an introduction to waters an introduction to waters ... · most ms-compatible hplc columns on the...

©2012 Waters Corporation 1

An Introduction to Waters An Introduction to Waters HPLCHPLC and and ACQUITYACQUITY UPLCUPLC®® Column TechnoColumn Technologieslogies

for small molecule analysisfor small molecule analysis

Esa LehtorinneEsa [email protected][email protected]: +358Tel: +358--99--5659 62885659 6288Fax: +358Fax: +358--99--5659 62825659 6282

Waters FinlandWaters FinlandKutomotie 16Kutomotie 1600380 Helsinki00380 Helsinki


AgendaAgenda

� Column Manufacturing and Platform Definitions

� New HPLC & ACQUITY UPLC Column Chemistries

� New 2.5 µm eXtended Performance (XP) Columns

� Summary


What makes an LC column reproducible?

C) Engineering B) Packing A) Sorbents


Developing a Waters Column:Developing a Waters Column:Quality By DesignQuality By Design

� Behave like our customers in regulated industries– US Food and Drug Administration registeredo cGMP’so Class 1 medical devices– ISO 9000:2001– ISO 13485:2003

� Own and control every step of the process– Raw materials to final producto Very few suppliers do this or are capable of doing this– Understand the parameters that make a difference….. and then

control them

� So what does this mean………


Developing a Waters ColumnDeveloping a Waters Column� Hardware

– Materials– Design– Optimization

� Sorbents– Chemistries– Synthesis

� Packing– Equipment Considerations– Optimization– Product QC and testing


Developing a Waters Column:Developing a Waters Column:Hardware ManufactureHardware Manufacture� Waters produces most of our hardware from raw materials

� Each step of the machining process is quality controlled

� Product hardware must pass: – Material specifications– Engineering tolerances– Cosmetic Standards


Developing a Waters Column:Developing a Waters Column:Particle and Sorbent Manufacture Particle and Sorbent Manufacture

� All Waters modern chemistry products are synthesized from raw materials allowing completeprocess control and traceability� Waters manufacturing philosophy:

– Small batches ensures process understanding

– Provide synthesis batches that reflect product need requirements• <2 kg scale (nano, capillary, analytical)• 10+ kg process scale(preparative, bulk)

� Completely scalable and predictable through state-of-the-art process control


Developing a Waters Column:Developing a Waters Column:Column Packing 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� Equals over 800 columns per working day


Developing a Waters Column:Developing a Waters Column:Final QC and InspectionFinal 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


AgendaAgenda




� Summary


Waters Column Product Waters Column Product HistoryHistory

Styragel®µBondapak™

DeltaPak®

1964 197919731992

Symmetry®

Spherisorb®

Atlantis®

Symmetry® 300

XTerra®

XTerraPrep®

1998

Nova-Pak®

ProteinPak™Pico-Tag™

SymmetryShield®

19841986

200219991994 2003

Atlantis® HILIC SilicaPrep OBD™Intelligent Speed™BioSuite™NanoEase™

2004

ACQUITY UPLC® BEHSunFire™ Columns

PrepPak®

1976

1958

Atlantis® T3ACQUITY UPLC® HSS T3AccQTag™ Ultra

20062005

XBridge™

2007

ACQUITY UPLC®

HSS C18 and HSS C18 SB

2008

XBridge™ HILIC

2009

ACQUITY UPLC® BEH AmideACQUITY UPLC® BEH GlycanXBridge AmideXSelect™ HSS HPLC Columns

2010

ACQUITY UPLC® BEH200 SECXSelect CSH HPLC columns

ACQUITY CSH ColumnsViridis SFC Columns

ProteinPak High Rs IEX

AccQTag ™

2011

ACQUITY UPLC® HSS Cyano & PFP columns

XSelect™ HSS Cyano & PFP columns

XP 2.5 µm Columns

2012

ACQUITY UPLC®

BEH125 SECBEH450 SECACQUITY UPC2


Begin with Begin with Two Fully Scalable Two Fully Scalable Column Column PlatformsPlatforms

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 & 10 µm BEH1.8 [UPLC], 2.5, 3.5 & 5 µm HSS1.7 [UPLC], 2.5, 3.5 & 5 µm CSH


Designed for Industry-Leading Chemical (pH)

Stability

Column Particle Platform Column Particle Platform DefinitionsDefinitions

Designed for Increased

Retentivity and Selectivity

Designed for Maximum

Selectivity and Sample Loading

BEH ParticleEthylene-Bridged Hybrid

130Å, 200Å & 300Å

HSS ParticleHigh Strength Silica

100Å

CSH ParticleCharged Surface Hybrid

130Å

All three substrates are fully scalable between UPLC and HPLC platforms


XBridge HPLC ColumnsXBridge 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


Ethylene Bridged Hybrid Ethylene Bridged Hybrid [BEH] [BEH] ParticleParticle

Anal. Chem. 2003, 75, 6781-6788

U.S. Patent No. 6,686,035 B2 Bridged Ethanes within a silica matrix


Ethylene Bridged Hybrid Ethylene Bridged Hybrid [BEH] [BEH] ChemistriesChemistries� 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.


Industry Leading pH stabilityIndustry Leading pH stabilityColumn lifetimes in acidic mobile phasesColumn lifetimes in acidic mobile phases

Extremely acidic mobile phases hydrolyse the bonded ligand of

conventional LC sorbentsReduces analyte retention

Test conditions: 1 % TFA in water (pH 1.0) at 80oC. Comparative separations may not be representative of all applications.


Industry Leading pH stabilityIndustry Leading pH stabilityColumn lifetimes in alkaline mobile phasesColumn lifetimes in alkaline mobile phases

� High pH mobile phases (>pH 10) rapidly dissolve silica-based stationary phases

� Only modern hybrid-based sorbents, [BEH] and [CSH], extend the usable mobile phase pH range from 1-12

Comparative separations may not be representative of all applications.


The importance of mobile phase pH: The importance of mobile phase pH: Rapid Method DevelopmentRapid 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


High Strength Silica High Strength Silica [HSS][HSS] ChemistriesChemistries� First silica-based particle (1.8 µm) designed for UPLC applications� Fully scalable manufacturing process enabled HPLC particle sizes(2.5, 3.5 & 5 µm)� Three general-purpose C18 columns for low to neutral pH reversed-phase separations– 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

� PFP and 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


Charged Charged Surface Hybrid Surface Hybrid [CSH] [CSH] ParticlesParticlesStep 1 Step 2 Step 3

CHARGED SURFACE HYBRID PROCESS


Charged Surface Hybrid [CSH] Charged Surface Hybrid [CSH] Chemistries 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


Charged Surface Hybrid [CSH] Charged Surface Hybrid [CSH] Particles 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)


Benefits of CSH Technology:Benefits of CSH Technology:Loading ComparisonLoading Comparison

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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)



Comparative separations may not be representative of all applications


EverEver--Growing Selectivity RangeGrowing Selectivity RangeEthylene Bridged Hybrid [Ethylene Bridged Hybrid [BEHBEH]:]: 20042004

ACQUITY BEH C18XBridge C18


EverEver--Growing Selectivity RangeGrowing Selectivity RangeEthylene Bridged Hybrid [Ethylene Bridged Hybrid [BEHBEH]:]: 20052005

ACQUITY BEH PhenylXBridge Phenyl


ACQUITY BEH Shield RP18XBridge Shield RP18



EverEver--Growing Selectivity RangeGrowing Selectivity Range[[BEH + BEH + HSSHSS]: ]: 20082008





ACQUITY HSS C18 SBXSelect HSS C18 SB

ACQUITY HSS C18XSelect HSS C18

ACQUITY HSS T3XSelect HSS T3


EverEver--Growing Selectivity RangeGrowing Selectivity Range[BEH + HSS + [BEH + HSS + CSHCSH]: ]: 20102010








ACQUITY CSH C18XSelect CSH C18

ACQUITY CSH Phenyl-HexylXSelect CSH Phenyl-Hexyl

ACQUITY CSH Fluoro-PhenylXSelect CSH Fluoro-Phenyl


EverEver--Growing Selectivity RangeGrowing Selectivity Range[BEH + CSH + HSS]:[BEH + CSH + HSS]: 20112011








ACQUITY CSH C18XSelect CSH C18

ACQUITY CSH Phenyl-HexylXSelect CSH Phenyl-Hexyl

ACQUITY CSH Fluoro-PhenylXselect CSH Fluoro-Phenyl

ACQUITY HSS CNXSelect HSS CN

ACQUITY HSS PFPXSelect HSS PFP

©2012 Waters Corporation 30Minutes0.0 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 mmMobile Phase A: 0.1 % CF3COOH in H2OMobile Phase B: 0.08 % CF3COOH in ACNFlow Rate: 0.5 ml/min Gradient: Time Profile Curve

(min) %A %B0.0 92 8 60.1 92 8 64.45 50 50 74.86 10 90 65.0 92 8 66.0 92 8 6

Injection Volume: 1.0 µlSample Diluent: 50:50 H2O: MeOH

with 0.05 % CF3COOH Sample Conc.: 100 µg/mlTemperature: 40 oCDetection: UV @ 330 nmSampling rate: 40 pts/secTime Constant: 0.1Instrument: Waters ACQUITY UPLC®, with

ACQUITY UPLC® TUV

Compounds1. Caftaric acid2. Chlorogenic acid3. Cynarin4. Echinacoside5. Cichoric acid

1 2 4 53BEH C18

BEH C8

BEH Shield RP18

BEH Phenyl

1 2 4 53

1 2 4 53

1 2 453

1 2 4 53HSS T3

HSS C181 43 52

Column Selectivity Column Selectivity ChoicesChoicesCaffeicCaffeic Acid Acid DerivativesDerivatives

1 2 453 HSS C18 SB

HSS CN

HSS PFP1 23

45

1 2

4

35

1 2 4 3 5 CSH Phenyl-Hexyl

1 2

4

3

5

CSH Fluoro-Phenyl

CSH C181 2

3,4

5

Comparative separations may not be representative of all applications


Expanding Retention Capability:Expanding Retention Capability:XSelect HSS/ACQUITY UPLC HSS PFP 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


Retention Comparison Retention Comparison -- Basic Compounds:Basic Compounds:CSH FluoroCSH Fluoro--Phenyl vs. HSS PFP Phenyl vs. HSS PFP

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Compounds Compounds 1. 1. AminopyrazineAminopyrazine2. 2. PindololPindolol3. Quinine 3. Quinine 4. 4. LabetalolLabetalol5. 5. VerapamilVerapamil6. 6. DiltiazemDiltiazem7. 7. AmitriptylineAmitriptyline

Conditions:Columns: 2.1 x 50 mm Mobile Phase A: 10 mM NH4COOH, pH 3.0Mobile Phase B: MeOHFlow 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 70 304.50 70 30

Inj. Volume: 1 µlSample Diluent: H2OTemperature: 30 °CDetection: UV @ 260 nm Sampling Rate: 40 points/secFilter Response: NormalInstrument: ACQUITY UPLC®

ACQUITY UPLC® PDA

2

1

4

6

3

7

5

HSS PFP

CSH Fluoro-Phenyl2

13

6

7

5

4

BASIC COMPOUNDSBASIC COMPOUNDS

HSS PFP Retains Basic Compounds Longer than CSH Fluoro-PhenylComparative separations may not be representative of all applications


SummarySummary� Waters has an ongoing commitment to developing high-performing column chemistries

� The XBridge columns family was developed to be the standard column for reliability and reproducibility

� The XSelect column family was developed for the method developer to maximize selectivity choice

� Continue to expect new products that are designed for purpose to solve the next generation LC challenges


AgendaAgenda




� Summary


Maximize HPLC ProductivityMaximize HPLC Productivity

� Increasingly, more organizations realize the business and scientific advantages UPLC Technology

� However, this technology shift has led companies to evaluate how to best utilize their existing HPLC instruments as they continue to invest in and transition to, newer UPLC systems.– Smaller particles (2.5 – 3.5 µm)– Core-shell columns

How to Best Use Existing HPLC Systems While Transitioning to UPLC Technology


� 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 & 3.0 mm IDs compatible with

UPLC pressures

� Flexibility in configurations– 2.1, 3.0 & 4.6 mm ID

o 2.1 & 3.0 mm incorporating eCord™ technology

– 30, 50, 75, 100 & 150 mm lengths

� Packed with XBridge [BEH] and XSelect[CSH & HSS] 2.5 µm particles and chemistries– 14 scalable stationary phases

eeXXtended tended PPerformance 2.5 µm Columnserformance 2.5 µm Columns


Challenges Still Arise:Challenges Still Arise:HPLC, UHPLC and UPLCHPLC, UHPLC and UPLC

•Need to utilize existing LC assets while improving throughput•Method transfer between different LC platforms•Selectivity choice for challenging separations•Solvent cost reduction is minimal due to the requirement of 3.0 – 4.6 mm ID columns to overcome system dispersion.•Transition strategy between different LC platforms?•Cannot transfer regulated methods without full revalidation•Would like to reduce backpressure when possible

What are the challenges?


Flexible Options to Improve LC Flexible Options to Improve LC Productivity Productivity


� Eddy dispersion (A-Term)– Increase bed homogeneity

o Improve column packingo Narrow particle size distribution

– Decrease particle size

� Axial Dispersion (B-Term)– Increase speed of mobile phase

� Mass transfer kinetics (C-Term)– Increase diffusion speed (elevated temperature)

– Decrease diffusion distance (smaller particles)– Limit diffusion distance (core-shell particles)

Understanding Column Performance:Understanding Column Performance:Improving Separations in the ColumnImproving Separations in the Column

Cuu

BAH ++=


Understanding Mass Transfer [Diffusion]:Understanding Mass Transfer [Diffusion]:Influence of Particle SizeInfluence of Particle Size

Diffusion distance is shorter with decreasing particle size resulting in a narrower, more efficient, chromatographic band


Understanding Mass Transfer [Diffusion]: Understanding Mass Transfer [Diffusion]: Superficially Porous ParticlesSuperficially Porous Particles

What SPP producers claim :Diffusion distance is short because the analyte band can only diffuse

into the porous layer of material Let’s do the test !


Understanding Performance:Understanding Performance:FullyFully--Porous Porous vs. vs. CoreCore--Shell ParticlesShell Particles

Linear Velocity, u [cm/sec]

Similar Efficiencies


Understanding Performance:Understanding Performance:FullyFully--Porous Porous vs. vs. CoreCore--Shell ParticlesShell Particles

Similar Backpressures


Performance Comparison:Performance Comparison:FullyFully--Porous Porous vs. vs. CoreCore--Shell ParticlesShell Particles

Imipramine concentration held constant at 0.5 mg/ml; 0.1 % formic acid moible phase

XSelect CSH C18 XP2.5 µm, 4.6 x 75 mm

Kinetex C182.6 µm, 4.6 x 75 mmIm

ipram

ineIm

ipram

ine


Impact of Particle Size on Impact of Particle Size on Column PerformanceColumn Performance

1.7 µm ACQUITY UPLC columns provide

15 % improvement in resolution and peak capacity and 20 %

higher sensitivitycompared to XP 2.5 µm

columns

Both columns were run on an ACQUITY UPLC H-Class system

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XSelect CSH C18 XP2.1 x 75 mm, 2.5 µmFlow rate = 0.54 ml/min

Pressure = 5,300 PSIPeak Capacity = 34

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ACQUITY UPLC CSH C182.1 x 75 mm, 1.7 µmFlow rate = 0.54 ml/min

Pressure = 9,200 PSIPeak Capacity = 40

Tolm

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Rs = 5.5

Rs = 6.3

15 % increase in Rs and Pc

20 % higher sensitivity

40 % lower backpressure


Performance Comparison:Performance Comparison:Goldenseal Root ExtractGoldenseal Root Extract

Mobile Phase A: 0.1 % FA in H2OMobile Phase B: 0.1 % FA in ACNFlow Rate: 0.6 ml/min Gradient: 0 to 40 % B in 6 min, holdInjection Volume: 0.5 µlColumn Temperature: 30 °CDetection: UV @ 210/254 nm Instrument: Waters ACQUITY UPLC®ACQUITY UPLC® TUV detector

Sample: Goldenseal extractColumn: 2.1 x 50 mm, 1.7 µm

ACQUITY UPLC® BEH C181.7 µm 2.1 x 50 mm

Core-Shell C18

1.7 µm 2.1 x 50 mm

1. Hydrastine2. Canadine3. Canadaline4. Berberine

Particle size alone does not dictate separation

performance.

Interaction between stationary phase and

analytes is critical.


Impact of Instrument Dispersion on Impact of Instrument Dispersion on Column PerformanceColumn Performance

Ultra-low UPLC instrument dispersion

enhances column performance, even with

larger 4.6 mm ID columns

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HPLC SystemXSelect CSH C18 XP4.6 x 75 mm, 2.5 µm

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ACQUITY UPLC H-ClassXSelect CSH C18 XP4.6 x 75 mm, 2.5 µm

Tolm

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15 % decrease in peak width

30 % increase in peak height


Summary: Matching Column Summary: Matching Column Configurations with LC SystemsConfigurations with LC Systems

System HPLC UPLCParticle Size 3.5 µm, 5 µm XP 2.5 µm 1.7/1.8 µm UPLC,

XP 2.5 µm, 3.5 µm, 5 µm1.7/1.8 µm UPLC,

XP 2.5 µm, 3.5 µm, 5 µmRoutine 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


SummarySummary� 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

an introduction to waters an introduction to waters ... · most ms-compatible hplc columns on the...

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