Transferring Yesterdays Methods to Tomorrows Technology: The ...

Transferring Yesterdays Methodsto Tomorrows Technology:

The Evolution from HPLC to UPLCTM

Eric S. Grumbach

Thomas E. Wheat

Chuck Phoebe

Joe Arsenault

Jeffrey R. Mazzeo

Diane M. Diehl

©2005 Waters Corporation

UPLC™ TECHNOLOGY

• 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 without compromise

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


Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

HPLC vs. UPLCTM

Speed, Sensitivity and Resolution

2.1 x 150 mm, 5 µmRs (2,3) = 4.29

12 3

HPLC

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2.1 x 50 mm, 1.7 µmRs (2,3) = 4.281

23

8X Speed3.4X SensitivitySame Resolution

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UPLCTM

Faster, More Sensitive Methods

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2.1 x 100 mm, 1.7 µmRs (2,3) = 6.38

1

2

4.5X Speed2X Sensitivity1.5X Resolution

4.50

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UPLCTM

Faster, More Sensitive, Higher Resolution Methods


What Does Ultra Performance LC™ Bring to the Chromatographic Laboratory?

Choices available:• Increased speed and sensitivity

with the same resolution

• Increased sensitivity and speed with resolution

• Increased resolution and sensitivity at the same speed

Speed Resolution

Sensitivity


HPLC to Optimized UPLC™

Abso

rban

ce 2

54 n

m

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1

23

Abso

rban

ce 2

54 n

m

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HPLC

Optimized UPLC™

Magnification ofOptimized UPLC™

Abso

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m

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An Example of aSuccessful Method Transfer


Methods Transfer Considerations

• Classes of methods transfer– Replace a column brand with another– Replace an instrument with another– Replace both the instrument and the column

• The benefits of UPLC™ can only be realized with a new column on a new instrument

• As with any project to transfer a Liquid Chromatographic method, proper consideration of all the factors which control the separation process is essential for success


UPLCTM Column SelectionInternal diameter and length

• Internal diameter2.1 mm or 1.0 mm

• Length– If primary goal is speed, choose 50 mm length – If primary goal is resolution, choose 100 mm length


Ratio of Column Length to Particle Size

If you keep the L/dp ratio the SAME for 2 columns, you will obtain the SAME Resolution. With smaller particle sizes in shorter columns, you will achieve the same separation, but in LESS TIME!

100mm1.7µm

300mm10 µm = 30,000

30,000150mm =5µm100mm =3µm 33,300

50mm1.7µm

= 29,500

= 58,820

1970’s ~ 30+ min.

1990’s ~ 5-10 min.

2004 ~1- 2 min.

1980’s ~10-15 min.

L/dp RATIO Typical

Run Times

2x Maximum Resolution Capability


UPLCTM Column SelectionLigand Selection

Available Ligands:• ACQUITY UPLCTM BEH C18

– Straight chain alkyl C18

• ACQUITY UPLCTM BEH Shield RP18

– Embedded polar group (carbamate)

• ACQUITY UPLCTM BEH C8

– Straight chain alkyl C8

• ACQUITY UPLCTM BEH Phenyl

Why Multiple Ligands?:• Changes in hydrophobicity

• Changes in silanol activity

• Changes in hydrolytic stability

• Changes in ligand density

• Changes in selectivity


Reversed-Phase Column Selectivity Chart

Retentivity(ln [k] acenaphthene)

012005

SunFire ™ C18

YMC-Pack™ PolymerC18™

Hypersil® CPS Cyano

YMC-Pack™ CN

Waters Spherisorb® S5 P

Hypersil® BDS PhenylNova-Pak® Phenyl

YMC-Pack™ Phenyl

Hypersil® PhenylInertsil® Ph-3

YMC-Pack™ Pro C4™

YMCbasic™

Symmetry® C8YMC-Pack™ Pro C8™

Nova-Pak® C8

XTerra® MS C18 Symmetry® C18

YMC-Pack™ Pro C18™

Inertsil® ODS-3

YMC-Pack™ ODS-A™

Nova-Pak®C18

YMC J'sphere™ODS–L80 Nucleosil® C18

Waters Spherisorb® ODS2

Waters Spherisorb® ODS1Resolve® C18

µBondapak™ C18

YMC-Pack™ ODS–AQ™

YMC J'sphere™ ODS–H80YMC J'sphere™ ODS–M80

Inertsil® CN-3

Waters Spherisorb® S5CN

Nova-Pak® CN HP

SymmetryShield™ RP8

SymmetryShield™ RP18

XTerra® RP8

XTerra® RP18

-0.6

-0.3

0

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

2.7

3

3.3

3.6

-1.5 -0.5 0.5 1.5 2.5 3.5

Sele

ctiv

ity(ln

[α] a

mitr

ipty

line/

acen

apht

hene

)

XTerra® MS C8

Luna ®C18 (2)

ACQUITY UPLC™ BEH C18

XTerra ®Phenyl Luna ™

Phenyl Hexyl

ChromolithTM

RP-18

Atlantis® dC18

Zorbax® XDB C18ACT Ace® C18

Zorbax® SB C18

SunFire ™ C8

Luna®C8 (2)

ACQUITY UPLC™ Shield RP18

ACQUITY UPLC™ BEH C8

ACQUITY UPLC™ BEH Phenyl


Method Transfer Consideration

• Solvent delivery– Scale flow rate appropriate to column dimensions– Scale linear velocity appropriate to particle size– Adjust all segments of method, including equilibration

• Sample injection– Scale injection volume

• Detection– Ensure proper data sampling rate– Ensure proper time constant


Original Gradient Profile

1

1

6

*

Curve

9551.5152

5951.5304

9551.5203

5951.50Initial

%B%AFlow Rate

Time Since Injection

Gradient Step

4.6 x 150 mm, 5 µm HPLC Column


Target Conditions –Gradient Profile

• Express gradient duration in % change per column volume (cv) units

• Calculate each segment as a number of column volumes

• Calculate time required to deliver the same number of column volumes to the UPLCTM column at the chosen flow rate.


Express Gradient Segments (Steps) in Units of Column Volumes

For 15 min at 1.5 mL/min on a 4.6 x 150 mm column

Gradient Volume = Flow Rate x Time = 1.5 mL/min x 15 min = 22.5 mL

Column Volume = π x r2 x L = 3.14 x 2.32 x 150 = 2.49 mL

Gradient Duration (cv) = Gradient VolumeColumn Volume

Gradient Duration = 22.5 mL2.49 mL

= 9.03 cv


Original Gradient Profile for Scaling

1

1

6

*

Curve

10

5

15

0

Segment Duration

(min)

9.039551.5152

6.025951.5304

3.019551.5203

05951.50Initial

Segment Duration (Col.Vol.)

%B%AFlow

RateTime Since

InjectionGradient

Step

4.6 x 150 mm, 5 µm HPLC Column


Estimate Optimum UPLC™ Flow Rate

Consider 1.7 µm target particle (2.1 mm i.d. column)Assume temperature and viscosity transferredAdjust flow rate based on van Deemter curve and

approximate molecular weight

~0.6 mL/min for average 500 dalton (molecular weight) molecules

~0.1 mL/min for larger molecules because diffusion is slower, e.g., ~2,000 dalton peptides


Scaling Gradient Step Time for UPLC™ Flow Rate - Maintain Duration (cv)

Original Step 2: 15 min @ 1.5 mL/min with Duration of 9.03cv

Calculate Target Step 2: (keeping duration @ 9.03cv)

Target Column Volume (2.1 x 50) = 0.17 mL

Gradient Step Volume = Duration (cv) x Target Column Volume

= 9.03cv x 0.17 mL = 1.54 mL

Gradient Step Time = Gradient Step Volume / UPLC™ Flow Rate

= 1.54 mL / 0.60 mL/min. =

2.6 min.


Optimized Gradient Profile

0015950.60InitialHold

1

1

6

*

Curve

1.74

0.87

2.61

0

Segment Duration

(min)

9.039550.62.612

6.025950.65.224

3.019550.63.483

05950.60Initial

Segment Duration (Col.Vol.)

%B

%A

Flow Rate

Time Since

Injection

Gradient

Step

2.1 x 50 mm, 1.7 µm UPLCTM Column


Comparing Results:Does the new method meet resolution criteria?

4.6 x 150 mmResolution 1/2 = 12Resolution 2/3 = 28

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Resolution 1/2 = 11Resolution 2/3 = 26


Comparing Results:Does the new method meet resolution criteria?

4.6 x 150 mm, 5µmResolution 1/2 = 12Resolution 2/3 = 28

Abs

orba

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254

nm

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12

3

0 10 20 30UPLC™ optimized 2.1 x 50 mm

Resolution 1/2 = 11Resolution 2/3 = 26

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Original HPLC Method:Caffeic Acid Derivatives in Echinacea Purpurea

Chromatographic Conditions :Columns: XTerra® MS C18 4.6 x 150 mm, 5.0 µmMobile Phase A: 0.1% CF3COOH in H2OMobile Phase B: 0.08% CF3COOH in ACNFlow Rate: 1.0 mL/min Gradient: Time Profile Curve

(min) %A %B0.0 92 8 62.0 92 8 732.0 50 50 635.0 10 90 636.0 92 8 641.0 92 8 6

Injection Volume: 10.0 µLWeak Needle Wash: 0.1% CF3COOH in 8% ACNSample: Caffeic Acid Derivatives in Echinacea PurpureaSample Diluent: 50:50 H2O: MeOH with 0.05% CF3COOH Sample Concentration: 100 µg/mLTemperature: 40 oCDetection: UV @ 330 nmSampling rate: 10 pts/secTime Constant: 0.1Instrument: Alliance® 2695 Separations Module with

2996 PDA detector

Analyte1. Caftaric acid2. Chlorogenic acid3. Cynarin4. Echinacoside5. Cichoric acid

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Enhanced BaselineE.G.

35 min.


HPLC-to-UPLCTM Method Transfer:Chromatographic Conditions

Chromatographic Conditions :Columns: XTerra® MS C18 4.6 x 150 mm, 5.0 µmMobile Phase A: 0.1% CF3COOH in H2OMobile Phase B: 0.08% CF3COOH in ACNFlow Rate: 1.0 mL/min Gradient: Time Profile Curve

(min) %A %B0.0 92 8 62.0 92 8 7

32.0 50 50 635.0 10 90 636.0 92 8 641.0 92 8 6

Injection Volume: 10.0 µLSample: Caffeic Acid Derivatives in Echinacea PurpureaSample Diluent: 50:50 H2O: MeOH with 0.05% CF3COOH Sample Concentration: 100 µg/mLTemperature: 40 oCDetection: UV @ 330 nmSampling rate: 10 pts/secTime Constant: 0.1Instrument: Alliance® 2695 Separations Module with

2996 PDA detector

Chromatographic Conditions :Columns: ACQUITY UPLCTM BEH C18 2.1 x 50 mm, 1.7 µmMobile Phase A: 0.1% CF3COOH in H2OMobile Phase B: 0.08% CF3COOH in ACNFlow Rate: 0.5 mL/min Gradient: Time Profile Curve

(min) %A %B0.0 92 8 60.1 92 8 7

4.45 50 50 64.86 10 90 65.0 92 8 66.0 92 8 6

Injection Volume: 1.0 µLWeak Needle Wash: 0.1% CF3COOH in 8% ACNSample: Caffeic Acid Derivatives in Echinacea PurpureaSample Diluent: 50:50 H2O: MeOH with 0.05% CF3COOH Sample Concentration: 100 µg/mLTemperature: 40 oCDetection: UV @ 330 nmSampling rate: 40 pts/secTime Constant: 0.1Instrument: Waters ACQUITY UPLCTM, with TUV detector

Original HPLC Method Final UPLCTM Method


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HPLC-to-UPLCTM Method Transfer:Caffeic Acid Derivatives in Echinacea Purpurea

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

Original HPLC Method = 35 minXTerra® MS C184.6 x 150 mm, 5 µm

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HPLC-to-UPLCTM Method Transfer:Caffeic Acid Derivatives in Echinacea Purpurea

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Original HPLC Method = 35 minXTerra® MS C184.6 x 150 mm, 5 µm

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

35.00

5.00


Conclusions

• Methods can be moved directly from HPLC to ACQUITY UPLC™• Improved resolution• Improved speed• Improved detectability

• Many parameters can and must be transferred to preserve results

• Attention to detail leads to success

Transferring Yesterdays Methods to Tomorrows Technology: The ...

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