GC HPLC SFC Capillary GC UPLC UPC - Waters Corporation · 3/15/2013 2 Why The Name? Giddings, J.C....

3/15/2013

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UPCUPC22

UltraPerformanceUltraPerformance Convergence ChromatographyConvergence Chromatography

Expanding Selectivity for the

Chromatographic Laboratory

©2012 Waters Corporation 1

Chromatographic Laboratory

Evolution of Separation TechnologyEvolution of Separation Technology

Gas Chromatography Liquid Chromatography Convergence Chromatography


GC

Capillary GC

HPLC

UPLC

SFC

UPC2

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Why The Name?Why The Name?

Giddings, J.C. (1965) A critical evaluation of the theory of gas chromatography. In Gas Chromatography. 1964, edited by A. Goldup, p. 3-24. Elsevier, Amsterdam

In this article Dr. Giddings stated “One of the most interesting features of ultra high pressure gas chromatography would be convergence with classical liquid chromatography.”


Prof. Calvin Giddings (1930-1996)

UltraPerformanceUltraPerformance Convergence ChromatographyConvergence Chromatography

Convergence Chromatography is a category of separation science that provides orthogonal and increased separation power, compared to that provides orthogonal and increased separation power, compared to liquid or gas chromatography, to solve separation challenges.

UltraPerformance Convergence Chromatography [UPC2] is a holistically designed chromatographic system that utilizes liquid CO2 as a mobile phase to leverage the chromatographic principles and selectivity of normal phase chromatography while providing the ease-of-use of reversed-phase LC.


The ACQUITY UPC2 System is built utilizing proven UPLC Technology to enable scientists the ability to address routine and complex separation challenges while delivering reliability, robustness, sensitivity and throughput never before possible for this analytical technique.

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Built upon proven UPLC Technology– Quantifiable increase in productivity

Exceptional increase in available selectivity– Solve routine and complex separation

challenges


How an ACQUITY UPCHow an ACQUITY UPC22 System WorksSystem Works

PDA detector

Splitter

Inject valve

AuxiliaryInject valve

Column Manager

Back Pressure Regulator(Dynamic and Static)

Make-upPump

Mass Spec


Waste Modifier CO2Supply CO2

PumpModifier

Pump

mixerThermo-electric heat exchanger

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What is a Supercritical Fluid?What is a Supercritical Fluid?

Liquid/gas Critical point Supercritical

Increase temp and pressure

Increase temp and pressure

Liquid/gas Critical point Supercritical fluid


High diffusivity, and low viscosity, same solvatation power result in fast, efficient chromatography

Sample PreparationSample PreparationMajor Source of Laboratory CostsMajor Source of Laboratory Costs

Sample preparation is the most often cited area of improvement to save time and operating costs

Most sample preparation involves being in an organic phase – Liquid/Liquid, PPT, Soxhlet, Distillation, Evaporation and

Reconstitution

Many matrices will respond best to organic phases (gels, blisters, ointments, synthesis solvents, etc.)


image from dyapharma.com image from sefetec.net image from tasnee.com

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Improving Workflow with Improving Workflow with Convergence ChromatographyConvergence Chromatography

GasGas ChromatographyChromatographyGasGas ChromatographyChromatography

pyrethroidspyrethroids

ReversedReversed--phase LCphase LCReversedReversed--phase LCphase LC

carbamatescarbamates

Convergence Convergence ChromatographyChromatography

Convergence Convergence ChromatographyChromatography

SPE Extraction(Florisil)STEP 1

Elution in hexane/ethyl acetateSTEP 2

Evaporate to drynessSTEP 3

SPE Extraction(Oasis HLB)

Elution in methanol

Evaporate to dryness

Direct Analysis on UPC2

Convergence Chromatography


Reconstitute in cyclohexaneSTEP 4

Derivatize sample

Ready for analysis

STEP 5

STEP 6

Reconstitute in water

Ready for analysis

g p y

Eliminate lengthy evaporation and

reconstitution steps

No need for derivatization

Separation Technology OverviewSeparation Technology Overview

Separation achieved by a temperature gradient

•High efficiency [N]• Virtually no limitation on column length•Low choices of optimization parameters (Flow Rate, Tp)GC

Gas Chromatography

•Limited selectivity [α]• Limited stationary phase options

Separation achieved by a solvent gradient

•High efficiency [N]• Limited to pressure drop across column

•High selectivity [α]• Different modes: reversed‐phase, normal‐phase, SEC, IEX, affinity, ion pair, HILIC, GPC…etc.

LC


Liquid Chromatography

Convergence Chromatography

p , ,

Separation achieved by density/solvent gradient

•High efficiency [N]• Very low viscosity enables longer columns and smaller particles

•Ultimate selectivity [α]• Wide variety of stationary phase and mobile phase co‐solvent and modifier options

CC

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Applicability of UPCApplicability of UPC22: : Fast Fast ChiralChiral ScreeningScreening

Fast Chiral ScreeningProviding meaningful impact to scientists from discovery to QC based on the reducing of non-value adding steps in analytical workflow process

Reduces the time consuming solvent mixing and

0.36

0.48 UPC²0.3 min

Reduces the time consuming solvent mixing and sample preparation so can reallocate resources to other value adding analytical work

Increases the column lifetime so can reallocate consumable budget

Reduces the cost of solvent investments of purchase and removal

Reduces the complexity of instrument multi-method use so can reduce the capital investments, or increase value added human resources

AU

0.00

0.12

0.24

Minutes0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

NPLC


Key advantages of moving to UPC2

– Results that are equal to or better

– 30X reduction in analysis time

– Nearly 75X reduction in solvent• 135 µL of MeOH vs 10 mL of hexane/ethanol

AU

0.00

0.30

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

11 min

Applicability of UPCApplicability of UPC22: : Normal Phase ReplacementNormal Phase Replacement

ACQUITY UPC² 15 min

Cost per run ~ $0.05

Normal Phase ReplacementProviding meaningful impact to scientists from discovery to QC based on the reducing of non-value adding steps in analytical workflow process

Reduces the time consuming solvent mixing and

30.8

56

0.0016

0.0018

0.0020

Normal Phase HPLC50 min

Reduces the time consuming solvent mixing and sample preparation so can reallocate resources to other value adding analytical work

Increases the column lifetime so can reallocate consumable budget

Reduces the cost of solvent investments of purchase and removal

Reduces the complexity of instrument multi-method use so can reduce the capital investments, or increase value added human resources


6.23

7

10.8

55

20.8

50

26.6

32

35.8

19

AU

-0.0004

-0.0002

0.0000

0.0002

0.0004

0.0006

0.0008

0.0010

0.0012

0.0014

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

50

Cost per run ~ $5.89Key advantages of moving to UPC2

Results that are equal to or better

Over 3X reduction in analysis time

Cost per analysis just from solvent use from $5.89 to 5 cents

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Diverse Applicability of Diverse Applicability of Convergence ChromatographyConvergence Chromatography

A 4-hydroxyacetophenoneB p-nitrophenolC 2-hydroxyacetophenoneD 4-chloroacetanilideE acetanilideF phenacetin

Pharmaceutical Impurity ProfileChemical Materials

Non-ionic surfactants without derivatization compared to GCParacetamol and impurities

F phenacetinG 3-aminophenolH paracetamolI 2-aminophenol

Environmental Food and Beverage

Chiral pesticides Vitamin E isomers


Permethrin

Understanding Convergence Understanding Convergence ChromatographyChromatography

Chromatographic technique similar to HPLC– Instead of mobile phase A being aqueous, it is replaced with CO2

Mobile phase is supercritical fluid + one or more co solventsMobile phase is supercritical fluid + one or more co-solvents– CO2 is the most common supercritical fluid

– MeOH is the most common co-solvent

Gives normal phase like selectivity

Substance Critical Temp oC Critical Pressure(bar)

Comments


Carbon Dioxide 31 74 Physical state easily changed

Water 374 221 Extreme conditions needed

Methanol 240 80 Extreme temperature needed

Ammonia 132 111 Highly corrosive

Freon 96 49 Environmentally unfriendly

Nitrous Oxide 37 73 Oxidizing agent

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Solvent Strength

Solvent EluotropicValue [Eo]

Polarity [P’]

Pentane, Hexane, 0 0 1

Solvent Selectivity ChoicesSolvent Selectivity Choices

Supercritical

The supercritical CO2 used in convergence chromatography is miscible with the entire

Pentane, Hexane, Heptane 0 0.1

Xylene 0.22 2.5

Toluene 0.22 2.4

Diethyl ether 0.29 2.8

Dichloromethane 0.30 3.1

Chloroform 0.31 4.1

Acetone 0.43 5.1

Dioxane 0.43 4.8

THF 0.48 4.0

Convergence chromatography

Wea

km

al p

has

e

Supercritical CO2

eluotropic series, opening up a wide range of solvent selectivity choices to develop a separation

Normal phase


MTBE 0.48 2.5

Ethyl acetate 0.48 4.4

DMSO 0.50 7.2

Acetonitrile 0.52 5.8

Isopropanol 0.60 3.9

Ethanol 0.68 4.3

Methanol 0.73 5.1

Water 10.2

Reversed-phaseStr

ong

Norm

Stationary Phase Stationary Phase Selectivity ChoicesSelectivity Choices

Stationary Phase Choices

Convergence chromatography can utilize both traditional normal phase and reversed-

Silica / BEH

2-ethylpyridine

Cyano

Aminopropyl

Diol

Normal phase range

Convergence Chromatography range

phase column chemistries, opening up a wide range of selectivity choices to develop a separation


Amide

PFP

Phenyl

C18 < C8

Reversed-phase range

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Addressing Selectivity:Addressing Selectivity:RP and NP ChromatographyRP and NP Chromatography

SolventPentane, Hexane, Heptane

Xylene

Stationary Phase

Silica / BEH

Toluene

Diethyl ether

Dichloromethane

Chloroform

Acetone

Dioxane

THF

2-ethylpyridine

Cyano

Aminopropyl

Diol

Normal Phase Selectivity

Space

limited miscibility between solvents


MTBE

Ethyl acetate

DMSO

Acetonitrile

Isopropanol

Ethanol

Methanol

Amide

PFP

Phenyl

C18 < C8

Reversed-phase

SelectivitySpace

Addressing Selectivity:Addressing Selectivity:Convergence ChromatographyConvergence Chromatography

SolventPentane, Hexane, Heptane

Xylene

Stationary Phase

Silica / BEH

ConvergenceChromatography Selectivity Space

Unlimited solvent

Toluene

Diethyl ether

Dichloromethane

Chloroform

Acetone

Dioxane

THF

2-ethylpyridine

Cyano

Aminopropyl

Diol


Unlimited solvent and stationary

phase selectivity

MTBE

Ethyl acetate

DMSO

Acetonitrile

Isopropanol

Ethanol

Methanol

Amide

PFP

Phenyl

C18 < C8

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Addressing Selectivity:Addressing Selectivity:Convergence ChromatographyConvergence Chromatography

SolventPentane, Hexane,

Heptane

X l

Stationary Phase

Silica / BEHSupercritical

CO2

ConvergenceChromatography Selectivity Space

Unlimited solvent

Xylene

Toluene

Diethyl ether

Dichloromethane

Chloroform

Acetone

Dioxane

THF

2-ethylpyridine

Cyano

Aminopropyl

Diol

Wea

k


Unlimited solvent and stationary phase

selectivityMTBE

Ethyl acetate

DMSO

Acetonitrile

Isopropanol

Ethanol

Methanol

Amide

PFP

Phenyl

C18 < C8

Str

ong

OrganicModifier

ACQUITY UPC² ACQUITY UPC² AchiralAchiral ColumnsColumns

ACQUITY UPC² BEH 2-EP• Good retention, peak shape and selectivity• Lipids, steroids, pesticides

Fully scalable to larger particle sizes

ACQUITY UPC² BEH (1.7, 3.5 and 5 µm)

ACQUITY UPC² BEH• Heightened interaction with polar groups such as phospholipids • OLED’s, polymer additives, pesticides

ACQUITY UPC² CSH Fluoro-Phenyl• Good retention of weak bases•Alternate elution for acidic and neutral compounds• Vitamin D metabolites, steroids, natural products

(1.7, 3.5 and 5 µm)

ACQUITY UPC² BEH 2-EP(1.7, 3.5 and 5 µm)

ACQUITY UPC² CSH Fluoro-phenyl (1.7, 3.5 and 5 µm)

ACQUITY UPC² HSS C18 SB(1.8 and 3.5 µm)


ACQUITY UPC² HSS C18 SB• Reversed-phase-like selectivity • Fat soluble vitamins, lipids (Free fatty acids)

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ACQUITY UPC² System:ACQUITY UPC² System:Expanding the Selectivity SpaceExpanding the Selectivity Space

: CSH PFP

U 0 024

0.036

0.048

A D(1 2)

CB

ACQUITY UPC2 CSH Fluoro-Phenyl 1.7 µm

AP

I

AU

0.000

0.012

0.024

: HSS C18 SB

AU

0.000

0.012

0.024

0.036

0.048

: BEH HILIC

0.036

0.048 ACQUITY UPC2 Hybrid 1.7 µm

GA D(1,2) HFE

GA D

C

HF

B

E

GDCB

ACQUITY UPC2 HSS C18 SB 1.7 µm

AP

I

AP

I©2012 Waters Corporation 21

AU

0.000

0.012

0.024

: 2-EP

AU

0.000

0.012

0.024

0.036

0.048

Minutes0.00 0.60 1.20 1.80 2.40 3.00 3.60 4.20 4.80 5.40 6.00

ACQUITY UPC2 Hybrid 2-EP 1.7 µm

GA

DC

H F

BE

GA HF*E

AP

I

Convergence Chromatography:Convergence Chromatography:Leveraging Selectivity to Solve Separation ChallengesLeveraging Selectivity to Solve Separation Challenges

Stationary Phase

Silica / BEHWeakWeak

Supercritical CO2

2-ethylpyridine

Cyano

Aminopropyl

Diol

% M

od

ifie

r

Fat solublevitamins


Amide

PFP

Phenyl

C18 < C8

StrongStrongOrganicModifier

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Convergence Chromatography:Convergence Chromatography:Applied to Foods and Pharmaceutical Life ScienceApplied to Foods and Pharmaceutical Life Science

Fat Soluble Vitamins

Rapid assay time in comparison to

0.48

9

0.78

1.04

α-tocopherol Vitamin E isomers

ACQUITY UPC2 BEH

reversed-phase and normal-phase LC

UPC2 can easily address multiple formulationso Oil-filled, powder-filled and pressed tablets

Sample extraction solvent directly injected

retinol0.60

0.65

0.70

Vitamin ApalmitateVitamin A

acetate

1.00

0.024

trans-vitamin A palmitate

0.69

5

0.76

3

0.89

3

AU

0.00

0.26

0.52

Minutes0.00 0.12 0.24 0.36 0.48 0.60 0.72 0.84 0.96 1.08 1.20

β-tocopherol

δ-tocopherol

γ-tocopherol

ACQUITY UPC2 HSS C18 SB ACQUITY UPC2 HSS C18 SB


AU

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

Minutes0.00 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 3.00

acetate

AU

0.00

0.25

0.50

0.75

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

6.86

2

AU

0.000

0.006

0.012

0.018

Minutes4.204.40 4.60 4.80 5.005.20 5.40 5.60 5.806.00 6.20 6.40 6.606.80 7.00 7.20 7.407.60 7.80 8.00 8.208.40 8.60 8.80 9.00

cis-vitamin Apalmitate

Vitamin D3cholecalciferol

Vitamin D3cholecalciferol


Stationary Phase


Supercritical CO2

2-ethylpyridine

Cyano

Aminopropyl

Diol

Lipids

% M

od

ifie

r


Amide

PFP

Phenyl

C18 < C8


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Convergence Chromatography:Convergence Chromatography:Applied to Neutral and Polar LipidsApplied to Neutral and Polar Lipids

Neutral and Polar Lipids

Unlike RPLC and GC which can only analyze lipids within a single class, UPC2 provides a single methodology to separate complex lipids by class

PC

PE

TAG

Exceptional separation of inter and intra class moieties

Selective for a number of biological extracts


SMLPC

PE

TAG: Triacylglycerides PE: Phosphotidylethanolamine PC: PhosphotidylcholineSM: Sphynogomyelin LPC: Lysophosphotidylcholine

FFA ESI‐

Convergence Chromatography:Convergence Chromatography:Applied to Neutral LipidsApplied to Neutral Lipids

TAG mix

ESI+


CE mix

All mix

ESI+

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Stationary Phase


Supercritical CO2

2-ethylpyridine

Cyano

Aminopropyl

Diol

Natural Products% M

od

ifie

r


Amide

PFP

Phenyl

C18 < C8

Natural Products


Convergence Chromatography:Convergence Chromatography:Applied to Natural ProductsApplied to Natural Products

Roman Chamomile

Characterization of natural products are ti l l h ll i d t l

Anthemis nobilis(Roman Chamomile)

AU

0 10

0.15

0.20

particularly challenging due to sample complexity

Orthogonal separation techniques are often needed to ensure proper sample characterization

Sample extract directly compatible with UPC21

3


0.00

0.05

0.10

Minutes0.00 2.20 4.40 6.60 8.80 11.00 13.20 15.40 17.60 19.80 22.00

1. apigenin-7-O-glucoside2. chamaemeloside3. apigenin

2

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Stationary Phase


Supercritical CO2

2-ethylpyridine

Cyano

Aminopropyl

Diol

% M

od

ifie

r


Amide

PFP

Phenyl

C18 < C8

Organic LED’s


Convergence Chromatography:Convergence Chromatography:Applied to Chemical MaterialsApplied to Chemical Materials

“RED Q” “green” “Red T” “Blue”

Light emitting dopantsOrganic Light Emitting Diode

Ir2+

N

Ir(piq)3

Ir2+

N

Ir(ppy)3

Ir+

S

N+

Fac-Ir(btpy)3

Ir+

F

F

N+

3 3 3 3

Ir(Fppy)3

Materials of construction


N+

NN

CH3

CH3CH3

TAZ“ETL and hole blocker”

O

OO

N+

N+

N

Al3-

Alq3“green EM and ETL”

N N+

N

N

N+

N

N

NTi O

Titanyl phthalocyanine“sensitizer”

“Hole injection material”

TCTAHole transport

material

N N

N

N

N

N

NPDHole transport

material

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Convergence Chromatography:Convergence Chromatography:Degradation Profile of Blue Emitter MaterialDegradation Profile of Blue Emitter Material

Ir(Fppy)3

Organic LED’s

Improved characterization of Ir(Fppy)3, a phosphorescent emitter material that is important for providing blue electroluminescence longevity for use in OLED devices

1.01

9

AU

0.06

0.09

0.12

( ppy)3

N

FF

N

F

F

Ir

N

F

F

N

F

F

Ir

N

F

F

N

F

F

Ir(Fppy)3Isomer

Ir

N

F

F

N

F Ir(Fppy)3 -2F

Ir(Fppy)3 -F

electroluminescence longevity for use in OLED devices10X reduction in analysis compared to published LC methodsDirectly compatible with normal phase extraction solvent


1.36

0

1.46

3

1.61

9

1.93

5

2.74

6

3.62

2

4.12

4

0.00

0.03

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

N

F F

Ir(Fppy)3 –FIsomers

N

F

Ir

N

F

N

F

F

( ppy)3

Assessing SpecificityAssessing Specificityin Presence of OLED materialsin Presence of OLED materials

Organic LED’s

Representative of an OLED extraction containing the blue light emitting dopant and other materials of

1.00

9

1.42

1

1.92

0

28

2.47

1

0.050

0.075

0.100

Ir(Fppy)3Area = 0.4%Rt = 0.1%

TCTAArea = 1.1%Rt = 0.1% Alq3

Area = 1.1%Rt = 0.1%

NPDArea = 0.4%Rt = 0.1%

blue light emitting dopant and other materials of construction

Directly compatible with normal phase extraction solvent


2.02

AU

0.000

0.025

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

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Convergence Chromatography:Convergence Chromatography:OLED Component MixtureOLED Component Mixture

0.100

TAZ Organic LED’s

Single assay to characterize light emitting dopants, degradants and materials of construction of the OLED

AU

0.050

0.075

3(btp

y) 3

Ir(F

pp

y) 3

NPD

Fppy)

3Is

om

er

TCTA Ir

(pp

y) 3

3-F

cyan

ine

F ated

degradants and materials of construction of the OLED

Directly compatible with normal phase extraction solvent


0.000

0.025

Minutes0.00 0.60 1.20 1.80 2.40 3.00 3.60 4.20 4.80 5.40 6.00

Ir(p

iq) 3

Fac-

Ir(

ALq

3

Ir( F

Ir(F

ppy)

3

Titan

ylphth

aloc

Ir(F

ppy)

3-2

F

Ir(F

ppy)

3re

la

UPCUPC22 Applications by MarketApplications by Market

Pharmaceutical Food/Env Chemical Materials ClinicalChiral drugs

Transfer from USP methodsFat‐soluble vitamins

Lipid analysisipid analysisAchiral impurity analysis

Chiral pesticidesExplosives

OLEDsAzo dyes

Non‐ionic surfactants

Polymer additivesDrugs of abuse

Vitamin D metabolitesCarotenoids

Positional isomers


Positional isomersSteroids Steroids

Extractables and leachablesReaction monitoringLibrary screeningDMPK/BioanalysisNatural productsMetabolomics

GlyceridesPAH

Lubricants

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ConclusionConclusion

ACQUITY UPC2 Technology, using compressed carbon dioxide (CO2) as the primary mobile phase, is a separation tool that solves both routine and complex chromatographic problems, especially for samples possessing a wide range of polaritiespolarities

UPC2 offers scientists unique workflow, application, and environmental impact benefits compared to LC and GC platforms

Because UPC2 is built utilizing UPLC Technology, customers are assured of its optimized performance through holistic design of instrumentation, detectors, software data systems and chemistries.


UPC2 provides an exceptional increase in available selectivity, making this technology widely applicable to a diverse range of compound types

– 80-85% overlap of compounds that can be analyzed by CC and RPLC

– Any compound soluble in an organic solvent


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N GC

EfficiencySelectivityVolatiles only

NPLC

Volatiles onlySimplified workflow

RPLC

UPLC

ProductivityROISensitivity

SFC

UPC2

ProductivityRobustnessEfficiency

©2012 Waters Corporation 37α

SelectivitySimplified WorkflowOrganic injections

SolventsRobustnessNon-volatiles onlyGradientsMS compatibility

RobustGradientsSolventsMS Compatibility

SelectivityNon-volatiles only

Simplified WorkflowOrthogonalityStructural SimilarityOrganic injectionsROISolventsVolatiles & Non-volatilesGradientsMS Compatibility

ProductivityRobustnessEfficiency

GC HPLC SFC Capillary GC UPLC UPC - Waters Corporation · 3/15/2013 2 Why The Name? Giddings, J.C....

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Transcript of GC HPLC SFC Capillary GC UPLC UPC - Waters Corporation · 3/15/2013 2 Why The Name? Giddings, J.C....