[ CARE AND USE MANUAL ]
Atlantis Premier BEH C18 AX Columns
CONTENTS I. INTRODUCTION
Thank you for choosing a Waters™ Atlantis™ Premier
I. INTRODUCTION
BEH C18 AX Column. The Atlantis Premier BEH C18 AX
II. GETTING STARTED Column packing material is manufactured in a cGMP,
a. eCord Installation ISO 9001 certified plant using ultra-pure reagents. Each
b. Column Installation batch is tested chromatographically and the results are
c. Column Equilibration held to narrow specification ranges to assure excellent,
d. Initial Column Efficiency Determination reproducible performance. Every column is individually
tested, and a Performance Chromatogram and Certificate
III. COLUMN USE
of Batch Analysis are provided in the column box or on the
a. Sample Preparation
eCord™ Intelligent Chip.
b. pH Range
c. Solvents The Atlantis Premier BEH C18 AX Column is offered with
d. Pressure or without a VanGuard™ Fully Integrated Technology [FIT]
e. Temperature Cartridge. To address the desire to extend the operating
f. Replacing the VanGuard FIT Cartridge lifetimes of analytical columns, the VanGuard FIT Cartridge
is designed to prevent the non-desired introduction of sample
IV. COLUMN CLEANING, REGENERATION, matrix or particulates onto the column without degrading
AND STORAGE the separation. The VanGuard FIT Cartridge can be easily
a. Cleaning and Regeneration replaced to restore separation performance and extend the
b. Storage analytical column’s lifetime.
V. eCORD INTELLIGENT CHIP TECHNOLOGY The Atlantis Premier BEH C18 AX Column utilizes MaxPeak™
a. Introduction High Performance Surfaces, which is an innovative technology
b. Installation designed to increase analyte recovery, sensitivity, and
c. Manufacturing Information reproducibility by minimizing analyte/surface interactions
d. Column Use Information that can lead to sample losses.
VI. TIPS AND TRICKS
VII. CAUTIONARY NOTE
1
[ CARE AND USE MANUAL ]
II. GETTING STARTED 3. When the mobile phase is flowing freely from the column
A Certificate of Analysis and Performance Test Chromatogram outlet, stop the flow and attach the column outlet to the
are available with each Atlantis Premier BEH C18 AX Column detector. This prevents entry of air into the detection system.
in the column box or located on the column’s eCord Intelligent 4. Gradually increase the flow rate as described in Step 2.
Chip. The Certificate of Analysis is specific to each batch of
5. Once a steady backpressure and baseline have been
packing material and includes the batch number, analysis
achieved, proceed to the next section.
of unbonded particles, analysis of bonded particles, and
chromatographic results and conditions. The Performance
c. Column equilibration
Test Chromatogram is specific to each individual column
It is important to ensure mobile-phase compatibility before
and contains the batch number, column serial number, USP
changing to a different mobile-phase system. Equilibrate the
tangent efficiency, USP tailing factor, retention factor, and
column with a minimum of 10-column volumes of the mobile
chromatographic conditions. These data should be stored for
phase to be used (refer to Table 1 for a list of column volumes).
future reference. For those not able to access the information
The column may be considered thermally equilibrated once a
on the eCord Intelligent Chip, the Certificate of Analysis and
constant backpressure is achieved.
Performance Test Chromatogram are available on request at
www.waters.com/coa. Note: These columns may require longer initial equilibration times
than conventional reversed-phase columns.
a. eCord installation
(May not be available for all column configurations) Table 1. Empty Column Volumes in mL
The eCord Intelligent Chip button is designed for use on (multiply by 10 for flush solvent volumes)
ACQUITY™ UPLC™ and ACQUITY Arc™ Systems and should Internal diameter
Column length (mm)
be attached to the side of the instruments’ column heater 2.1 mm 4.6 mm
module. The eCord button is magnetized and does not require 30 0.10 –
specific orientation. For more information on eCord Intelligent 50 0.17 0.83
Chip functionality, go to section V in this care and use manual. 75 0.26 1.25
100 0.35 1.66
b. Column installation
150 0.52 2.49
(with or without a VanGuard FIT Cartridge)
250 – 4.15
Note: Prior to handling Atlantis Premier BEH C18 AX Columns
and any chemical, consult with your safety department and/or To avoid precipitating mobile-phase buffers on your column
local regulations on the use of proper protective equipment. or in your system, flush the column with five column volumes
of a water/organic solvent mixture, using the same or lower
Atlantis Premier BEH C18 AX Columns are shipped in 100%
solvent content as in the desired buffered mobile phase.
acetonitrile. The flow rates given in the procedure below are
(For example, flush the column and system with 60% methanol
for 2.1 mm I.D. columns. They should be multiplied by 4.8 for
in water prior to introducing 60% methanol/40% buffer
4.6 mm I.D. columns.
mobile phase.)
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.
Atlantis Premier BEH C18 AX Columns 2
[ CARE AND USE MANUAL ]
d. Initial column efficiency determination III. COLUMN USE
1. Perform an efficiency test on the column before using it. To ensure the continued high performance of Atlantis Premier
This test may consist of: BEH C18 AX Columns, follow these guidelines:
a. An analyte test mixture that is commonly used in your
laboratory, and/or a. Sample preparation
1. Sample impurities often contribute to column
b. The analyte mixture as found on the “Performance Test
contamination. One option to avoid this is to use Oasis™
Chromatogram” that accompanied your column.
Solid-Phase Extraction Cartridges/Columns or Sep-Pak™
Note: If (b) is performed, the isocratic efficiencies Cartridges of the appropriate chemistry to clean up the
measured in your laboratory may be less than those given sample before analysis. For more information,
on the Waters “Performance Test Chromatogram.” This is visit www.waters.com/sampleprep
normal. The Waters isocratic column testing systems have
2. It is preferable to prepare the sample in the operating mobile
been modified to achieve extremely low system volumes.
phase or a solvent that is weaker than the mobile phase
This presents a more challenging test of how well the
for the best peak shape and sensitivity. Acetone should
column was packed. This guarantees the highest quality
not be used as a sample solvent/diluent unless a Hexane
packed column. These special testing systems have been
Tetrahydrofuran Compatibility Kit has been installed.
modified to such an extent that they are not commercially
viable and have limited method flexibility other than 3. If the sample is not dissolved in the mobile phase, ensure
isocratic column testing. that the sample, solvent, and mobile phases are miscible to
avoid sample and/or buffer precipitation.
2. Determine the number of theoretical plates (N) and use
this value for periodic comparisons. 4. Filter sample with a 0.2 µm membrane to remove
particulates. If the sample is dissolved in a solvent that
3. Repeat the test at predetermined intervals to track column
contains an organic modifier (e.g., acetonitrile, methanol,
performance over time. Slight variations may be observed
etc.) ensure that the membrane material does not dissolve
due to differences in the quality of the connections,
in the solvent. Contact the membrane manufacturer with
operating environment, system electronics, reagent
solvent compatibility questions. Alternatively, consider
quality, column condition, and operator technique.
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.
Atlantis Premier BEH C18 AX Columns 3
[ CARE AND USE MANUAL ]
Table 2. Buffer Recommendations for Use with Atlantis Premier BEH C18 AX Columns
Buffer Used for
Additive/Buffer pKa Volatility Comments
range Mass Spec
TFA 0.3 — Volatile Yes Ion pair additive, can suppress MS signal.
Maximum buffering obtained when
Acetic acid 4.76 — Volatile Yes
used with ammonium acetate salt.
Maximum buffering obtained when
Formic acid 3.75 — Volatile Yes
used with ammonium formate salt.
Note that sodium or potassium salts
Acetate (NH4 CH3CO2) 4.76 3.76–5.76 Volatile Yes
are not volatile.
Note that sodium or potassium salts
Formate (NH4HCO2) 3.75 2.75–4.75 Volatile Yes
are not volatile.
Phosphate 1 2.15 1.15–3.15 Non-volatile No Traditional low pH buffer, good UV transparency.
Above pH 7, reduce temperature/concentration
Phosphate 2 7.2 6.2–8.2 Non-volatile No
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 (NH4 OH) 9.2 8.2–10.2 Volatile Yes For MS work keep source >150 ˚C.
Ammonium For MS work keep source >150 ˚C. Adjust pH
Bicarbonate 1 6.4 5.4–7.4 Volatile Yes with ammonium hydroxide or acetic acid.
Good buffering capacity at pH 10.
Ammonium
10.3 9.3–11.3 Volatile Yes Note: Use ammonium bicarbonate (NH4HCO3), not
Bicarbonate 2 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.
Reduce temperature/concentration and use a
Borate 9.2 8.2–10.2 Non-volatile No
guard column to maximize lifetime.
Zwitterionic buffer, compatible with acetonitrile.
CAPSO 9.7 8.7–10.7 Non-volatile No
Low odor.
Zwitterionic buffer, can give longer lifetimes
Glycine 2.4, 9.8 8.8–10.8 Non-volatile No
than borate buffer.
c. Solvents e. Temperature
To maintain maximum column performance, use high quality Temperatures between 4 °C – 60 °C are recommended for
chromatography grade solvents. Filter all aqueous buffers operating Atlantis Premier BEH C18 AX Columns to enhance
prior to use through a 0.2 µm filter. Solvents containing selectivity, lower solvent viscosity, and increase mass
suspended particulate materials will generally clog the inlet transfer rates. When operating near the pH limits, lower
frit of the column. This will result in higher operating pressure operating temperatures are recommended for longer column
and poorer performance. lifetime. Working at higher temperatures (e.g., >60 °C) may
result in shorter column lifetimes.
d. Pressure
2.1 mm I. D. Atlantis Premier BEH C18 AX 1.7 µm and 2.5 µm f. Replacing the VanGuard FIT Cartridge
columns can tolerate operating pressures up to 18000 psi When desired, the VanGuard FIT Cartridge can be replaced
(1241 bar or 124 MPa). The 4.6 mm i.d. columns containing on the Atlantis Premier BEH C18 AX Columns using two 3/8”
2.5 µm and 5 µm particles can tolerate pressures up to wrenches. Simply apply the wrenches to the flats on the guard
10,000 psi (700 bar or 70 MPa). and column end nut and turn in a counter clockwise direction
(see Figure 1). This will allow the VanGuard FIT Cartridge to be
Note: Working at the extremes of pressure, pH and/or
removed and appropriately discarded when following good
temperature may result in shorter column lifetimes.
laboratory practices.
Atlantis Premier BEH C18 AX Columns 4
[ CARE AND USE MANUAL ]
A new VanGuard FIT Cartridge can now be used to replace the IV. COLUMN CLEANING, REGENERATION,
discarded one. Extra cartridges can be obtained separately AND STORAGE
as needed. Hand-tighten the new cartridge in a clockwise
direction, then tighten using two 3/8” wrenches. Proper a. Cleaning and regeneration
sealing should not require more than a 1/4 turn past the hand- Changes in peak shape, peak splitting, shoulders on the
tightened position. 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
Flats used to tighten problem, purge the column using the following cleaning
guard to column 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
Figure 1. Recommended 3/8” wrench placement to remove the VanGuard
FIT Cartridge from the Atlantis Premier BEH C 18 AX Column. 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.
Table 3. Column Cleaning Instructions
Polar contaminants Non-polar contaminants** Proteinaceous contaminants
1.Isopropanol (or an appropriate Option 1: Inject repeated aliquots of
1. Water
isopropanol/ water mixture*) dimethylsulfoxide (DMSO)
2. Methanol 2. Tetrahydrofuran (THF) Option 2: Gradient of 10% to 90% B where:
3. Tetrahydrofuran (THF) 3. Dichloromethane A = 0.1% trifluoroacetic acid (TFA) in water
4. Methanol 4. Hexane B = 0.1% trifluoroacetic acid (TFA) in acetonitrile (CH3CN)
5. Isopropanol (followed by
Option 3: Flush column with 7 M guanidine hydrochloride,
5. Water an appropriate isopropanol/
or 7 M urea
water mixture*)
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.
Atlantis Premier BEH C18 AX Columns 5
[ CARE AND USE MANUAL ]
V. eCORD INTELLIGENT CHIP TECHNOLOGY b. Installation
(Not available for all column configurations) Install the column into the column heater and plug the eCord
into the side of the column heater. Once the eCord is inserted
a. Introduction into the column heater the identification and overall column
The eCord Intelligent Chip stores the history of a column’s usage information will be available in the ACQUITY™ Console,
performance throughout its lifetime. The eCord is permanently allowing access to column information.
attached to the column to ensure the column’s performance
history is maintained if the column is moved from one c. Manufacturing information
instrument to another. 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
eCord Intelligent Chip
Figure 2. Waters eCord Intelligent Chip.
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 The eCord Intelligent Chip stores column use data. The top
automatically download key parameters into a column history of the screen identifies the column including chemistry type,
file stored on the chip. In this section, we explain how the column dimensions, and serial number. The overall column
eCord provides a solution for easily tracking the history of the usage information includes the total number of samples,
column, reduces the frustration of paperwork trails, and gives total number of injections, total sample sets, date of first
reassurance that a well performing column is installed. 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.
eCord inserted into side of column heater
Figure 3. eCord inserted into side of column heater.
Atlantis Premier BEH C18 AX Columns 6
[ CARE AND USE MANUAL ]
VI. START UP: TIPS AND TRICKS FOR SUCCESS 1. Thiourea
2. 5-Fluorouracil
Atlantis Premier BEH C18 AX Columns are reversed-phase (RP) 3. Nicotinamide
4. Procainamide
columns. They differ from other C18 bonded, reversed-phase 5. Resorcinol
6.Adenosine 5'-monophosphate
columns by utilizing an additional positive charge on the
0.105 4
surface of the stationary phase. This surface charge allows 2
3
5 mM Ammonium Formate pH 3.00 aq
0.070
AU
you to utilize another interaction mechanism: anion exchange 0.035
1
5
6
(AX) in addition to hydrophobic interaction to manipulate 0.000
selectivity and retention. 0.105 10 mM Ammonium Formate pH 3.00 aq
2
3 4
0.070
The surface charge of the Atlantis Premier BEH C18 AX
AU
1
0.035 6 5
stationary phase is influenced by the pH of the mobile phase. 0.000
The anion-exchange groups of this stationary phase are
0.105 2 3 20 mM Ammonium Formate pH 3.00 aq
ionized from pH 2–8.5. Analytes that are negatively charged will 0.070 4
AU
have increased retention when operating in this pH window. 0.035
1 6
5
Conversely, if the analyte is positively charged, the retention 0.000
0.0 1.5 3.0 4.5 6.0 7.5 9.0 10.5 12.0 min
could be reduced due to ionic repulsion. The following tips
on mobile phase provide guidance when using the Atlantis Figure 4. Separation of acidic, basic, and neutral compounds varying
Premier BEH C18 AX mixed-mode columns. buffer concentration.
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.
18
A. Buffer concentration 16
An important mobile phase variable is the concentration of the 14
buffer, which strongly affects the ion-exchange mechanism. 12
Figure 4 shows the separation of a polar mixture using a
Retention factor
10
series of mobile phases containing pH 3 ammonium formate 8
at concentrations ranging from 5-20 mM. Two of the analytes,
6
adenosine 5’-monophosphate (AMP) and procainamide, show
4
a strong dependence of retention on buffer concentration,
with opposite trends. The retention factor of negatively 2
charged AMP decreases with increasing buffer concentration, 0
0 10 20 30 40 50
due to the formate anions displacing the AMP from the AX mM Ammonium Formate pH 3.00 aq
sites on the stationary phase. In contrast, the retention factor Procainamide
of positively charged procainamide increases with increasing Adenosine 5'-monophosphate
buffer concentration. This is attributed to a weakening of
Nicotinamide
the ionic repulsion of protonated procainamide as the buffer
concentration increases. Figure 5 shows retention factors vs
Figure 5. The impact of buffer concentration on the retention of positively
buffer concentration for the three charged analytes. and negatively charged compounds.
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
Atlantis Premier BEH C18 AX Columns 7
[ CARE AND USE MANUAL ]
B. Mobile phase pH C. Organic modifier
Although pH has little affect on the retention of neutral Hydrophobic retention is markedly affected by organic
molecules, it does affect the retention of ionizable molecules modifier concentration in the mobile phase. In general, all
significantly. For example, with a pH decrease, molecules types of molecules (acids, bases, and neutrals) are less
containing carboxyl groups may become less negatively retained on this column with increased organic content in
charged, giving rise to decreased ion-exchange retention. the mobile phase, when keeping other conditions constant
(e.g., ionic strength, pH, temperature, etc).
Knowing the ionization state of the analyte as a function of pH
will help determine the optimal mobile phase pH (see Table 2
D. Isocratic vs. gradient
for buffer and mobile phase additive considerations). Figure 6
For many applications that involve small numbers of analytes,
shows the ionization states for acids and bases with different
it is usually easier to develop an isocratic method on an Atlantis
pKa values as a function of pH. The anion-exchange groups
Premier BEH C18 AX Column versus a gradient method. For
on the stationary phase are active from pH 2-8.5, and will
more complicated separations, such as one that involves a
provide increased retention for negatively charged analytes in
mixture of molecules with different types and numbers of
this pH range due to interactions with the positively charged
charged groups, as well as different hydrophobicities, a
stationary phase.
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
Bases: pKa < 7 BEH C18 AX Columns.
Bases: pKa 7–9
Bases: pKa > 9
VII. CAUTIONARY NOTE
Acids: pKa > 4
Depending on the user’s application, these products may
Acids: pKa 2–4
be classified as hazardous following their use, and as such
Acids: pKa < 2
are intended to be used by professional laboratory personnel
Neutrals trained in the competent handling of such materials.
1 2 3 4 5
pH
6 7 8 9 10 11 Responsibility for the safe use and disposal of products rests
Positively charged Neutral
entirely with the purchaser and user. The Safety Data Sheet
(SDS) for this product is available at www.waters.com/sds.
Negatively charged Anion exchange region
Figure 6. Ionization states of acids and bases with different pK a values as
a function of pH.
Research only. Not for IVD use.
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
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.
T: 1 508 478 2000
F: 1 508 872 1990
©2021 Waters Corporation. Produced in the U.S.A. May 2021 720006576EN Rev A IH-PDF www.waters.com
