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©2011 Waters Corporation 1
Nuevos Desarrollos en Columnas ACQUITY UPLC® & HPLC
Bàrbara Bagó
Bilbao, 12 Julio 2012
©2011 Waters Corporation 2
Agenda
Column Manufacturing and Platform Definitions
New HPLC & ACQUITY UPLC Column Chemistries
Upcoming Changes to USP Chromatography <621>
New 2.5 µm eXtended Performance (XP) Columns
Summary
©2011 Waters Corporation 3
What makes an LC column reproducible?
0
5
10
15
20
25
0.0 0.5 1.0 1.5 2.0 2.5 3.0
1 . 7 µm ACQUITY UPLC® BEH C18
2 . 5 µm XBridge™ BEH C18 XP
2 . 6 µm Core-Shell C18
Linear Velocity, u (cm/sec)
Plat
e H
eigh
t (H
, 4 s
igm
a)
Acenaphthene, 2.1 x 50 mm columns
70/30 MeCN/H2O, 30 °C, 254 nm
A) Sorbents B) Packing C) Engineering
©2011 Waters Corporation 4
Developing a Waters Column: cGMP Registration
All Waters manufacturing facilities are registered with the FDA for the manufacture and distribution of class I medical devices, and are ISO 9001:2000 certified
The GMP Quality Systems Regulations cover such items as — Quality management and organization
— Production and process controls
— Packaging and labeling controls
— Document and record controls
As part of the registration process, the FDA may inspect the facilities every two years for compliance Taunton, MA, USA:
Particle Synthesis Milford MA, USA:
Hardware Manufacture Wexford, Ireland:
Device Manufacturing
©2011 Waters Corporation 5
Developing a Waters Column
Hardware — Materials
— Design
— Optimization
Sorbents — Chemistries
— Synthesis
Packing — Equipment Considerations
— Optimization
— Product QC and testing
©2011 Waters Corporation 6
Developing a Waters Column: Particle Synthesis and Bonding
Manufacture of a particle through bonding takes between 4-6 months — Each step of the synthesis process quality controlled
©2011 Waters Corporation 7
Developing a Waters Column: Column Packing
Computer controlled real time packing optimization
Each column is packed on a dedicated custom designed packing station
Optimized for slurry flow
Optimized for packing pressure
200 000+ columns packed annually
©2011 Waters Corporation 8
Developing a Waters Column: Final QC and Inspection
Each column Passes:
Efficiency testing using a test mixture
©2011 Waters Corporation 9
Developing a Waters Column: Final QC and Inspection
Each column Passes:
Efficiency testing using a test mixture
COA tracking and QC checks of product conformity
All column eCord information is verified against original manufacturing data and
testing
©2011 Waters Corporation 10
Developing a Waters Column: Final QC and Inspection
Each column Passes:
Rigorous cosmetic standards
Efficiency testing using a test mixture
COA tracking and QC checks of product conformity
Final packaging and documentation
©2011 Waters Corporation 11
Agenda
Column Manufacturing and Platform Definitions
New HPLC & ACQUITY UPLC Column Chemistries
Upcoming Changes to USP Chromatography <621>
New 2.5 µm eXtended Performance (XP) Columns
Summary
©2011 Waters Corporation 12
Waters Column Product History
Styragel®
µBondapak™
DeltaPak®
1964 1979 1973
1992
Symmetry®
Spherisorb®
Atlantis ®
Symmetry® 300
XTerra®
XTerraPrep®
1998
Nova-Pak®
ProteinPakTM
Pico-TagTM
SymmetryShield®
1984
1986
2002
1999 1994 2003
Atlantis ® HILIC Silica Prep OBD™ Intelligent SpeedTM
BioSuite™ NanoEase™
2004
ACQUITY UPLC® BEH SunFire™ Columns
PrepPak®
1976
1958
Atlantis® T3 ACQUITY UPLC® HSS T3 AccQTagTM Ultra
2006
2005
XBridge™
2007
ACQUITY UPLC ® HSS C18 and HSS C18 SB
2008
XBridge™ HILIC
2009
ACQUITY UPLC® BEH Amide ACQUITY UPLC® BEH Glycan
XBridge Amide XSelect HSS HPLC Columns
2010
ACQUITY UPLC® BEH200 SEC XSelect CSH HPLC columns
ACQUITY CSH Columns Viridis SFC Columns
ProteinPak High Rs IEX
AccQTagTM
2011
ACQUITY UPLC® HSS Cyano/PFP columns XSelectTM HSS Cyano/PFP columns 2.5 µm XP columns
©2011 Waters Corporation 13
Begins with Two Fully-Scalable HPLC Column Platforms
Family designed and optimized for pH stability
Most MS-compatible HPLC
columns on the market
Family designed and optimized for selectivity
Multiple particle substrates to
solve multiple chromatographic problems
1.7 [UPLC], 2.5, 3.5, 5 and 10 µm 1.8 [UPLC], 2.5, 3.5 and 5 µm HSS 1.7 [UPLC], 2.5, 3.5 and 5 µm CSH
©2011 Waters Corporation 14
Designed for Industry-Leading
Chemical (pH) Stability
Column Particle Platform Definitions
Designed for Increased
Retentivity and Selectivity
Designed for Maximum
Selectivity and Sample Loading
BEH Particle Ethylene-Bridged Hybrid
130Å, 200Å and 300Å
HSS Particle High Strength Silica
100Å
CSH Particle Charged Surface Hybrid
130Å
All three substrates are fully scalable between UPLC and HPLC platforms
©2011 Waters Corporation 15
Begin with Two Fully-Scalable HPLC Column Platforms
Family designed and optimized for pH stability
Most MS-compatible HPLC columns
on the market
Family designed and optimized for selectivity
Multiple particle substrates to
solve multiple chromatographic problems
1.7 [UPLC], 2.5, 3.5, 5 and 10 µm 1.8 [UPLC], 2.5, 3.5 and 5 µm HSS 1.7 [UPLC], 2.5, 3.5 and 5 µm CSH
©2011 Waters Corporation 16
XBridge HPLC Columns
General-purpose HPLC and UPLC columns for small molecule separations — Broadest range of compound classes
— When buffers are exclusively used
— Where high pH stability is most important
— Where ultra-low MS bleed is desired
— Direct scalability between UPLC Technology, analytical HPLC and preparative HPLC
©2011 Waters Corporation 17
Ethylene Bridged Hybrid [BEH] Particles
Anal. Chem. 2003, 75, 6781-6788
U.S. Patent No. 6,686,035 B2 Bridged Ethanes within a silica matrix
©2011 Waters Corporation 18
Ethylene Bridged Hybrid [BEH] Chemistries
Rugged, reproducible, fully-scalable column chemistries for reversed-phase and HILIC separations — BEH C18: First column choice, widest pH range, LC/MS
— BEH C8: For hydrophobic compounds, widest pH range, LC/MS
— BEH Shield RP18: Embedded carbamate group, alternate selectivity
— BEH Phenyl: Most stable phenyl column, wide pH range
— BEH HILIC: Unbonded, rugged BEH particle for HILIC separation of very polar bases
— BEH Amide: General-purpose HILIC column for very polar compounds such as sugars, saccharides, carbohydrates, etc.
©2011 Waters Corporation 19
The importance of mobile phase pH: Rapid Method Development
Using a wide mobile phase pH range is an effective approach to change compound selectivity
Increase selectivity for: — Acids (Green/Blue)
— Bases (Red/Yellow)
Neutrals (Peak 2) are largely unaffected by mobile phase pH
©2011 Waters Corporation 20
Begin with Two Fully-Scalable HPLC Column Platforms
Family designed and optimized for pH stability
Most MS-compatible HPLC columns
on the market
Family designed and optimized for selectivity
Multiple particle substrates to
solve multiple chromatographic problems
1.7 [UPLC], 2.5, 3.5, 5 and 10 µm 1.8 [UPLC], 2.5, 3.5 and 5 µm HSS 1.7 [UPLC], 2.5, 3.5 and 5 µm CSH
©2011 Waters Corporation 21 Minutes 0.0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Conditions : Columns: 2.1 x 50 mm Mobile Phase A: 0.1% CF3COOH in H2O Mobile Phase B: 0.08% CF3COOH in ACN Flow Rate: 0.5 mL/min Gradient: Time Profile Curve (min) %A %B 0.0 92 8 6 0.1 92 8 6 4.45 50 50 7 4.86 10 90 6 5.0 92 8 6 6.0 92 8 6 Injection Volume: 1.0 µL Sample Diluent: 50:50 H2O: MeOH with 0.05% CF3COOH Sample Conc.: 100 µg/mL Temperature: 40 oC Detection: UV @ 330 nm Sampling rate: 40 pts/sec Time Constant: 0.1 Instrument: Waters ACQUITY UPLC®, with ACQUITY UPLC® TUV
Compounds 1. Caftaric acid 2. Chlorogenic acid 3. Cynarin 4. Echinacoside 5. Cichoric acid
1 2 4 5 3 BEH C18
BEH C8
BEH Shield RP18
BEH Phenyl
1 2 4 5 3
1 2 4 5 3
1 2 4 5 3
1 2 4 5 3 HSS T3
HSS C18 1 4 3 5 2
Column Selectivity Choices Caffeic Acid Derivatives
1 2 4 5 3 HSS C18 SB
HSS CN
HSS PFP 1 2 3
4 5
1 2
4
3 5
1 2 4 3 5 CSH Phenyl-Hexyl
1 2
4
3
5
CSH Fluoro-Phenyl
CSH C18 1 2
3,4
5
Comparative separations may not be representative of all applications
©2011 Waters Corporation 22
Designed for Industry-Leading
Chemical (pH) Stability
Column Particle Platform Definitions
Designed for Increased
Retentivity and Selectivity
Designed for Maximum
Selectivity and Sample Loading
BEH Particle Ethylene-Bridged Hybrid
130Å, 200Å and 300Å
HSS Particle High Strength Silica
100Å
CSH Particle Charged Surface Hybrid
130Å
©2011 Waters Corporation 23
[CSH] and [HSS] Chemistries
FOUR general-purpose C18 columns for low to neutral pH reversed-phase separations — CSH C18: Rapid pH switching for method development
— HSS C18: Superior peak shape & acid stability
— HSS C18 SB: Alternate Selectivity for Basic (SB) compounds
— HSS T3: Enhanced reversed-phase retention of polar compounds
NEW HSS PFP and HSS CN Chemistries — Designed for alternate selectivities vs. C18 chemistries
— HSS PFP: Exceptional selectivity and retention for positional isomers, halogenated compounds and polar basic compounds
— HSS CN: Stable, normal phase-compatible, reproducible CN chemistry
©2011 Waters Corporation 24
First silica-based particle (1.8 μm) designed for UPLC applications
Fully scalable manufacturing process enabled HPLC particle sizes (2.5, 3.5 and 5 μm)
Available with FIVE bonded phases
HSS particles benefits: — Alternate selectivities (vs. BEH &
CSH particle columns)
— Increased retention
— Seamless transferability
— Highest pressure tolerance
Ever-Growing Selectivity Range High Strength Silica [HSS] Particles
5 μm 3.5 μm 2.5 μm 1.8 μm
©2011 Waters Corporation 25
Designed for Industry-Leading
Chemical (pH) Stability
Column Particle Platform Definitions
Designed for Increased
Retentivity and Selectivity
Designed for Maximum
Selectivity and Sample Loading
BEH Particle Ethylene-Bridged Hybrid
130Å, 200Å and 300Å
HSS Particle High Strength Silica
100Å
CSH Particle Charged Surface Hybrid
130Å
©2011 Waters Corporation 26
Ever-Growing Selectivity Range Charged Surface Hybrid [CSH] Particles
Step 1 Step 2 Step 3
CHARGED SURFACE HYBRID PROCESS
©2011 Waters Corporation 27
Charged Surface Hybrid [CSH] Chemistries
Three innovative CSH chemistries that provide alternate selectivity and improved performance low ionic strength mobile phases
— CSH C18: Superior peak shape for bases, increased loadability, alternate selectivity vs. conventional C18 columns
— CSH Phenyl-Hexyl: High performance phenyl column, best in additive-based mobile phases, different selectivity vs BEH Phenyl
— CSH Fluoro-Phenyl: Truly different selectivity, optimized for low pH separations, enhanced retention of acidic compounds
©2011 Waters Corporation 28
Charged Surface Hybrid [CSH] Particles
Designed for LC/MS
— Use mobile phase additives at low ionic strength
— Formic acid versus TFA exposure
Ideal for isolation/purification
— Superior peak shape for basic compounds at high mass load
— Use volatile additives versus ion-pairing reagents and buffers
Rapid method development
— Prefer to work at low pH with occasional high pH work
— Switch back/forth between low & high pH (additives)
©2011 Waters Corporation 29
Benefits of CSH Technology: Loading Comparison
AU
0.0
1.0
2.0
3.0
AU
0.0
1.0
2.0
3.0
AU
0.0
1.0
2.0
3.0
Minutes 0.6 0.8 1.0 1.2 1.4 1.6
ACQUITY UPLC® CSH C18
2.1 x 50 mm, 1.7 µm
Fully porous silica C18
2.1 x 50 mm, 1.8 µm
Kinetex C18
2.1 x 50 mm, 1.7 µm
Quetiapine (base)
Quetiapine (base)
Quetiapine (base)
Propiophenone (neutral)
Propiophenone (neutral)
Propiophenone (neutral)
Comparative separations may not be representative of all applications
©2011 Waters Corporation 30
Ever-Growing Selectivity Range Ethylene Bridged Hybrid [BEH]: 2004
ACQUITY BEH C18 XBridge C18
©2011 Waters Corporation 31
Ever-Growing Selectivity Range Ethylene Bridged Hybrid [BEH]: 2005
ACQUITY BEH Phenyl XBridge Phenyl
ACQUITY BEH C18 XBridge C18
ACQUITY BEH Shield RP18 XBridge Shield RP18
ACQUITY BEH C8 XBridge C8
©2011 Waters Corporation 32
Ever-Growing Selectivity Range [BEH + HSS]: 2008
ACQUITY BEH Phenyl XBridge Phenyl
ACQUITY BEH C18 XBridge C18
ACQUITY BEH Shield RP18 XBridge Shield RP18
ACQUITY BEH C8 XBridge C8
ACQUITY HSS C18 SB XSelect HSS C18 SB
ACQUITY HSS C18 XSelect HSS C18
ACQUITY HSS T3 XSelect HSS T3
©2011 Waters Corporation 33
Ever-Growing Selectivity Range [BEH + HSS + CSH]: 2010
ACQUITY BEH Phenyl XBridge Phenyl
ACQUITY BEH C18 XBridge C18
ACQUITY BEH Shield RP18 XBridge Shield RP18
ACQUITY BEH C8 XBridge C8
ACQUITY HSS C18 SB XSelect HSS C18 SB
ACQUITY HSS C18 XSelect HSS C18
ACQUITY HSS T3 XSelect HSS T3
ACQUITY CSH C18 XSelect CSH C18
ACQUITY CSH Phenyl-Hexyl XSelect CSH Phenyl-Hexyl
ACQUITY CSH Fluoro-Phenyl XSelect CSH Fluoro-Phenyl
©2011 Waters Corporation 34
Ever-Growing Selectivity Range [BEH + CSH + HSS]: 2011
ACQUITY BEH Phenyl XBridge Phenyl
ACQUITY BEH C18 XBridge C18
ACQUITY BEH Shield RP18 XBridge Shield RP18
ACQUITY BEH C8 XBridge C8
ACQUITY HSS C18 SB XSelect HSS C18 SB
ACQUITY HSS C18 XSelect HSS C18
ACQUITY HSS T3 XSelect HSS T3
ACQUITY CSH C18 XSelect CSH C18
ACQUITY CSH Phenyl-Hexyl XSelect CSH Phenyl-Hexyl
ACQUITY CSH Fluoro-Phenyl Xselect CSH Fluoro-Phenyl
ACQUITY HSS CN XSelect HSS CN
ACQUITY HSS PFP XSelect HSS PFP
©2011 Waters Corporation 35
New XSelect/ACQUITY UPLC Column Chemistries
HSS PFP —Exceptional selectivity and retention for
positional isomers, halogenated compounds and polar compounds
HSS CN —Stable, NP-compatible, reproducible CN
chemistry available in HPLC and 1.8 µm (UPLC) particle sizes
Two Robust and Fully Scalable Chemistries that Increase the Selectivity Range of our XSelect
HPLC & ACQUITY UPLC Column Families
©2011 Waters Corporation 36
Expanding Retention Capability: XSelect HSS/ACQUITY UPLC HSS PFP
Provides chromatographer with multiple selectivity mechanisms including: — hydrogen bonding — dipole-dipole — aromatic (pi-pi) — hydrophobic (reversed-phase)
Target Analyses:
positional isomers, halogenated compounds and polar compounds
Compared to Similar Fluorinated ligands: — HSS PFP: LONGER retention of basic compounds — CSH Fluoro-Phenyl: SHORTER retention of basic compounds — HSS PFP exhibits TRUE PFP behavior
©2011 Waters Corporation 37
Retention Comparison - Basic Compounds: CSH Fluoro-Phenyl vs. HSS PFP
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50
Compounds 1. Aminopyrazine 2. Pindolol 3. Quinine 4. Labetalol 5. Verapamil 6. Diltiazem 7. Amitriptyline
Conditions: Columns: 2.1 x 50 mm Mobile Phase A: 10mM NH4COOH, pH 3.0 Mobile Phase B: MeOH Flow Rate: 0.4 mL/min Gradient: Time Profile (min) %A %B 0.00 70 30 3.00 15 85 3.50 15 85 3.51 70 30 4.50 70 30 Inj. Volume: 1 µL Sample Diluent: H2O Temperature: 30°C Detection: UV @ 260 nm Sampling Rate: 40 points/sec Filter Response: Normal Instrument: ACQUITY UPLC® ACQUITY UPLC® PDA
2
1
4
6
3
7
5
HSS PFP
CSH Fluoro-Phenyl 2
1 3
6 7
5
4
BASIC COMPOUNDS
HSS PFP Retains Basic Compounds Longer than CSH Fluoro-Phenyl
Comparative separations may not be representative of all applications
©2011 Waters Corporation 38
Selectivity at pH 3.0: Ligands that improve chromatographic diversity
XSelect HSS C18
XSelect HSS T3
XSelect HSS C18 SB
XSelect HSS PFP
XSelect HSS CN
XSelect CSH Fluoro-Phenyl
XSelect CSH Phenyl-Hexyl XSelect CSH C18
XBridge Shield RP18
XBridge C18
XBridge C8
XBridge Phenyl
Hydrophobicity
Sele
ctiv
ity
HSS PFP: Increased Retentivity vs. CSH Fluoro-Phenyl
©2011 Waters Corporation 39
PFP Column Performance Comparison Basic Compound Mixture
Compounds 1. Trimethoprim 2. Nordoxepin 3. Doxepin 4. Nortriptyline 5. Imipramine 6. Amitriptyline 7. Trimipramine
ACQUITY UPLC HSS PFP, 1.8 µm, 2.1 X 50mm
Phenomenex Kinetex PFP, 1.7 µm, 2.1X50 mm
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00
1
2
3 4 5 6 7
1
2 3
4 6 7
0.000
0.005
0.010
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
5
RPHAve = 3.04
RPHAve = 17.77
System: ACQUITY UPLC® H-Class with
ACQUITY UPLC ® PDA Detector
Mobile Phase A: 10mM NH4COOH, pH3.0
Mobile Phase B: ACN
Isocratic Mobile Phase: 60:40; A:B
Flow Rate: 0.5 mL/min
Injection Volume: 1 µL
Column Temperature: 30 °C
UV Detection: 254 nm
Comparative separations may not be representative of all applications
©2011 Waters Corporation 40
XSelect HSS/ACQUITY UPLC HSS CN: A Phase for Non-Polar Compounds
Provides the chromatographer with a phase for: — Low hydrophobicity for rapid elution of hydrophobic analytes
— Unique selectivity
— Normal-phase compatible
Chromatographic benefits: — Significantly less retention than C18 (typically requires lower %
organic mobile phase)
— Compatible with highly aqueous phases
— Stable, low-bleed in LC-MS separations
©2011 Waters Corporation 41
Reversed-Phase Retention: Steroids Separation
0.00
0.10
0.20
1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
0.00
0.10
0.20
0.40 0.60 0.80 1.00 1.20 1.40
XSelect HSS CN 5 µm 2.1 x 150 mm
ACQUITY UPLC HSS CN 1.8 µm 2.1 x 50 mm
XSelect HSS CN 3.5 µm 2.1 x 100 mm
1. Hydrocortisone 2. Corticosterone 3. β - Estradiol 4. Progesterone 0.00
0.10
0.20
3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0
1 2
3
4
Isocratic: 40% ACN
©2011 Waters Corporation 42
Normal-Phase Retention: Steroids Separation
0.00
0.05
0.10
0.15
Minutes
1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
0.00
0.05
0.10
0.15
0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.0 1.10 1.20
XSelect HSS CN 5 µm 2.1 x 150 mm
XSelect HSS CN 3.5 µm 2.1 x 100 mm
ACQUITY UPLC HSS CN 1.8 µm 2.1 x 50 mm
0.00
0.05
0.10
0.15
3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0
3
4 2 1
Isocratic: 83% Hexane / 5% DCM / 12% IPA
1. Hydrocortisone 2. Corticosterone 3. β - Estradiol 4. Progesterone
©2011 Waters Corporation 43
Launch Date
3 Particles and 14 Chemistries ACQUITY UPLC, XBridge, XSelect
Jan 2008
Ligand Type Available Particle Sizes
Ligand Density
Carbon Load
Endcap Style
pH range
Trifunctional C18 1.7,2.5,3.5,5,10 3.1 µmol/m2 18% Proprietary 1-12
Trifunctional C8 1.7,2.5,3.5,5,10 3.2 µmol/m2 13% Proprietary 1-12
Monofunctional Embedded Polar Group
1.7, 2.5, 3.5, 5 3.3 µmol/m2 17% TMS 2-11
Trifunctional C6 Phenyl 1.7,2.5,3.5,5,10 3.0 µmol/m2 15% Proprietary 1-12
Unbonded 1.7, 2.5, 3.5, 5 --- --- --- 1-8
Trifunctional Amide 1.7, 2.5, 3.5, 5 7.5 µmol/m2 12% None 2-11
Trifunctional C18 1.8, 2.5, 3.5, 5 1.6 µmol/m2 11% Proprietary 2-8
Trifunctional C18 1.8, 2.5, 3.5, 5 3.2 µmol/m2 15% Proprietary 1-8
Trifunctional C18 1.8, 2.5, 3.5, 5 1.6 µmol/m2 8% None 2-8
Trifunctional Propylfluorophenyl 1.8, 2.5, 3.5, 5 3.3 µmol/m2 7.5% None 2-8
Monofunctional Diisopropyl Cyano 1.8, 2.5, 3.5, 5 2.0 µmol/m2
5.5%
None
2-8
Trifunctional C18 1.7, 2.5, 3.5, 5 2.3 µmol/m2 15% Proprietary 1-11
Trifunctional Propylfluorophenyl 1.7, 2.5, 3.5, 5 2.3 µmol/m2 10% None 1-8
Trifunctional C6 Phenyl 1.7, 2.5, 3.5, 5 2.3 µmol/m2 14% Proprietary 1-11
©2011 Waters Corporation 44
What is HILIC?
HILIC - Hydrophilic Interaction Chromatography — Term coined in 1990 to distinguish from normal-phase*
HILIC is a variation of normal-phase chromatography without the
disadvantages of using solvents that are not miscible in water — “Reverse reversed-phase” or “aqueous normal-phase”
chromatography
Stationary phase is a POLAR material — Silica, hybrid, cyano, amino, diol, amide
The mobile phase is highly organic (> 80% ACN) with a smaller
amount of aqueous mobile phase — Water (or the polar solvent(s)) is the strong, eluting solvent
*Alpert, A. J. J.Chromatogr. 499 (1990) 177-196.
©2011 Waters Corporation 45
Benefits of HILIC
Retention of highly polar analytes not retained by reversed-phase — Less interference from non-polar matrix components
Complementary selectivity to reversed-phase
— Polar metabolites/impurities/degradants retain more than parent compound
Enhanced sensitivity in mass spectrometry — High organic mobile phases (> 80% ACN) promotes enhanced
ESI-MS response — Direct injection of PPT supernatant without dilution — Facilitates use of lower volume samples
Improved sample throughput
— Direct injection of high organic extracts from PPT, LLE or SPE without the need for dilution or evaporation and reconstitution
©2011 Waters Corporation 46
When To Use HILIC
When to Use HILIC:
Need improved retention of hydrophilic or ionizable compounds
Need improved MS response for polar or ionizable compounds
Need improved sample throughput for assays using organic extraction
Reversed-phase
polar non-polar
Compound Index
Normal-phase
ESI-M
S R
espo
nse
exce
llent
poor
HILIC
©2011 Waters Corporation 47
Stationary Phases for HILIC separations
ACQUITY UPLC® BEH Amide XBridgeTM Amide
ACQUITY UPLC® BEH HILIC XBridgeTM HILIC
Atlantis® HILIC Silica
Silica HILIC Columns (pH range 1 – 5)
Hybrid HILIC Columns (pH range 1 – 11)
©2011 Waters Corporation 48
Waters HILIC screening strategy
pH 3
ACQUITY UPLC® BEH HILIC 2.1 x 50 mm, 1.7 µm
Opt
imiz
atio
n
pH 9
ACQUITY UPLC® BEH Amide 2.1 x 50 mm, 1.7 µm
Atlantis HILIC Silica 2.1 x 50 mm, 3 µm
Where do I start? • Initial scouting gradient from 95 to 50% acetonitrile over 5 minutes • At least 5% should be a polar solvent (i.e., water or methanol)
©2011 Waters Corporation 49
Implementing the Approach: Example 1, Water Soluble Vitamins
O
OH OH
O
OH
OH
Ascorbic acid
O
N
NH2
Nicotinamide
N
O
OHOH
CH3
Pyridoxal
N N
NNH
O
OCH3
CH3
OH
OHOH
OH
Riboflavin
O
N
OH
Nicotinic acid
N+
CH3
SN NH2
N
OHCH3
Thiamine
Cl-
N N
N N
CH3
CH3
NH2
O
CH3
NH2
O
O NH2
O
NH2
O
NH2
CH3CH3
O
NH2
NH
PO
O
CH3
CH3
O-
O
O
O
OH
N
OH
Co+
N
N CH3
CH3
HCH3
CH3
B12
N
NH
N
N
NH
O
O
O
NH2
NH
O
OHOH
Folic Acid
©2011 Waters Corporation 50
Stationary Phase Selectivity at Low pH: Water-soluble Vitamins
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
BEH Amide
BEH HILIC
Atlantis HILIC Silica
1
2
4
3 6
5
8 7
1,6
3,2
4
8
5
7
1
6 3,2
4
8
5
7
Compounds 1. Nicotinamide 50 µg/mL 2. Pyridoxine 50 µg/mL 3. Riboflavin 30 µg/mL 4. Nicotinic acid 50 µg/mL 5. Thiamine 50 µg/mL 6. Ascorbic Acid 50 µg/mL 7. B12 50 µg/mL 8. Folic Acid 25 µg/mL
pH 3 All 3 columns yield different selectivity BEH Amide has greatest resolution of all peaks
©2011 Waters Corporation 51
Commercial Food Samples Using XBridge BEH Amide Column
XBridge BEH Amide, 3.5µm, 4.6 x 250mm Isocratic: 75% ACN with 0.2% TEA 1.0mL/min, 15 µL injection volume
Minutes 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00
Lamb Curry Meal
Food Sugar Standard 1 2 3 4 5 6
White Bread
Hot Cross Buns
Strawberry Smoothie
1) p-Toluamide 2) Fructose 3) Glucose 4) Sucrose 5) Maltose 6) Lactose
©2011 Waters Corporation 52
Agenda
Column Manufacturing and Platform Definitions
New HPLC & ACQUITY UPLC Column Chemistries Upcoming Changes to USP Chromatography <621> New 2.5 µm eXtended Performance (XP) Columns
Summary
©2011 Waters Corporation 53
What is the USP?
The United States Pharmacopeia (USP) is an independent, official public standards-setting authority — Prescription and OTC medicines
— Healthcare products
— Food ingredients
— Dietary supplements
The USP-NF is the official authority – FDA-enforceable standards — Enforcement of USP standards is the responsibility of FDA and
other government authorities in the U.S. and elsewhere
— USP has no role in enforcement
The U.S. Federal Food, Drug, and Cosmetics Act designates the USP–NF as the official compendia for drugs marketed in the
United States
©2011 Waters Corporation 54
The Beginning of A Significant Change to Chromatography <621>
Chromatography <621> describes the adjustments allowed in the chromatography system when system suitability test fails
In early 2009, Waters was approached to write a ‘Stimuli Article’ to modernize/add more flexibility to selecting LC columns in Chromatography <621>
In the summer of 2009 Dr. Uwe Neue et. al. wrote a paper (article) that basically describes UPLC technology and method transfer — Dr. Neue’s calculations are also the basis of the ACQUITY UPLC
Columns Calculator
©2011 Waters Corporation 55
The Beginning of A Significant Change to Chromatography <621>
Stimuli Article PF 35(6) [Nov-Dec 2009]
Are you aware of this Article? What does the Stimuli Article propose?
©2011 Waters Corporation 56
What is the PF 35(6) [Nov-Dec 2009] Stimuli Article?
Stimuli Article PF 35(6) [Nov-Dec 2009] allows the flexibility to change the column dimensions and/or particle size as long as an equivalent (or better) separation is achieved
— The ability to change LC columns in a method was restrictive and
outdated
— What if the prescribed column is no longer available? What if a
faster (more modern) separation can be obtained with another column (e.g., UPLC)?
— When particle size and/or diameter of the column is changed in the method, the flow rate may need adjustment.
©2011 Waters Corporation 57
Allowable Adjustments in Chromatography <621>
Variable Allowable Changes
Before Stimuli Article PF 35(6) After Stimuli Article PF 35(6) Particle Size -50% Any allowed as long as column length to
particle size (l/dp) is maintained (±25%) Column Length ±70% Flow Rate ±50% Adjust for length, particle size and column ID Column ID Any allowed Any allowed
Injection Volume Any reduction Any allowed Column Temperature ±10% ±10%
Mobile Phase pH ±0.2 unit ±0.2 unit
When Stimuli Article PF 35(6) [Nov-Dec 2009] Officially Becomes Part of Chromatography <621>, Analysts Will Have More Flexibility to Utilize Modern Chromatographic Techniques
(e.g., UPLC technology)
©2011 Waters Corporation 58
Effect of Stimuli Article PF 35(6) [Nov-Dec 2009] Change
EXAMPLE: If a monograph specifies a 4.6 x 150 mm, 5-µm column operated at 1.5 mL/min, the same separation may be expected with a 2.1 x 75 mm, 2.5-µm column operated at 1.5 mL/min x 0.4 = 0.6 mL/min, along with a pressure increase of about 4 times and a reduction in run time to about 30% of the original.1
OVERVIEW: Adjustments in column length, internal diameter, particle size, and flow rate can be used in combination to give equivalent conditions (same N), but with differences in pressure and run time. The table below lists some of the more popular column configurations to give equivalent efficiency (N), by adjusting these variables.1
Column Length (mm)
Column ID (mm)
Particle Size (dp, µm)
Relative Values
l/dp F N Pressure Run Time
250 4.6 10 25,000 0.5 0.8 0.2 3.3 150 4.6 5 30,000 1.0 1.0 1.0 1.0 150 2.1 5 30,000 0.2 1.0 1.0 1.0 100 4.6 3.5 28,600 1.4 1.0 1.9 0.5 100 2.1 3.5 28,600 0.3 1.0 1.9 0.5 75 4.6 2.5 30,000 2.0 1.0 4.0 0.3 75 2.1 2.5 30,000 0.4 1.0 4.0 0.3 50 4.6 1.7 29,400 2.9 1.0 8.5 0.1 50 2.1 1.7 29,400 0.6 1.0 8.5 0.1
1http://www.usppf.com/pf/pub/data/v373/CHA_IPR_373_c621.html#CHA_IPR_373_c621
©2011 Waters Corporation 59
How Might These Changes Benefit Chromatographers and Organizations?
Chromatographic Benefits — More flexibility to change the column dimensions and/or particle
size as long as an equivalent (or better) separation is achieved
— A more restrictive, science-based approach to change a column or method that requires an equivalent separation (performance) be obtained
Organizational Benefits — Analyses can continue even if the prescribed column is no longer
available
— A faster, greener and more sustainable separation can be obtained using a more modern separation technique
©2011 Waters Corporation 60
What Does This Revision Status Change Mean?
It means: — Sufficient changes to revised General Chapter <621>
Chromatography were made that require public comment (again)
— Comment period will begin 01-Mar-2012 in PF 38(2)
— No changes to <621> will occur in 2012
— eXtended Performance (XP) Columns are compliant with current <621> guidelines NOW
©2011 Waters Corporation 61
Agenda
Column Manufacturing and Platform Definitions
New HPLC & ACQUITY UPLC Column Chemistries Upcoming Changes to USP Chromatography <621> New 2.5 µm eXtended Performance (XP) Columns
Summary
©2011 Waters Corporation 62
Packed in ultra-low dispersion hardware to minimize band spreading
Designed to withstand high pressure — 4.6 mm ID capable of 9,000 PSI
— 2.1 and 3.0 mm IDs compatible with UPLC pressures
Flexibility in configurations — 2.1, 3.0 and 4.6 mm ID (2.1 and 3.0mm
incorporating eCord™ technology)
— 30, 50, 75 and 100 mm lengths
— 14 scalable stationary phases
Packed with XBridge [BEH] and XSelect [CSH and HSS] 2.5 µm particles and chemistries
— BEH C18, Shield RP18, C8, Phenyl, HILIC and Amide
— CSH C18, Phenyl-Hexyl and Fluoro-Phenyl
— HSS C18, T3, C18 SB, Cyano and PFP
eXtended Performance 2.5 µm Columns
©2011 Waters Corporation 63
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XSelect CSH C18 XP 4.6 x 75 mm, 2.5 µm
HPLC System
2X Faster than 3.5 µm HPLC
2.5 µm XP
Improved Productivity
ACQUITY UPLC CSH C18 2.1 x 50 mm, 1.7 µm
UPLC System
9X Faster than 3.5 µm HPLC
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1.7 µm
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XSelect CSH C18 4.6 x 100 mm, 3.5 µm
HPLC System 3.5 µm
5X Faster than 2.5 µm HPLC
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©2011 Waters Corporation 64
Resolution, Throughput and Sensitivity: Comparing 2.5 µm XP and 1.7 µm UPLC Columns
1.7 µm ACQUITY UPLC columns provide
2X increased speed and 20% higher sensitivity
compared to 2.5 µm XP columns while
maintaining selectivity
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XSelect CSH C18 XP 2.1 x 75 mm, 2.5 µm Flow rate: 0.54 mL/min
A: water with 0.1% formic acid B: acetonitrile with 0.1% formic acid NSAID mixture (50-100 ug/mL) UV detection at 270 nm Gradient (2.5µm): 35%B to 65%B over 4.85 min, hold for 0.66 min, re-equilibrate at 35%B. Gradient (1.7µm): 35%B to 65%B over 2.20 min, hold for 0.3 min, re-equilibrate at 35%B. Both columns were run on an ACQUITY UPLC H-Class system
ACQUITY UPLC CSH C18 2.1 x 50 mm, 1.7 µm
Flow rate: 0.8 mL/min
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©2011 Waters Corporation 65
Impact of Particle Size on Column Performance
1.7 µm ACQUITY UPLC columns provide
15% improvement in resolution and peak
capacity and 20% higher sensitivity compared to
XP 2.5 µm columns
Both columns were run on an ACQUITY UPLC H-Class system
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XSelect CSH C18 XP 2.1 x 75 mm, 2.5 µm Flow rate = 0.54 mL/min
Pressure = 5,300 PSI
Peak Capacity = 34
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ACQUITY UPLC CSH C18 2.1 x 75 mm, 1.7 µm Flow rate = 0.54 mL/min
Pressure = 9,200 PSI
Peak Capacity = 40
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Rs = 6.3
15% increase in Rs and Pc
20% higher sensitivity
40% lower backpressure
©2011 Waters Corporation 66
Impact of Instrument Dispersion on Column Performance
Ultra-low UPLC instrument dispersion
enhances column performance, even with
larger 4.6 mm ID columns
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4.6 x 75 mm, 2.5 µm
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ACQUITY UPLC H-Class XSelect CSH C18 XP
4.6 x 75 mm, 2.5 µm
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15% decrease in peak width
30% increase in peak height
©2011 Waters Corporation 67
Efficiency vs. Flow Rate
0
2000
4000
6000
8000
10000
12000
14000
16000
0.0 0.5 1.0 1.5 2.0 2.5
Flow Rate (mL/min)
Plat
e Co
unt (
4 si
gma)
1.7 µm ACQUITY UPLC® BEH C18
2.5 µm XBridge™ BEH C18 XP
2.6 µm Core-Shell C18
Acenaphthene, 2.1 x 50 mm columns
70/30 MeCN/H2O, 30 °C, 254 nm
Observations: When plotting efficiency vs. flow rate, it is clear that a smaller particle size will always perform better than a larger particle size 2.5 µm fully-porous and 2.6 µm core-shell particles provide the same efficiency
What about back pressure?
©2011 Waters Corporation 68
Back Pressure Comparison: Fully Porous vs. Core-Shell
0
1000
2000
3000
4000
5000
6000
7000
8000
0.0 0.5 1.0 1.5 2.0 2.5
Flow Rate (mL/min)
Pres
sure
(psi
)
2 . 5 µm XBridge™ BEH C18 XP
2 . 6 µm Core-Shell C18
2.1 x 50 mm columns
70/30 MeCN/H2O, 30 °C, 254 nm
Observations: 2.5 µm fully-porous and 2.6 µm superficially porous produce the same back pressure
©2011 Waters Corporation 69
Summary: Matching Column Configurations with LC Systems
System HPLC UPLC
Particle Size 3.5 μm, 5 μm XP 2.5 μm 1.7/1.8 μm UPLC, XP 2.5 μm, 3.5 μm, 5 μm
1.7/1.8 μm UPLC, XP 2.5 μm, 3.5 μm, 5 μm
Routine Pressure < 4000 psi < 4000 psi < 15000 psi (H-Class) < 18000 psi (I-Class)
Column ID 4.6 mm 4.6 mm 2.1 & 3.0 mm* 1.0 & 2.1 mm*
Column Length ≤ 250 mm ≤ 75 mm ≤ 150 mm* ≤ 150 mm*
(*) – additional UPLC-based bioseparation column configurations also available
©2011 Waters Corporation 70
Summary
Waters ongoing commitment to high-quality consumables manufacturing ensures reproducible results year after year
XP 2.5 µm columns bridge the gap between HPLC and UPLC and are compatible with HPLC, UHPLC and UPLC system platforms — Wide range of scalable chemistries, column lengths and IDs
Use 4.6 mm ID XP columns with Alliance HPLC systems and 2.1 mm ID XP and UPLC columns with ACQUITY UPLC systems — Maximize the efficiency, performance and throughput of ANY LC
system
©2011 Waters Corporation 71
Resources that Enable Success: Online Tools
Column selection tools and advisors
©2011 Waters Corporation 72
Quickly compare columns based on: — Retention
— Selectivity
— Similarity
— USP Designation
Map method development kits based on: — Waters MD kits
— Non-Waters columns
Also available as a web-based tool http:\\www.waters.com\selectivitychart
Resources that Enable Success: Columns Selectivity Chart
©2011 Waters Corporation 73
Resources that Enable Success: Waters Column Advisor
Make informed decisions based on: — Method requirements
— Column attributes
Choose recommended columns based on your most important criteria
Visit: http:\\www.waters.com\columnadvisor
©2011 Waters Corporation 74
Resources that Enable Success: Technology Primers
715001531
715001940
715002099
715002531
Four easy to read and understand books covering the basic concepts of:
©2011 Waters Corporation 75
Resources that Enable Success: LC Columns Wall Chart
Easily compare and relevant column data based on – Ligand type
– Usable pH range
– Temperature limits
– Carbon Load
Contains data for both HPLC and all UPLC column configurations.
720002241EN
©2011 Waters Corporation 76
Resources that Enable Success: LC Column Family Brochures
XP Columns XBridge™
Columns XSelect™ Columns ACQUITY UPLC®
Columns
720001140EN
720001255EN
720004195EN
720004178EN
©2011 Waters Corporation 77
Adding a New Business Competency
©2009 Waters Corporation | COMPANY CONFIDENTIAL Waters Corporation Confidential
An Introduction
www.waters.com/standards
WATERS ANALTYICAL STANDARDS
& REAGENTS
©2011 Waters Corporation 79
Benefits To Laboratory
Increased Productivity — Save time on preparation by using pre-made, ready-to-use products
— Achieve consistent, repeatable results and reduce assay re-runs
— Add reassurance to your analyses through consistent practice and trending
— Maintain optimum performance for equipment and improve system up-time
— Eliminate raw material sourcing, stocking, storage, and inventory management
— Minimize variations across instruments, users, labs, site locations, geographies
Enhanced Analytical Confidence — Reassurance in your analytical results by using Waters products manufactured,
traceable, and certified to exacting specifications.
— Confidence in reproducibility as Waters products offer consistency from vial to vial and batch to batch.
Built-In Compliance — Our products are developed, manufactured and documented to the highest
accreditation levels in a state-of-the-art custom built facility. o ISO 9001, ISO/IEC 17025, ILAC Guide 13, ISO 43-1, ISO Guide 34
— Standardizing your processes with Waters fully compliant products will simplify your compliance burdens
©2011 Waters Corporation 80
The Products
©2011 Waters Corporation 81
Thank You For Your Time and Attention
©2011 Waters Corporation 82
APPENDIX: ADDITIONAL SLIDE RESOURCES
AND INFORMATION
©2011 Waters Corporation 83
First range of sub-2-µm column chemistries — Designed and tested for the ACQUITY UPC2 system
— High efficiency
— Speed
— Resolution
ACQUITY UPC2 Columns offered in 3.5 µm column chemistries — Maximum flexibility to tackle challenging routine analyses
— Maximize plate counts and selectivity
Columns
©2011 Waters Corporation 84
Why SFC ?
Reduce Solvent Costs
Green Chemistry
Need for Orthogonal Separations
Increase Speed &
Throughput
Better, Faster &
Easier Chiral Separations
Issues with Range of Polarity
Limit Exposure to
Toxic Solvents
©2011 Waters Corporation 85
ACQUITY UPC2 Columns
BEH 2-EP (2-Ethylpyridine) • Good retention, peak shape and selectivity
BEH • Heightened interaction with polar groups such as phospholipids
CSH Fluoro-Phenyl • Good retention of weak bases • Alternate elution orders for acidic and neutral compounds
HSS C18 SB • Analysis of glycerides across vertical markets (Pharmaceutical, Food, Chemical Materials)
©2011 Waters Corporation 86
Different Selectivity
1. Coumarin 2. Flavone 3. Caffeine 4. Thymine
5. Papaverine 6. Sulfamethoxazole 7. Cytosine 8. Sulfamethizole
CSH Fluoro-Phenyl
BEH
BEH 2-EP
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5 6 7
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Column: 4.6 x 150mm, 5 µm 5-40% MeOH without additive 3.0mL/min at 40°C, 150 bar Injection Volume: 3µL
©2011 Waters Corporation 87
Batch Testing
All ACQUITY UPC2 Columns have SFC batch test results reported on the Certificate of Analysis (CoA)
All ACQUITY UPC2 Columns have an attached e-Cord™
©2011 Waters Corporation 88
HILIC COLUMN CHEMISTRIES
©2011 Waters Corporation 89
Stationary Phases for HILIC separations
ACQUITY UPLC® BEH Amide XBridgeTM Amide
ACQUITY UPLC® BEH HILIC XBridgeTM HILIC
Atlantis® HILIC Silica
Silica HILIC Columns (pH range 1 – 5)
Hybrid HILIC Columns (pH range 1 – 11)
©2011 Waters Corporation 90
Waters HILIC screening strategy
pH 3
ACQUITY UPLC® BEH HILIC 2.1 x 50 mm, 1.7 µm
Opt
imiz
atio
n
pH 9
ACQUITY UPLC® BEH Amide 2.1 x 50 mm, 1.7 µm
Atlantis HILIC Silica 2.1 x 50 mm, 3 µm
Where do I start? • Initial scouting gradient from 95 to 50% acetonitrile over 5 minutes • At least 5% should be a polar solvent (i.e., water or methanol)
©2011 Waters Corporation 91
Implementing the Approach: Example 1, Water Soluble Vitamins
O
OH OH
O
OH
OH
Ascorbic acid
O
N
NH2
Nicotinamide
N
O
OHOH
CH3
Pyridoxal
N N
NNH
O
OCH3
CH3
OH
OHOH
OH
Riboflavin
O
N
OH
Nicotinic acid
N+
CH3
SN NH2
N
OHCH3
Thiamine
Cl-
N N
N N
CH3
CH3
NH2
O
CH3
NH2
O
O NH2
O
NH2
O
NH2
CH3CH3
O
NH2
NH
PO
O
CH3
CH3
O-
O
O
O
OH
N
OH
Co+
N
N CH3
CH3
HCH3
CH3
B12
N
NH
N
N
NH
O
O
O
NH2
NH
O
OHOH
Folic Acid
©2011 Waters Corporation 92
Stationary Phase Selectivity at Low pH: Water-soluble Vitamins
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BEH Amide
BEH HILIC
Atlantis HILIC Silica
1
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4
3 6
5
8 7
1,6
3,2
4
8
5
7
1
6 3,2
4
8
5
7
Compounds 1. Nicotinamide 50 µg/mL 2. Pyridoxine 50 µg/mL 3. Riboflavin 30 µg/mL 4. Nicotinic acid 50 µg/mL 5. Thiamine 50 µg/mL 6. Ascorbic Acid 50 µg/mL 7. B12 50 µg/mL 8. Folic Acid 25 µg/mL
pH 3 All 3 columns yield different selectivity BEH Amide has greatest resolution of all peaks
©2011 Waters Corporation 93
Carbohydrate Analysis Using the XBridge Amide Column
©2011 Waters Corporation 94
Commercial Food Samples Using XBridge BEH Amide Column
XBridge BEH Amide, 3.5µm, 4.6 x 250mm Isocratic: 75% ACN with 0.2% TEA 1.0mL/min, 15 µL injection volume
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Lamb Curry Meal
Food Sugar Standard 1 2 3 4 5 6
White Bread
Hot Cross Buns
Strawberry Smoothie
1) p-Toluamide 2) Fructose 3) Glucose 4) Sucrose 5) Maltose 6) Lactose
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