Waters Atlantis Premier BEH C18 AX Columns Guide

1
[ CARE AND USE MANUAL ]
CONTENTS
I. INTRODUCTION
II. GETTING STARTED
a. eCord Installation
b. Column Installation
c. Column Equilibration
d. Initial Column Efficiency Determination
III. COLUMN USE
a. Sample Preparation
b. pH Range
c. Solvents
d. Pressure
e. Temperature
f. Replacing the VanGuard FIT Cartridge
IV. COLUMN CLEANING, REGENERATION,
AND STORAGE
a. Cleaning and Regeneration
b. Storage
V. eCORD INTELLIGENT CHIP TECHNOLOGY
a. Introduction
b. Installation
c. Manufacturing Information
d. Column Use Information
VI. TIPS AND TRICKS
VII. CAUTIONARY NOTE
I. INTRODUCTION
Thank you for choosing a Waters™ Atlantis™ Premier
BEH C18 AX Column. The Atlantis Premier BEH C18 AX
Column packing material is manufactured in a cGMP,
ISO 9001 certified plant using ultra-pure reagents. Each
batch is tested chromatographically 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 in the column box or on the
eCord™ Intelligent Chip.
The Atlantis Premier BEH C18 AX Column is offered with
or without a VanGuard™ Fully Integrated Technology [FIT]
Cartridge. To address the desire to extend the operating
lifetimes of analytical columns, the VanGuard FIT Cartridge
is designed to prevent the non-desired introduction of sample
matrix or particulates onto the column without degrading
the separation. The VanGuard FIT Cartridge can be easily
replaced to restore separation performance and extend the
analytical column’s lifetime.
The Atlantis Premier BEH C18 AX Column utilizes MaxPeak™
High Performance Surfaces, which is an innovative technology
designed to increase analyte recovery, sensitivity, and
reproducibility by minimizing analyte/surface interactions
that can lead to sample losses.
Atlantis Premier BEH C18 AX Columns

2Atlantis Premier BEH C18 AX Columns
[ CARE AND USE MANUAL ]
II. GETTING STARTED
A Certificate of Analysis and Performance Test Chromatogram
are available with each Atlantis Premier BEH C18 AX Column
in the column box or located on the column’s eCord Intelligent
Chip. The Certificate of Analysis is specific to each batch of
packing material and includes the 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 the batch number, column serial number, USP
tangent efficiency, USP tailing factor, retention factor, and
chromatographic conditions. These data should be stored for
future reference. For those not able to access the information
on the eCord Intelligent Chip, the Certificate of Analysis and
Performance Test Chromatogram are available on request at
www.waters.com/coa.
a. eCord installation
(May not be available for all column configurations)
The eCord Intelligent Chip button is designed for use on
ACQUITY™ UPLC™ and ACQUITY Arc™ Systems and should
be attached to the side of the instruments’ column heater
module. The eCord button is magnetized and does not require
specific orientation. For more information on eCord Intelligent
Chip functionality, go to section V in this care and use manual.
b. Column installation
(with or without a VanGuard FIT Cartridge)
Note: Prior to handling Atlantis Premier BEH C18 AX Columns
and any chemical, consult with your safety department and/or
local regulations on the use of proper protective equipment.
Atlantis Premier BEH C18 AX Columns are shipped in 100%
acetonitrile. The flow rates given in the procedure below are
for 2.1 mm I.D. columns. They should be multiplied by 4.8 for
4.6 mm I.D. columns.
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 five 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.
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.
c. Column equilibration
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.
Note: These columns may require longer initial equilibration times
than conventional reversed-phase columns.
Table 1. Empty Column Volumes in mL
(multiply by 10 for flush solvent volumes)
Column length (mm) Internal diameter
2.1 mm 4.6 mm
30 0.10 –
50 0.17 0.83
75 0.26 1.25
100 0.35 1.66
150 0.52 2.49
250 –4.15
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.)

3Atlantis Premier BEH C18 AX Columns
[ CARE AND USE MANUAL ]
d. 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. The analyte mixture as found on the “Performance Test
Chromatogram” that 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 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.
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 observed
due to differences in the quality of the connections,
operating environment, system electronics, reagent
quality, column condition, and operator technique.
III. COLUMN USE
To ensure the continued high performance of Atlantis Premier
BEH C18 AX 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 solvent 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 has been installed.
3. If the sample is not dissolved in the mobile phase, ensure
that the sample, solvent, and mobile phases are miscible 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 material does not dissolve
in the solvent. Contact the membrane manufacturer with
solvent compatibility questions. Alternatively, consider
centrifugation for 20 minutes at 8000 rpm, followed by the
transfer of the supernatant liquid to an appropriate vial.
b. pH range
The recommended operating pH range for Atlantis Premier
BEH C18 AX Columns is 2–10. A listing of commonly used
buffers and additives is provided in Table 2. Note that
the column lifetime varies depending upon the operating
temperature, type, and concentration of buffer.

4Atlantis Premier BEH C18 AX Columns
[ CARE AND USE MANUAL ]
Table 2. Buffer Recommendations for Use with Atlantis Premier BEH C18 AX Columns
Additive/Buffer pKaBuffer
range Volatility Used for Mass Spec Comments
TFA 0.3 —Volatile Yes Ion pair additive, can suppress MS signal.
Acetic acid 4.76 —Volatile Yes Maximum buffering obtained when
used with ammonium acetate salt.
Formic acid 3.75 —Volatile Yes Maximum buffering obtained when
used with ammonium formate salt.
Acetate (NH4CH3CO2)4.76 3.76–5.76 Volatile Yes Note that sodium or potassium salts
are not volatile.
Formate (NH4HCO2)3.75 2.75–4.75 Volatile Yes 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 6.2–8.2 Non-volatile No 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.
Ammonia (NH4OH) 9.2 8.2–10.2 Volatile Yes For MS work keep source >150 ˚C.
Ammonium
Bicarbonate 1
Ammonium
Bicarbonate 2
6.4
10.3
5.4–7.4
9.3–11.3
Volatile
Volatile
Yes
Yes
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)
Ammonium (Acetate) 9.2 8.2–10.2 Volatile Yes For MS, use in the 1-30 mM range.
Ammonium (Formate) 9.2 8.2–10.2 Volatile Yes For MS, use in the 1-30 mM range.
Borate 9.2 8.2–10.2 Non-volatile No Reduce temperature/concentration and use a
guard column to maximize lifetime.
CAPSO 9.7 8.7–10.7 Non-volatile No Zwitterionic buffer, compatible with acetonitrile.
Low odor.
Glycine 2.4, 9.8 8.8–10.8 Non-volatile No Zwitterionic buffer, can give longer lifetimes
than borate buffer.
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. Solvents containing
suspended particulate materials will generally clog the inlet
frit of the column. This will result in higher operating pressure
and poorer performance.
d. Pressure
2.1 mm I. D. Atlantis Premier BEH C18 AX 1.7 µm and 2.5 µm
columns can tolerate operating pressures up to 18000 psi
(1241 bar or 124 MPa). The 4.6 mm i.d. columns containing
2.5 µm and 5 µm particles can tolerate pressures up to
10,000 psi (700 bar or 70 MPa).
Note: Working at the extremes of pressure, pH and/or
temperature may result in shorter column lifetimes.
e. Temperature
Temperatures between 4 °C – 60 °C are recommended for
operating Atlantis Premier BEH C18 AX Columns to enhance
selectivity, lower solvent viscosity, and increase mass
transfer rates. When operating near the pH limits, lower
operating temperatures are recommended for longer column
lifetime. Working at higher temperatures (e.g., >60 °C) may
result in shorter column lifetimes.
f. Replacing the VanGuard FIT Cartridge
When desired, the VanGuard FIT Cartridge can be replaced
on the Atlantis Premier BEH C18 AX Columns using two 3/8”
wrenches. Simply apply the wrenches to the flats on the guard
and column end nut and turn in a counter clockwise direction
(see Figure 1). This will allow the VanGuard FIT Cartridge to be
removed and appropriately discarded when following good
laboratory practices.

5Atlantis Premier BEH C18 AX Columns
[ CARE AND USE MANUAL ]
A new VanGuard FIT Cartridge can now be used to replace the
discarded one. Extra cartridges can be obtained separately
as needed. Hand-tighten the new cartridge in a clockwise
direction, then tighten using two 3/8” wrenches. Proper
sealing should not require more than a 1/4 turn past the hand-
tightened position.
IV. COLUMN CLEANING, REGENERATION,
AND STORAGE
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
contaminants. 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 what
you believe is contaminating the column (see Table 3). Flush
columns with 20-column volumes of solvent. Increasing column
temperature increases cleaning efficiency. If the column
performance is poor after regenerating and cleaning, call
your local Waters office for additional support.
b. Column storage
Do not store columns in buffered eluents. If the mobile phase
contained a buffer salt, flush Atlantis Premier BEH C18 AX
Columns with 10-column volumes of HPLC grade water (see
Table 1) and then with 10-column volumes of 100% acetonitrile
prior to storage. Failure to perform this intermediate step
could result in precipitation of the buffer salt in the column
when 100% acetonitrile is introduced. Completely seal column
to avoid evaporation and drying out of the bed.
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.
Flats used to tighten
guard to column
Figure 1. Recommended 3/8” wrench placement to remove the VanGuard
FIT Cartridge from the Atlantis Premier BEH C18 AX Column.
Table 3. Column Cleaning Instructions
Polar contaminants Non-polar contaminants** Proteinaceous contaminants
1. Water 1.Isopropanol (or an appropriate
isopropanol/ water mixture*)
Option 1: Inject repeated aliquots of
dimethylsulfoxide (DMSO)
2. Methanol 2. Tetrahydrofuran (THF) 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)
3. Tetrahydrofuran (THF) 3. Dichloromethane
4. Methanol 4. Hexane
5. Water
5. Isopropanol (followed by
an appropriate isopropanol/
water mixture*)
Option 3: Flush column with 7 M guanidine hydrochloride,
or 7 M urea
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, using solvents such as THF or hexane should only be considered when
the column cannot be cleaned by using neat, reversed-phase organic solvents such as acetonitrile. When using THF and/or hexane, reduce the flow rate, use a low
operating temperature, and limit the time to avoid damage to the system.

6Atlantis Premier BEH C18 AX Columns
[ CARE AND USE MANUAL ]
V. eCORD INTELLIGENT CHIP TECHNOLOGY
(Not available for all column configurations)
a. Introduction
The eCord Intelligent Chip stores the history of a column’s
performance throughout its lifetime. The eCord is permanently
attached to the column to ensure the column’s performance
history is maintained if the column is moved from one
instrument to another.
At the time of manufacture, tracking and quality control
information is downloaded to the eCord. Storing this information
on the chip eliminates the need for a paper Certificate
of Analysis. Once the column is installed, the software will
automatically download key parameters into a column history
file stored on the chip. In this section, we explain how the
eCord provides a solution for easily tracking the history of the
column, reduces the frustration of paperwork trails, and gives
reassurance that a well performing column is installed.
b. Installation
Install the column into the column heater and 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 the ACQUITY™ Console,
allowing access to column information.
c. Manufacturing information
The eCord Intelligent Chip retains the results for the
Performance Test Chromatogram and the Certificate of
Analysis. In addition, the chromatographic test conditions
used to determine the results are given. They include mobile
phases, analytes, and system parameters used.
d. Column use information
Figure 2. Waters eCord Intelligent Chip.
eCord Intelligent Chip
Figure 3. eCord inserted into side of column heater.
eCord inserted into side of column heater
The eCord Intelligent Chip stores 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.

7Atlantis Premier BEH C18 AX Columns
[ CARE AND USE MANUAL ]
VI. START UP: TIPS AND TRICKS FOR SUCCESS
Atlantis Premier BEH C18 AX Columns are reversed-phase (RP)
columns. They differ from other C18 bonded, reversed-phase
columns by utilizing an additional positive charge on the
surface of the stationary phase. This surface charge allows
you to utilize another interaction mechanism: anion exchange
(AX) in addition to hydrophobic interaction to manipulate
selectivity and retention.
The surface charge of the Atlantis Premier BEH C18 AX
stationary phase is influenced by the pH of the mobile phase.
The anion-exchange groups of this stationary phase are
ionized from pH 2–8.5. Analytes that are negatively charged will
have increased retention when operating in this pH window.
Conversely, if the analyte is positively charged, the retention
could be reduced due to ionic repulsion. The following tips
on mobile phase provide guidance when using the Atlantis
Premier BEH C18 AX mixed-mode columns.
METHOD DEVELOPMENT OVERVIEW
To optimize chromatographic methods, mobile phase buffer
concentration, pH, and organic modifier are three key
variables that can be adjusted independently or concurrently.
A. Buffer concentration
An important mobile phase variable is the concentration of the
buffer, which strongly affects the ion-exchange mechanism.
Figure 4 shows the separation of a polar mixture using a
series of mobile phases containing pH 3 ammonium formate
at concentrations ranging from 5-20 mM. Two of the analytes,
adenosine 5’-monophosphate (AMP) and procainamide, show
a strong dependence of retention on buffer concentration,
with opposite trends. The retention factor of negatively
charged AMP decreases with increasing buffer concentration,
due to the formate anions displacing the AMP from the AX
sites on the stationary phase. In contrast, the retention factor
of positively charged procainamide increases with increasing
buffer concentration. This is attributed to a weakening of
the ionic repulsion of protonated procainamide as the buffer
concentration increases. Figure 5 shows retention factors vs
buffer concentration for the three charged analytes.
An increase in buffer concentration can create the
following results:
1. Retention decreases for negatively charged molecules
2. Retention increases for positively charged molecules
3. Minimal effect for neutral molecules
Figure 4. Separation of acidic, basic, and neutral compounds varying
buffer concentration.
AU
0.000
0.035
0.070
0.105
AU
0.000
0.035
0.070
0.105
AU
0.000
0.035
0.070
0.105
0.01.53.04.56.07.59.010.512.0 min
1
1
1
2
2
2
3
3
3
4
4
4
5
5
5
6
6
6
5 mM Ammonium Formate pH 3.00 aq
10 mM Ammonium Formate pH 3.00 aq
20 mM Ammonium Formate pH 3.00 aq
1. Thiourea
2. 5-Fluorouracil
3. Nicotinamide
4. Procainamide
5. Resorcinol
6.Adenosine 5'-monophosphate
Figure 5. The impact of buffer concentration on the retention of positively
and negatively charged compounds.
0
2
4
6
8
10
12
14
16
18
01020304050
Retention factor
mM Ammonium Formate pH 3.00 aq
Procainamide
Adenosine 5'-monophosphate
Nicotinamide

[ CARE AND USE MANUAL ]
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
[ CARE AND USE MANUAL ]
Waters, Atlantis, eCord, ACQUITY, UPLC, ACQUITY Arc, MaxPeak , Oasis, and Sep-Pak are trademarks
of Waters Corporation. All other trademarks are the property of their respective owners.
©2021 Waters Corporation. Produced in the U.S.A. May 2021 720006576EN Rev A IH-PDF
Research only. Not for IVD use.
B. Mobile phase pH
Although pH has little affect on the retention of neutral
molecules, it does affect the retention of ionizable molecules
significantly. For example, with a pH decrease, molecules
containing carboxyl groups may become less negatively
charged, giving rise to decreased ion-exchange retention.
Knowing the ionization state of the analyte as a function of pH
will help determine the optimal mobile phase pH (see Table 2
for buffer and mobile phase additive considerations). Figure 6
shows the ionization states for acids and bases with different
pKavalues as a function of pH. The anion-exchange groups
on the stationary phase are active from pH 2-8.5, and will
provide increased retention for negatively charged analytes in
this pH range due to interactions with the positively charged
stationary phase.
Figure 6. Ionization states of acids and bases with different pKavalues as
a function of pH.
2345678910
pH
Bases: pKa< 7
111
Positively charged
Negatively charged
Neutral
Anion exchange region
Bases: pKa7–9
Bases: pKa> 9
Acids: pKa> 4
Acids: pKa2–4
Acids: pKa< 2
Neutrals
C. Organic modifier
Hydrophobic retention is markedly affected by organic
modifier concentration in the mobile phase. In general, all
types of molecules (acids, bases, and neutrals) are less
retained on this column with increased organic content in
the mobile phase, when keeping other conditions constant
(e.g., ionic strength, pH, temperature, etc).
D. Isocratic vs. gradient
For many applications that involve small numbers of analytes,
it is usually easier to develop an isocratic method on an Atlantis
Premier BEH C18 AX Column versus a gradient method. For
more complicated separations, such as one that involves a
mixture of molecules with different types and numbers of
charged groups, as well as different hydrophobicities, a
gradient method could be advantageous. Gradients of organic
solvent concentration, buffer concentration and/or buffer pH
are all options for optimizing separations for Atlantis Premier
BEH C18 AX Columns.
VII. CAUTIONARY NOTE
Depending on the user’s application, these products may
be classified as hazardous following their use, and as such
are intended to be used by professional laboratory personnel
trained in the competent handling of such materials.
Responsibility for the safe use and disposal of products rests
entirely with the purchaser and user. The Safety Data Sheet
(SDS) for this product is available at www.waters.com/sds.