Introduction to Waters Sample Preparation and

©2013 Waters Corporation 1

Introduction to Waters Sample Preparation

and Chromatography Columns Tools and

Techniques

Robert Frost

Consumables Private Market Manager UK


Agenda

Tools and Techniques for Sample Preparation

Column Technologies


Tools and techniques for Sample Preparation

The Importance of Solid Phase Extraction

How SPE Works

Device Design and Tips for Processing Samples

Chromatographic Sorbent Choices


The New SPE “Textbook”

Part #: 715003405

212 pages, paperback

Size: 8.25 x 11”

>150 full color figures and diagrams

Chapter Titles

– Benefits of SPE in Sample Preparation

– SPE is LC

– Key Terms and Calculations

– In the Lab

– Method Development

– Troubleshooting

– Appendix: Glossary of SPE and LC Terms

– Appendix: Oasis Sorbent Technology for SPE

– Appendix: Applications

– Appendix: Additional Reference Materials


Why do Sample Preparation?

Issue: Plugging

Cause: solid particulates

Impact: may limit instrument/system up-time


Sample Prep Tools

For solid particulates:

– Filters

– Centrifuge

For sample matrix components:

– Precipitation

– Liquid-Liquid Extraction {LLE}

– Solid-Phase Extraction {SPE}


Why do Sample Prep?

Issue: Complicated analytical results / too much variability and

potential resolution issues

Cause: analytes contained in a complex sample matrix

Impact:

Quantitation errors: slight changes might resolute in loss of

resolution of critical pairs

May reduce instrument/system up-time

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Improve Analytical Results: Example #1

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Only

need to analyze

these

peaks

Improvement: Remove unnecessary peaks



PD

A

(0.0

1 A

UFS)

1 2

Sample Prep

0 10 5

1

2

Sample

Minutes

Improvement: Remove Baseline interferences



Spiked Sample

Blank Sample Matrix

1 2

3 0.004 AU

10 8 6 4 2 0 min.

0.0

0

0.0

0

2.0

0

2.0

0

4.0

0

4.0

0

6.0

0

6.0

0

8.0

0

8.0

0

10.0

0

10.0

0

Min

ute

s

Min

ute

s

Spiked Sample

Blank Sample Matrix

1

3

2 0.004 AU

Peak 1 has baseline contamination from sample matrix

Peaks 2 and 3 are clean

Peak 1

is clean

With SPE Clean-up

Improvement: Remove Baseline interferences


Why do Sample Prep?

Issue: not enough sensitivity

Cause:

Analyte concentration in original sample matrix

TOO LOW to measure by instrument

Impact:

Quantitation errors



Concentration of Analyte in

Original Sample TOO LOW

(difficult to quantitate)

Time 0 1 2 3 4 5



Concentration of Analyte

in Original Sample

TOO LOW

(difficult to quantitate )

Time 0 1 2 3 4 5

Utilize SPE Chromatographic Bed

to Trace Concentrate the Original Sample

for that Analyte -

Obtain Good Response


Why do Sample Prep?

Issue:

– Not enough sensitivity

Cause:

– For MS detection: minimize ion suppression/enhancement

Impact:

– Quantitation Errors


260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

591.7

354.4 518.5

472.6 485.5 609.6

Scan ES+ 354.4

260.2

291.3

609.6

485.6472.6

411.5591.6

50/50 Water/ACN + human plasma supernatant

50/50 Water/ACN

260.2 - 97 %

291.2 - 96 %

354.4 - 86 %

411.4 - 93 %

472.6 - 93 %

485.6 - 95 %

591.6 - 89 %

609.5 - 93 %

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

591.7

354.4 518.5

472.6 485.5 609.6

Scan ES+ 354.4

260.2

291.3

609.6

485.6472.6

411.5591.6


50/50 Water/ACN

260 280 300 320 340 360 380 400 420 440 460 480260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

591.7

354.4 518.5

472.6 485.5 609.6

Scan ES+ 354.4

260.2

291.3

609.6

485.6472.6

411.5591.6


50/50 Water/ACN

260.2 - 97 %

291.2 - 96 %

354.4 - 86 %

411.4 - 93 %

472.6 - 93 %

485.6 - 95 %

591.6 - 89 %

609.5 - 93 %

Analytes in human plasma with only

Protein Precipitation Standards in aqueous solution give acceptable response

What is Ion Suppression?


260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

591.7

354.4 518.5

472.6 485.5 609.6

Scan ES+ 354.4

260.2

291.3

609.6

485.6472.6

411.5591.6


50/50 Water/ACN

260.2 - 97 %

291.2 - 96 %

354.4 - 86 %

411.4 - 93 %

472.6 - 93 %

485.6 - 95 %

591.6 - 89 %

609.5 - 93 %

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

591.7

354.4 518.5

472.6 485.5 609.6

Scan ES+ 354.4

260.2

291.3

609.6

485.6472.6

411.5591.6


50/50 Water/ACN

260 280 300 320 340 360 380 400 420 440 460 480260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0

100

%

500 520 540 560 580 600 620m/z0

100

%

0

100

%

Scan ES+

591.7

354.4 518.5

472.6 485.5 609.6

Scan ES+ 354.4

260.2

291.3

609.6

485.6472.6

411.5591.6


50/50 Water/ACN

260.2 - 97 %

291.2 - 96 %

354.4 - 86 %

411.4 - 93 %

472.6 - 93 %

485.6 - 95 %

591.6 - 89 %

609.5 - 93 %

Analytes in human plasma with only Protein Precipitation

Analyte standards in aqueous solution

% Loss

Complex Sample Matrix: Ion Suppression



80% ion suppression

Minimal ion suppression

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

%

0

100 MRM

472.2 > 436.4 1.27e6

1.89

MRM 472.2 > 436.4

1.27e6 1.91

SPE with Oasis

® MCX

Protein Precipitation (PPT)

Note: These samples are dried and reconstituted. Gradient time = 1.5 min

MRM for Terfenadine

Significant ion suppression observed for analytes that co-elute with residual matrix

components using just PPT.


Do all Sample Preparation Techniques Give the Same Result?

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

%

0

100

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

%

0

100 MRM

472.2 > 436.4 1.27e6

1.89

MRM 472.2 > 436.4

1.27e6 1.91

80% ion suppression

Minimal ion suppression B (SPE) (Oasis® MCX)

A (PPT)

Note: These samples are dried and reconstituted. Gradient time = 1.5 min

No loss in signal observed for analytes when the interferences, which cause the suppression,

are removed by SPE.

MRM for Terfenadine


The Ultimate Goal is to make your analytical lab more productive

- Lower Limits of Detection

- Run more samples with less time

- Minimize costs in manpower and equipment maintenance

Goals of Sample Preparation: Summary

To remove interferences for

– Better chromatography

– More confident analytical results

– Longer column lifetime

– Less instrument downtime

To enrich sample for

– Higher detection sensitivity

To make sample more compatible for separation and detection

– Matching solvent strength

– Eliminating ion-suppression in LC/MS analysis


Agenda


How SPE Works




x

Yellow is the earliest eluting analyte “band”, it “likes” the mobile phase,

has very low ‘k” (little retention)

Blue is well retained, it “likes” the particles, has high “k” (high

retention - “captured”)

Mix Yellow, Red and Blue Dyes together to create what appears,

to our eyes as a “Black” Sample

Food

Dyes

How a Chromatographic Column Works -- “BANDS”


Yellow is the earliest eluting analyte “band” - it “likes” the mobile phase,

has very low ‘k” (little retention)

Blue is well retained - it “likes” the particles,

has high “k” (high retention - “captured”)

How a Chromatographic Column Works -- “BANDS”


Band Migration on an SPE Cartridge

SPE follows the same principles as LC

Blue

Red Yellow

Sample


Agenda


How SPE Works




SPE Configurations from Waters

Syringe Barrel Cartridge

Standard 96-Well Plate

On-line SPE Devices

melution 96-Well Plate


Sample Processing: Gravity


Stop Cock

flow control valves-

to prevent

drying – out effect

Male-male Adapter

Syringe Adapter

Vacuum Manifold

Sample Processing: Glass Vacuum Manifold


Vacuum Manifold

Sample Processing: Large Liquid Volumes


Sample Processing: 96-well Plate

Set-up for typical 96-well plates Under vacuum, a properly seated well-plate should move down and the tips will extend into the wells of the collection plate.

Collection Plate


Sample Processing: Positive Pressure


Tip: Cartridge Stacking

Multiple cartridges can be stacked for

a mixed-mode approach

2 of the SAME

for More Capacity

2 Different

Sorbents for

2 Dimensional

Selectivity


Sample Processing: Positive Pressure Manifold


Agenda


How SPE Works




SPE Terminology

Sorbent -- Chromatographic packing material (Stationary Phase)

Retention Mechanisms: 1. Reversed Phase (RP) 2. Normal Phase (NP) 3. Ion-Exchange (IEX)

Syringe Barrel Style

Sorbent

Filter/Frit

Filter/Frit


Sorbents for SEP-PAK® Products Silica and Alumina Based


Sorbents for SEP-PAK® Products Reversed-Phase


Sorbents for SEP-PAK® Products Normal Phase


Sorbents for SEP-PAK® Products Ion-Exchange (Silica Based)


Sorbents for SEP-PAK® Products Specialty Phases


Hydrophilic

monomer

Lipophilic

monomer

N O

Reversed-phase Retention

Hydrophilic-Lipophilic Balanced Copolymer

Polymeric SPE Sorbent Chemistry: Oasis® HLB

• Water wettable

• Polar retention

• Stable across pH 1-14

• No silanol interactions

• High recoveries for acids, bases and neutrals

Retention of Polars


Polymeric SPE Sorbent Chemistries: Oasis® Mixed-mode Ion Exchangers


Polymeric SPE Sorbent Chemistries: Oasis® Mixed-mode Ion Exchangers

Strong Cation

RP

Strong Anion

RP

Weak Cation

RP

Weak Anion

RP

Reversed Phase

RP


Oasis® 2x4 Method: Acids and Bases

Neutrals

For Bases:

pKa 2-10

Use Oasis® MCX

For Strong Acids

pKa <1.0

Use Oasis® WAX

For Strong Bases

pKa >10

Use Oasis® WCX

For Acids

pKa 2-8

Use Oasis® MAX

Prepare Sample

Condition/Equilibrate

Load Sample

Wash:

5% NH4OH

Elute 1:

100% MeOH

Elute 2:

2% Formic Acid in MeOH

Protocol 2 Prepare Sample


Load Sample

Wash:

2% Formic acid

Elute 1:

100% MeOH

Elute 2:

5% NH4OH in MeOH

Protocol 1

Bases Strong

Acids

Strong

Bases Acids


Oasis® 2x4 Method: Neutrals

Neutrals

For Bases:

pKa 2-10

Use Oasis® MCX

For Strong Acids

pKa <1.0

Use Oasis® WAX

For Strong Bases

pKa >10

Use Oasis® WCX

For Acids

pKa 2-8

Use Oasis® MAX

Prepare Sample


Load Sample

Wash:

5% NH4OH

Elute 1:

100% MeOH

Elute 2:

2% Formic Acid in MeOH

Protocol 2 Prepare Sample


Load Sample

Wash:

2% Formic acid

Elute 1:

100% MeOH

Elute 2:

5% NH4OH in MeOH

Protocol 1

Bases Strong

Acids

Strong

Bases Acids

Reversed-phase

Backbone


Ostro™ Plate Technology Pass Through Strategy

Specifically Designed for

Phospholipid Removal which, when present,

results in Significant

Ion-Suppression


Methodology

Precipitate

Proteins

Held up by

Filters

Phospholipids

Retained

by SPE

Sorbent Bed,

Analytes

Pass Through


It is possible to work with lower sample volumes (such as

25µL). When doing so you will need a higher

organic solvent to sample ratio, such as

10:1 or 20:1.

The well volume is 1.9 mL, however in order to mix by aspiration, the

maximum volume is 1.4 mL. This translates to a maximum sample size

of 350µL.

Place Ostro plate onto

collection plate

Pipette 50-200µL of

plasma into wells

Forcefully add 2% formic acid in acetonitrile,

3:1 solvent:plasma

(methanol not recommended)

Mix thoroughly by aspirating 3x with pipette

Filter samples using vacuum manifold or

positive pressure manifold

Analyze samples


OSTRO™ Plate

- Phospholipid

Removal

- No Method

Development

- Superior

Performance

Competitors



Competitive Techniques: Phospholipids Remaining

MRM of m/z 184-184

Time 0.20 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

%

0

100

0.20 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

%

0

100

0.20 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

%

0

100

0.20 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

%

0

100 184.4 > 184.4 (Lipid 184) 2.00e8

2.88 2.29 2.21

2.10

1.90

2.60 2.78 2.72

184.4 > 184.4 (Lipid 184) 2.00e8

2.80 2.27

1.90

2.62 2.56 2.68

184.4 > 184.4 (Lipid 184) 2.00e8

1.96 1.90 1.77

184.4 > 184.4 (Lipid 184) 2.00e8

2.84 2.21 1.96 1.75 1.42 1.38

1.32

1.63 1.51

PPT

Ostro™

LLE with MTBE

LLE with 5%

NH4OH in MTBE


Agenda

Tools and Techniques for Sample Preparation

Column Technologies


Agenda

Introduction

Modern HPLC and UPLC Columns

Maximizing HPLC Performance with XP 2.5 µm Columns

NEW CORTECS UPLC 1.6 & 2.7 µm 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

BEH130 Columns BEH300 Columns

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 XP 2.5 µm Columns

2012

ACQUITY UPLC® BEH125 SEC ACQUITY UPC2™ Columns

2013

CORTECS™ Columns ACQUITY UPLC® BEH450 SEC ACQUITY APC™ Columns CSH130 Columns


What makes an LC column

reproducible?

A) Sorbents

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)

Pla

te H

eigh

t (H

, 4 s

igm

a)

Acenaphthene, 2.1 x 50 mm columns

70/30 MeCN/H2O, 30 °C, 254 nm

B) Packing C) Engineering

All are CRITICAL!


Why Choose a Waters Column?


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. Single source for all Waters columns.

The GMP Quality Systems Regulations cover items such as

— Quality management and organization

— Production, process, 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 Manufacture

Waters produces most of our hardware from raw materials

– Each incoming lot is controlled for material conformity and specification

Each step of the machining process is quality controlled

– The first milling operation automatically:

• Machines the column body to dimension

• Cuts the threads

• Creates unique identifying number

Product hardware must pass: – Material specifications

– Engineering tolerances

– Cosmetic Standards


Enabling Expertise Primary Manufacturing for Reproducibility

All modern chromatographic media is manufactured from starting monomers to finished product

Waters has set the industry benchmark for batch-to-batch reproducibility

Stringent monitoring of key parameters assure repeatable and reproducible results year after year

Minimize the risk of method variation due to differences caused by chromatographic media batch or column inconsistency

Process control charts for 85 batches of ACQUITY UPLC BEH C18 demonstrating manufacturing control and long-term repeatability

between material batches over the course of 7 years.


Quality Systems Minimize Risk with Dependable Column Performance

Method Validation kits provide

three batches of chromatographic

media [derived from different base

particles] to judge the quality,

reliability and consistency of an

analytical method

Waters uniquely positioned as an

industry partner to minimize risk


Developing a Waters Column: Column Packing

Computer controlled real time packing

optimization

Each column configuration is packed on a

dedicated custom designed packing station

– Preparative, analytical and UPLC columns

require different conditions

– Optimized for slurry flow and packing pressure

Every column passes:

– Efficiency test using a specific test mixture


The Result


Agenda

Introduction


– Benchmarking System Performance

– Particle Platforms

– Understanding Stationary Phase



Summary


Benchmarking System Performance

Typical Criteria

1. Retention time range or

reproducibility

2. Peak area range or reproducibility

3. Peak tailing range

4. Peak resolution

5. Response

6. System Pressure

Principle of benchmarking:

–Use of a Quality Control Reference Material (QCRM) to evaluate or

check key performance criteria by comparison with data generated

when the system is known to be in good working order

Importance of benchmarking

–Routine use on the analytical system and control charting the data

allows for an understanding of the capability of your system to provide

reliable results and is a useful troubleshooting tool.

//upload.wikimedia.org/wikipedia/commons/f/f7/ControlChart.svg


Quality Control Reference Materials

Requirements for a QCRM Material

– Reproducible Lot to Lot

– Accurate

– Appropriate to analysis type (LCMS, LC-UV)

The quality of the reference material is critical to the

evaluation of analytical data and instrument performance.

Waters has an extensive portfolio of QCRMs designed to

allow scientists to determine if their system is in working

order

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Aceto

ne

Naphth

ale

ne

Acenaphth

ene

Compound Type

Acetone (V0) Void Marker

Naphthalene Neutral

Acenaphthene Neutral

Neutrals QCRM: 186006360


Versatility of Neutrals QCRM

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10mM Ammonium Formate pH 3; Acetonitrile (50/50, v/v)

20mM Potassium Phosphate pH 7; Acetonitrile (50/50, v/v)

10mM Ammonium Bicarbonate pH 10; Acetonitrile (50/50, v/v)

Peak ID: Acetone, Naphthalene, Acenaphthene

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ACQUITY UPLC CSH™ C18 1.7 µm

XSelect™ CSH™ C18 3.5 µm

XSelect™ CSH™ C18 5 µm

pH Particle Size


Versatility of Neutrals QCRM

Peak ID: Acetone, Naphthalene, Acenaphthene

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ACQUITY UPLC® HSS C18

ACQUITY UPLC® CSH C18

AU

0.00

0.10

0.20 ACQUITY UPLC® BEH C18

Hybrid Particle

Silica Particle

Charged Surface Hybrid Particle

CORTECS™ UPLC® C18 1.6 µm

Solid-Core Particle

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ACQUITY UPLC BEH C18

ACQUITY UPLC BEH C8

ACQUITY UPLC BEH Shield RP18

ACQUITY UPLC BEH Phenyl

Stationary Phase Particle Substrate


Widest Selection of Unique Particle Offering

CORTECS

Solid-Core

Technology

Higher efficiency and

resolution

Increased throughput

at similar resolution

Higher performance

at same backpressure

BEH Technology

Unparalleled pH stability

Mobile phase and

temperature versatility

Seamless scalability

UPLC to HPLC

HSS Technology

Maximum retention

Particle and ligand

selectivity


UPLC to HPLC

CSH Technology

Exceptional loading capacity

Superior peak shape

for basic analytes


UPLC to HPLC

Waters is the ONLY company that has

fully porous and solid-core sub-2-µm particle columns


Column Naming

HPLC UPLC


Two Fully-Scalable LC Column Platforms

Family designed and optimized for pH stability

Family designed and optimized for selectivity

1.7 [UPLC], 2.5 XP, 3.5, 5 and 10 µm 1.8 [UPLC], 2.5, 3.5 and 5 µm HSS

1.7 [UPLC], 2.5 XP, 3.5, 5 and 10 µm CSH


Excellent general-purpose

HPLC and UPLC columns for

small molecule separations

– Broadest range of compound

classes

– Widest pH range (1 – 12)

– Widest temperature range

– Ultra-low MS bleed

Direct scalability between UPLC

Technology, analytical HPLC and

preparative HPLC

XBridge/BEH Family

Family designed and optimized for

pH stability

1.7 [UPLC], 2.5 XP, 3.5, 5 and 10 µm


Ethylene Bridged Hybrid [BEH] Particle

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.


Industry Leading pH Stability Column lifetimes in acidic mobile phases

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

Shorter bar equals longer lifetime under

low pH conditions


Industry Leading pH stability Column 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: 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


XSelect HSS and CSH Family

Family designed and optimized for

selectivity

Multiple particle substrates to solve multiple chromatographic

problems

1.8 [UPLC], 2.5 XP, 3.5 and 5 µm HSS

1.7 [UPLC], 2.5 XP, 3.5, 5 and 10 µm CSH

Highest pressure tolerance; first silica-based particle (1.8 μm) designed for UPLC applications

Alternate selectivities (vs. BEH & CSH particle columns)

Increased retention

Designed for LC/MS

Improved peak shape for basic analytes under low-ionic strength acidic mobile phases

Reduces overload effect

Rapid equilibration; prevents retention time drift with changes in pH


Charged Surface Hybrid [CSH] Particles

Step 1 Step 2 Step 3

CHARGED SURFACE HYBRID PROCESS


Benefits of CSH Technology: Loading Comparison

AU

0.0

1.0

2.0

3.0

AU

0.0

1.0

2.0

3.0

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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

Competitive Core-Shell 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


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

Alternative Selectivity Stationary Phases

– 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

– HSS PFP: Exceptional selectivity and retention for positional isomers,

halogenated compounds and polar basic compounds

– HSS CN: Stable, normal phase-compatible, reproducible CN chemistry

©2013 Waters Corporation 78 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


Agenda

Introduction




Summary


Maximize HPLC Performance

Increasingly, more organizations realize the business and scientific

advantages of UPLC Technology

Increased availability of UHPLC instruments provides vendor choice

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 particle (2.5 – 3.0 µm) 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 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, 100 and 150 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


AU

0.00

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0.20

0.30

Minutes 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00

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


AU

0.00

0.10

0.20

0.30

0.00 0.50 1.00 1.50 2.50 3.00 3.50 Minutes 2.10

1.7 µm

AU

0.00

0.10

0.20

0.30

Minutes 0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00

XSelect CSH C18 4.6 x 100 mm, 3.5 µm

HPLC System 3.5 µm


Tolm

eti

n

Nap

roxe

n

Fen

op

rofe

n

Ind

om

eth

acin

Dic

lofe

nac


Addressing the Challenges: 2.5 µm eXtended Performance Columns

Utilize XP 2.5 µm columns to improve productivity or achieve higher resolution

Addressing the challenges

Compatible with bandspread volumes of UHPLC systems

Implement XP 2.5 µm columns to decrease backpressure or increase flow rate


Flexible Options to Improve LC Productivity


Agenda

Introduction




Summary


CORTECS 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

– Increased throughput

3 Chemistries:

– C18+

– C18

– HILIC


The CORTECS Solid-Core Particle

Only thin outer layer contains the

pores with the chromatographic

surface

The center core is nonporous

The outer shell is typically “bumpy”

not pretty

The particle size distribution is

narrower

CORTECS

Solid-core

dcore = 1.1 µm

dp =

1.6

µm

Rho, r = 1.1/1.6 = 0.7

66% Porous Volume

ρ = 0 → fully porous particle

ρ = 1 → nonporous particle

ρ = core diameter / particle diameter

Compared to Fully Porous Particles

G. Guiochon, F. Gritti, J. Chromatogr. A 1218 (2011) 1915–1938 Omamogho et al., J. Chromatogr. A 1218 (2011) 1942-1953


Where We Were…

14,150

4,000

8,000

12,000

16,000

0.00 0.25 0.50 0.75 1.00 1.25

Pla

tes (

4 s

igm

a)

Flow Rate (mL/min)

Since 2004, fully porous ACQUITY UPLC 1.7 µm BEH C18

Has been our most efficient particle

Started us down the path of sub-2-µm particles and the development

of UPLC Technology, which was needed to demonstrate its efficiency

ACQUITY UPLC 1.7 µm BEH C18

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…

19,700

14,150

4,000

8,000

12,000

16,000

20,000

0.00 0.25 0.50 0.75 1.00 1.25

Pla

tes (

4 s

igm

a)

Flow Rate (mL/min)

39% higher efficiency

or up to 3x faster!

CORTECS UPLC 1.6 µm C18+

ACQUITY UPLC 1.7 µm BEH C18

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


Similar Backpressure

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

0.00 0.25 0.50 0.75 1.00 1.25

Pre

ssure

(psi)

Flow Rate (mL/min)

Specific Permeability

CORTECS UPLC C18+ 1.6 µm: k0 = 2.22E-15 m²

ACQUITY UPLC BEH C18 1.7 µm: k0 = 2.28E-15 m²

Conditions: Tested on an ACQUITY UPLC I-Class using 2.1 x 50 mm columns, 70% Acetonitrile at 30 °C


How do CORTECS Columns Achieve Higher Efficiency?

1.6 µm solid-core particle morphology

– The solid-core decreases diffusion within the column

– Rough particle surface yields better packed-bed uniformity

Optimized column hardware

– Low dispersion

– Pressure-tolerant for UPLC separations

Advanced packing techniques

– More than 10 years experience packing sub-2-µm particles

– Proprietary packing instrumentation


Overview: CORTECS 2.7 µm Columns

HPLC/UHPLC columns featuring 2.7 µm solid-core silica particles

Key benefits

– High efficiency at reduced backpressure

o Resolution

o Speed

– Scalability to/from UPLC

3 chemistries:

– C18+

– C18

– HILIC


Porous Particle Equivalence

CORTECS Solid-Core Particle Columns demonstrate an efficiency

equivalent to that of smaller fully porous particles and a

backpressure equivalent to that of larger fully porous particles:

CORTECS Particle Size

Equivalent Porous Particles

Efficiency Backpressure

1.6 µm 1.3 µm 1.8 µm

2.7 µm 2.2 µm 3.1 µm


Backpressures on 4.6 x 150 mm Columns Using Maximum Viscosity ACN Mobile Phase

Maximum performance at HPLC-optimized backpressures!

Comparative separations may not be representative in all applications.

Fully Porous C18, 2.5 µm

Competitive Solid-Core C18, 2.6 µm

CORTECS C18, 2.7 µm

Traditional HPLC Pressure

Limit

20% acetonitrile:water mixture


High Efficiency at HPLC Backpressures

AU

0.00

0.02

0.04

0.06

0.08

0.10

AU

0.00

0.02

0.04

0.06

0.08

0.10

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes

1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

1

2 3 4

Rs 4.9

Rs 6.3

Rs 8.2

Fully Porous C18, 5 µm

Psi: 1900 N peak 4: 10,400

Fully Porous C18, 3.5 µm

Psi: 2900 N peak 4: 17,600

CORTECS C18, 2.7 µm

Psi: 3200 N peak 4: 23,600

1. Estradiol 2. Ethinyl estradiol 3. Estrone 4. Levonorgestrel

Configuration: 4.6 x 150 mm


Effect of System Dispersion on Column Performance

In 2004, key component of UPLC introduction was the

relationship between observed efficiency of small particle

columns and the dispersion of the system

As system dispersion decreases, observed efficiency of columns

increases

In order to realize full potential of CORTECS Columns (all small

particle columns), low-dispersions systems should be used


Where Does Band Spreading / System Dispersion Occur?

Band Spreading: 1) From the Injector 2) Into, through and out

of the column 3) Into the Detector

Extra Column

Within Column


Band Spreading, Peak Height

and Resolution

LC systems (column and instrument) capable of producing

narrower/sharper bands create narrower/sharper peaks

This results in better resolution, taller peaks and better sensitivity

Better separation More concentrated “Bands” Higher Sensitivity

Both analytes (blue and red) are not separated [a partial co-

elution – shown as a “purple” band]

System with

MORE Band Spreading

System with

LESS Band Spreading


Impact of System Dispersion on Observed Efficiency of CORTECS Columns

ACQUITY UPLC I-Class

5.5 µL

ACQUITY UPLC H-Class

12 µL

Acetonitrile/ Water (70/30 v/v), 0.4 mL/ min, 30oC, 0.5 µL injection. Peak i.d.: Acetone, Naphthalene, Acenaphthene

2.1 x 50 mm CORTECS C18 Column

USP N: 18,000

USP N: 11,700

53% Increase in Observed CORTECS Column Efficiency on

ACQUITY UPLC I-Class System


Common UHPLC and UPLC System Bandspreads

System Band Spread (µL) 5 sigma

Shimadzu UFLC 41

Agilent 1200 28

Shimadzu Nexera (with microbore f low cell) 26

Agilent 1290 (Configured for Dual Column) 23

Thermo Accela 21

Agilent 1290 (Configured for Single Column) 20

Dionex Ultimate 3000 17

ACQUITY UPLC H-Class with Column Manager 12

ACQUITY UPLC H-Class with Column Heater 9

ACQUITY UPLC (with 1μL loop) 8

ACQUITY UPLC I-Class SM-FTN 7.5

ACQUITY UPLC I-Class SM-FL 5.5

As system bandspread decreases, observed CORTECS Column efficiency and performance increase.



C18+ C18 HILIC

Chemistry

Intended Use

General purpose, high-efficiency, reversed-phase column. A positively

charged surface delivers excellent peak shape for basic compounds at low pH.

General purpose, high-efficiency, reversed-phase column. Balanced

retention of acids, bases and neutrals at low and mid-range pH.

High-efficiency column designed for retention of extremely polar analytes. Offers

orthogonal selectivity vs. C18 columns.

Ligand 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

CORTECS Chemistry Overview


ACQUITY UPLC BEH C18 1.7 µm

CORTECS™ UPLC C18 1.6 µm

CORTECS™ UPLC C18+ 1.6 µm

Competitor B Solid-Core C18 1.7 µm

% Loss in k' for Methylparaben after Exposure to Aqueous 0.5% TFA at 60 °C for 21 hours

Competitor C Solid-Core C18 2.7 µm

Accelerated Acid Stability Data


Shorter bar

equals longer

lifetime

under low pH conditions

0 5 10 15 20 25 30 35


Improved Peak Shape for Tricyclic Antidepressants

1. Nordoxepin (10 µg/mL) 2. Protriptyline (10 µg/mL)

3. Nortriptyline (10 µg/mL) 4. Amitriptyline (10 µg/mL)

5. Trimipramine (10 µg/mL)

AU

0.000

0.010

0.020

0.030

0.040

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

1

2 3

4

5

CORTECS UPLC C18+ 2.1 x 50 mm 1.6 µm

Minutes

AU

0.000

0.010

0.020

0.030

0.040

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

Competitor Solid-Core C18 2.1 x 50 mm 1.7 µm

1

2 3

4 5

Significant

improvement in peak shape and

peak capacity for

basic compounds!

ACQUITY UPLC H-Class with ACQUITY PDA Mobile Phase A: 0.1% Formic Acid in Water Mobile Phase B: 0.1% Formic Acid in ACN Gradient: 28% B to 35% B in 3 min Flow Rate: 0.6 mL/min Column Temp: 30° C UV Detection: 254 nm Injection Volume: 1.0 µL


Pc= 47

Pc= 123


Loading Capacity Comparison

Imip

ram

ine Amitriptyline

Imip

ram

ine

Amitriptyline

Imip

ram

ine

Amitriptyline ACQUITY UPLC CSH C18 2.1 x 50 mm 1.7 µm

CORTECS C18+ 2.1 x 50 mm 1.6 µm

Competitive Solid-Core C18

2.1 x 50mm 1.7 µm

System: ACQUITY UPLC I-Class with ACQUITY PDA Mobile Phase A: 0.1% Formic Acid in Water Mobile Phase B: 0.1% Formic Acid in Acetonitrile Gradient: See Table Flow Rate: 0.6 mL/min Column Temperature: 30° C Detection: 254nm Injection Volume: 5.0 µL

Highest Loading

Capacity on ACQUITY UPLC

CSH C18



Time0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40

%

0

Time0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40

%

0

9

3

2

1

4

5

6

7

8

10

3

2

1

4

5

6

7

8

10

9

1) AM 2223 2) RCS-4, M10 3) RCS-4, M11 4) AM 1248 5) JWH-073 4-butanoic acid

metabolite 6) JWH-073 4-hydroxybutyl

metabolite 7) JWH-018 5-pentanoic acid

metabolite 8) JWH-073 (+/-) 3-

hydroxybutyl metabolite 9) JWH-018 5-hydroxypentyl

metabolite 10) JWH-018 (+/-) 4-

hydroxypentyl metabolite

CORTECS UPLC C18 2.1 x 100 mm 1.6 µm

ACQUITY UPLC BEH C18 2.1 x 100 mm 1.7 µm

Resolution of peaks 5/6, 7/8 and 9/10 (isobaric) allows accurate quantitation on

CORTECS C18 vs ACQUITY UPLC BEH C18

Excellent Retention and Resolution of Synthetic Cannabinoids on CORTECS C18

Minutes

ACQUITY UPLC with Xevo TQD MS


ACh

HA

t-MIAA

t-MHA

iso-ACh Ch

Analysis of Neurotransmitters in Artificial CSF Using CORTECS HILIC

HA: Histamine t-MHA: tele-methylhistamine

t-MIAA: tele-methylimidazoleacetic acid ACh: Acetylcholine

Ch: Choline iso-ACh: iso-Acetylcholine

CORTECS UPLC HILIC 2.1 x 100 mm 1.6 µm ACQUITY UPLC with Xevo TQ-S MS

Minutes

These neurotransmitters are highly polar and poorly retained in RP-LC

Iso-ACh, an isobaric endogenous interference of ACh in CSF, is

chromatographically resolved using the CORTECS column.


Practical Benefits of Increased Efficiency Better Resolution

AU

0.00

0.05

0.10

0.15

0.20

AU

-0.05

0.00

0.05

0.10

0.15

0.20

Minutes 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

1

2

3

4

5

ACQUITY BEH HILIC 2.1 x 50mm 1.7 µm

CORTECS UPLC HILIC 2.1 x 50 mm 1.6 µm

1

2

3

4

5

1. Lidocaine

2. Butacaine

3. Tetracaine

4. Procaine

5. Procainamide

USP Resolution2,3: 1.2

USP Resolution2,3: 2.2


Practical Benefits of Increased Efficiency Higher Throughput – Double Flow Rate

Comparable peak capacities (Pc) in

half the time

Note: the gradient is scaled to account for the change in f low rate


AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

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

Competitor Fully-porous C18 at 0.5 mL/min 2.1 x 50 mm 1.8 µm Gradient time: 4.2 min Runtime: 6 min

1

2 3

4

5

6

7

1. Sulfathiazole 2. Sulfamerazine

3. Sulfamethazine 4. Sulfamethoxypyridazine

5. Sulfachloropyridazine 6. Sulfamethoxazole 7. Sulfasoxazole

CORTECS UPLC C18+ at 1.0 mL/min 2.1 x 50 mm 1.6 µm Gradient time: 2.1 min Runtime: 3 min

Pc= 131 31 peaks per minute (gradient)



Practical Benefits of Increased Efficiency Higher Throughput – Shorter Column

Comparable peak capacities (Pc) in

half the time

Note: the gradient is scaled to account for the change in column length


2.1 x 100 mm 1.7 µm Competitor Solid-Core C18

Gradient time: 8.4 min Runtime: 12 min

2.1 x 50 mm 1.6 µm CORTECS UPLC C18+ Gradient time: 4.2min Runtime: 6 min




Widest Selection of UPLC Particle Offering

CORTECS

Solid-Core

Technology

Higher efficiency and

resolution

Increased throughput

at similar resolution

Higher performance

at same backpressure

BEH Technology

Unparalleled pH stability

Mobile phase and

temperature versatility


to HPLC

HSS Technology

Maximum retention

Particle and ligand

selectivity


to HPLC

CSH Technology

Exceptional loading capacity

Superior peak shape

for basic analytes


to HPLC

Waters is the ONLY company that has

fully porous and solid-core sub-2-µm particle columns


Agenda

Introduction




Summary


Summary

Waters is committed to manufacturing high-quality

consumables that ensure reproducible results year after year

System benchmarking with QCRMs ensures confidence in

your analytical results

XBridge BEH Columns designed for pH stability and method

flexibility

XSelect HSS and CSH Columns provide maximum retention,

selectivity and excellent peak shape for basic analytes

2.5 µm XP columns bridge the gap between HPLC and UPLC

– Compatible with HPLC, UHPLC and UPLC system platforms

– Maximize HPLC productivity

CORTECS UPLC Columns enable highest efficiency,

resolution and throughput


Thank you for your attention

Questions?


Documents

Introduction to Waters Sample Preparation and