
12
Table 4. Reversed-Phase Column Cleaning Sequence
Polar Samples Non-polar S amples
1. water
1. isoproanol (or an appropriate isopropanol/
water mixture*)
2. methanol 2. tetrahydrofuran (THF)
3. tetrahydrofuran (THF) 3. dichloromethane
4. methanol 4. hexane
5. water
6. mobile phase 6. mobile phase
* Use low organic solvent content to avoid precipitating buffers.
** Unless a Hexane Tetrahydrofuran Compatibility Kit (P/N: 205000464) has been installed, running solvents suc h as THF or hexane should only be considered when the
column cannot be cleaning by running neat, reversed-phase organic solvents such as acetonitrile. Reduce flow rate, lower operating temperatures and limit system exposure
to THF and/or hexane.
5. isopropanol (followed by an appropriate
isopropanol/water mixture*)
**
Proteinaceous Samples
Option 1: Inject repeated aliquots of
dimethylsulfoxide (DMSO)
Option 2: gradient of 10% to 90% B where:
A = 0.1% trifluoroacetic acid (TFA) in water
B = 0.1% trifluoroacetic acid (TFA) in
acetonitrile
(CH3CN)
Option 3: Flush column with 7M guanidine
hydrochloride, or 7M urea
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[ CARE AND USE MANUAL ]
b. Storage
For periods longer than four days at room temperature, store
reversed-phase columns in 100% acetonitrile. For elevated
temperature applications, store immediately after use in 100%
acetonitrile for the best column lifetime. Do not store columns in
buffered eluents. If the mobile phase contained a buffer salt, flush
reversed-phase ACQUITY UPLC BEH Columns with 10 column
volumes of HPLC grade water (see Table 1 for common column
volumes) and replace with 100% acetonitrile for storage. Failure
to perform this intermediate step could result in precipitation
of the buffer salt in the column when 100% acetonitrile is
introduced. Run a gradient to 100% ACN in order to flush all
aqueous solvent from an ACQUITY UPLC BEH Amide Column
prior to storage in 100% ACN. Completely seal column to avoid
evaporation and drying out of the bed.
For periods longer than four days, store ACQUITY UPLC BEH
HILIC Columns in 95:5 acetonitrile:water. Do not store in buffered
solvent. If the mobile phase contained a buffered salt, flush the
column with 10 column volumes of 95:5 acetonitrile:water (see
Table 1 for common column volumes).
Figure 1. Waters eCord Intelligent Chip
Waters eCord intelligent chip
At the time of manufacture, tracking and quality control
information will be downloaded to the eCord. Storing this
information on the chip will eliminate the need for a paper
Certificate of Analysis. Once the user installs the column, the
software will automatically download key parameters into a
column history file stored on the chip. In this manual, we explain
how the eCord will provide a solution for easily tracking the
history of the columns, reduce the frustration of paperwork trails,
and give customers the reassurance that a well-performing column
is installed onto their instruments.
Figure 2. eCord Inserted into Side of Column Heater
Note: If a column has been run with a mobile phase that contains
formate (e.g., ammonium formate, formic acid, etc.) and is then
flushed with 100% acetonitrile, slightly longer equilibration times
may be necessary when the column is re-installed and run again
with a formate-containing mobile phase.
IV. INTRODUCING eCORD INTELLIGENT
CHIP TECHNOLOGY
a. Introduction
The eCord Intelligent Chip is a new technology that will provide
the history of a column’s performance throughout its lifetime. The
eCord will be permanently attached to the column to assure that
the column’s performance history is maintained in the event that
the column is moved from one instrument to another.
eCord inserted into
side of column heater
b. Installation
Install the column into the column heater. Plug the eCord into
the side of the column heater. Once the eCord is inserted into
the column heater the identification and overall column usage
information will be available in Empower® and MassLynx®
software allowing the user to access column information on
their desktop.
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[ CARE AND USE MANUAL ]
c. Manufacturing Information
The eCord chip provides the
user with an overview of the
bulk material QC test results.
V. ADDITIONAL INFORMATION
The eCord chip provides the
user with QC test conditions
and results on the column
run by the manufacturer. The
information includes mobile
phases, running conditions
and analytes used to test
the columns. In addition the
QC results and acceptance is
placed onto the column.
d. Column Use Information
The eCord chip provides the customer with column use data. The
top of the screen identifies the column including chemistry type,
column dimensions and serial number. The overall column usage
information includes the total number of samples, total number
of injections, total sample sets, date of first injection, date of last
injection, maximum pressure and temperature. The information
also details the column history by sample set including date
started, sample set name, user name, system name, number of
injections in the sample set, number of samples in the sample
set, maximum pressure and temperature in the sample set and if
the column met basic system suitability requirements. Up to 50
sample sets can be stored on the eCord chip.
a. Tips for Maximizing ACQUITY UPLC BEH Column Lifetimes
1. To maximize ACQUITY UPLC column lifetime, pay close
attention to:
Water quality (including water purification system)
Solvent quality
Mobile-phase preparation, storage and age
Sample, buffer and mobile-phase solubilities
Sample quality and preparation.
2. When problems arise, often only one improper practice must
be changed.
3. Always remember to:
Use in-line filter unit or, preferably, a VanGuard
Pre-Column.
Discourage bacterial growth by minimizing the use of
100% aqueous mobile phases where possible.
Change aqueous mobile phase every 24 – 48 hours (if
100% aqueous mobile phase use is required).
Discard old 100% aqueous mobile phases every 24-48
hours to discourage bacterial growth.
Add 5%-10% organic modifier to mobile phase A and
adjust gradient profile.
Filter aqueous portions of mobile phase through 0.2 µm
filter.
Maintain your water purification system so that it is in good
working order.
Only use ultra pure water (18 megohm-cm) water and
highest quality solvents possible. HPLC grade water is not
UPLC grade water.
Consider sample preparation (e.g., solid-phase extraction,
filtration, etc).
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[ CARE AND USE MANUAL ]
4. Avoid (where possible):
100% aqueous mobile phases (if possible).
HPLC-grade bottled water.
“Topping off” or adding “new” mobile phase to “old” mobile phase.
Old aqueous mobile phases. Remember to rinse bottles thoroughly and prepare fresh every 24 to 48 hours.
Using phosphate salt buffer in combination with high ACN concentrations (e.g., > 70%) due to precipitation.
5. Don’t: assume a “bad” column is the culprit when high backpressure or split peaks are observed:
Investigate cause of column failure
Backpressure
Mobile phase(s), bacteria, precipitation and/or samples
Peak splitting
Sample quality
Injection solvent strength.
6. Remember: the diameter of UPLC columns (1.0, 2.1 and 3.0 mm ID) are often lower than that of a conventional HPLC column and
therefore, mobile phases last much longer. To reduce the chances of mobile-phase contamination or degradation, only prepare what you
need for analysis or store excess bulk quantities in a refrigerated environment.
7. Mobile phase-related questions to ask:
Am I using 100% aqueous mobile phases? Am I able to add a small amount of organic modifier to my mobile phase A?
Do I filter my aqueous mobile phases through 0.2 µm filters?
How old is my mobile phase? Do I label the bottle with preparation date?
Do I “top off” or do I prepare fresh mobile phases every 24 – 48 hours?
What is the quality of my water? Has the quality recently changed? How is my water purification system working? When was it
last serviced?
Am I working with pH 7 phosphate buffer (which is VERY susceptible to bacterial growth)?
8. Sample-related questions to ask:
If I inject neat standards prepared in mobile phase do I observe these problems?
If I prepare my standards in water and prepare them like samples (e.g., SPE, filtration, etc.) do I still observe these problems?
Has the quality of my samples changed over time?
b. Rcommended Flow Rates and Backpressures for Reversed-Phase ACQUITY UPLC BEH Columns
1.0 mm i.d. Columns (40 °C)
UPLC Linear Velocity
(mm /se c)
Column Dimensions
1.0 x 50 mm 0.1 4300 0.13 5600 0.17 7400 0.2 8700
1.0 x 100 mm 0.1 8600 0.13 1120 0 0.17 14600 0.2 17200
1.0 x 150 mm 0.1 12800 0.13 16700 0.17 21800 0.2 25600
Flow Rate
(mL/min)
3 4 5 6
Backpressure
(psi)
Flow Rate
(mL/min)
Backpressure
(psi)
Flow Rate
(mL/min)
Backpressure
(psi)
Flow Rate
(mL/min)
Backpressure
(psi)
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[ CARE AND USE MANUAL ]
2.1 mm i.d. Columns (40 °C)
UPLC Linear Velocity
(mm /se c)
Column Dimensions
2.1 x 30 mm 0.45 3000 0.60 4100 0.75 5100 0.9 6100
2.1 x 50 mm 0.45 4800 0.60 6400 0.75 8000 0.9 9500
2.1 x 100 mm 0.45 9100 0.60 12100 0.75 15200 0.9 18200
2.1 x 150 mm 0.45 13400 0.60 17900 0.75 22400 0.9 26900
Flow Rate
(mL/min)
Note: 1) ACQUITY BEH UPLC 1.7 µm particle reversed-phase columns
2) ACN/Aqueous gradient, Pmax at ~30% ACN
3) Approximate maximum total system backpressure given
UPLC Linear Velocity
(mm /se c)
Column Dimensions
3.0 x 30 mm 0.9 3400 1.17 4400 1.53 5800 1.8 6800
3.0 x 50 mm 0.9 5100 1.17 6600 1.53 8700 1.8 10200
3.0 x 100 mm 0.9 9300 1.17 12100 1.53 15900 1.8 18700
3.0 x 150 mm 0.9 13600 1.17 176 00 1.53 23100 1.8 27100
Flow Rate
(mL/min)
3 4 5 6
Backpressure
(psi)
3 4 5 6
Backpressure
(psi)
Flow Rate
(mL/min)
3.0 mm i.d. Columns (40 °C)
Flow Rate
(mL/min)
Backpressure
(psi)
Backpressure
(psi)
Flow Rate
(mL/min)
Flow Rate
(mL/min)
Backpressure
(psi)
Backpressure
(psi)
Flow Rate
(mL/min)
Flow Rate
(mL/min)
Backpressure
(psi)
Backpressure
(psi)
c. Getting Started With ACQUITY UPLC BEH HILIC Columns
1. Because ACQUITY UPLC BEH HILIC Columns do not posses a
bonded phase, the pH operating range is 1 to 9, and they can
be operated at temperatures up to 45 °C.
2. As with any LC column, operating at the extremes of pH, pressures
and temperatures will result in decreased column lifetime.
Column Equilibration
1. When column is first received, flush in 50% acetonitrile:
50% water with 10 mM final buffer concentration for 50
column volumes.
2. Equilibrate with 20 column volumes of initial mobile-phase
conditions before making first injection.
3. If gradient conditions are used, equilibrate with 8-10 column
volumes between injections.
4. Failure to appropriately equilibrate the column could result in
drifting retention times.
Mobile-Phase Considerations
1. Always maintain at least 3% polar solvent in the mobile phase
or gradient (e.g., 3% aqueous/3% methanol or 2% aqueous/
1% methanol, etc.). This ensures that the ACQUITY UPLC BEH
particle is always hydrated.
2. Maintain at least 40% organic solvent (e.g., acetonitrile) in
your mobile phase or gradient.
3. Avoid phosphate salt buffers to avoid precipitation in HILIC
mobile phases. Phosphoric acid is okay.
4. Buffers such as ammonium formate or ammonium acetate
will produce more reproducible results than additives such as
formic acid or acetic acid. If an additive (e.g., formic acid, etc.)
must be used instead of a buffer, use 0.2% (v:v) instead of
0.1%.
5. For best peak shape, maintain a buffer concentration of 10 mM
in your mobile phase/gradient at all times.
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[ CARE AND USE MANUAL ]
Injection Solvents
1. If possible, injection solvents should be 95% acetonitrile. The
polar solvent (i.e., water, methanol, isopropanol) should be
minimized to 25% of the total volume.
2. A generic injection solvent is 75:25 acetonitrile:methanol. T his is
a good compromise between analyte solubility and peak shape.
3. Avoid water and dimethylsulfoxide (DMSO) as injection solvents.
These solvents will produce very poor peak shapes.
4. Exchange water or DMSO with acetonitrile by using reversedphase solid-phase extraction (SPE). If this is not possible, dilute
the water or DMSO with organic solvent.
Miscellaneous Tips
1. ACQUITY UPLC BEH HILIC Columns are designed to retain very
polar bases. Acidic, neutral and/or non-polar compounds will
have limited retention.
2. Optimal flow rates for small (<200 daltons) very polar bases
are in the 0.4 to 0.8 mL/min range with the ACQUITY UPLC
BEH HILIC Columns.
3. As compared to Atlantis® HILIC Silica HPLC Columns, the
ACQUITY UPLC BEH HILIC Columns are approximately 20%
less retentive for gradient analysis and 35 to 65% less
retentive for isocratic analysis. This is due to the lower residual
surface silanol concentration of the BEH particle.
4. In HILIC, it is important to remember that water is the strongest
solvent. Therefore, it must be eliminated or minimized in the
injection solvent.
5. For initial scouting conditions, run a gradient from 95%
aceto-nitrile to 50% acetonitrile. If no retention occurs, run
isocratically with 95:3:2 acetonitrile:methanol:aqueous
buffer.
6. Alternate polar solvents such as methanol, acetone or
isopropanol can also be used in place of water to increase
retention.
7. If using an ACQUITY UPLC System, the weak needle wash
should closely match the % organic present in the initial
mobile-phase conditions, otherwise, analyte peak shape or
retention may suffer.
d. Getting Started with ACQUITY UPLC BEH Amide Columns
Operating Ranges
1. ACQUITY UPLC BEH Amide Columns can be used routinely
under HILIC conditions between pH 2 to 11, and they can be
operated at temperatures up to 90 °C.
2. As with any LC column, operating at the extremes of pH,
pressures and temperatures will result in decreased column
lifetime.
Column Equilibration
1. When column is first received, flush in 60% acetonitrile:
40% aqueous (or initial starting conditons) for 50 column
volumes.
2. Equilibrate with 20 column volumes of initial mobile phase
conditions before making first injection.
3. If gradient conditions are used, equilibrate with 8-10 column
volumes between injections.
4. Failure to appropriately equilibrate the column could result in
drifting retention times.
Mobile Phase Considerations
1. Always maintain at least 3% polar solvent in the mobile
phase or gradient (e.g., 3% aqueous, 3% methanol or 2%
aqueous/1% methanol, etc.).
2. Maintain at least 40% organic solvent (e.g., acetonitrile) in
your mobile phase or gradient.
3. At aqueous concentrations greater than 60%, lower flow rates
should be used due to high backpressure. This includes all
aqueous wash procedures.
4. Avoid phosphate salt buffers to avoid precipitation in HILIC
mobile phases. Phosphoric acid is OK.
Injection Solvents
1. If possible, injection solvents should be as close to the mobile
phase composition as possible (if isocratic) or the starting
gradient conditions. Acetone should not be used as a sample
solvent/diluent unless a Hexane Tetrahydrofuran Compatibility
Kit (P/N: 205000464) has been installed.
2. A generic injection solvent is 75:25 acetonitrile:methanol.
This is a good compromise between analyte solubility and peak
shape. When separating saccharides with limited solubility in
in organic solvents, higher concentrations of aqueous solvent
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[ CARE AND USE MANUAL ]
in the sample are acceptable. 50:50 acetonitrile:water can
provide satisfactory results.
3. The injection solvent’s influence on peak shape should be
determined experimentally. In some cases, injections of water
(or highly aqueous solutions) may not adversely affect peak
shape.
Miscellaneous Tips
1. For initial scouting conditions, run a gradient from 95%
acetonitrile to 50% acetonitrile. If no retention occurs, run
isocratically with 95:3:2 acetonitrile:methanol:aqueous
buffer.
2. Alternate polar solvents such as methanol, acetone or
isopropanol can also be used in place of water to increase
retention.
3. Ensure that your weak needle wash solvent/purge solvent is
your starting mobile phase (i.e., high organic), or your peak
shapes will suffer. Typical needle wash conditions: 800 µL
Strong wash in 20:80 ACN/H2O, 500 µL Weak wash in 75:25
ACN/H2O.
4. Acetone should not be used as a sample solvent/diluent
unless a Hexane Tetrahydrofuran Compatibility Kit (P/N:
205000464) has been installed.
Tips for Separating Sugars/Saccharides/Carbohydrates
1. If separating sugars or sugar-containing compounds that
do not include reducing sugars (see below) follow generic
‘Getting Started with ACQUITY UPLC BEH Amide Columns’
recommendations described above.
a. Operate at a slow flow rate (e.g., 0.10 - 0.13 mL/min on
2.1 x 50 mm column) to facilitate anomer collapse.
b. With longer columns, increased flow rates (e.g., up to
0.3 mL/min) can be used. As with all LC separations,
optimal flow rates should be determined experimentally.
c. Add triethylamine (TEA) or ammonium hydroxide (NH4OH)
modifiers to both mobile phase (e.g., A2, B2, etc) reservoirs.
d. For UPLC/ELSD separations of mono- and/or disaccharides,
typical isocratic UPLC conditions include:
i. 75% acetonitrile (ACN) with 0.2% TEA, 35 °C,
0.13 mL/min, 2.1 x 50 mm BEH Amide column;
ii. 77% acetone with 0.05% TEA, 85 °C, 0.15 mL/min,
2.1 x 50 mm BEH Amide column;
iii. 75% ACN with 0.2% TEA, 35 °C, 0.2mL/min,
2.1 x 100 mm BEH Amide column.
e. For UPLC/ELSD separations of more complex sugar
mixtures (e.g., polysaccharides), typical gradient UPLC
conditions include (add T EA modifier to both mobile
phases A and B):
i. Gradient going from 80% to 50% ACN with 0.2% TEA in
10 min, 35 °C, 0.13 mL/min, 2.1 x 100 mm BEH Amide
column or up to 0.3 mL/min flow rate with 2.1 x 150 mm
BEH Amide column;
ii. 80%-55% Acetone with 0.05% TEA in 10 min,
85 °C, 0.15 mL/min, 2.1 x 100 mm BEH Amide column.
f. For UPLC/MS separations of mono- and disaccharides,
typical isocratic UPLC conditions include:
2. If separating reducing sugars, please review the following
information.
3. Reducing sugars can undergo mutarotation which produces the
undesired separation of the α and β ring forms (anomers).
4. Collapsing anomers into one peak is accomplished through the
use of a combination of elevated temperature and high pH:
a. Use of 35 °C with high pH (0.2% triethylamine (TEA) or
0.1% ammonium hydroxide (NH4OH)) and/or
b. Use of >80 °C with 0.05% TEA high temperature (>80 °C)
5. When separating reducing sugars (e.g., fructose, glucose,
maltose, lactose, arabinose, glyceraldehyde, etc.) please pay
attention to the following suggestions. Failure to do so will
result in the appearance of split peaks (anomer separation) for
these analytes:
i. 75% ACN with 0.1% NH
OH, 35 °C, 0.13 mL/min,
4
2.1 x 50 mm BEH Amide column.
g. For UPLC/MS separations of more complex sugar mixtures
(e.g., polysaccharides), typical gradient UPLC conditions
include (add NH
OH modifier to both mobile phases A
4
and B):
i. Gradient going from 75% to 45% ACN with 0.1% NH4OH
in 10 min, 35 °C, 0.2 mL/min, 2.1 x 100 mm BEH Amide
column.
6. More complex sample mixtures may require the use of gradient
conditions and/or longer UPLC column lengths.
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7. If acetone is used in one or more mobile phases, do not use
acetone as a sample diluent or needle wash solvent. Refer to
injection solvents section for sample diluent recommendations
and miscellaneous tip (#3) for needle wash solvent/purge
solvent recommendations.
8. Typical sample preparation suggestions for samples that contain
sugars/saccharides/carbohydrates:
a. Liquid Samples
i. Dilute with 50:50 ACN/H2O
ii. Filter using 0.45 µm or 0.22 µm syringe filter
(if necessary)
b. b. Solid Samples
i. Weigh out sample (~3 g) into 50 mL centrifuge tube
ii. Add 25 mL of 50:50 ACN/H
O and homogenize
2
(mechanically)
iii. Centrifuge at 3200 rpm for 30 minutes
iv. Collect supernatant and filter using 0.45 µm or
0.22 µm syringe filter (if necessary)
c. Depending on sample and/or analyte concentrations,
additional sample dilutions may be necessary.
d. More complex samples and/or lower analyte
concentrations may require additional sample
preparation steps and/or procedures such as solid phase
extraction (SPE).
e. Consider VanGuard BEH Amide pre-columns for UPLC
column protection.
Waters, The Science of W hat’s Possible, ACQUITY UPLC, Oasis, Sep-Pak, Empower, MassLynx, Atlantis, and UPLC are registered
tradmarks of Waters Corporation. eCord and VanGuard are trademarks of Waters Corporation. All other trademarks are the property of
their respective owners.
©2014 Waters Corporation. Produced in the U.S.A. November 2014 Rev H 715001371 KP-PDF
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
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