using uplc for hplc method dev - american chamber of...
TRANSCRIPT
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
©2006 Waters Corporation
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∝
©2006 Waters Corporation
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
©2006 Waters Corporation
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
©2006 Waters Corporation
Smaller ParticlesThe Enabler of Productivity
Optimal velocity range
©2006 Waters Corporation
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
©2006 Waters Corporation
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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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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
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UPLCTM
Faster, More Sensitive, Higher Resolution Methods
ESG
©2006 Waters Corporation
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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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ESG
©2006 Waters Corporation
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 ?
©2006 Waters Corporation
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)
©2006 Waters Corporation
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
©2006 Waters Corporation
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
©2006 Waters Corporation
ACQUITY UPLCTM Columns
ACQUITY UPLC™ BEH C18
ACQUITY UPLC™ BEH C8
ACQUITY UPLC™ BEH Shield RP18
ACQUITY UPLC™ BEH Phenyl
©2006 Waters Corporation
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
©2006 Waters Corporation
Solvent Properties
• Methanol– Weaker eluent– Proton donor
• Acetonitrile– Proton acceptor– Stronger eluent– Lower viscosity
©2006 Waters Corporation
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
©2006 Waters Corporation
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
©2006 Waters Corporation
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
©2006 Waters Corporation
Flow rate/ Linear Velocity
• Optimal flow rate dependance– Diffusion constants– Compound characteristics
• Limits on adjustment– System– Particle size
• UPLC™ gives widest usable range
©2006 Waters Corporation
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
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%
1
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1
Method Optimization:Gradient Slope
*
**
BGC
Rate of Change0.75%/ col. vol.
Rate of Change1.5%/ col. vol.
©2006 Waters Corporation
Method Optimization:Gradient Slope
• 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
©2006 Waters Corporation
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
©2006 Waters Corporation
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)
©2006 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
©2006 Waters Corporation
Experimental MatrixpH 3, Acetonitrile pH 10, Acetonitrile
C18
Shield RP18
C8
Phenyl
pH 3, Methanol pH 10, Methanol
©2006 Waters Corporation
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
©2006 Waters Corporation
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
©2006 Waters Corporation
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
©2006 Waters Corporation
Column Selection:pH 3, acetonitrile
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Observations:•Peak shape indicates there might be additional coeluting species
Shield RP18
C8
ESG
Actions:•Shield RP18 column selected
©2006 Waters Corporation
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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
©2006 Waters Corporation
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
Acetonitrile, pH 3.0
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Shield RP18
Acetonitrile, pH 10.0
ESG
Actions:•Selection of Shield RP18 at pH3
©2006 Waters Corporation
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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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©2006 Waters Corporation
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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
©2006 Waters Corporation
Minutes4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
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Isolate a Specific Impurity Peak
•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
©2006 Waters Corporation
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
Isolate a Specific Impurity Peak
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
Imp
1
Imp
2
ESG, FX
©2006 Waters Corporation
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
©2006 Waters Corporation
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?
©2006 Waters Corporation
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+=
©2006 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
©2006 Waters Corporation
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