Post on 17-Apr-2018
UPLC Column Positioning UPLC Column Positioning UPLC Column Positioning UPLC Column Positioning JurmalaJurmala September 3September 3--55
Anne Anne DyrdalDyrdal CBUCBU
©2010 Waters Corporation
Waters Column Product Waters Column Product HistoryHistoryyyACQUITY UPLC® BEH AmideACQUITY UPLC® BEH Glycan
XBridge AmideXSelect HSS HPLC Columns ACQUITY UPLC® HSS
Cyano & PFP columns
Styragel®
µBondapak™
DeltaPak®
Symmetry®
Spherisorb® XTerra®
XTerraPrep®
SymmetryShield®
ACQUITY UPLC® BEHSunFire™ Columns
PrepPak® XBridge™
ACQUITY UPLC®
HSSC18 and HSSC18 SB
y
XSelectTM HSS Cyano & PFP columns
XP 2.5 µm Columns
1964 19791973
1992
Symmetry p
1998
®
1984
1986
2002
19991994 2003 20041976
1958
2006
2005 2007
2008
2009
2010
2011
2012
Atlantis®
Symmetry® 300
Nova‐Pak®
ProteinPakTM
Pico‐TagTM
Atlantis® HILIC Silica
Atlantis® T3ACQUITY UPLC® HSS T3
AccQTagTM UltraAccQTagTM
ACQUITY UPLC®
BEH125 SEC
ACQUITY UPC2
Prep OBD™Intelligent SpeedTM
BioSuite™NanoEase™
XBridge™ HILIC
ACQUITY UPLC® BEH200 SEC
©2010 Waters Corporation |2
ACQUITY UPLC BEH200 SECXSelect CSH HPLC columns
ACQUITY CSH ColumnsViridis SFC Columns
ProteinPak High Rs IEX
The Widest UPLC Column OfferingThe Widest UPLC Column Offeringgg
BEH C18
BEH C8
Five particle substrates • 130Å, 200Å and 300Å BEH [Ethylene Bridged Hybrid], HSS [High
Strength Silica] and CSH [Charged Surface Hybrid]
BEH Phenyl
BEH Shield RP18
g g y
• All are available in HPLC and UPLC particle sizes
Wide and growing selection of column chemistries• 15 stationary phases
BEH HILIC
BEH Amide
HSS T3
• BEH 130Å C18, C8, Shield RP18, Phenyl, HILIC and Amide
• BEH 300Å C18 and C4• BEH 200Å SEC
• HSS C18, T3, C18 SB HSS T3
HSS C18
HSS C SB
HSS C18, T3, C18 SB
• CSH C18, Fluoro‐Phenyl and Phenyl‐Hexyl
Proven application‐based solutions • AAA, OST, PST, PrST and Glycan
CSH C18
HSS C18 SB Transferability between HPLC and UPLC
XBridge HPLC and ACQUITY UPLC BEH columns
HSS HPLC and ACQUITY UPLC HSS columns
S l C d CQ CS l
Si
O
O
O
F
F
FO
©2010 Waters Corporation |3
CSH Fluoro‐Phenyl XSelect HPLC and ACQUITY CSH columns
VanGuard Pre‐columns
eCord Technology CSH Phenyl‐Hexyl
SiF
F
O
O
O
Si
O
O
O
Positioning Attributes Positioning Attributes to select the right columnto select the right columngg
Retentivity
Selectivity
Stability, Low pH
Stability, high pH
Polar Analyte Retention
Loadability
Peak Shape Low pH (Base)
MS Bleed
pH Drift Tolerant
Re-Equilibration Ease Peak Shape Low pH (Base) Re-Equilibration Ease
©2010 Waters Corporation |4
Waters Waters UPLC UPLC ColumnColumn PlatformsPlatforms
Three major column platforms
Application‐Directed Column Chemistries
High Purity Silica Columns
Hybrid Columns
•ACQUITY UPLC® HSS T3•ACQUITY UPLC® BEH HILIC•ACQUITY UPLC® BEH Amide
•ACQUITY UPLC® HSS C18•ACQUITY UPLC® HSS C18SB•ACQUITY UPLC® HSS T3
•ACQUITY UPLC® BEH
•ACQUITY UPLC® CSH™
High Added Value Products
ACQUITY UPLC HSS T3Q
•ACQUITY UPLC® PST•ACQUITY UPLC® PrST•ACQUITY UPLC® OST•ACQUITY UPLC® Glycan•AccQ•Tag™ Ultra UPLC•ACQUITY UPLC® BEH200 SEC•ACQUITY UPLC® BEH300 C4ACQUITY UPLC® BEH300 C
©2010 Waters Corporation |5
•ACQUITY UPLC® BEH300 C18
ACQUITY UPLCACQUITY UPLC®® High Strength Silica High Strength Silica (HSS) Particles(HSS) Particles( )( )
Why develop a 100% silica UPLC® particle?Why develop a 100% silica UPLC particle?— Different application needs require different selectivities
— Different particles impart selectivity differences
Customers asked for:— Customers asked for:
1. A column that retains and separates small, water-soluble organic molecules
HSS T3 S 06• HSS T3
2. A column that offers superior peak shape, increased retentivity and superior acid stability
Sep-06
• HSS C18
3. A column that is truly “different” in terms of selectivity for basic compounds
Jun-07
©2010 Waters Corporation |6
• HSS C18 SB Jan-08
HSS [High Strength Silica ] ChemistriesHSS [High Strength Silica ] Chemistries[ g g ][ g g ]
HSS C18— High coverage, trifunctionally ‐ bonded C18 [pH 1 – 8]
— Universal, high performance C18
— Proprietary endcapping technique for superior peak shape and lifetimelifetime
HSS C18 SB [Selectivity for Bases]— Low ligand density, trifunctionally ‐ bonded C18 [pH 2 – 8]
1.8 µm
UPLC
— Non‐endcapped C18 designed for silanophilic interactions and alternate selectivity with exceptional peak shape for bases
HSS T3
HPLC
3.5 and5 µm
HSS T3— Low ligand density, trifunctionally ‐ bonded C18 [pH 2 – 8]
— Aqueous ‐ compatible C18 chemistry with long column lifetimes
— Recommended for maximum polar compound retention by
µ
©2010 Waters Corporation |7
RPLC
HSS [High Strength Silica ] Chemistries HSS [High Strength Silica ] Chemistries PositioningPositioninggg
HSS C18 and HSS T3 :G l l f l l H— General purpose columns for low to neutral pH
— Alternate selectivity to BEH particles
HSS C18 SB : — Alternate selectivity ,especially for basic compounds, to BEH
particlesp
HSS T3 :E h d RP t ti f l l l— Enhanced RP retention for polar molecules
Direct scalability between UPLC Technology and HPLC (no
©2010 Waters Corporation |8
prep)
Waters UPLC Waters UPLC ColumnColumn PlatformsPlatforms
Three major column platforms
Application‐Directed Column Chemistries
High Purity Silica Columns
Hybrid Columns
•ACQUITY UPLC® HSS T3•ACQUITY UPLC® BEH HILIC•ACQUITY UPLC® BEH Amide
•ACQUITY UPLC® HSS•ACQUITY UPLC® BEH
•ACQUITY UPLC® CSH™
High Added Value Products
Q
•ACQUITY UPLC® PST•ACQUITY UPLC® PrST•ACQUITY UPLC® OST•ACQUITY UPLC® Glycan•AccQ•Tag™ Ultra UPLC•ACQUITY UPLC® BEH200 SEC•ACQUITY UPLC® BEH300 C4ACQUITY UPLC® BEH300 C
©2010 Waters Corporation |9
•ACQUITY UPLC® BEH300 C18
WhatWhat isis HILIC?HILIC?
HILIC ‐ Hydrophilic Interaction Chromatography
Retention of highly polar analytes not retained by reversed-phase— Less interference from matrix components
Complementary selectivity to reversed-phase— Polar pesticides retain more than hydrophobic interferences
Enhanced sensitivity in mass spectrometry Enhanced sensitivity in mass spectrometry— High organic mobile phases (> 80% ACN) promotes enhanced
ESI-MS response— Direct injection of high organic extracts without dilution— Facilitates use of lower volume samples
Improved sample throughput— Direct injection of high organic eluates from PPT LLE or SPE
©2010 Waters Corporation |10
— Direct injection of high organic eluates from PPT, LLE or SPE without the need for dilution or evaporation and reconstitution
BEH HILIC BEH HILIC ChemistriesChemistries
ACQUITY UPLC BEH HILIC— Unbonded ethylene bridged hybrid particle
— Particle size: 1.7 µm [UPLC]; 2.5, 3.5 and 5 µm [HPLC]
— pH range: 1 – 9; Temp. limit: 45°C
— Exceptional peak shape and retention for polar basic analytes
ACQUITY UPLC BEH Amide
1.7 µm
UPLC
ACQUITY UPLC BEH Amide— Alternative selectivity for HILIC separations
— Particle size: 1.7 µm [UPLC]; 3.5 µm [HPLC]
— pH range: 2 11; Temp limit: 90°C
HPLC
2.5, 3.5,5 & 10— pH range: 2 – 11; Temp. limit: 90 C
— Designed for retention of polar acidic, neutral and basic
compounds.
— Ideal column for carbohydrates and glycans
5 & 10 µm
©2010 Waters Corporation |11
Ideal column for carbohydrates and glycans
Selectivity ComparisonSelectivity ComparisonBEH Amide vs. BEH HILICBEH Amide vs. BEH HILIC
Compounds1. Methacrylic Acid 2. Cytosine3. Nortriptyline4. Nicotinic Acid
0.60 1122 ACQUITY UPLC BEH HILIC
2.1 x 100 mm, 1.7 µm
4. Nicotinic Acid
AU
0.20
0.4033
44
33
0.00
ACQUITY UPLC BEH Amide
AU
0.40
0.60
11 22
ACQUITY UPLC BEH Amide 2.1 x 100 mm, 1.7 µm
0.00
0.2044
©2010 Waters Corporation |12
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
Isocratic mobile phase of 90/5/5 ACN/EtOH/10 mM CH3COONH4 in H2O with 0.02% CH3COOH, flow rate 0.5 mL/min, column temp. 25 °C, 1.5 µL injection, 60 µg/mL each compound, sample diluent 75/25 ACN/MeOH, UV 210 nm
Blueberry Jam on Blueberry Jam on 1.7µm BEH Amide Column1.7µm BEH Amide Columnµµ
ACQUITY ELSD using 0.29mL/min at 35°C, Isocratic with 0.2%TEA in 75% ACN
1
3
mid
e
2
5
P-T
olu
am
Food Sugars(1mg/mL)
4
Blueberry Jam Extract
(5mg/mL)
1 2
4
©2010 Waters Corporation |131) Fructose, 2) Glucose, 3) Sucrose, 4) Maltose, 5) Lactose
Minutes0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
BEH [Ethylene Bridged Hybrid] BEH [Ethylene Bridged Hybrid] HILIC Chemistries HILIC Chemistries -- PositioningPositioninggg
For enhanced retention of polar compounds when RP For enhanced retention of polar compounds when RP columns do not work
BEH A id f lt t l ti it t BEH HILIC l BEH Amide for alternate selectivity to BEH HILIC column and Sugar/Saccharides/Carbohydrates separation
Direct scalability between UPLC Technology to HPLC to Prep (only for BEH HILIC)
©2010 Waters Corporation |14
Waters UPLC Waters UPLC ColumnColumn PlatformsPlatforms
Three major column platforms
Application‐Directed Column Chemistries
High Purity Silica Columns
Hybrid Columns
•ACQUITY UPLC® HSS T3•ACQUITY UPLC® BEH HILIC•ACQUITY UPLC® BEH Amide
•ACQUITY UPLC® HSS•ACQUITY UPLC® BEH C18•ACQUITY UPLC® BEH C8•ACQUITY UPLC® BEH Phenyl
High Added Value Products
Q y•ACQUITY UPLC® BEH Shield RP18
•ACQUITY UPLC® CSH™
•ACQUITY UPLC® PST•ACQUITY UPLC® PrST•ACQUITY UPLC® OSTQ•ACQUITY UPLC® Glycan•AccQ•Tag™ Ultra UPLC•ACQUITY UPLC® BEH200 SEC•ACQUITY UPLC® BEH300 C4ACQUITY UPLC® BEH300 C
©2010 Waters Corporation |15
•ACQUITY UPLC® BEH300 C18
BEH [Ethylene Bridged Hybrid] BEH [Ethylene Bridged Hybrid] Reversed Phase ChemistriesReversed Phase Chemistries
BEH C18— Trifunctionally ‐ bonded C18— Widest pH range for maximum selectivity [pH 1 ‐ 12]— Superior peak shape and efficiency in buffered mobile phases
BEH C8— Trifunctionally ‐ bonded C8— Wide pH range [pH 1‐12]— Lower hydrophobicity than C18
1.7 µm
UPLC
BEH Shield RP18— Monofunctionally bonded, embedded carbamate phase [pH 2 – 11]— Alternate selectivity compared to alkyl chain columns
HPLC
2.5, 3.5,5 & 10 µm
BEH Phenyl— Trifunctionally ‐ bonded C6‐Phenyl— Wide pH range [1‐12]
C l t l ti it f ti i
©2010 Waters Corporation |16
— Complementary selectivity for aromatic species
BEH RP Chemistries BEH RP Chemistries -- PositioningPositioninggg
General purpose columns applicable General purpose columns applicable
— to a broadest range of compound classes
h b ff l i l d— when buffers are exclusively used
— where high pH stability is most important
Direct scalability between UPLC Technology to HPLC to Prep
©2010 Waters Corporation |17
Designed for pH Stability
Industry Trends:Industry Trends:Current State of ReversedCurrent State of Reversed--phase Separationsphase Separationsp pp p
Advances in stationary phase design— Hybrid particle technology
o Extended usable pH range [1-12]
o Exceptional peak shape and efficiency
o Rugged and reliable column life
— Sub 2 µm particle technology
o Improvements in resolution, sensitivity and speed of analysis
— Pellicular [core-shell] particles
Instrument platform of choice— UltraPerformance LC with UV and mass spectrometry [UPLC/MS/[MS]]p y [ / /[ ]]
o Requires volatile mobile phases
• Excludes typical UV-based buffers [i.e., phosphate buffers]
o Preference towards low ionic strength additives[i.e., formic acid,
©2010 Waters Corporation |18
g [ , ,acetic acid, ammonium hydroxide]
• Avoid preparation of buffers if possible
Defining the Problem:Defining the Problem:Modern High Purity Stationary PhasesModern High Purity Stationary Phasesg y yg y y
Lack of selectivity choices for method development
Poor mass loading of charged cationic [basic] solutes in low ionic strength additives under low pH mobile phases due to limited sample capacitylimited sample capacity— High tailing factors
— Poor signal intensity
Elution [retention] time shift after exposure to a higher pH mobile phase*1
Irreproducible assay performance when performing method screening— Irreproducible assay performance when performing method screening
o Low/high pH switching with un-buffered mobile phases
©2010 Waters Corporation |19
*1 Marchand, D.H., et al., J. Chromatogr. A 2003, 1015, 53-64
Waters UPLC Waters UPLC ColumnColumn PlatformsPlatforms
Three major column platforms
Application‐Directed Column Chemistries
High Purity Silica Columns
Hybrid Columns
•ACQUITY UPLC® HSS T3•ACQUITY UPLC® BEH HILIC•ACQUITY UPLC® BEH Amide
•ACQUITY UPLC® HSS•ACQUITY UPLC® BEH
•ACQUITY UPLC® CSH™C18
High Added Value Products
Q 18•ACQUITY UPLC® CSH™ Phenyl‐Hexyl•ACQUITY UPLC® CSH™ Fluoro‐Phenyl •ACQUITY UPLC® PST
•ACQUITY UPLC® PrST•ACQUITY UPLC® OST•ACQUITY UPLC® Glycan•AccQ•Tag™ Ultra UPLC•ACQUITY UPLC® BEH200 SEC•ACQUITY UPLC® BEH300 C4ACQUITY UPLC® BEH300 C
©2010 Waters Corporation |20
•ACQUITY UPLC® BEH300 C18
Charged Surface Hybrid Charged Surface Hybrid (CSH™) Technology(CSH™) Technology( ) gy( ) gy
CSH™ Technology
Ch d S f H b id—Charged Surface Hybrid
©2010 Waters Corporation |21
CSH [CSH [ChargedCharged Surface Surface HybridHybrid] ] ChemistriesChemistries
ACQUITY UPLC CSH C18
— Mid/high coverage trifunctional C18
— End capped
— High pH stable
— Superior peak shape for bases
— Lower retentivity for bases (vs. BEH C18)
— pH range: 1 – 11pH range: 1 11
ACQUITY UPLC CSH Fluoro‐Phenyl
— Most ‘different’ selectivity (vs. CSH C18 & Phenyl‐Hexyl)
— Excellent reproducibility
1.7 µm
UPLC
— Not end capped
— Superior peak shape and lower retentivity for bases
— pH range: 1 – 8
ACQUITY UPLC CSH Phenyl‐Hexyl
HPLC
3.5,5ACQUITY UPLC CSH Phenyl Hexyl
— Trifunctional phenylhexyl
— End capped
— High pH stable
5 µm
©2010 Waters Corporation |22
— Superior peak shape for bases
— Different selectivity (vs. BEH Phenyl)
— pH range: 1 – 11
New Way to think about selectivityNew Way to think about selectivity
14
Si il l i ien
yl
pH 3 ammonium formate/acetonitrile gradient
10
12S-value = 6
Similar selectivity
R² = 0.574Flu
oro
-Ph
e
6
8
R² = 0.997 S-value = 65Different selectivity
C8
or
CS
H F
R² = 0.997
0
2
4
go
n B
EH
C
Higher S-values = larger selectivity differences
00 2 4 6 8 10 12
kg on BEH C18(kg: gradient retention factor)
k
©2010 Waters Corporation |23BAA JET PCI
21100 R*)S(ySelectivit without berberine and PSA
Wide Selectivity Range for ACQUITY Wide Selectivity Range for ACQUITY CSH ColumnsCSH Columns
11
8
101
2
16
5
14
4
9
15
18
0
2
17
9
XBridge™ C18 3.5µm
136 12
3 7
1
19
8 11
3 16
5
14
9 15
18
2 XSelect CSH™ C18 3.5µm
S XB C18 11
10 13
1
6
12
1
3 7
1
40 17
19
11 16
15 XSelect CSH™
Fl Ph l 3 5
S XBr C18 11
XSelect™ / CSH™ Columns Were Designed for Selectivity8
1
6
12
3
57
14
4
9 18
0
2
10
17
19
Fluoro-Phenyl 3.5µm
S XBr C18 36
S XS CSH C18 59
Designed for Selectivity8
11
13
1
16
5
4
9
15
182
XSelect CSH™Phenyl-Hexyl 3.5µm
S XBr C18 20 S XS CSH C18 16
©2010 Waters Corporation |24
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
6 12
3 7
14
40
10 17
19 S XS CSH Fluoro-Phenyl 48
ImprovingImproving LC LC ColumnColumn performance :performance :Unexpectedly High Unexpectedly High TfTf For Basic CompoundsFor Basic Compoundsp y gp y g pp
Current State of Reversed-Phase Separations— Unexpectedly high tailing factors for analytical mass loads of basic analytes in low ionic strength, low
pH mobile phases due to mass overloadp p
— Slow equilibration at low pH
— Retention time shifts in low pH mobile phases after exposure to high pH (e.g., > pH 7) mobile phases
Introducing Charged Surface Hybrid (CSH™) Technology
Achieving Performance in Acidic Low-Ionic-Strength Mobile D V McCalley / J Chromatogr A 1075 (2005) 57 64 Achieving Performance in Acidic, Low-Ionic-Strength Mobile Phases
— Tailing Factors and Loading Capacity for Bases.
— Retention Time Changes after High pH Exposure.
St bilit
D.V. McCalley / J. Chromatogr. A 1075 (2005) 57–64
1.25 µg on a 4.6 x 150 mm
Stability— Acid stability
— Base Stability
Overlaid chromatograms for 1.25, 0.25 and 0.05 µg nortriptyline in A= 0.02M formic acid in water pH 2.75, B= acetonitrile–0.04M formic 2.75, B acetonitrile 0.04M formic
acid in water 50:50 (v/v) on 4.6 mm x 150 mm column.
©2010 Waters Corporation |25
Implementing Mobile Phase pH Switching:Monitoring Column Performanceg
Most systematic screening protocol in method development Most systematic screening protocol in method development evaluates high and low pH mobile phases.— Screen multiple columns and organic modifiers at pH 3 and pH 10
— Stationary phase must be re-equilibrated when exposed to a new set of Stationary phase must be re equilibrated when exposed to a new set of conditions
With low ionic strength mobile phases [i e formic acid With low ionic strength mobile phases [i.e., formic acid, ammonium hydroxide], column performance [retention and selectivity] can change *1
Slow surface equilibration at low pH— Slow surface equilibration at low pH
— Inconsistent selectivity can impact open access systems and method transfer
©2010 Waters Corporation |26
*1 Marchand, D.H., et al., J. Chromatogr. A 2003, 1015, 53-64
Low pH to High pH to Low pH Low pH to High pH to Low pH Method DevelopmentMethod Development
I n l e t m e t h o d w e l l 2 : 1
2 4 - S e p - 2 0 0 8 1 5 : 0 4 : 5 8P u r p l e L C / M S T r a c eL N B R e f Q C 1 3 8 6N a m e P u r p l e
1 . 6 e + 2
1 . 8 e + 2
Q C 1 3 8 6 3 : D i o d e A r r a y R a n g e : 2 . 1 1 7 e + 20 . 9 4
1 . 4 91 . 0 4
0.1% Formic AcidNew Column ty
line
pp
AU
6 . 0 e + 1
8 . 0 e + 1
1 . 0 e + 2
1 . 2 e + 2
1 . 4 e + 2
0 . 3 9
0 1 80 . 8 2
1 . 7 9New Column
Am
itript
1. Low pH
T i m e0 . 2 0 0 . 4 0 0 . 6 0 0 . 8 0 1 . 0 0 1 . 2 0 1 . 4 0 1 . 6 0 1 . 8 0 2 . 0 0
0 . 0
2 . 0 e + 1
4 . 0 e + 10 . 1 8
1 5 - O c t - 2 0 0 8 1 5 : 3 6 : 3 7P u r p l e L C / M S T r a c eN a m e T o n y W C o o k
Meth
oD
evelo
p
I n l e t m e t h o d w e l l 2 : 1
1 5 - O c t - 2 0 0 8 1 5 : 3 6 : 3 7P u r p l e L C / M S T r a c eL N B R e f T M h p h 4N a m e T o n y W C o o k
1 . 0 e + 2
1 . 1 e + 2
1 . 2 e + 2
1 . 3 e + 2
J S B 1 1 1 6 6 - 5 3 : D io d e A r r a y R a n g e : 1 . 3 3 3 e + 20 . 6 1
1 . 0 7
1 . 5 3
1 . 8 4
mitripty
line10 mM Ammonium
BicarbonateNew Column
od
m
en
t
1.25e+2
1.5e+2
JSB11379-2 3: Diode Array Range: 1.891e+20.93
1.02 1.481.78
0.1% Formic AcidColumn Previously Exposed to High pH
Note:pty
line
AU
4 . 0 e + 1
5 . 0 e + 1
6 . 0 e + 1
7 . 0 e + 1
8 . 0 e + 1
9 . 0 e + 1
0 . 3 8 1 . 3 8
Am
2. High pHAU
2.5e+1
5.0e+1
7.5e+1
1.0e+20.38
0.17
0.96
Increased RT & Peak Shape
Loss for Amitriptyline
Am
itrip
3. Low pH
©2010 Waters Corporation |27
T im e0 . 2 0 0 . 4 0 0 . 6 0 0 . 8 0 1 . 0 0 1 . 2 0 1 . 4 0 1 . 6 0 1 . 8 0 2 . 0 0
0 . 0
1 . 0 e + 1
2 . 0 e + 1
3 . 0 e + 1 0 . 1 8
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
0.0
Implementing Mobile Phase pH Switching:Implementing Mobile Phase pH Switching:Monitoring Column PerformanceMonitoring Column Performancegg
Performance in 0.1% formic acid/acetonitrile gradient— MAINTAINS Retention Time and Peak Shape After High pH Exposure.
XSelect CSH C18 3.5 µm
0 12
0.18AU
Before high pH exposureAft hi h H
0 00
0.06
0.12 After high pH exposure
l h h l0.00
0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
0.18Before high pH exposure
Gemini-NX C18 3 µmAU
min
neutral phthalates
25% RT change81% peak height loss
20% RT change64% peak height loss
Metoprolol Amitriptyline
0.06
0.12
Before high pH exposureAfter high pH exposure
81% peak height lossp g
©2010 Waters Corporation |28
0.00
0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 min
neutral phthalatesMetoprolol Amitriptyline
Why Haven’t I Seen This Before?Why Haven’t I Seen This Before?yy
Remember: when using low pH buffers as part of your Remember: when using low pH buffers as part of your method development protocols (e.g., ammonium formate, sodium phosphate, etc.) these ‘pH switching’ effects do notoccuroccur
‘pH switching’ effects only occur when using acidic, low ionic strength additives
ALL high pH-capable columns EXCEPT ACQUITY CSH and ALL high pH capable columns EXCEPT ACQUITY CSH and XSelect Columns exhibit this behavior
©2010 Waters Corporation |29
CSH Technology:Controlled Surface Charge Yields High Performanceg g
1
0.10
ACQUITY CSH C18 1.7 µm0.05 % formic acid
Peak capacity =71
23 4
Column: 2.1 x 50 mmMP A: water
MP B: acetonitrileMP C: 2% formic acid
Gradient: 25-35%B in 2
AU
0.05
Peak capacity =7145 6
min, 35 – 95%B from 2 -3 min; [2.5% C held
constant]Inj. Vol.: 5 µL
Sample Conc.: 10 µg/mL
Detection UV @ 254 nmSampling rate: 20 Hz
0.00
Minutes
0.00 1.00 2.00 3.00 4.00
Filter: 0.1 secSystem: ACQUITY UPLC H-Class with ACQUITY UPLC PDA and SQD
Tricyclicantidepressants:1. Doxepin
Minutes
0.10
Fused Core C18 1.7 µm 0.05 % formic acid
Peak capacity = 262. Desipramine3. Imipramine4. Nortriptyline5. Amitriptyline6. Trimipramine
AU 0.05 1 2 3 45 6
©2010 Waters Corporation |30
0.00
Minutes
0.00 1.00 2.00 3.00 4.00
CSH CSH [Charged Surface Hybrid] [Charged Surface Hybrid] Chemistries Chemistries -- PositioningPositioninggg
Use additives AND buffersUse additives AND buffers
Isolation/purification
Prefer to work at low pH with occasional high pH work
Switch back/forth between low & high pH (additives)
LC/MS laboratory
Seeking additional ACQUITY UPLC column selectivitiesSeeking additional ACQUITY UPLC column selectivities
Direct scalability between UPLC Technology to HPLC to Prep
©2010 Waters Corporation |31
Designed for Selectivity
Waters Waters UPLC UPLC ColumnColumn PlatformsPlatforms
Three major column platforms
Application‐Directed Column Chemistries
High Purity Silica Columns
Hybrid Columns
•ACQUITY UPLC® HSS T3•ACQUITY UPLC® BEH HILIC•ACQUITY UPLC® BEH Amide
•ACQUITY UPLC® HSS•ACQUITY UPLC® BEHHigh Added Value Products
•ACQUITY UPLC® CSH™ •ACQUITY UPLC® PST•ACQUITY UPLC® PrST•ACQUITY UPLC® OST•ACQUITY UPLC® Glycan•AccQ•Tag™ Ultra UPLC•ACQUITY UPLC® BEH200 SEC•ACQUITY UPLC® BEH300 C4ACQUITY UPLC® BEH300 C
©2010 Waters Corporation |32
•ACQUITY UPLC® BEH300 C18
BioseparationBioseparation ColumnsColumnspp
Sub‐2 µm particle specifically designed For UPLCFor UPLC
QC Tested with relevant biomoleculesto achieve unmatched:
S i i i Sensitivity Resolution Method Reproducibility
Characterization of: Amino Acids Peptides Proteins Glycans Synthetic oligonucleotides
©2010 Waters Corporation |33
BioseparationBioseparation ColumnsColumnspp
ACQUITY UPLC BEH200 SEC Column
Determines aggregation levels for monoclonal antibodies • up to 10X faster than HPLC SEC methods
F ll ti i d l h i t Fully optimized column chemistry• Eliminates high salt concentration mobile phases
QC tested with relevant proteins, h d b h b h i • unmatched batch-to-batch consistency
• increased confidence in validated QC release methods
2Analyte pI MW
1. Thyroglobulin, 3 mg/mL 4.6 669,000
2. IgG, 3 mg/mL (Vicam) 6.7 150,000
3. BSA, 5 mg/mL 4.6 66,400
4. Myoglobin, 2 mg/mL 6.8, 7.2 17,0001
2
34
5
©2010 Waters Corporation |34
5. Uracil, 0.1 mg/mL N/A 112
Minutes0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
BioseparationBioseparation ColumnsColumnspp
Protein Separation Technology: BEH 300Å C4 and C18 300Å pores 300Å pores C4 and C18 Ligands available Minimal undesirable secondary interactions Minimal detectable carryover at elevated temperature separations
BEH 300Å C
y p p
B
D
E
BEH 300Å C18
A. Ribonuclease
AB
CF
E
BEH 300Å C4
A. RibonucleaseB. Cytochrome cC. BSAD. β-LactoglobulinE. EnolaseF Phosphorylase b
A
BC D
F
©2010 Waters Corporation |35
Minutes0.00 4.00 8.00 12.00 16.00 20.00
F. Phosphorylase b
Shorter chain gives narrower peaks, particularly for the later eluting components.
TranferabilityTranferability fromfrom UPLC UPLC TechnologyTechnologyto HPLC to HPLC
©2010 Waters Corporation |36
UPLC and HPLC Column EquivalencyUPLC and HPLC Column Equivalency
UPLC 1.7/ 1.8 µm
HPLC
ACQUITY UPLC BEH C18 XBridge C18
ACQUITY UPLC BEH C8 XBridge C8
ACQUITY UPLC BEH Shield RP18 XBridge Shield RP18
===
ACQUITY UPLC BEH Phenyl XBridge Phenyl
ACQUITY UPLC BEH HILIC XBridge HILIC
ACQUITY UPLC BEH Amide XBridge Amide
==
=ACQUITY UPLC BEH300 C18 XBridge BEH300 C18
ACQUITY UPLC BEH300 C4 XBridge BEH300 C4
ACQUITY UPLC HSS C18 HSS C18
===
ACQUITY UPLC HSS C18 SB HSS C18 SB
ACQUITY UPLC HSS T3 HSS T3
ACQUITY UPLC CSH C18 XSelect C18
===
©2010 Waters Corporation |37
ACQUITY UPLC CSH Phenyl Hexyl XSelect Phenyl Hexyl
ACQUITY UPLC CSH Fluoro Phenyl XSelect Fluoro Phenyl
==
Introducing CORTECS™ Columns
Featuring 1 6 µm Solid Core ParticlesFeaturing 1.6 µm Solid-Core Particles
©2010 Waters Corporation |38
IntroducingIntroducing
CORTECS™ Columns herald an unprecedented level of efficiency and sensitivity for UPLC columns.
Featuring 1.6 μm silica-based solid-core particles
and ultra-low dispersion hardware, p ,
CORTECS UPLC® Columns enable higher peak capacities
and thereby greater resolution and improved throughput
h h ll d dto meet even the most challenging demands.
©2010 Waters Corporation |39
CORTECS ColumnsCORTECS Columns
UPLC Columns featuring 1.6 µm solid-core silica particles
Key benefits: – Highest efficiency UPLC Column (>35% vs fully porous sub-2-µm
columns)– Improved performance at similar backpressure– Improved performance at similar backpressure– Increased throughput
3 Chemistries:
– C18+
– C18
– HILIC
©2010 Waters Corporation |40
10 Year Evolution of UPLC 10 Year Evolution of UPLC Particle TechnologyParticle Technologygygy
BEH Technology™BEH Technology™
First ACQUITY UPLC
HSS TechnologyHSS Technology
Enhanced retention
CSH™ TechnologyCSH™ Technology
Designed for First ACQUITY UPLC particle
Wide pH and temperature range
Enhanced retention
Particle and ligandselectivity
Designed for selectivity
Improved basic peak shape at low pHtemperature range
2004
selectivity
2006
shape at low pH
2010
©2010 Waters Corporation |41
10 Year Evolution of UPLC 10 Year Evolution of UPLC Particle OfferingParticle Offeringgg
CORTECS CORTECS SolidSolid--Core Core TechnologyTechnology
BEH TechnologyBEH Technology HSS TechnologyHSS Technology CSH TechnologyCSH Technology
Increased efficiency and resolution
Higher throughput
First ACQUITY UPLC particle
Wide pH and temperature range
Enhanced retention
Particle and ligandselectivity
Designed for selectivity
Improved basic peak shape at low pH
2013
temperature range
2004
selectivity
2006
shape at low pH
2010
©2010 Waters Corporation |42
What allowed us to take solid-core particle technology to the next level of performance?
The CORTECS SolidThe CORTECS Solid--Core ParticleCore Particle
Compared to Fully Porous Particles
Only thin outer layer contains the pores with the chromatographic surface
CORTECSSolid-core
dp
= 1
.6 µ
m
The center core is nonporous
The outer shell is typically “bumpy” not pretty
The particles size distribution is d
The particles size distribution is narrower
dcore = 1.12 µm
Rho, = 1.12/1.60 = 0.70
ρ = core diameter / particle diameter
,
65% Porous Volume
ρ = 0 → fully porous particle
©2010 Waters Corporation |43
ρ y p p
ρ = 1 → nonporous particle
Enabling ExpertiseEnabling ExpertiseDifferentiating Advancements in UPLC ColumnsDifferentiating Advancements in UPLC Columnsgg
Bulk SynthesisRugged sub-2-µm solid-core particlesgg µ p
Highest Efficiencies
EngineeringUPLC column hardwareUPLC column hardwareLow band broadening
Column PackingUltra-stable packed column beds Software
©2010 Waters Corporation |44
Proprietary packing processes Paperless tracking of column history witheCordTM technology
Where we were…Where we were…
Since 2004, fully porous ACQUITY UPLC 1.7 µm BEH C18
Has been our most efficient particle
16,000
Started us down the path of sub-2-µm particles and the development of UPLC Technology, which was needed to demonstrate its efficiency
14,150
12,000
,
4 s
igm
a)
8,000Plat
es (
4
ACQUITY UPLC 1 7 µm BEH C 8
4,0000.00 0.25 0.50 0.75 1.00 1.25
ACQUITY UPLC 1.7 µm BEH C18
©2010 Waters Corporation |45
Flow Rate (mL/min)
2.1 x 50 mm column. A standard ACQUITY UPLC I-Class using 70% Acetonitrile in H2O at 30 °C with 0.5 µL injections from a 1 µL FL injector
However, as of today…However, as of today…, y, y
19,70020 000
39% higher efficiency
016,000
20,000
14,150
12,000
,
4 s
igm
a)
or up to 3x faster!
8,000Plat
es (
ACQUITY UPLC 1 7 µm BEH C18
4,0000.00 0.25 0.50 0.75 1.00 1.25
CORTECS UPLC 1.6 µm C18+
ACQUITY UPLC 1.7 µm BEH C18
©2010 Waters Corporation |46
Flow Rate (mL/min)
2.1 x 50 mm column. A standard ACQUITY UPLC I-Class using 70% Acetonitrile in H2O at 30 °C with 0.5 µL injections from a 1 µL FL injector
CORTECS Chemistry OverviewCORTECS Chemistry Overview
C18+ C18 HILIC
Chemistry
yy
y
Intended Use
General purpose, high-efficiency, reversed-phase column. A positively
General purpose, high-efficiency, reversed-phase column. Balanced
High-efficiency column designed for retention of extremely polar Intended Use charged surface
delivers excellent peak shape for basic compounds at low pH.
retention of acids, bases and neutrals at low and mid-range pH.
analytes. Offers orthogonal selectivity vs. C18 columns.
Ligand Type Trifunctional C18 Trifunctional C18 NoneLigand Type Trifunctional C18 Trifunctional C18 None
Surface Charge Modification + None None
Endcap Style Proprietary Proprietary None
Carbon Load 5.7% 6.6% None
Ligand Density 2.4 µmol/m2 2.7 µmol/m2 None
pH Range 2 – 8 2 – 8 1 - 5
©2010 Waters Corporation |47
Temperature Limits Low pH = 45 °C High pH = 45 °C
Low pH = 45 °C High pH = 45 °C
Low pH = 45 °C High pH = 45 °C
Increased Efficiency of Increased Efficiency of CORTECS ColumnsCORTECS Columns
©2010 Waters Corporation |48
Practical Benefits of Practical Benefits of Increased EfficiencyIncreased Efficiencyyy
Benefits of higher efficiency separations include:– Improved peak shape and resolutionImproved peak shape and resolution
– Higher sample throughput:
o Comparable separations using faster flow rates
oror
o Comparable separations using a shorter column
©2010 Waters Corporation |49
Higher Efficiency leads to Sharper Peaks, Higher Efficiency leads to Sharper Peaks, Better Resolution of Local AnestheticsBetter Resolution of Local Anesthetics
0.201
2 4
5
ACQUITY BEH HILIC
1. Lidocaine 2. Butacaine 3 Tetracaine
AU0.10
0.153
4 ACQUITY BEH HILIC2.1 x 50mm 1.7 µm
3. Tetracaine4. Procaine
5. Procainamide
USP Resolution2,3: 1.2
0.00
0.05
1
AU
0.10
0.15
0.20CORTECS UPLC HILIC
2.1 x 50 mm 1.6 µm
2
3
4
5
USP Resolution2,3: 2.2A
-0.05
0.00
0.05
©2010 Waters Corporation |50
Minutes0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00
Higher Throughput Higher Throughput –– Sulfa Drugs:Sulfa Drugs:Double the Flow RateDouble the Flow Rate
0.10
0.12 Competitor Fully-porous C18 at 0.5 mL/min2.1 x 50 mm 1.8 µm
Gradient time: 4.2 minRuntime: 6 min
1. Sulfathiazole2. Sulfamerazine3. Sulfamethazine
4. Sulfamethoxypyridazine5 Sulfachloropyridazine
AU
0.04
0.06
0.08
1
23
4
5
6
5. Sulfachloropyridazine6. Sulfamethoxazole
7. Sulfasoxazole Pc= 13131 peaks per minute (gradient)
Comparable peak capacities (Pc) in
half the time0.00
0.02
0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00
7
CORTECS UPLC C18+ at 1.0 mL/min2.1 x 50 mm 1.6 µm
Gradient time: 2.1 minRuntime: 3 min
P = 136Pc= 13665 peaks per minute (gradient)
©2010 Waters Corporation |51
Note: the gradient is scaled to account for the change in flow rate
Comparative separations may not be representative in all applications.
Higher Throughput Higher Throughput –– Sulfa Drugs:Sulfa Drugs:Using a Shorter ColumnUsing a Shorter Columngg
2.1 x 100 mm 1.7 µmCompetitor Solid-Core C18
Gradient time: 8.4 minRuntime: 12 min
Pc= 17521 peaks per minute (gradient)
Comparable peak capacities (Pc) in
half the time2.1 x 50 mm 1.6 µmCORTECS UPLC C half the timeCORTECS UPLC C18+
Gradient time: 4.2minRuntime: 6 min
Pc= 16740 peaks per minute (gradient)40 peaks per minute (gradient)
©2010 Waters Corporation |52
Note: the gradient is scaled to account for the change in column length
Comparative separations may not be representative in all applications.
Impact of System Dispersion on Impact of System Dispersion on CORTECS Column EfficiencyCORTECS Column Efficiencyyy
ACQUITY UPLC I-Class5.5 µL USP N: 18,000
ACQUITY UPLC H-Class12 µL
USP N: 11,700
53% Increase in CORTECS Column Efficiency on the ACQUITY UPLC I Class System
©2010 Waters Corporation |53
Acetonitrile/ Water (70/30 v/v), 0.4 mL/ min, 30oC, 0.5 µL injection. Peak i.d.: Acetone, Naphthalene, Acenaphthene2.1 x 50 mm CORTECS C18 Column
SummarySummaryyy
CORTECS Column family features 1.6 µm solid-core silica particlesp
Enabled by over 10 years of designing, synthesizing and packing sub 2 µm particles packing sub-2-µm particles
Set the bar as the new efficiency standard for UPLC columns
Three unique chemistries for selectivity and exceptional peak shapeshape
©2010 Waters Corporation |54
SummarySummaryyy
Higher efficiency leads to improved resolution and faster throughputg p
Lowest dispersion systems result in ultimate efficiency and performanceperformance
CORTECS 1.6 µm particle size chosen to maximize efficiency while pairing seamlessly with UPLC systems
Most recent addition to an ever evolving sub 2 µm UPLC Most recent addition to an ever-evolving sub-2-µm UPLC particle family
©2010 Waters Corporation |55
©2010 Waters Corporation