The Application of Ultra Performance LC™ for High Speed, High … · 2007-11-21 · The...

The Application of Ultra Performance LCTM

for High Speed, High Resolution Chromatographic Methods

Eric S. Grumbach

Thomas E. Wheat

Jeffrey R. Mazzeo

Eastern Analytical SymposiumNovember 16th, 2005

©2005 Waters Corporation

Ultra Performance LC™

• A new class of separation science– Based on chromatography columns with very small particles– Based on instruments designed to take advantage of the small

particles

• Provides improved Resolution, Speed, and Sensitivity with no compromises

• Suitable for chromatographic applications in general– Appropriate for developing new methods– Appropriate for improving existing methods


More Resolution Faster

• How can I improve sample throughput without compromising resolution?

• Improve productivity by improving Speed

(faster flow rates, reducing run times)

• It is necessary to maintain resolution, while reducing the run times


Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

HPLC vs. UPLC™Speed, Sensitivity and Resolution

2.1 x 150 mm, 5 µmRs (2,3) = 4.29

12 3

HPLC

20.00

0.26

Abs

orba

nce

at 2

70 n

m

0.00

Minutes0.40 0.80 1.20 1.60 2.00 2.50

2.1 x 50 mm, 1.7 µmRs (2,3) = 4.281

23

8X Speed3.4X SensitivitySame Resolution

0.26

Abs

orba

nce

at 2

70 n

m

0.00

UPLCTM

Faster, More Sensitive Methods

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

3

2.1 x 100 mm, 1.7 µmRs (2,3) = 6.38

1

2

4.5X Speed2X Sensitivity1.5X Resolution

4.50

0.26

Abs

orba

nce

at 2

70 n

m

0.00

UPLCTM

Faster, More Sensitive, Higher Resolution Methods

ESG


Criteria for Success:High Resolution and High Speed Separations

• Mechanically and chemically robust column chemistry

• Uncompromised chromatographic data while improving throughput

• Transferability and Scalability

• Low dispersion, higher pressure instrumentation

• Fast injection-to-injection cycle times

• High speed, high sensitivity, low dispersion optical and mass detectors


Agenda

• UPLCTM column chemistry

• Scaling UPLCTM separations to isolation and purification

• Influence of optical detectors on sensitivity and dispersion


Chromatography PrinciplesMass Transfer / Diffusion

Porous Particle

Analyte Molecules

Mobile Phase


Smaller Particles:The Enabler of Productivity

UPLC™ Separations-Chromatography of the Future


Bridged Ethyl Hybrid Chromatographic Particles

OSi

OSi

OSi

OSi

OSi

OSi

OSi

OSi

OSi

O

OH OH OH OH OH H2C CH2OH OH

O O O O O O O

OSi O Si

OSi

OSi

OSi

OSi

OSi

OSi

OSi

OH2C CH2 O O O O O O O

Si

SiO

O

O

OH

O

CH2H2C

H2CCH2

OEtSi

EtO OEtOEt

CH2SiEtO

OEtEtO

+

Tetraethoxysilane(Inorganic)

Bis(triethoxysilyl)ethane(Organic)

CH2

SiOEt

OEtOEt

•Superior mechanical strength•Stability exceeding 65,000 PSI*1

•Improved chemical resistance•Routine operation pH 1 -12

•Improved efficiencies•Tightly centered particle distribution

•Improved peak shapes•Lower residual silanol content

J. Scott Mellors, James W. Jorgenson. Anal Chem 2004, 76, 5441 - 5450


Mechanical Stability

Minutes0.00 1.00 2.00 3.00 4.00

Minutes0.00 1.00 2.00 3.00 4.00

1

1000

2000

Injection

60oC8,100 psi

5.00

ACQUITY UPLC™ BEH C18, 2.1 x 50 mm30% ACN (v/v) with 0.1% TFA (pH 2.0) at 0.9 mL/min

Analytes: protriptyline, amitriptyline, butyrophenone

30oC11,400 psi

5.00

Direct Overlays


Long UPLC™ Column LifetimesCustomer Testing

Initial injection

Injection 4,000

AU 0.00

0.20

0.40

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

AU

0.00

0.20

0.40

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

Accelerated Column Aging Experiment:1. Install and equilibrate column.2. Set flow rate to 0.5 mL/min (6,000 psi), inject and record data.3. Increase flow rate to 1.25 mL/min to achieve backpressure of 13,000 psi.4. Inject 200 to 400 times.5. Reduce flow rate to 0.5 mL/min (6,000 psi), inject and record data.6. Cycle and repeat steps 2 – 5.

Compounds1. Ketorolac (208 µg/mL) in MeOH2. Naproxen (13 µg/mL) in MeOH

1 2

21

NO loss in efficiency or peak shape after 4,000 injections at

13,000 psi and 65oC

Data courtesy of Dr. Ken Wehmeyer, The Proctor & Gamble Company

ACQUITY UPLC™ BEH Shield RP18 2.1 x 50 mm, 1.7 µm


AU

0.00

0.02

0.04

0.06

0.08

Minutes0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

AU

-0.02

0.00

0.02

0.04

0.06

0.08

Minutes0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

Injection 1Peak Capacity = 44

Lifetime of UPLCTM Columns: Protein Precipitated Plasma 12,000 PSI

Conditions 2.1 x 50 mmACQUITY UPLCTM BEH Shield RP18, 2.1 x 50 mm, 1.7 µmMobile Phase A: 0.1% TFA in H2OMobile Phase B: 0.08% TFA in ACNFlow Rate: 1.0 mL/minGradient: Time Profile

(min) %A %B curve0 90 10

0.7 60 40 70.77 5 95 61.05 5 95 61.12 90 10 61.47 90 10 6

Injection Volume: 5.0 µLTemperature: 30 oCDetection: UV @ 272 nm

PSIInitial = 11,800PSIFinal = 12,6006.8% increase in backpressure

- Different Plasma Lots

Injection 4225Peak Capacity = 43

Ate

nolo

l Pin

dolo

l

Met

opro

lol

Ate

nolo

l

Pin

dolo

l

Met

opro

lol


UPLCTM Methods

• UPLCTM improves resolution by reducing band broadening

• Useful separations still require retention and selectivity


Factors That Control Retentivity and Selectivity

• Stationary phase– Base particle– Bonded phase– Secondary interactions

• Mobile phase– Solvents– Proportions of solvent– pH and ionic strength

• Operating conditions– Flow rate– Gradient slope– Temperature


Different Ligands:Different Selectivity

• Changes in hydrophobicity

• Changes in silanol activity

• Changes in hydrolytic stability

• Changes in ligand density


ACQUITY UPLCTM Columns

ACQUITY UPLC™ BEH C18

ACQUITY UPLC™ BEH C8

ACQUITY UPLC™ BEH Shield RP18

ACQUITY UPLC™ BEH Phenyl


Stationary Phase SelectivityFungicides in Apple Juice

pH 3.0, Methanol

AU

0.00

0.10

0.20

0.30

Minutes0.50 1.00 1.50 2.00 2.50 3.00

C18

AU

0.00

0.10

0.20

0.30

Minutes0.50 1.00 1.50 2.00 2.50 3.00

Shield RP18

AU

0.00

0.10

0.20

0.30

Minutes0.50 1.00 1.50 2.00 2.50 3.00

C8

AU

0.00

0.10

0.20

0.30

Minutes0.50 1.00 1.50 2.00 2.50 3.00

Phenyl

1 2

1 2

12

1 2

Analyte1 carbendazim2 thiabendazole

thiabendazolem.w. 201.24

carbendazimm.w. 191.19

ESG


pH Selectivity:Basic and Neutral Compounds

pH 3.0acetonitrile

pH 10.0acetonitrile

B1

B1

B2

B2

N1

N1

N2

N2

Test Probes:B1 imipramineB2 amitriptylineN1 flavoneN2 octanophenone

ACQUITY UPLCTM BEH Phenyl

Minutes

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

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

•Neutrals unaffected by pH•At alkaline pH, bases are in there unionized form resulting in greater retention

ESG


pH Selectivity:Acidic Compounds

pH 10.0acetonitrile

Test Probes:A1 1-pyrenesulfonic acidA2 diclofenacA3 dinoseb

A1A2

A3

ACQUITY UPLCTM BEH C8

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

A1A2

A3

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

pH 3.0acetonitrile

•At acidic pH, acids are in their unionized form resulting in greater retention•Same elution order•Dramatic change in α

ESG


pH SelectivityFungicides in Apple Juice

Analyte1 carbendazim2 thiabendazole

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Minutes0.20 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

AcetonitrilepH 3.0

1 2

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Minutes0.20 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

AcetonitrilepH 10.0

1 2

ACQUITY UPLCTM BEH C18

•Alkaline pH moves acidic matrix constituents away from basic fungicides resulting in less potential matrix interferences

ESG


Minutes

4.00 5.00 6.00 7.00 8.00 9.00 10.00

Method Optimization:Influence of Temperature

36 oC12,080 PSI

37 oC11,920 PSI

38 oC11,735 PSI

39 oC11,570 PSI

40 oC11,430 PSI

Minutes

4.00 5.00 6.00 7.00 8.00 9.00 10.00

41 oC11,295 PSI

42 oC11,160 PSI

43 oC11,010 PSI

44 oC10,875 PSI

45 oC10,700 PSI

ESG


UPLC Systematic Screening

• Four ACQUITY UPLC Chemistries 2.1 x 50 mm, 1.7 µm: – ACQUITY UPLCTM BEH C18

– ACQUITY UPLCTM BEH Shield RP18

– ACQUITY UPLCTM BEH C8

– ACQUITY UPLCTM BEH Phenyl

• Solvents: – Acetonitrile– Methanol

• Buffers: – pH 3 ammonium formate– pH 10 ammonium bicarbonate


Experimental MatrixpH 3, Acetonitrile pH 10, Acetonitrile

C18

Shield RP18

C8

Phenyl

pH 3, Methanol pH 10, Methanol


Develop Methods Faster with UPLC:Time Savings

EQUIVALENT HPLC Methods Development Protocol4.6 x 150 mm, 5 µmpH 3/ acetonitrile TimeFlow ramp 5 minColumn conditioning (2 blank gradients) 80 minSample injection (2 replicates) 80 minpH 3/ methanolFlow ramp 5 minColumn conditioning (2 blank gradients) 80 minSample injection (2 replicates) 80 minColumn purge 35 minpH 10/ acetonitrileFlow ramp 5 minColumn conditioning (2 blank gradients) 80 minSample injection (2 replicates) 80 minpH 10/ methanolFlow ramp 5 minColumn conditioning (2 blank gradients) 80 minSample injection (2 replicates) 80 minColumn purge 35 min

730 min

SCREENING TIME 12.2 Hours/columnx 4 columns

TOTAL SCREENING TIME 48.8 HOURS

UPLC Methods Development Protocol2.1 x 50 mm, 1.7 µmpH 3/ acetonitrile TimeFlow ramp 5 minColumn conditioning (2 blank gradients) 11 minSample injection (2 replicates) 11 minpH 3/ methanolFlow ramp 5 minColumn conditioning (2 blank gradients) 11 minSample injection (2 replicates) 11 minColumn purge 6 minpH 10/ acetonitrileFlow ramp 5 minColumn conditioning (2 blank gradients) 11 minSample injection (2 replicates) 12 minpH 10/ methanolFlow ramp 5 minColumn conditioning (2 blank gradients) 11 minSample injection (2 replicates) 11 minColumn purge 6 min

120 min

SCREENING TIME 2 Hours/columnx 4 columns

TOTAL SCREENING TIME 8 HOURS


Agenda





Scalability

First Introduced at Pittcon 2004 asACQUITY UPLC™ BEH Columns

Chemistry Offerings Expanded at Pittcon 2005 First Introduced at HPLC 2005

Ease of Migration from HPLC to UPLC™

Simplified Purification and Isolation


Ratio of Column Length to Particle Size

100 mm1.7 µm

30,000150mm =5µm100mm =3.5µm 28,571

50 mm1.7 µm

= 29,500

= 58,820

L/dp RATIO

150 mm1.7 µm

= 88,235

•L/dp ratio is a good measure of the resolution power of a column.


Same Resolution and Selectivity withIncreased Speed - Constant L / dp

2.5 µm – 75 mmF = 500 µL/min

Injection = 2.5 µLRs (2,3) = 2.34

5 µm – 150 mmF = 200 µL/min

Injection = 5.0 µLRs (2,3) = 2.28

3.5 µm – 100 mmF = 300 µL/min

Injection = 3.3 µLRs (2,3) = 2.32

1.7 µm – 50 mmF = 600 µL/min

Injection = 1.7 µLRs (2,3) = 2.29

AU

0.00

0.10

0.20

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

AU

0.00

0.10

0.20

Minutes0.00 2.00 4.00 6.00 8.00 10.00

AU

0.00

0.10

0.20

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

AU

0.00

0.10

0.20

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.10


Minutes4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

ACQUITY UPLCTM

BEH Shield RP182.1 x 50 mm, 1.7 µm10 µL injectionF = 0.5 mL/minTG 15 minutespH 3.05 – 90% acetonitrile

ResolutionNimodipineImp1 2.42Imp2 2.72N

imod

ipin

e

Imp

1 Imp

2

Scaling From UPLCTM – to - IsolationIsolate a Specific Impurity Peak

• Need to isolate impurity to characterize in another technique

• Use larger column to isolate sufficient amount of material

• Must use same method to preserve peak profile

• Scale from 1.7 µm UPLCTM material to 5 µm XBridgeTM material

ESG, FX


Minutes4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Minutes26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00

Scaling From UPLCTM – to - IsolationIsolate a Specific Impurity Peak

ACQUITY UPLCTM

BEH Shield RP182.1 x 50 mm, 1.7 µm10 µL injectionF = 0.5 mL/minTG 15 minutespH 3.05 – 90% acetonitrile

Nim

odip

ine

Imp

1 Imp

2

XBridgeTM Shield RP1819 x 100 mm, 5 µm850 µL injectionF = 17.06 mL/minTG 71 minutespH 3.05 – 90% acetonitrile

Nim

odip

ine

Imp

1 Imp

2

ESG, FX


Agenda





Flow Cell Considerations for UPLCTM:Light Guided Flow Cells

• ACQUITY UPLCTM columns produce small volume peaks– To avoid band spreading and maintain concentration, the flow

cell volume must be correspondingly low– If you use conventional flow cells, the path length must be

reduced which results in loss of sensitivity– Waters has specifically designed low volume light guided flow

cells that have an optimal path length and high light throughput

• ACQUITY UPLCTM columns produce peaks that are narrow in time– Fast data rates– Exceptional signal-to-noise to minimize smoothing


Flow Cell Considerations for UPLCTM

• Absorbance detectors measure concentration. Signal depends on path length (Beer’s law)

• Mass sensitivity for a narrow peak requires flow cell volume reduction to avoid dispersion. This is traditionally done by using a shorter path length. However, a shorter path length results in sensitivity loss

• A smaller cross-sectional area reduces the amount of light transmission and generally increases baseline noise

• How do we increase light throughput and maintain pathlengthwithout increasing volume?


Light-Guided Flow Cell Technology

• A light guiding flow cell is essentially an optical fiber whose core material is a fluid

• Light rays entering the liquid core of the flow cell are internally reflected when they meet the Teflon AF boundary. These rays are transmitted through the cell without loss, except for absorption by the sample.

Light Path

α Mobile Phase

TeflonAF

TeflonAF


Flow Cell Considerations for UPLCTM:Light Guided Flow Cells

• Analytical Flow Cell – 10 mm path length, 500 nL illuminated volume– Better chromatographic resolution

• High Sensitivity Flow Cell– 25 mm path length, 2400 nL illuminated volume– Higher peak height (Beer’s law)– Better signal-to-noise


AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Minutes4.40 4.45 4.50 4.55 4.60 4.65 4.70 4.75 4.80 4.85 4.90

AU

-0.06

-0.04

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

1.0380.0420330.040497Peak Width at 13.4%

2.24821113893942Height

2.319326881140955Area

DeltaHigh SensitivityAnalytical

Flow Cell Comparison:Sensitivity


Fast UPLCTM Data Rate AcquisitionUPLC ConditionsSystem: ACQUITY UPLC (BSM, SM, TUV)Column: 2.1 X 50 mm BEH C18, 1.7 µm Mobile Phase: 50% of A: 10 mMol Ammonium Bicarbonate pH=10

50% of B: AcetonitrileFlow Rate: 1.2 mL/minInjection Volume: 1 µL (5 uL Loop, 30 uL PEEK Needle,

PLUNO, 1 uL air gaps)Strong Wash = 75% ACN/25% Water (250 uL)Weak Wash = 50% ACN/25%Water (1000 uL)Sample Temperature: 15 °CColumn Temperature: 45 °C Detection: ACQUITY TUV @ 301 nm, Various data rates, FTC=OffSample = ~ 0.05 mg/mL Benzocaine, Butamben, and Tetracaine

in 1:1 Buffer/ACNData: Empower 2

1 Point per Second2 Points per Second5 Points per Second

10 Points per Second20 Points per Second40 Points per Second80 Points per Second

AU

0.00

0.05

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0.15

0.20

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0.30

0.35

0.40

0.45

0.50

Minutes0.12 0.14 0.16 0.18 0.20 0.22 0.24 0.26 0.28 0.30 0.32 0.34 0.36 0.38 0.40 0.42 0.44 0.46 0.48


Pharmaceutical Impurity Profile:Quantitation of Trace Level Impurities

2.20.00342.714Impurity 5

5.80.01062.549Impurity 4

2.60.00372.504Impurity 3

6454499.9542.263Butamben

16.40.02292.231Impurity 2

4.10.00542.174Impurity 1

Signal-to-NoiseArea %

Retention Time

(Minutes)Compound


Conclusions

• Achieve more resolution, faster by utilizing UPLCTM

– Not just speed, speed and resolution

• BEH particle technology is mechanically and chemically stable to provide long column lifetimes and robust separations• BEH particle technology present in both ACQUITY

UPLCTM columns and XBridgeTM HPLC columns, allow for transferability and scalability from HPLC - to – UPLC and UPLC to preparative separations• High speed, low dispersion UPLCTM optical detectors

allow for best achievable sensitivity, linearity and peak definition


Acknowledgements

• Eric Grumbach

• Tom Wheat

• Jeffrey Mazzeo

• Tanya Jenkins

• Andy Aubin

• Beth Hazel

• Jeannine Jordan

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