HPLC COLUMNS - Continuing the Legacy of HPLC Column Performance
Continuing the Legacy of
[ HPLC COLUMNS ]
HPLC Column Performance
Creating Exceptional Chromatography
Waters® reputation is based on chromatography, but we do not create
chromatography — you do. Innovative thinking within your laboratory
creates the chromatographic methods and assays that sustain your
business. The metric of your success is monitored by the methods and
results that you produce, and the HPLC column that you choose today
needs to support your success for the future. Waters full line of
state-of-the-art, reversed-phase and HILIC HPLC columns are chosen
by scientists who understand that performance and innovation are linked
and their success depends on them
.
HPLC Columns
2
CORTECS
XBridge
XSelect
Atlantis
SunFire
Symmetry
XTerra
Waters Spherisorb
Nova-Pak
Resolve
Delta-Pak
µBondaPak/BondaPak
µPorasil/Porasil
VanGuard
Waters Analytical
Standards and Reagents
3
O
Si
O
O
O
Si
O
O
O
Si
O
O
O
Si
O
O
* Expected or approximate values.
All CORTECS Columns are available in UPLC particle sizes.
O
Si
CH
Polar Group
3
CH 3
O
Si
O
O
Solid-core particle packing materials combine a fully-porous surface layer
that has been bonded to a solid-core substrate. This combination creates a
highly efficient particle substrate that maintains chromatographic resolution
while offering the advantage of lower column back pressures.
CORTECS® 2.7 µm Solid-Core Particle Columns maximize resolution and peak
capacity for all LC separations and are optimized to extend HPLC or UHPLC
instrument performance. The innovative solid-core technology and bonding
chemistry used in CORTECS Columns helps you by:
■
■
Reducing Operational Backpressure: Lower backpressure without
sacrificing efficiency
■
■
Increasing Sensitivity: Improved signal-to-noise performance for
LC-MS applications
■
■
Simplifying Method Transfers: Compatible with a wide range
of chromatographic systems
The selection of CORTECS 2.7 µm Columns in both reversed-phase and HILIC
phases gives you the flexibility to rapidly separate a wide range of compound
classes. The improved efficiency of CORTECS 2.7 µm Solid-Core Columns
produces sharper, narrower peaks compared to columns using fully-porous
substrates and allows you to run faster flow rates to improve sample throughput.
CORTECS
C18+
C18
T3
Shield RP18
C8
Phenyl
HILIC
Ligand Type
Trifunctional C18
Trifunctional C18
Trifunctional C18
Monofunctional
Embedded Polar
Trifunctional C8
Trifunctional C6
Phenyl
None
Ligand Density*
2.4 µmol/m2
2.7 µmol/m2
1.6 µmol/m2
3.2 µmol/m2
3.4 µmol/m2
3.2 µmol/m2
N/A
Carbon Load*
5.7%
6.6%
4.7%
6.4%
4.5%
5.9%
Unbonded
End-capped
Proprietary
Proprietary
Proprietary
Proprietary
Proprietary
Proprietary
No
pH Range
2–8
2–8
2–8
2–8
2–8
2–8
1–5
Low pH Temp. Limit
45 °C
45 °C
45 °C
45 °C
45 °C
45 °C
45 °C
High pH Temp. Limit
45 °C
45 °C
45 °C
45 °C
45 °C
45 °C
45 °C
Pore Diameter
90Å
90Å
90Å
90Å
90Å
90Å
90Å
Surface Charge Modification
+
None
None
None
None
None
None
USP Classification
L1
L1
L1
L1
L7
L11
L3
4
Reduced Backpressure
CORTECS Columns reduce operational backpressure allowing you to run methods using conventional LC instrumentation without
compromising efficiency or resolution. Additionally, longer columns can be used to improve resolution for co-eluting peaks in complex
sample mixtures.
Backpressure Advantages of CORTECS 2.7 µm Columns
Flow
Rate
(mL/min)
2.1 x 50 mm
0.17
0.35
0.52
0.69
0.87
1.04
3.0 x 50 mm
0.35
0.71
1.06
1.41
1.77
2.12
4.6 x 50 mm
0.83
1.66
2.49
3.32
4.15
4.99
CORTECS 2.7 µm Columns offer a 25% reduction in operating backpressure—
without sacrificing efficiency. Data conditions—Columns: 2.1 x 50 mm;
Mobile phase: water/acetonitrile (25/75, v/v); Column temperature: 30 ˚C.
Comparative separations may not be representative in all applications.
Efficiency Advantages of CORTECS 2.7 µm Columns
Flow
Rate
(mL/min)
2.1 x 50 mm
0.17
0.35
0.52
0.69
0.87
1.04
3.0 x 50 mm
0.35
0.71
1.06
1.41
1.77
2.12
4.6 x 50 mm
0.83
1.66
2.49
3.32
4.15
4.99
CORTECS 2.7 µm Columns exhibit excellent efficiency compared to similarly-sized,
fully-porous and solid-core particle columns. Data conditions—Columns: 2.1 x 50 mm;
Mobile phase: water/acetonitrile (25/75, v/v); Column temperature: 30 ˚C; Compounds:
acenaphthene (200 µg/mL), octanophenone (100 µg/mL).
Comparative separations may not be representative in all applications.
Increased Sensitivity
Charged surface technology improves peak shape and compound loading when using low-ionic strength mobile phases such as formic acid.
The permanent low-level surface charge used during the C18+ bonding process enhances signal-to-noise performance by eliminating
ion-pairing reagents and additives that would otherwise negatively impact LC-MS applications.
Superior Peak Shape for Low Level Impurity Analysis
HPLC-UV/MS analysis of the low-ionic basic antidepressant imipramine reveals a low level impurity. Using a CORTECS C18+, 2.7 µm Column designed for use with low ionic
strength acidic mobile phases results in narrower peaks and improved signal-to-noise.
Comparative separations may not be representative in all applications.
LC Conditions
Columns:
3.0 x 50 mm
Mobile phase A:
0.1% formic acid in water
Mobile phase B:
0.1% formic acid in acetonitrile
Gradient:
25 to 35% B in 4.6 min
Flow rate:
0.8 mL/min
Column temp.:
30 °C
Detection:
254 nm and ESI+ MS
Injection volume: 10 μL
Compounds
Imipramine (0.5 mg/mL)
Impurity at 0.1% spike (0.5 μg/mL)
%
0
100
1: SIR of 1 Channel ES+
TIC (Impurity at 0.1% spike)
3.68e7
1: SIR of 1 Channel ES+
TIC (Impurity at 0.1% spike)
3.68e7
0.4
0.6
0.8
1.0
1.2
1.4
1.6 min
(1) PDA Ch1 [email protected]
Impurity at 0.1% spike
PW13.4%: 0.089 min
S/N Ratio: 180
S/N Ratio: 113
0.4
0.6
0.8
1.0
1.2
1.4
1.6 min
0.4 0.6
0.8
1.0
1.2
1.4
1.6 min
0.4
0.6
0.8
1.0
1.2
1.4
1.6 min
AU
0.0
5.0e-3
1.0e-2
1.5e-2
2.0e-2
2.5e-2
3.0e-2
3.5e-2
4.0e-2
(1) PDA Ch1 [email protected]
Imipramine
Imipramine
Impurity at 0.1% spike
PW13.4%: 0.132 min
Competitor Solid-Core C18, 2.6 µm
Pressuremax: 3250 psi
CORTECS C
18+, 2.7 µm
Pressuremax: 2750 psi
60% Increase
in S/N Ratio
35% Decrease
in Peak Width
5
www.waters.com/cortecs
The CORTECS Family
A dedicated selection of 7 phases can be used to separate a wide array of compound classes. CORTECS C18 provides a balanced retention
profile for acidic, basic, and neutral compounds. CORTECS C18+ gives the best peak shape and increased sensitivity of basic analytes when
using low ionic strength mobile phases such as formic acid. CORTECS T3 is an excellent phase to use when separating compounds of
various polarity. The lower C18 ligand density provides balance retention for both polar and nonpolar compounds and the 120Å pore
diameter allows for the use of 100% aqueous mobile phase. CORTECS C8, being less hydrophobic than a typical C18 bonded phase, is
an excellent choice for the separation of strongly hydrophobic compounds. CORTECS Phenyl offers alternative selectivity to C8 and C18
due to analyte interactions with the benzyl ring; selectivity differences for this phase are particularly noticed for aromatic compounds
especially when using methanol as the organic modifier. The CORTECS Shield RP18 also provides alternative selectivity over typical C8
and C18 bonded phases due to the embedded polar group, and is a great choice for method development, especially for phenolic and basic
compounds. The orthogonal unbonded CORTECS HILIC phase provides superior peak shape and retention of polar analytes. With particle
sizes that are compatible with HPLC, UPLC,® and UHPLC platforms, any method that you develop can be simply and seamlessly transferred
without limitation to particle size, column configuration, or instrument manufacturer.
Methods developed on 5 µm fully-porous columns can be scaled and transferred to shorter 2.7 µm columns.
For further efficiency gains and productivity improvements, sub-2-µm UPLC columns can be used, enabling
greater flexibility in method consistency when transitioning between laboratories within an organization
or to contract partners.
1
2
3
4
5
AU
0.00
0.01
0.02
AU
0.00
0.01
0.02
AU
0.00
0.01
0.02
12.0
14.0
16.0
18.0
20.0
22.0
24.0
26.0
28.0
30.0 min
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0 min
AU
0.00
0.01
0.02
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0 min
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6 min
Column: Fully-porous C
18, 5 µm, 4.6 x 150 mm
Flow rate: 1.00 mL/min
Injection volume: 8 µL
LC system: Alliance® HPLC
Column: CORTECS C
18, 2.7 µm, 4.6 x 75 mm
Flow rate: 1.85 mL/min
Injection volume: 4 µL
LC system: Alliance HPLC
Column: CORTECS C
18, 2.7 µm, 4.6 x 75 mm
Flow rate: 1.85 mL/min
Injection volume: 4 µL
LC system: ACQUITY® Arc
Column: CORTECS UPLC C
18, 1.6 µm, 2.1 x 50 mm
Flow rate: 0.65 mL/min
Injection volume: 0.6 µL
LC system: ACQUITY UPLC® H-Class
Original
method
Transfer to 2.7 µm
4x faster method
2x less solvent
Compatible with
UHPLC systems
4x less solvent
Transfer to 1.6 µm
9x faster method
15x less solvent
USP Method Transfer of Abacavir with Time and Solvent
Comparative separations may not be representative in all applications.
LC Conditions
Mobile phase A:
0.1% trifluoroacetic acid in water
Mobile phase B:
85% methanol in water
Column A:
Fully-Porous C18, 5 μm, 4.6 x 150 mm
Column B:
CORTECS C18, 2.7 μm, 4.6 x 75 mm
Column C:
CORTECS C18, 2.7 µm, 3.0 x 75 mm
Column D:
CORTECS C18, 1.6 µm, 2.1 x 50 mm
Geometrically-scaled gradients (i.e., same column volumes
per gradient step):
Column A:
5 to 30% B in 23.6 min and
30 to 90% B in 14.8 min
Column B:
5 to 30% B in 6.4 min and
30 to 90% B in 4.0 min
Column C:
5 to 30% B in 6.4 min and
30 to 90% B in 4.0 min
Column D:
5 to 30% B in 2.5 min and
30 to 90% B in 1.6 min
Compounds
1. Dicyclopropyl Abacavir
2. Abacavir
3. 1R,4R trans-Abacavir
4. o-(4-Chloro-2,5-diaminopyimidnyl)-abacavir
5. o-t-Butyl-abacavir
6
7
* Expected or approximate value.
Based on BEH Technology
Ethylene Bridged Hybrid (BEH) Technology synthesis creates particles
that ensure extreme column performance and long column lifetimes
under harsh operating conditions. The particle is prepared from two
high purity monomers: tetraethoxysilane (TEOS) and bis(triethoxysilyl)
ethane (BTEE), which results in a highly stable, pH resistant, and
mechanically strong particle.
Particle Synthesis
Tetraethoxysilane
(T EOS)
Polyethoxysilane
(BPEOS)
Bis(triethoxysilyl)ethane
(BTE E)
+
4
Si
Et O
Et O
OEt
Et O
Si
Et O
Et O
Et O
Si
OEt
OEt
OEt
Si
Et O
O
CH 2 CH2
CH2
CH2
Si
O Si
Et O
OE t
Si
O
O
OEt
Si
O
Si
O
O
Et
O
O
O
Et
Et
Et
n
* US Patents 6,686,035; 7,223,473;
7,250,214 Refer to “Ethylene-Bridged [BEH
Technology] Hybrids and Their Use in Liquid
Chromatography” whitepaper (720001159EN)
for further detail.
XBridge® BEH HPLC Columns are designed for one purpose,
to maximize your productivity. Whether your goal is to create
a quality control method or develop a leading edge LC-MS assay,
XBridge BEH Columns are designed to help you by:
■
■
Improving pH Stability: Increased column lifetime
■
■
Improving Column Reliability: Assay ruggedness
■
■
Maximizing Particle Efficiency: Unmatched peak
shape and peak capacity
With a selection of 10 general purpose and application specific
sorbents in the widest range of particle sizes available, no other
HPLC column family gives you the tools you need for the most
demanding chromatographic challenges. Whether you require
robust HPLC methods, seamless UPLC transferability, or
preparative scaling for product isolation, you can count on
the versatility of an XBridge Column.
O
Si
O
O
O
Si
O
O
O
Si
O
O
O
Si
CH
Polar Grou
p
3
CH3
O
Si
O
O
O
Si
O
O
O
Si
O
O
O
Si
CH
Polar Grou
p
3
CH3
O
Si
O
O
O
Si
O
O
O
Si
O
O
O
Si
CH
Polar Grou
p
3
CH3
O
Si
O
O
O
Si
O
O
O
Si
O
O
O
Si
CH
Polar Grou
p
3
CH3
XBridge
C18
C8
Shield RP18
Phenyl
HILIC
Amide
Peptide BEH C18, 130Å
Peptide BEH C18, 300Å
Protein BEH C4, 300Å
Oligo BEH C18
SEC
Ligand Type
Trifunctional C18
Trifunctional C8
Monofunctional
Embedded Polar
Trifunctional
Phenyl-Hexyl
Unbonded
BEH Particle
Amide
Trifunctional C18
Trifunctional C18
Monofunctional C4
Trifunctional C18
SEC
Ligand Density*
3.1 µmol/m2
3.2 µmol/m2
3.3 µmol/m2
3.0 µmol/m2
N/A
7.5 µmol/m2
3.1 µmol/m2
3.1 µmol/m2
2.4 µmol/m2
3.1 µmol/m2
N/A
Carbon Load*
18%
13%
17%
15%
Unbonded
12%
18%
12%
8%
18%
12%
End-capped
Proprietary
Proprietary
TMS
Proprietary
No
No
Proprietary
Proprietary
No
Proprietary
No
USP Classification
L1
L7
L1
L11
L3
N/A
L1
L1
L26
L1
L33
pH Range
1–12
1–12
2–11
1–12
1–9
2–1
1–12
1–12
1–10
1–12
1–8
Low pH Temp. Limit
80 °C
60 °C
50 °C
80 °C
45 °C
90 °C
80 °C
80 °C
80 °C
80 °C
45 °C
High pH Temp. Limit
60 °C
60 °C
45 °C
60 °C
45 °C
90 °C
60 °C
60 °C
50 °C
60 °C
45 °C
Pore Diameter*
130Å
130Å
130Å
130Å
130Å
130Å
130Å
300Å
300Å
130Å
125, 200, 450Å
Surface Area*
185 m2/g
185 m2/g
185 m2/g
185 m2/g
185 m2/g
185 m2/g
185 m2/g
90 m2/g
90 m2/g
185 m2/g
220 m2/g
Particle Size
2.5, 3.5, 5, 10 µm
2.5, 3.5, 5, 10 µm
2.5, 3.5, 5, 10 µm
2.5, 3.5, 5 µm
2.5, 3.5, 5 µm
2.5, 3.5, 5 µm
3.5, 5, 10 µm
3.5, 5, 10 µm
3.5 µm
2.5 µm
3.5 µm
8
Column Reliability
Much of the cost when developing a chromatographic method is associated with the rigorous testing and validation of the final
method. We understand that revalidation of your method is not an option, so we thoroughly test each batch of sorbent and final
column product to ensure that you get the most reproducible columns available. With an XBridge BEH Column, you have the
confidence that the method you develop today will be repeatable for the lifetime of your assay.
XBridge Family USP Tailing Factors
1
2
3
C
8
Phenyl
Shield RP18
C
18
Zorbax®
SB-C18
Desipramine
Amitriptyline
pH 3
pH 7
Nortriptyline
Amitriptyline
Tailing Factor (
T
f)
The combination of excellent particle and ligand stability as well as high
chromatographic efficiencies makes XBridge BEH Columns an ideal choice for
low and intermediate pH methods.
Comparative separations may not be representative in all applications.
O
Si
O
O
O
NH
2
Linker
O
Si
O
O
O
Si
O
O
O
Si
O
O
O
Si
CH
Polar Grou
p
3
CH3
O
Si
O
O
O
Si
O
O
O
Si
O
O
O
Si
CH
Polar Grou
p
3
CH3
O
Si
CH
3
CH3
O
Si
CH
3
C 4
C 4
H 9
H 9
CH3
O
Si
O
O
O
Si
O
O
O
Si
O
O
O
Si
CH
Polar Grou
p
3
CH3
Particle Efficiency
BEH Technology™ offers many advantages over conventional
silica-based particles, including the ability to control the silanol
activity with great precision. By controlling the silanol activity,
you control and reduce unwanted silanol interactions that
increase peak tailing.
XBridge
C18
C8
Shield RP18
Phenyl
HILIC
Amide
Peptide BEH C18, 130Å
Peptide BEH C18, 300Å
Protein BEH C4, 300Å
Oligo BEH C18
SEC
Ligand Type
Trifunctional C18
Trifunctional C8
Monofunctional
Embedded Polar
Trifunctional
Phenyl-Hexyl
Unbonded
BEH Particle
Amide
Trifunctional C18
Trifunctional C18
Monofunctional C4
Trifunctional C18
SEC
Ligand Density*
3.1 µmol/m2
3.2 µmol/m2
3.3 µmol/m2
3.0 µmol/m2
N/A
7.5 µmol/m2
3.1 µmol/m2
3.1 µmol/m2
2.4 µmol/m2
3.1 µmol/m2
N/A
Carbon Load*
18%
13%
17%
15%
Unbonded
12%
18%
12%
8%
18%
12%
End-capped
Proprietary
Proprietary
TMS
Proprietary
No
No
Proprietary
Proprietary
No
Proprietary
No
USP Classification
L1
L7
L1
L11
L3
N/A
L1
L1
L26
L1
L33
pH Range
1–12
1–12
2–11
1–12
1–9
2–1
1–12
1–12
1–10
1–12
1–8
Low pH Temp. Limit
80 °C
60 °C
50 °C
80 °C
45 °C
90 °C
80 °C
80 °C
80 °C
80 °C
45 °C
High pH Temp. Limit
60 °C
60 °C
45 °C
60 °C
45 °C
90 °C
60 °C
60 °C
50 °C
60 °C
45 °C
Pore Diameter*
130Å
130Å
130Å
130Å
130Å
130Å
130Å
300Å
300Å
130Å
125, 200, 450Å
Surface Area*
185 m2/g
185 m2/g
185 m2/g
185 m2/g
185 m2/g
185 m2/g
185 m2/g
90 m2/g
90 m2/g
185 m2/g
220 m2/g
Particle Size
2.5, 3.5, 5, 10 µm
2.5, 3.5, 5, 10 µm
2.5, 3.5, 5, 10 µm
2.5, 3.5, 5 µm
2.5, 3.5, 5 µm
2.5, 3.5, 5 µm
3.5, 5, 10 µm
3.5, 5, 10 µm
3.5 µm
2.5 µm
3.5 µm
9
www.waters.com/xbridge
LC Conditions
LC system:
ACQUITY UPLC with TUV Detector
Columns:
XBridge BEH C18, 5 μm, 2.1 x 150 mm
XBridge BEH C18, 3.5 μm, 2.1 x 100 mm
XBridge BEH C18, XP, 2.5 μm, 2.1 x 75 mm
ACQUITY UPLC BEH C18, 1.7 μm,
2.1 x 50 mm
Mobile phase A:
0.1% formic acid in water
Mobile phase B:
0.1% formic acid in acetonitrile
Isocratic:
95% A:5% B
Sample conc.:
25 µg/mL
Column temp.: 38 °C
Detection:
280 nm
1.7 µm – 50 mm
Injection: 1.7 µL
Flow rate: 0.6 mL/min
5 µm – 150 mm
Injection: 5.0 µL
Flow rate: 0.2 mL/min
2.5 µm – 75 mm
Injection: 2.5 µL
Flow rate: 0.5 mL/min
3.5 µm – 100 mm
Injection: 3.3 µL
Flow rate: 0.3 mL/min
Methods Transfer Using 2.5 µm Columns
All XBridge and XSelect® HPLC Columns (discussed on the next page) are offered in eXtended Performance [XP] 2.5 µm UHPLC
column formats to help you transfer methods from HPLC to UPLC instrumentation. The XP 2.5 µm columns improve the performance
of your current HPLC and UHPLC instrumentation and provide you with a pathway to gain maximum separation efficiency using
sub-2-µm ACQUITY® UPLC Technology.
Columns of different lengths and particle sizes were used to successfully reduce run times
and maintain resolution.
Scalable Separations
Accelerated High pH Stability Test of Competitive Columns
0
50
100
150
200 hours
Hours in 50 mM TEA, pH 10, 50 °C
Analyte: Acenaphthene
30
50
70
90
110
% Initial
N
5s
XBridge BEH C
18
XTerra® MS C
18
Gemini™ C
18
Luna® C18(2)
YMC™ Pro C
18
Zorbax® Extend C
18
Chromatograms, run at regular intervals during the high-pH lifetime study, verify that
86% of the original XBridge Column efficiency remains after 300 hours at pH 10 and
elevated temperature, with little change in peak shape or retention time.
pH Stability
XBridge BEH Columns have been specifically designed to
contain the most chemically-stable chromatographic sorbent
available, allowing you to explore the full benefits of a wide
pH (1–12) mobile-phase range. Chemical stability, especially
for the extremes of pH, is built into the particle during
the synthesis process and it cannot be duplicated using a
conventional silica-based bonding process. No other column
can match the chemical stability of an XBridge Column.
0.0
2.0
4.0
6.0
8.0
10.0 min
0.0
0.2
0.4
0.6
0.8
1.0
1.1 min
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0 min
10
[ FOLIO INSIDE HEADER ]
11
XSelect HPLC Columns are designed for the method development
scientist who demands the most diverse selection of sorbents to
easily separate the most difficult analyte co-elutions. XSelect
Columns are tools that are:
■
■
Designed for Selectivity: Improve your ability to separate
closely eluting peaks
■
■
Intended for Isolation and Purification: Highest analyte
mass loading available
■
■
Ideal for Rapid Method Development: Reduce the time
and cost spent developing methods
The XSelect HPLC Column family features two base particles with
a unique blend of optimized ligands to provide highly selective
chromatographic phases while maintaining the reproducibility
expected from modern high performance LC columns. With more
than 500 column configurations that combine 8 selectivity-
optimized bonded phases and 3 scalable particles sizes, XSelect
Columns are your first choice for method development.
O
Si
F
F
F
F
F
O
O
O
Si
O
O
O
Si
O
O
O
Si
O
O
O
Si
O
O
O
Si
O
O
O
Si
F
F
F
F
F
O
O
O
Si
CN
* Expected or approximate value.
Charged Surface Hybrid (CSH™) particles incorporate a low level
surface charge that improves sample loading and peak symmetry
when using low ionic strength mobile phases. The CSH particle is
the next evolution of hybrid particle technology that maintains the
mechanical and chemical stability inherent in BEH particle technology.
Unbonded BEH
Particle
Apply Controlled
Surface Charge
Bond and
Endcap
The Charged-Surface Particle
Many silica-based particles do not have the mechanical stability
to withstand the high operational pressures used with modern LC
instrumentation. High Strength Silica (HSS) is the first and only 100%
silica-based particle substrate that has been designed and tested for
mechanical stability up to 18,000 psi (1240 bar).
OEt
Si
O
O
EtO
Si
OEt
OEt
OEt
Si
OEt
EtO
EtO
EtO
Si
EtO
OEt
EtO
Polyethoxysilane
(PEOS)
Tetraethoxysilane
(TEOS)
XSelect
CSH C18
CSH
Phenyl-Hexyl
CSH
Fluoro-Phenyl
HSS T3
HSS C18
HSS C18 SB
HSS PFP
HSS CN
Ligand Type
Trifunctional
C18
Trifunctional
C6 Phenyl
Trifunctional
Propylfluoro-
phenyl
Trifunctional
C18
Trifunctional
C18
Trifunctional
C18
Trifunctional
Pentafluoro-
phenyl
Monofunctional
Cyano-propyl
Ligand Density*
2.3 µmol/m2
2.3 µmol/m2
2.3 µmol/m2
1.6 µmol/m2
3.2 µmol/m2
1.6 µmol/m2
3.2 µmol/m2
2.0 µmol/m2
Carbon Load*
15%
14%
10%
11%
15%
8%
7%
5%
End-capped
Proprietary
Proprietary
No
Proprietary
Proprietary
No
No
No
USP Classification
L1
L11
L43
L1
L1
L1
L43
L10
pH Range
1–11
1–11
1–8
2–8
1–8
2–8
2–8
2–8
Low pH Temp. Limit
80 ˚C
80 ˚C
60 ˚C
45 ˚C
45 ˚C
45 ˚C
45 ˚C
45 ˚C
High pH Temp. Limit
45 ˚C
45 ˚C
45 ˚C
45 ˚C
45 ˚C
45 ˚C
45 ˚C
45 ˚C
Pore Diameter*
130Å
130Å
130Å
100Å
100Å
100Å
100Å
100Å
Surface Area*
185 m2/g
185 m2/g
185 m2/g
230 m2/g
230 m2/g
230 m2/g
230 m2/g
230 m2/g
Particle Size
2.5, 3.5, 5, 10 µm
2.5, 3.5, 5 µm
2.5, 3.5, 5 µm
2.5, 3.5, 5 µm
2.5, 3.5, 5 µm
2.5, 3.5, 5 µm
2.5, 3.5, 5 µm
2.5, 3.5, 5 µm
12
Comparative separations may not be representative in all applications.
XSelect
CSH C18
CSH
Phenyl-Hexyl
CSH
Fluoro-Phenyl
HSS T3
HSS C18
HSS C18 SB
HSS PFP
HSS CN
Ligand Type
Trifunctional
C18
Trifunctional
C6 Phenyl
Trifunctional
Propylfluoro-
phenyl
Trifunctional
C18
Trifunctional
C18
Trifunctional
C18
Trifunctional
Pentafluoro-
phenyl
Monofunctional
Cyano-propyl
Ligand Density*
2.3 µmol/m2
2.3 µmol/m2
2.3 µmol/m2
1.6 µmol/m2
3.2 µmol/m2
1.6 µmol/m2
3.2 µmol/m2
2.0 µmol/m2
Carbon Load*
15%
14%
10%
11%
15%
8%
7%
5%
End-capped
Proprietary
Proprietary
No
Proprietary
Proprietary
No
No
No
USP Classification
L1
L11
L43
L1
L1
L1
L43
L10
pH Range
1–11
1–11
1–8
2–8
1–8
2–8
2–8
2–8
Low pH Temp. Limit
80 ˚C
80 ˚C
60 ˚C
45 ˚C
45 ˚C
45 ˚C
45 ˚C
45 ˚C
High pH Temp. Limit
45 ˚C
45 ˚C
45 ˚C
45 ˚C
45 ˚C
45 ˚C
45 ˚C
45 ˚C
Pore Diameter*
130Å
130Å
130Å
100Å
100Å
100Å
100Å
100Å
Surface Area*
185 m2/g
185 m2/g
185 m2/g
230 m2/g
230 m2/g
230 m2/g
230 m2/g
230 m2/g
Particle Size
2.5, 3.5, 5, 10 µm
2.5, 3.5, 5 µm
2.5, 3.5, 5 µm
2.5, 3.5, 5 µm
2.5, 3.5, 5 µm
2.5, 3.5, 5 µm
2.5, 3.5, 5 µm
2.5, 3.5, 5 µm
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5 min
1
1
2
2
2
3
3
4
4
5,6
7
5,6
7
7
3
4
3
4
5
6
7
1
2
3
4
5
6
7
1
2
3
4
5
6
7
5,6
1
2
3
4
3
4
5,6
7
7
HSS C
18 SB
HSS C
18
HSS T3
HSS CN
HSS PFP
CSH Fluoro-Phenyl
CSH Phenyl-Hexyl
CSH C
18
1
2
1
5 6
1
2
LC Conditions
LC system:
ACQUITY UPLC with
ACQUITY UPLC PDA Detector
Columns:
2.1 x 50 mm
Mobile phase A:
10 mM ammonium formate, pH 3.0
Mobile phase B:
Methanol
Flow rate:
0.4 mL/min
Injection volume: 1 μL
Sample diluent:
Water
Column temp.:
30 °C
Gradient:
Time
(min)
%A
%B
0.00
70
30
3.00
15
85
3.50
15
85
3.51
70
30
4.50
70
30
Detection:
260 nm
Enhanced Selectivity
Selectivity and retentivity are the most powerful tools a method developer has to
influence chromatographic behavior. The XSelect family offers a diverse range of
reversed-phase C18 columns (e.g., CSH C18, HSS C18, HSS C18 SB) for general purpose
separations; as well as columns that offer improved polar retention (T3) and greater
selectivity options (phenyl-hexyl, fluoro-phenyl, and cyano) for method development.
XSelect Columns Provide Diverse Analyte Selectivity
AU
0.00
0.02
0.04
0.06
0.08
Imipramine
Imipramine
2.0%
1.0%
0.5%
0.1%
2.0%
1.0%
0.5%
0.1%
Amitriptyline
Imipramine concentration held
constant at 0.5 mg/mL
Amitriptyline
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0 min
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0 min
ACQUITY UPLC CSH C
18
AU
0.00
0.02
0.04
0.06
0.08
Kinetex® C
18
LC Conditions
LC system:
ACQUITY UPLC H-Class with
ACQUITY UPLC PDA Detector
Columns:
2.1 x 50 mm
Mobile phase A:
Water
Mobile phase B:
Acetonitrile
Mobile phase C:
2% formic acid in water
Gradient:
Time
(min)
Flow
(mL/min) %A
%B
%C
Initial
0.6
70
25
5
2.0
0.6
60
35
5
5.0
0.6
70
25
5
Injection volume:
5 μL
Sample diluent:
Water
Sample conc.:
Imipramine: 0.5 mg/mL; amitriptyline:
as indicated (% of imipramine)
Column temp.:
40 °C
Detection:
254 nm
Isolation and Purification
High mass loading applications like compound purification, impurity profiling,
and dissolution testing demand superior column performance. For these types of
applications, column loading is limited by its inability to maintain symmetrical peak
shape. This manifests itself as severe broadening of the main compound peak, which often
overwhelms the trace impurities that you are trying to remove in the purification. XSelect
CSH Columns consistently provide narrow peaks under high loading conditions that allow
the chromatographer the ability to separate trace-level impurities or degradants giving you
more loading capacity with less time and solvent.
Maintaining Peak Shape with High Mass Loading
Observed selectivity differences for a mixture of basic analytes. Compounds: [1] aminopyrazine, [2] pindolol, [3]
quinine, [4] labetalol, [5] verapamil, [6] diltiazem, [7] amitriptyline.
T he improved mass loading of XSelect Columns permits the separation, identification, and quantification
of closely eluting impurities or degradants
.
13
Method Development and Transfer
When developing methods, skilled chromatographers realize that any method
developed using uniquely selective columns must be easily transferable across
laboratories, independent of the LC system platform used. XSelect Columns are
engineered for method development and are fully compatible with all modern
detection modes.
LC Conditions
LC system:
ACQUITY UPLC with
ACQUITY UPLC PDA Detector
Columns:
2.1 x 50 mm
Flow rate:
0.5 mL/min
Mobile phase A:
15.4 mM ammonium formate, pH 3.0
Mobile phase B:
Acetonitrile
Gradient:
5 to 90% B linear in 5 minutes
Injection volume: 5 μL
Column temp.:
30 °C
Detection:
254 nm
Compounds
1. Thiourea
2. Resorcinol
3. Metoprolol
4. 3-Nitrophenol
5. 2-Chlorobenzoic acid
6. Amitriptyline
7. Diethylphthalate
8. Fenoprofen
9. Dipropylphthalate
10. Pyrenesulfonic acid
Reproducible and Scalable Separations
3.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
4.0 min
1
2
3
4
5
6
7
8
9
10
XSelect CSH Fluoro-Phenyl, 5 µm
XSelect CSH Fluoro-Phenyl, 3.5 µm
ACQUITY UPLC CSH Fluoro-Phenyl, 1.7 µm
Reproducibility and scalability for gradient separations on 2.1 x 50 mm columns containing nine
different batches of CSH fluoro-phenyl representing three (1.7,- 3.5,- and 5-µm) particle sizes.
www.waters.com/xselect
14
15
* Expected or approximate value.
O
Si
O
O
Atlantis
T3
dC18
HILIC Silica
Ligand Density*
1.6 µmol/g
1.6 µmol/g
N/A
Carbon Load*
14%
12%
N/A
End-capped
Proprietary
Proprietary
No
USP Classification
L1
L1
L3
pH Range
2–8
3–7
1-5
Low pH Temp. Limit
45 ˚C
45 ˚C
45 ˚C
High pH Temp. Limit
45 ˚C
45 ˚C
45 ˚C
Pore Diameter*
100Å
100Å
100Å
Surface Area*
330 m2/g
330 m2/g
330 m2/g
Particle Size
3, 5, 10 µm
3, 5, 10 µm
3, 5, 10 µm
Atlantis® HPLC Columns provide exceptional performance,
versatility, and retention for polar compounds, while also
affording balanced retention for broad analyte mixtures.
Compatibility with 100% Aqueous
Mobile Phases
To maximize polar compound retention in reversed-phase
methods, it is possible to use Atlantis Reversed-phase HPLC
Columns with highly aqueous mobile phases and buffers without
the risk of pore dewetting and hydrophobic collapse of the
stationary phase.
Long Column Lifetimes Using
Low-pH Mobile Phases
Atlantis Columns resist ligand hydrolysis when using strongly
acidic mobile phases, thus maintaining method efficiency,
compound retention, and critical analyte selectivity.
0
10
20
30
40
50
60
70
80
90
20 Hour Exposure to 0.5% TFA at 60 °C
Atlantis T3 SunFire C
18
Phenomenex
®
Synergi™ Hydro-RP
Shiseido Capcell PAK
®
AQ
Phenomenex
® Synergi™ Polar-RP
Agilent Zorbax Eclipse
® Plus
Atlantis dC
18
Phenomenex
®
Aqua
YMC Hydrosphere™ C18
GL Sciences Inertsil
®
ODS-SP
YMC-Pack
™ ODS AQ
™
During this accelerated test, the columns were exposed to low pH and high
temperature conditions to determine the affect of ligand loss due to hydrolysis.
The Atlantis T3 bonding resists ligand hydrolysis to maintain analyte retention
using extremely harsh mobile-phase conditions.
% Retention Loss for Methylparaben
Polar Compound Retention
Comparative separations may not be representative in all applications.
16
Polar Compound Retention
without Ion-Pairing Reagents
Eliminating ion-pairing reagents improves detection limits,
method reproducibility, and robustness, while reducing instrument
maintenance due to harsh mobile-phase environments.
LC Conditions
LC system:
Alliance 2695 with 2487 Dual-
Wavelength Absorbance Detector
Column:
4.6 x 150 mm
Mobile phase:
10 mM ammonium formate, pH 3.0
Flow rate:
1.3 mL/min for 3 µm
Injection volume: 2.0 µL
Column temp.:
30 °C
Detection:
254 nm
Compounds
1. Thiourea
2. 5-Fluorocystine
3. Adenine
4. Guanosine-5’-monophosphate
5. Thymine
Polar Compound Retention
Separating highly polar analytes on the Atlantis T3 Column compared to
competitive brands. Scientists rely on the uncompromised peak shape and
retention that only Atlantis Columns provide.
1
2
3 4
5
1
2
4
3,5
1
2
3,4
5
1 2
4
3
5
1
2
3
4
5
0
1
2
3
4
5
6
7
8 min
Atlantis T3
Agilent Zorbax® SB-AQ
Thermo Hypersil Gold AQ™
Phenomenex ® Synergi™ Hydro-RP
Shiseido Capcell PAK® C
18 AQ
Comparative separations may not be representative in all applications.
www.waters.com/atlantis
17
* Expected or approximate value.
O
Si
O
CH 3
O
Si
O
CH 3
Isocratic Separation
LC system:
Alliance 2695 with 2487 Dual-
Wavelength Absorbance Detector
Mobile phase A: 35% 20 mM dipotassium phosphate/
20 mM monopotassium phosphate pH 7.0
Mobile phase B: 65% methanol
Wavelength:
254 nm
Flow rate:
1.0 mL/min
Injection vol.:
14 µL
Column temp.:
23 ˚C
Compounds
1. Uracil
2. Propranolol
3. Butylparaben
4. Naphthalene
0
1
2
3
4
5
6
5
10
15
20
25
30
35
40 min
TUSP – 1.26
TUSP – 1.35
TUSP – 1.79
SunFire C18
4.6 x 150 mm, 5 µm
Phenomenex® Luna® C18 (2)
4.6 x 150 mm, 5 µm
ACT® Ace® C18
4.6 x 150 mm, 5 µm
Comparative separations may not be representative in all applications.
SunFire
C8
C18
Silica
Ligand Density*
3.5 µmol/g
3.5 µmol/g
N/A
Carbon Load*
12%
16%
N/A
End-capped
Proprietary
Proprietary
No
USP Classification
L7
L1
L3
pH Range
2–8
2–8
2–8
Low pH Temp. Limit
40 ˚C
50 ˚C
55 ˚C
High pH Temp. Limit
40 ˚C
40 ˚C
45 ˚C
Pore Diameter*
100Å
100Å
100Å
Surface Area*
340 m2/g
340 m2/g
340 m2/g
Particle Size
2.5, 3.5, 5, 10 µm
2.5, 3.5, 10 µm
2.5, 3.5, 10 µm
SunFire™ Columns set the standard for state-of-the-art bonded
C18- and C8- silica HPLC columns. Benefiting from years of
research and product development, SunFire Columns represent
the best in particle and bonding expertise and deliver industry-
leading levels of chromatographic performance.
Excellent Low-pH Stability
Under low-pH mobile-phase conditions, SunFire Columns exhibit
superior column lifetimes that exceed many silica-based HPLC
column brands.
High Efficiency
A synergistic combination of particle synthesis, packing
technology, and hardware engineering is required for high
efficiency. SunFire Intelligent Speed™ (IS™) and Optimum Bed
Density (OBD™) Columns were developed specifically from
this knowledge.
Superior Peak Shapes
SunFire Columns provide symmetrical peaks for improved
resolution of acidic, neutral and basic compounds
at low and moderate pH ranges (2–8).
Peak Shape Comparison of SunFire Columns
18
www.waters.com/sunfire
0
5
10
15
20
25
30
35
40 min
Batch 112 (2005)
Batch 118 (2007)
Batch 130 (2007)
Batch 134 (2008)
Batch-to-Batch Reproducibility of SunFire Columns
This excellent reproducibility is a result of our commitment to maintaining the
tightest specifications in the HPLC column industry. SunFire Columns start with
high purity raw materials, and are produced using controlled manufacturing
processes and column packing procedures that provide today’s scientists with
the best, most reproducible HPLC columns available.
Batch-to-Batch Reproducibility
Waters is dedicated to maintain the tightest specifications in the
HPLC industry. Controlled manufacturing processes and column
packing procedures ensure that you receive the best, most
reproducible HPLC column available.
19
O
Si
CH 3
CH 3
O
Si
CH 3
CH 3
Si
CH 3
Polar Group
CH 3
O
Si
CH 3
Polar Group
CH 3
O
O
Si
CH 3
CH 3
O
Si
O
O
Symmetry
Symmetry and
SymmetryPrep
C18
Symmetry and
SymmetryPrep
C8
SymmetryShield
RP18
SymmetryShield
SymmetryPrep
RP8
Symmetry300
C18
Symmetry300
C4
Particle Size
3.5, 5, 7 µm
3.5, 5, 7 µm
3.5, 5, 7 µm
3.5, 5, 7 µm
3.5, 5 µm
3.5, 5 µm
Particle Shape
Spherical
Spherical
Spherical
Spherical
Spherical
Spherical
Pore Size
100Å
100Å
100Å
100Å
300Å
300Å
Carbon Load
19%
12%
17%
15%
8.5%
2.8%
End-capped
Proprietary
Proprietary
Proprietary
Proprietary
Proprietary
Proprietary
Symmetry® Columns are manufactured
using high purity silica and tightly
controlled manufacturing processes to
ensure that you receive a column that
exceeds the standards for HPLC column
performance. No other silica-based LC
column brand can match the column-to-
column and batch-to-batch reproducibility
of the Symmetry family. Symmetry
Columns are available in column,
cartridge, and guard formats:
■
■
Symmetry and SymmetryPrep™ Columns: Deliver maximum reproducibility
■
■
SymmetryShield™ RP18 and RP8 Columns: Provide superior peak shape
■
■
Symmetry300™ C18 and C4 Columns: Offer high recoveries of peptides and proteins
0.0
1
2
3
4
1.50
3.0
4.5
6.00
7.5
9.0
10.5
12.0
Batch 136
Batch 142
Batch 149
Batch 151
Batch 156
13.5
15.0 min
Unmatched year-to-year reproducibility.
Batch-to-Batch Reproducibility of Symmetry Columns
LC Conditions
Column:
Symmetry C18, 5 µm,
4.6 x 150 mm
Mobile phase A:
Water
Mobile phase B:
Acetonitrile
Mobile phase C:
pH 3.75; 100 mM
ammonium formate in water
Flow rate:
1.4 mL/min
Isocratic:
30% A; 60% B; 10% C
RSD’s for retention times
1. Terbinafine HCI 0.7%
2. Ibuprofen
0.8%
3. Lovastatin
0.6%
4. Simvastatin
0.7%
Symmetry Columns for Reproducibility
You can rely on a Symmetry HPLC Column for rugged and
reproducible performance. Narrow column specification ranges
minimize variation giving you the confidence that the methods
you use today will continue to be used in the future.
Injection vol.:
5.0 µL
Column temp.:
30 °C
Detection: 233nm
20
Symmetry
Symmetry and
SymmetryPrep
C18
Symmetry and
SymmetryPrep
C8
SymmetryShield
RP18
SymmetryShield
SymmetryPrep
RP8
Symmetry300
C18
Symmetry300
C4
Particle Size
3.5, 5, 7 µm
3.5, 5, 7 µm
3.5, 5, 7 µm
3.5, 5, 7 µm
3.5, 5 µm
3.5, 5 µm
Particle Shape
Spherical
Spherical
Spherical
Spherical
Spherical
Spherical
Pore Size
100Å
100Å
100Å
100Å
300Å
300Å
Carbon Load
19%
12%
17%
15%
8.5%
2.8%
End-capped
Proprietary
Proprietary
Proprietary
Proprietary
Proprietary
Proprietary
Symmetry Columns for Superior
Peak Shape
SymmetryShield Columns feature Waters’ patented Embedded Polar
Group Technology that shields the silica’s residual silanols from
highly basic analytes that improves overall peak shape. Additionally,
by placing the embedded polar group close to the silica surface,
the activity of the surface silanols is further reduced. This imparts
selectivity and retention that is different compared to the Symmetry
C18 ligand.
Unmatched year-to-year reproducibility.
Batch-to-Batch Reproducibility of Symmetry Columns
RSD’s for retention times
1. Terbinafine HCI 0.7%
2. Ibuprofen
0.8%
3. Lovastatin
0.6%
4. Simvastatin
0.7%
Embedded Polar Group Technology improves chromatographic peak shape
and selectivity.
SymmetryShield Columns Deliver Unique Selectivity
USP Tailing Factor = 1.2
SymmetryShield RP18
USP Tailing Factor = 1.9
Symmetry C18
0
5
10
15
20
25
1
2
3
4
5
7
6
0
5
10
15
20
25
1
2
3
4
5
6
7
AU
AU
30 min
30 min
LC Conditions
Columns:
SymmetryShield RP18, 5 µm, 3.9 x 150 mm
Symmetry C18, 5 µm, 3.9 x 150 mm
Mobile phase: 65% methanol; 35% 20 mM
monopotassium phosphate/
dipotassium phosphate at pH 7
Flow rate:
1.0 mL/min
Detection:
254 nm
Column temp.: 23 °C
Compounds
1.
Uracil
2. Propranolol
3. Butylparaben
4. Dipropyl phthalate
5. Naphthalene
6. Amitriptyline
7. Acenaphthene
Injection vol.:
5.0 µL
Column temp.:
30 °C
Detection: 233nm
www.waters.com/symmetry
21
XTerra® MS, Shield RP, and Phenyl Columns combine the best
properties of silica and polymeric bonded phases with patented
Hybrid Particle Technology that replaces one out of every three
silanols with a methyl group during particle synthesis. This can
only be achieved during the initial particle synthesis and the
inclusion of this methyl group is an integral part of the base
particle backbone. The result is a mechanically strong particle
that can be used for high pH separations that will improve loading
and peak shapes for basic compounds.
The Efficiency of Silica with
Stability of Polymers
The vast majority of reversed-phase HPLC separations take place
on silica-based stationary phases. Silica has long enjoyed such
attributes as high efficiency and mechanical strength. However,
XTerra
MS C18
Shield
RP18
Shield
RP8
Phenyl
Particle Size
2.5, 3.5, 5,
10 µm
3.5, 5,
10 µm
3.5, 5,
10 µm
3.5, 5 µm
Particle Shape
Spherical
Spherical
Spherical
Spherical
Pore Size
125Å
125Å
125Å
125Å
Carbon Load
15.5%
15.0%
13.5%
12.0%
End-capped
Proprietary
Proprietary
Proprietary
Proprietary
Traditional Silica Manufacturing Process
Bonded and End-capped Silica Particle
XTerra Manufacturing Process
Much more than a surface modification
Bonded and End-capped XTerra Particle
silica suffers from poor peak shape for bases and a limited pH
range. One way that chromatographers have attempted to overcome
these limitations is by turning to polymer-based stationary phases.
Polymers, however, have not enjoyed the acceptance of silica due
to poor efficiency, low mechanical strength, and unpredictable peak
elution order when transferring methods from polymeric to silica-
based columns.
Hybrid Particle Technology overcomes these limitations and
combines the best attributes of both these materials while
overcoming each material’s weaknesses. The result is a rugged
material that has high mechanical strength, high efficiency,
excellent peak shape for bases, and easy scale-up from analytical
to preparative chromatography.
Traditional Silica versus XTerra Manufacturing Process
Si
CH 3
Polar Group
CH 3
O
Si
CH 3
Polar Group
CH 3
O
O
Si
C H 3
C H
2
C H
C H
3
O
■
Highest, most
Homogenous Coverage
■
1/3 Less Silanols
■
Superior Peak Shape
■
pH Range 1-12
■
Lowest Surface Coverage
■
Poor Peak Shape
■
pH Range 2–8
Unbonded
XTerra
Particle
Methypolyethoxysilane
(MPEOS)
Tetraethoxysilane
(TEOS)
Methyltriethoxysilane
(MTEOS)
Unbonded
Silica Particle
Polythoxysilane
(PEOS)
Tetraethoxysilane
(TEOS)
* Expected or approximate value.
OH
Si
O
O
O
O
Si
O
OH
O
Si
O
OH
O
Si
O
O
O
Si
O
Si
O
OH
Si
OH
O
Si
O
O
Si
Si
Si OH
O
O
Si O
O
Si
Si
Si
Si
Si
Si
Si
HO
Si
Si
O
O
Si
O
O
Si O
HO
O
Si
Si
Si
Si
O
HO
O
Si
Si
O
HO
O
Si
O
HO
HO
Si
Si
O
Si
O
OH
O
Si
Si
OH
O
O
O
Si
OH
O
O
Si
Si
Si
OH
O
O
Si
O
O
Si OH
O
Si
Si
Si
Si
OH
Si
O
O
O
Si
O
O
Si
O
OH
O
Si O
O
O
Si
O
Si
O
OH
Si
O
Si
O
O
O
Si
Si
Si
O
O
Si O
O
Si
Si
Si
Si
Si
Si
Si
HO
Si
O
Si
O
O
Si
O
O
Si O
HO O
Si
Si
Si
Si
O
O
Si
Si
O
HO
O
Si
O
HO
Si
Si
O
Si
O
OH
O
Si
Si
OH
O
O
Si
O
O
Si
Si
Si OH
O
O
Si
O
O
Si OH
O
Si
Si
Si
Si
H3C
H3C
CH3
H3C
H3C
H3C
CH3
CH3
H3C
CH3
CH3
CH3
H3C
H3C
CH3
CH3
22
Silica Separations at Polymer pH
0
0.1
0.2
0.3
0.4
0.5
1
3
2
4
6
5
10
20
AU
6
5
4
3
2
1
AU
0
0.4
0.8
1.2
10
20
min
min
LC Conditions
LC system:
Alliance 2690 with 996 PDA Detector
Mobile phase A: 20 mM ammonium hydroxide, pH 10.7
Mobile phase B: Acetonitrile
Flow rate:
3 mL/min
Gradient:
Time Profile
(min) %A %B
0.0
70 30
25.0 40 60
Injection vol.:
5 µL
Column temp.:
Ambient
Detection:
220 nm
Compounds
1.
Codeine
2. Yohimbine
3. Thebaine
4. Cocaine
5. Resperine
6. Methadone
XTerra Shield RP18: 4.6 x 150 mm
pH 10.7
Polymer Column: 4.1 x 150 mm
pH 10.7
Traditional Silica versus XTerra Manufacturing Process
0
0.1
0.2
0.3
0.4
0.5
1
3
2
4
6
5
10
20
AU
6
5
4
3
2
1
AU
0
0.4
0.8
1.2
10
20
min
min
www.waters.com/xterra
23
WATERS SPHERISORB COLUMNS
Waters Spherisorb® Columns are one of the most widely referenced HPLC columns in the scientific literature. There are over
2,000 analytical abstracts published using Waters Spherisorb Columns, providing a tremendous range of validated methods and
applications to assist in your method development process.
Waters Spherisorb Columns are produced in a wide range of particle sizes (3-, 5-, and 10- µm) and bonded phases to meet your
chromatographic needs. In addition, Waters Spherisorb Columns’ high quality bonded phases give many different and unique
separation selectivities. Waters Spherisorb Analytical Columns are supplied with industry-standard Parker-style column
end fittings.
Ligand Type
Phenyl
CN (Nitrile)
OD/CN
W (Silica)
SCX
SAX
Particle Size
3, 5, 10 μm
3, 5, 10 μm
5 μm
3, 5, 10 μm
5, 10 μm
5, 10 μm
Surface Area
220 m2/g
220 m2/g
220 m2/g
220 m2/g
220 m2/g
220 m2/g
Particle Shape
Spherical
Spherical
Spherical
Spherical
Spherical
Spherical
Pore Size
80Å
80Å
80Å
80Å
80Å
80Å
Carbon Load
2.5%
3.1%
5%
N/A
4%
4%
Ligand Coverage
2.72 µmol/m2
3.29 µmol/m2
1.15 µmol/m2
N/A
N/A
N/A
End-capped
No
No
Proprietary
No
No
No
Spherisorb
Ligand Type
ODS2 (C18)
ODS1 (C18)
ODSB (C18)
C8
C6
C1
NH2 (Amino)
Particle Size
3, 5, 10 μm
3, 5, 10 μm
5 μm
3, 5, 10 μm
3, 5, 10 μm
3, 5, 10 μm
3, 5, 10 μm
Surface Area
220 m2/g
220 m2/g
220 m2/g
220 m2/g
220 m2/g
220 m2/g
220 m2/g
Particle Shape
Spherical
Spherical
Spherical
Spherical
Spherical
Spherical
Spherical
Pore Size
80Å
80Å
80Å
80Å
80Å
80Å
80Å
Carbon Load
11.5%
6.2%
11.5%
7.75%
4.7%
2.15%
1.9%
Ligand Coverage
2.98 µmol/m2
1.49 µmol/m2
2.98 µmol/m2
3.12 µmol/m2
3.36 µmol/m2
2.97 µmol/m2
2.64 µmol/m2
End-capped
Proprietary
No
Proprietary
Proprietary
Proprietary
No
No
www.waters.com/spherisorb
24
Ligand Type
Phenyl
CN (Nitrile)
OD/CN
W (Silica)
SCX
SAX
Particle Size
3, 5, 10 μm
3, 5, 10 μm
5 μm
3, 5, 10 μm
5, 10 μm
5, 10 μm
Surface Area
220 m2/g
220 m2/g
220 m2/g
220 m2/g
220 m2/g
220 m2/g
Particle Shape
Spherical
Spherical
Spherical
Spherical
Spherical
Spherical
Pore Size
80Å
80Å
80Å
80Å
80Å
80Å
Carbon Load
2.5%
3.1%
5%
N/A
4%
4%
Ligand Coverage
2.72 µmol/m2
3.29 µmol/m2
1.15 µmol/m2
N/A
N/A
N/A
End-capped
No
No
Proprietary
No
No
No
NOVA-PAK COLUMNS
The bonded phases of Nova-Pak® Columns are available in 4 µm and 6 µm particle sizes that offer high resolution as well
as faster and more efficient chromatography. The smaller particle size in conjunction with shorter column lengths can be used to
reduce solvent consumption while maintaining resolution for complex mixtures. Analytical columns with 4 µm particle size packing
are available in 75, 150, and 300 mm length steel columns. Semi-preparative Prep Nova-Pak HR Columns are packed with 6 µm
particle-size packings and provide an unparalleled range of separation possibilities. The high-efficiency packing of Prep Nova-Pak HR
Columns provides faster separations using less solvent with the added advantage of more concentrated fractions, all of which reduce
preparative chromatography cost. All Nova-Pak Columns are packed to stringent QC procedures in our cGMP manufacturing facility to
ensure batch-to-batch reproducibility.
Spherisorb
Ligand Type
ODS2 (C18)
ODS1 (C18)
ODSB (C18)
C8
C6
C1
NH2 (Amino)
Particle Size
3, 5, 10 μm
3, 5, 10 μm
5 μm
3, 5, 10 μm
3, 5, 10 μm
3, 5, 10 μm
3, 5, 10 μm
Surface Area
220 m2/g
220 m2/g
220 m2/g
220 m2/g
220 m2/g
220 m2/g
220 m2/g
Particle Shape
Spherical
Spherical
Spherical
Spherical
Spherical
Spherical
Spherical
Pore Size
80Å
80Å
80Å
80Å
80Å
80Å
80Å
Carbon Load
11.5%
6.2%
11.5%
7.75%
4.7%
2.15%
1.9%
Ligand Coverage
2.98 µmol/m2
1.49 µmol/m2
2.98 µmol/m2
3.12 µmol/m2
3.36 µmol/m2
2.97 µmol/m2
2.64 µmol/m2
End-capped
Proprietary
No
Proprietary
Proprietary
Proprietary
No
No
Nova-Pak
Chemistry
C18
C8
Phenyl
CN
Silica
Prep HR C18
Prep HR Silica
Particle Size
4 µm
4 µm
4 µm
4 µm
4 µm
6 µm
6 µm
Particle Shape
Spherical
Spherical
Spherical
Spherical
Spherical
Spherical
Spherical
Pore Size
60Å
60Å
60Å
60Å
60Å
60Å
60Å
Carbon Load
7%
4%
5%
2%
N/A
7%
N/A
End-capped
Proprietary
Proprietary
Proprietary
Proprietary
No
Proprietary
No
www.waters.com/nova-pak
25
RESOLVE COLUMNS
The non-endcapped Resolve™ packing is significantly different from Waters other packing materials. The change in chromatographic
behavior is most commonly noticed with polar compounds which are typically more retained. Basic compounds can be
chromatographed using mobile phase modifiers, such as ion-pairing reagents, which reduce excessive tailing.
DELTA-PAK COLUMNS
Delta-Pak™ Columns are ideal for separation and isolation of peptides, proteins, and natural products and are available in
two different pore sizes that are optimized for large molecule separations. Delta-Pak Columns are known for consistent and
predicable scaling between column formats, allowing purification scientists the ability to isolate target compounds from
the milligram to gram quantities. The highly stable Delta-Pak bonded silica is available in 5 μm and 15 μm
particle sizes.
Resolve
Ligand Type
Silica
C18
C8
CN
Particle Size
5, 10 µm
5, 10 µm
10 µm
10 µm
Particle Shape
Spherical
Spherical
Spherical
Spherical
Pore Size
90Å
90Å
90Å
90Å
Carbon Load
10 %
10%
5%
3%
End-capped
No
No
No
No
Delta-Pak
Ligand Type
C18
C18
C4
C4
Particle Size
5, 15 μm
5, 15 μm
5, 15 μm
5, 15 μm
Particle Shape
Spherical
Spherical
Spherical
Spherical
Pore Size
100Å
300Å
100Å
300Å
Carbon Load
17%
7%
7%
3%
End-capped
Proprietary
Proprietary
Proprietary
Proprietary
www.waters.com/delta-pak
www.waters.com/resolve
26
Irregular Particle Technology
The first HPLC packing materials were comprised of non-spherical and irregularly shaped particles. Typically, these columns have
reduced mechanical stability and lower efficiency compared to a column packed with spherical particles. However, even with these
limitations, there are many methods that require the use of these sorbents. As a primary manufacture of sorbents and bonded
materials, Waters has demonstrated consistent and reliable column performance for over 40 years and we will continue to support
these brands for the future.
µBONDAPAK/BONDAPAK COLUMNS
If your method calls for a μBondapak® Column, there is only one column that contains μBondapak C18 packing material. Many
companies claim “μBondapak-like” selectivity, but none have passed Waters stringent QC batch tests. μBondapak or BondaPak®
packing materials have demonstrated reproducibility from year-to-year since 1973, allowing μBondapak Columns to be the one
of the most widely referenced HPLC column brands.
µPORASIL/PORASIL COLUMNS
μPorasil™ and Porasil™ particles were one of the first commercially available fully porous packing materials used for LC
separations. In contrast to the reversed-phase separation ability of μBondapak C18, the non-bonded, silica-based material
in μPorasil Columns was produced to provide normal-phase separations for a wide array of sample types.
µBondapak/
Bondapak
Ligand Type
C18
Phenyl
CN
NH2
Particle Size
10 μm
10 μm
10 μm
10 μm
Particle Shape
Irregular
Irregular
Irregular
Irregular
Pore Size
125Å
125Å
125Å
125Å
Carbon Load
10%
8%
6%
3.5%
End-capped
Proprietary
Proprietary
Proprietary
No
www.waters.com/bondapak
µPorasil/Porasil
Ligand Type
Silica
Particle Size
10, 15-20 μm
Particle Shape
Irregular
Pore Size
125Å
Carbon Load
N/A
End-capped
No
www.waters.com/delta-pak
www.waters.com/resolve
www.waters.com/porasil
27
Column
Performance Monitoring
Intended Use
Detector
Performance Monitoring
Intended Use
Neutrals QC Reference
Material
Provides chromatographic performance
information under isocratic conditions
using 3 neutral probes.
QDa QC Reference Material
Provides chromatographic and mass
spectrometer information using an
8 component mixture in an optimized
format for the ACQUITY QDa® Detector.
This solution contains 1 critical pair to
measure chromatographic performance.
Reversed-Phase QC
Reference Material
Provides reversed-phase chromatographic
performance information under gradient
conditions using 1 void marker, 3 neutral,
1 acidic, and 2 basic probes.
Quad LCMS QC Reference
Material
Provides chromatographic and mass
spectrometer information using a 9 component
mixture in a format optimized for quadrupole
MS Systems. This solution contains 2
critical pairs to measure chromatographic
performance.
HILIC QC Reference Material
Provides chromatographic performance
information inclusive of mobile-phase pH
in HILIC mode using 1 void marker, 1 polar
neutral, and 2 polar basic probes.
LCMS QC Reference Material
Provides chromatographic and mass
spectrometer information using a 9 component
mixture in a format optimized for the highest
resolution Tof/QTof MS Systems. This
solution contains 2 critical pairs to measure
chromatographic performance.
How Do You Know Your Chromatographic System is in Proper Working Order?
Quality Control (QC) Reference Materials contain mixtures of standards specifically chosen to provide an
easy and reliable way to monitor the performance of any chromatographic system. By using a QC Reference
Materials, you can be assured that your column and system are ready to analyze your samples. Regular use
of QC Reference Materials also provides an opportunity to benchmark your chromatographic systems and
trend performance over time, making it easier to proactively identify problems and resolve them faster.
Since chromatographic analyses are complex and depend on many different variables, such as mobile-
phase composition, column type, and detection method, Waters has formulated specific QC Reference
Material mixtures designed to test systems with these differences in mind.
To locate additional information for standards specific to calibration, qualification, and tuning of instruments
and detectors, as well as a more comprehensive list of available standards and reagents, visit asr.waters.com
Extend Column Lifetime with VanGuard
Column Protection Products
VanGuard™ Pre-columns and Cartridges are optimized to protect
and prolong analytical column lifetimes without compromising
chromatographic performance. They are available in a wide selection of
particle sizes and stationary phases, making them ideally suited for the
physical and chemical protection for all Waters analytical columns.
■
■
Removes particulates and chemical contamination
■
■
Maintains UPLC, UHPLC, and HPLC separation efficiency
■
■
Provides cost effective protection for all Waters analytical columns
28
Column
Performance Monitoring
Intended Use
Detector
Performance Monitoring
Intended Use
Neutrals QC Reference
Material
Provides chromatographic performance
information under isocratic conditions
using 3 neutral probes.
QDa QC Reference Material
Provides chromatographic and mass
spectrometer information using an
8 component mixture in an optimized
format for the ACQUITY QDa® Detector.
This solution contains 1 critical pair to
measure chromatographic performance.
Reversed-Phase QC
Reference Material
Provides reversed-phase chromatographic
performance information under gradient
conditions using 1 void marker, 3 neutral,
1 acidic, and 2 basic probes.
Quad LCMS QC Reference
Material
Provides chromatographic and mass
spectrometer information using a 9 component
mixture in a format optimized for quadrupole
MS Systems. This solution contains 2
critical pairs to measure chromatographic
performance.
HILIC QC Reference Material
Provides chromatographic performance
information inclusive of mobile-phase pH
in HILIC mode using 1 void marker, 1 polar
neutral, and 2 polar basic probes.
LCMS QC Reference Material
Provides chromatographic and mass
spectrometer information using a 9 component
mixture in a format optimized for the highest
resolution Tof/QTof MS Systems. This
solution contains 2 critical pairs to measure
chromatographic performance.
Waters Part Selector and Selectivity Chart for iPad®
www.waters.com/apps
Electronic Tools
Waters Reversed-Phase Column Selectivity Chart
www.waters.com/selectivitychart
Waters Column Advisor
www.waters.com/columnadvisor
29
Waters, The Science of What’s Possible, CORTECS, UPLC, Alliance, ACQUITY UPLC, XBridge, XSelect,
Atlantis, Symmetry, XTerra, Waters Spherisorb, Nova-Pak, BondaPak, μBondaPak, QDa, SunFire, and
ACQUITY are registered trademarks of Waters Corporation. BEH Technology, CSH, Intelligent Speed,
IS, OBD, SymmetryShield, Symmetry300, μPorasil, Porasil, Resolve, Delta-Pak, and SymmetryPrep
are trademarks of Waters Corporation. All other trademarks are property of their respective owners.
©2014-16 Waters Corporation. Printed in the U.S.A.
December 2016 720003995EN KP-SIG
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 508 478 2000
F: 508 872 1990
www.waters.com
www.waters.com/hplccolumns
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