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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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
Agenda
• UPLCTM column chemistry
• Scaling UPLCTM separations to isolation and purification
• Influence of optical detectors on sensitivity and dispersion
©2005 Waters Corporation
Agenda
• UPLCTM column chemistry
• Scaling UPLCTM separations to isolation and purification
• Influence of optical detectors on sensitivity and dispersion
©2005 Waters Corporation
Chromatography PrinciplesMass Transfer / Diffusion
Porous Particle
Analyte Molecules
Mobile Phase
©2005 Waters Corporation
Smaller Particles:The Enabler of Productivity
UPLC™ Separations-Chromatography of the Future
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
UPLCTM Methods
• UPLCTM improves resolution by reducing band broadening
• Useful separations still require retention and selectivity
©2005 Waters Corporation
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
©2005 Waters Corporation
Different Ligands:Different Selectivity
• Changes in hydrophobicity
• Changes in silanol activity
• Changes in hydrolytic stability
• Changes in ligand density
©2005 Waters Corporation
ACQUITY UPLCTM Columns
ACQUITY UPLC™ BEH C18
ACQUITY UPLC™ BEH C8
ACQUITY UPLC™ BEH Shield RP18
ACQUITY UPLC™ BEH Phenyl
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
Experimental MatrixpH 3, Acetonitrile pH 10, Acetonitrile
C18
Shield RP18
C8
Phenyl
pH 3, Methanol pH 10, Methanol
©2005 Waters Corporation
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
©2005 Waters Corporation
Agenda
• UPLCTM column chemistry
• Scaling UPLCTM separations to isolation and purification
• Influence of optical detectors on sensitivity and dispersion
©2005 Waters Corporation
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
©2005 Waters Corporation
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.
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
Agenda
• UPLCTM column chemistry
• Scaling UPLCTM separations to isolation and purification
• Influence of optical detectors on sensitivity and dispersion
©2005 Waters Corporation
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
©2005 Waters Corporation
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?
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
0.10
0.15
0.20
0.25
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
©2005 Waters Corporation
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
©2005 Waters Corporation
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
©2005 Waters Corporation
Acknowledgements
• Eric Grumbach
• Tom Wheat
• Jeffrey Mazzeo
• Tanya Jenkins
• Andy Aubin
• Beth Hazel
• Jeannine Jordan