uplc column positioning jurmalajurmala september 3 ... · uplc column positioning jurmalajurmala...

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


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


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


•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


• 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


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


prep)

Waters UPLC Waters UPLC ColumnColumn PlatformsPlatforms




Hybrid Columns


•ACQUITY UPLC® HSS•ACQUITY UPLC® BEH



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



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


— 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


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


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)






Hybrid Columns


•ACQUITY UPLC® HSS•ACQUITY UPLC® BEH C18•ACQUITY UPLC® BEH C8•ACQUITY UPLC® BEH Phenyl


Q y•ACQUITY UPLC® BEH Shield RP18


•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



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


— 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


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,


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


*1 Marchand, D.H., et al., J. Chromatogr. A 2003, 1015, 53-64





Hybrid Columns


•ACQUITY UPLC® HSS•ACQUITY UPLC® BEH

•ACQUITY UPLC® CSH™C18


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



Charged Surface Hybrid Charged Surface Hybrid (CSH™) Technology(CSH™) Technology( ) gy( ) gy

CSH™ Technology

Ch d S f H b id—Charged Surface Hybrid


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


— 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


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.


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


*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


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


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


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


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


Designed for Selectivity

Waters Waters UPLC UPLC ColumnColumn PlatformsPlatforms




Hybrid Columns


•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



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



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


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


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


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


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

===


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


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.


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


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


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


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


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


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


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


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


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


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


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


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


P = 136Pc= 13665 peaks per minute (gradient)


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


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)


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


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


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


uplc column positioning jurmalajurmala september 3 ... · uplc column positioning jurmalajurmala...

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