using uplc for hplc method dev - american chamber of...

Using Ultra Performance LCTM

for Fast & Efficient HPLC Method Development

ESAC 2006Rune Buhl Frederiksen

©2006 Waters Corporation

What is 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


Efficiency (N), is inversely proportional to Particle Size , dp

To get Rs 1.7x, we need N 3x, this now means dp 3X

In summary, to get Rs 1.7x we must reduce particle size by 3X.

Improving Resolution with Smaller ParticlesAt Constant Column Length

dpN 1∝


Efficiency, N is inversely proportional to the square of Peak Width, W

Peak height is inversely proportional to peak width

What happens to Sensitivity as I increase Resolution?Constant Column Length

2

1w

N ∝

wHeight 1

∝

Remember the simple math;

To get Rs 1.7x we need N 3x, and, so dp 3x, but T 3x

Bonus: Sensitivity 1.7x


A Term(Particle size and how well bed was packed)

Hei

ght E

quiv

alen

t to

Theo

retic

al P

late

Linear Velocity

HET

P

u

Lowest HETP => Optimum Plate Count

{cm/sec}

HETP PLATES

HC Term

(Mass transfer)

B Term(Axial Diffusion)

Add the 3 terms to obtain final “van Deemter Curve”

Particle Size and Flow Ratevan Deemter Equation

A term + B term + C term

H = a(dp) + b + c(dp)2uu


Smaller ParticlesThe Enabler of Productivity

Optimal velocity range


Summary of Key System Attributes

At constant column length

At constant L/dp ratio

Resolution Improvement

Speed Improvement

Sensitivity Improvement Back Pressure

5 to 1.7µm particles 1.7X 3X 1.7X 27X3 to 1.7 µm particles 1.3X 2X 1.3X 6X

Resolution Improvement

Speed Improvement

Sensitivity Improvement Back Pressure

5 to 1.7µm particles Same 9X 3x 9X3 to 1.7 µm particles Same 3X 2x 3X


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HPLC vs. UPLC™Speed, Sensitivity and Resolution

2.1 x 150 mm, 5 µmRs (2,3) = 4.29

12 3

HPLC

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orba

nce

at 2

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2.1 x 50 mm, 1.7 µmRs (2,3) = 4.281

23

8X Speed3.4X SensitivitySame Resolution

0.26

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orba

nce

at 2

70 n

m

0.00

UPLCTM

Faster, More Sensitive Methods

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3

2.1 x 100 mm, 1.7 µmRs (2,3) = 6.38

1

2

4.5X Speed2X Sensitivity1.5X Resolution

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orba

nce

at 2

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UPLCTM

Faster, More Sensitive, Higher Resolution Methods

ESG


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Gradient Separations:Maintain Resolution, Improve Throughput

Final UPLCTM Method = 5 minACQUITY UPLCTM BEH C182.1 x 50 mm, 1.7 µm

Original HPLC Method = 35 min4.6 x 150 mm, 5 µm

17.1714.76510.019.67420.2819.1234.374.132

1FinalOriginalAnalyte

Resolution

1

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3

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5

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Ultra Performance LC™Expands analytical LC boundaries

Speed, sensitivity and resolution can all be increased without compromises

How can we use this to develop methods faster and more efficiently ?


Strategies for Methods Development

• Literature search, neighbor, speculation?

• Stepwise iterative procedure– Next step experimental design based on results from previous

experiment

• Systematic screening– Evaluate difficult and costly variables first (stationary phase)– Paired choices (solvents, pH)– Fine tuning (temperature, gradient slope)


Factors That Control Retention 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


Introducing 2nd Generation Hybrid:Bridged EthylSiloxane/Silica Hybrid Particles

Bridged EthanesIn Silica Matrix

Anal. Chem. 2003, 75, 6781-6788

Waters Patented TechnologyNo. 6,686,035 B2

Tetraethoxysilane(TEOS)

Bis(triethoxysilyl)ethane(BTEE)

+4

Polyethoxysilane(BPEOS)

Si

EtO

EtO OEtEtO

Si

EtOEtO

CH2EtO

CH2Si

OEt

OEtOEtSi

EtO

O

CH2 CH2

Si O

Si

EtO

OEt

Si O

O

OEtO

Si

O

Si

OEt

O

OOEt

Et

Et

n


ACQUITY UPLCTM Columns

ACQUITY UPLC™ BEH C18

ACQUITY UPLC™ BEH C8

ACQUITY UPLC™ BEH Shield RP18

ACQUITY UPLC™ BEH Phenyl


Solvent Properties

• Methanol– Weaker eluent– Proton donor

• Acetonitrile– Proton acceptor– Stronger eluent– Lower viscosity


Effect of Mobile Phase pH

• Affects only analytes with ionizable functional groups– Amines– Carboxylic acids– Phenols

• Some compounds contain one or more ionizable function

• Strongest selectivity effects can be caused by pH changes


Note: Column Particle,Temperature and % Organic Held Constant

Reversed-Phase Retention Map

pH

0

5

10

15

20

25

30

35

40

0 2 4 6 8 10 12

Ret

enti

on F

acto

r (k

) Acid

Base

Neutral

Note: Retention of neutral analytes not affected by pH

Increased, robust base retention

Increased acid retention

Silica pH Range

Hybrid Particle pH Range

Reversed Phase ChromatographyPolar Modifier Choices


Flow rate/ Linear Velocity

• Optimal flow rate dependance– Diffusion constants– Compound characteristics

• Limits on adjustment– System– Particle size

• UPLC™ gives widest usable range


Method Optimization:Gradient Slope

• Shallower gradient slope may improve resolution– Decreasing gradient slope will decrease sensitivity

• Steeper gradient slope may compress the peaks and often reduce the resolution – Increasing gradient slope will increase sensitivity

• Changing gradient slope is a balance between peak heights relative to resolution

• Changes in retention and selectivity

©2006 Waters CorporationTime

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00

%

1

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00

%

1


*

**

BGC

Rate of Change0.75%/ col. vol.

Rate of Change1.5%/ col. vol.



• Changes in gradient slope do not have a linear effect onresolution

• Changes in selectivity can occur due to differences in analyte thermodynamic effects and diffusivity


Method Optimization:Influence of Temperature

• Reduced viscosity

• Lower backpressure

• Changes in retention and selectivity

©2006 Waters CorporationMinutes

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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

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41 oC11,295 PSI

42 oC11,160 PSI

43 oC11,010 PSI

44 oC10,875 PSI

45 oC10,700 PSI

ESG


Strategies for Methods Development

• Literature search, neighbor, speculation?

• Stepwise iterative procedure– Next step experimental design based on results from previous

experiment

• Systematic screening– Evaluate difficult and costly variables first (stationary phase)– Paired choices (solvents, pH)– Fine tuning (temperature, gradient slope)


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


Complete Separation of a Sample:Impurity Profiling

• Intentionally overload parent compound to better quantitate trace level impurities– Parent concentration: 30 mg/mL in DMSO

• Resolve as many components as possible

NH

N+

O

O-

O

O

O

O

CH3 CH3O

CH3

CH3

CH3

Nimodipinem.w. 418.44


UPLC Systematic Screening:Experimental Details

Chromatographic Conditions :Columns: ACQUITY UPLCTM BEH C18, BEH Shield RP18, BEH C8, BEH PhenylColumn Dimensions: 2.1 x 50 mm, 1.7 µmMobile Phase A1: 20 mM NH4COOH in H2O, pH 3.0Mobile Phase A2: 20 mM NH4HCO3 in H2O, pH 10.0Mobile Phase B1: acetonitrileMobile Phase B2: methanolFlow Rate: 0.5 mL/min Gradient: Time Profile Curve

(min) %A %B0.0 95 5 65.0 10 90 65.01 95 5 65.5 95 5 6

Injection Volume: 10.0 µLWeak Needle Wash: 3% methanolStrong Needle Wash: 90% acetonitrileTemperature: 30 oCDetection: UV @ 254 nmSampling rate: 20 pts/secTime Constant: 0.1Instrument: Waters ACQUITY UPLCTM, with 2996 ACQUITY PDA detector


Stationary Phase Selectivity:pH 3.0, Acetonitrile

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C18

Shield RP18

C8

Phenyl

Observations:•These two columns appear to have the most potential in resolving a majority of the peaks

ESG

Actions:•Investigate the resolution of the low level impurities on Shield RP18 and C8 columns


Column Selection:pH 3, acetonitrile

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Observations:•Peak shape indicates there might be additional coeluting species

Shield RP18

C8

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Actions:•Shield RP18 column selected


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Solvent Selectivity

Observations:•The analyte is too hydrophobic for methanol to be used successfully as the organic modifier

Shield RP18

Acetonitrile, pH 3.0

Shield RP18

Methanol, pH 3.0

ESG

Actions:•Selection of acetonitrile as organic modifier


pH selectivityAU

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Observations:•In this case, there is little selectivity difference between the two pH values with acetonitrile as the organic modifier

Shield RP18


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Shield RP18


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Actions:•Selection of Shield RP18 at pH3


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Optimize for Greatest Number of Resolved Peaks

Initial Screening MethodTG 5 minutes5 – 90% acetonitrile, pH 3.0

Decreasing Gradient SlopeTG 15 minutes5 – 90% acetonitrile, pH 3.0

ACQUITY UPLCTM BEH Shield RP18

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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

Isolate 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


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•Scale to preparative dimensions to isolate impurity peak

•Less resolution on larger column

•Must adjust UPLC method to successfully isolate Impurity1

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

2XBridgeTM 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


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ACQUITY UPLCTM

BEH Shield RP182.1 x 50 mm, 1.7 µm10 µL injectionF = 0.5 mL/minTG 23 minutes5% ACN 1 min36% ACN 1 – 20 min86% ACN 21 – 23 min

Nim

odip

ine

Imp

1

Imp

2

XBridgeTM Shield RP1819 x 100 mm, 5 µm850 µL injectionF = 17.06 mL/minTG 108 minutes5% ACN 2.4 min36% ACN 2.4 – 93.6 min86% ACN 98.4 – 108 min

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odip

ine

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Imp

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Scale-up Guidelines:UPLC-to-Isolation

• May observe less resolution when scaling from UPLC to a larger column for isolation

• May require adjusting UPLC method to provide extra resolution for isolation method

• Adjusting the UPLC method and increasing resolution gave satisfactory isolation for direct scale up


Summary

• Principles of methods development remain the same– Acetonitrile, methanol– pH 3 and pH 10

• BEH particle technology enables the exploration of pHextremes in methods development– Stability from pH 1 - 12

• Large selectivity differences can occur between bonded phases with – Methanol as organic modifier– Analytes in their unionized state, enabled by BEH particle

technology

• Why UPLC for methods development?


Develop Methods Faster with UPLC:Time Savings

Equivalent HPLC Gradient Conditions :Column Dimensions: 4.6 x 150 mm, 5 µmFlow Rate: 1.0 mL/min Gradient: Time Profile Curve

(min) %A %B0.0 95 5 635.0 10 90 6

Peak Capacity (P) = 150

UPLC Gradient Conditions :Column Dimensions: 2.1 x 50 mm, 1.7 µmFlow Rate: 0.5 mL/min Gradient: Time Profile Curve

(min) %A %B0.0 95 5 65.0 10 90 6

Peak Capacity (P) = 150

UPLC screening method 7X Fasterthan directly scaled HPLC method

wt

1P g+=


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


Conclusions

• Achieve more resolution, faster by utilizing 1.7 µm UPLC columns

• Principles of methods development remain the same

• 4 UPLC column chemistries provide a broad range ofselectivity to successfully develop methods efficiently– C18, C8, Shield RP18 and Phenyl

• UPLC can significantly improve laboratory efficiency

• UPLC allows for faster methods development

using uplc for hplc method dev - american chamber of...

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