nuevos desarrollos en columnas acquity uplc & hplc · ©2011 waters corporation 1 nuevos...

©2011 Waters Corporation 1

Nuevos Desarrollos en Columnas ACQUITY UPLC® & HPLC

Bàrbara Bagó

Bilbao, 12 Julio 2012


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

http://www.waters.com/waters/nav.htm?locale=es_ES&cid=134618172�




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


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


Developing a Waters Column

Hardware — Materials

— Design

— Optimization

Sorbents — Chemistries

— Synthesis

Packing — Equipment Considerations

— Optimization

— Product QC and testing


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


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


Developing a Waters Column: Final QC and Inspection

Each column Passes:

Efficiency testing using a test mixture



Each column Passes:


COA tracking and QC checks of product conformity

All column eCord information is verified against original manufacturing data and

testing



Each column Passes:

Rigorous cosmetic standards


COA tracking and QC checks of product conformity

Final packaging and documentation


Agenda


New HPLC & ACQUITY UPLC Column Chemistries

Upcoming Changes to USP Chromatography <621>

New 2.5 µm eXtended Performance (XP) Columns

Summary





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


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


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


Begin with Two Fully-Scalable HPLC Column Platforms


Most MS-compatible HPLC columns

on the market






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


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


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.


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


Begin with Two Fully-Scalable HPLC Column Platforms


Most MS-compatible HPLC columns

on the market





©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










130Å, 200Å and 300Å


100Å


130Å


[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


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










130Å, 200Å and 300Å


100Å


130Å


Ever-Growing Selectivity Range Charged Surface Hybrid [CSH] Particles

Step 1 Step 2 Step 3

CHARGED SURFACE HYBRID PROCESS


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


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)


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)





Ever-Growing Selectivity Range Ethylene Bridged Hybrid [BEH]: 2004

ACQUITY BEH C18 XBridge C18


Ever-Growing Selectivity Range Ethylene Bridged Hybrid [BEH]: 2005

ACQUITY BEH Phenyl XBridge Phenyl


ACQUITY BEH Shield RP18 XBridge Shield RP18



Ever-Growing Selectivity Range [BEH + HSS]: 2008





ACQUITY HSS C18 SB XSelect HSS C18 SB

ACQUITY HSS C18 XSelect HSS C18

ACQUITY HSS T3 XSelect HSS T3


Ever-Growing Selectivity Range [BEH + HSS + CSH]: 2010








ACQUITY CSH C18 XSelect CSH C18

ACQUITY CSH Phenyl-Hexyl XSelect CSH Phenyl-Hexyl

ACQUITY CSH Fluoro-Phenyl XSelect CSH Fluoro-Phenyl


Ever-Growing Selectivity Range [BEH + CSH + HSS]: 2011








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


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


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


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



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


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



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


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


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


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 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 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


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.


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


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


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)


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)


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


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


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


Agenda


New HPLC & ACQUITY UPLC Column Chemistries Upcoming Changes to USP Chromatography <621> New 2.5 µm eXtended Performance (XP) Columns

Summary





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


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


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?


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.


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)


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


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


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


Agenda


New HPLC & ACQUITY UPLC Column Chemistries Upcoming Changes to USP Chromatography <621> New 2.5 µm eXtended Performance (XP) Columns

Summary





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


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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


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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


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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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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


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


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?


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


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


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


Resources that Enable Success: Online Tools

Column selection tools and advisors


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


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


Resources that Enable Success: Technology Primers

715001531

715001940

715002099

715002531

Four easy to read and understand books covering the basic concepts of:


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


Resources that Enable Success: LC Column Family Brochures

XP Columns XBridge™

Columns XSelect™ Columns ACQUITY UPLC®

Columns

720001140EN

720001255EN

720004195EN

720004178EN


Adding a New Business Competency

©2009 Waters Corporation | COMPANY CONFIDENTIAL Waters Corporation Confidential

An Introduction

www.waters.com/standards

WATERS ANALTYICAL STANDARDS

& REAGENTS


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


The Products


Thank You For Your Time and Attention


APPENDIX: ADDITIONAL SLIDE RESOURCES

AND INFORMATION


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


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


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)


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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3 4

5 6 7

8

1

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8

Column: 4.6 x 150mm, 5 µm 5-40% MeOH without additive 3.0mL/min at 40°C, 150 bar Injection Volume: 3µL


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™


HILIC COLUMN CHEMISTRIES


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)


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)


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


Stationary Phase Selectivity at Low pH: Water-soluble Vitamins

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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


Carbohydrate Analysis Using the XBridge Amide Column


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

nuevos desarrollos en columnas acquity uplc & hplc · ©2011 waters corporation 1 nuevos...

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