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
©2005 Waters Corporation
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
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
©2005 Waters Corporation
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
©2005 Waters Corporation
HPLC to Optimized UPLC™
Abso
rban
ce 2
54 n
m
Minutes0 10 20 30
1
23
Abso
rban
ce 2
54 n
m
Minutes0 2.6 5.2
1
23
HPLC
Optimized UPLC™
Magnification ofOptimized UPLC™
Abso
rban
ce 2
54 n
m
Minutes0 10 20 30
1
23
An Example of aSuccessful Method Transfer
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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.
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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.
©2005 Waters Corporation
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
©2005 Waters Corporation
Comparing Results:Does the new method meet resolution criteria?
4.6 x 150 mmResolution 1/2 = 12Resolution 2/3 = 28
Abs
orba
nce
254
nm
Minutes
12
3
0 10 20 30
Abs
orba
nce
254
nm
Minutes0 10 20 30
1
23 UPLC™ optimized 2.1 x 50 mm
Resolution 1/2 = 11Resolution 2/3 = 26
©2005 Waters Corporation
Comparing Results:Does the new method meet resolution criteria?
4.6 x 150 mm, 5µmResolution 1/2 = 12Resolution 2/3 = 28
Abs
orba
nce
254
nm
Minutes
12
3
0 10 20 30UPLC™ optimized 2.1 x 50 mm
Resolution 1/2 = 11Resolution 2/3 = 26
Abs
orba
nce
254
nm
Minutes0 2.6 5.2
1
23
©2005 Waters Corporation
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
AU
0.00
0.10
0.20
0.30
0.40
Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00
1 24
5
3
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00
Enhanced BaselineE.G.
35 min.
©2005 Waters Corporation
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
©2005 Waters Corporation
AU
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
HPLC-to-UPLCTM Method Transfer:Caffeic Acid Derivatives in Echinacea Purpurea
AU
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
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
1
1
2
2
3
3
4
4
5
5
35.00
5.00
©2005 Waters Corporation
HPLC-to-UPLCTM Method Transfer:Caffeic Acid Derivatives in Echinacea Purpurea
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
au
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
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
©2005 Waters Corporation
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