Waters AAPS 2007 Seminars November 12-13, 2007
©2007 Waters Corporation
Today’s Schedule
©2007 Waters Corporation
2
Tomorrow’s Schedule
©2007 Waters Corporation
3
An Introduction to UPLC® Technology: Improve Productivity and Data Quality Doug McCabe
©2007 Waters Corporation
Agenda
Introduction: What is UPLC® Technology? Migrating an HPLC Method to a UPLC® Method Efficient UPLC® Method Development and Validation Conclusion
©2007 Waters Corporation
5
UPLC® Technology & The Fundamental Resolution Equation
( )( )
N α −1 Rs = 4 α Physical
k k +1
Chemical
•In UPLC® systems, increasing N (efficiency) is the primary focus •Selectivity and retentivity are the same as in HPLC •Resolution, Rs, is proportional to the square root of N
Rs ∝ N If N ↑ 3x, Rs ↑ 1.7x ©2007 Waters Corporation
6
Improving Resolution with Smaller Particles
Constant Column Length
Efficiency (N), is inversely proportional to Particle Size, dp
∝
dp ↓ 3X, (e.g., 5 μm to 1.7 μm)
N ↑ 3X,
Rs ↑ 1.7X (i.e., Rs α √N)
©2007 Waters Corporation
7
UPLC® Particles, van Deemter Curves & Flow Rates 30 5 µm SunFire™ C18 3.5 µm SunFire™ C18
25
HETP (µm)
1.8 µm ACQUIT Y UPLC® HSS T 3 1.7 µm ACQUIT Y UPLC® BEH C18
20 15 10 5 0 0
1
2
3
Flow rate (mL/min)
4
5
6
7
8
9
10
11
12
Linear Velocity [mm/s]
1.0 mm ID
0.04
0.07
0.11
0.14
0.18
0.21
0.25
0.28
0.32
0.35
0.39
0.42
2.1 mm ID
0.15
0.30
0.45
0.60
0.75
0.90
1.05
1.20
1.35
1.50
1.65
1.80
4.6 mm ID
0.70
1.40
2.10
2.80
3.50
4.20
4.90
5.60
6.30
7.00
7.70
8.40
©2007 Waters Corporation
8
Resolution (and Speed) Constant Column Length
Plates, Flow Rate and Particle Size
Rs N
12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000
Smaller Particle
Smaller Particle Size *Increased N, *Higher, optimal u *Increased pressure
Optimal
Larger Particle
1.0
2.0
3.0
Flow Rate {mL/min}
Optimal flow rate is inversely proportional to dp
1 Fopt ∝ dp Isocratic analysis time is inversely proportional to F
dp ↓ 3X, N ↑ 3X, Rs ↑ 1.7X, T ↓ 3X (e.g., 5 μm to 1.7 μm)
(Rs α √N) ©2007 Waters Corporation
9
Peak Width and Sensitivity Constant Column Length
Efficiency, N is inversely proportional to the square of Peak Width, W
1 N∝ 2 w Peak height is inversely proportional to Peak Width
1 Height ∝ w
dp ↓ 3X, N ↑ 3X, Rs ↑ 1.7X, T ↓ 3X (e.g., 5 μm to 1.7 μm)
(Rs α √N)
Sensitivity ↑ 1.7X ©2007 Waters Corporation
10
Backpressure
Constant Column Length Backpressure is proportional to Flow Rate, FR, and inversely proportional to Particle Size squared
1 ΔP ∝ FR × 2 dp Optimal Flow Rate is inversely proportional to Particle Size (further to the right on van Deemter curve)
1 FRopt ∝ dp
dp ↓ 3X, (e.g., 5 μm to 1.7 μm)
P ↑ 27X (~1/dp3)
©2007 Waters Corporation
11
Constant Column Length
Flow Rate Proportional to Particle Size
1.7 µm, 0.6 mL/min, 7656 psi
0.050 0.040
0.020 0.010 0.000 0.00
1.00
2.00
3.00
4.00
5.00
Minutes
0.050
6.00
4.8 µm, 0.2 mL/min, 354 psi
0.040 0.030 AU
AU
0.030
0.020 0.010 0.000 0.00
2.00
4.00
6.00
8.00
10.00
12.00
Minutes
2.1 x 50 mm columns
15.00
Reality 1.5X Resolution 2.6X Faster 1.4X Sensitivity 22X Pressure Theory 1.7X Resolution 3X Faster 1.7X Sensitivity 25X Pressure Too Much Backpressure! ©2007 Waters Corporation
12
Column Length to Particle Size Ratio Indicates Maximum Resolution Capability IS® Columns (20 mm Length) dp
L/dp
2.5 μm
8,000
3.0 μm
6,667
3.5 μm
5,714
5.0 μm
4,000
Length (L) L/dp 30 mm
17,650
50 mm
29,500
100 mm 58,820 150 mm 88,235
L/dp RATIO
Typical Run Times
300mm = 10μm 150mm = 5 μm 100mm = 3 μm
30,000
1970’s
30,000
1980’s
~ 15 min.
1990’s
~ 10 min.
50mm 1.7μm
=
29,410
100mm 1.7μm
=
58,820
2x Max. Resolution Capability
150mm = 1.7μm
88,230
3x Max. Resolution Capability
33,300
2004
~ 30 min.
~1 - 2 min.
The SAME L/dp for 2 columns will produce the SAME Resolution. The difference: shorter columns will produce the separations©2007 faster. Waters Corporation
13
Scaling HPLC to UPLC® Separations Constant L/dp = Equivalent Resolution
UPLC®
Separation
HPLC
Separations
5 µm – 150 mm Injection = 5.0 µL Flow rate = 0.2 mL/min Rs (2,3) = 2.28 3.5 µm – 100 mm Injection = 3.3 µL Flow rate = 0.3 mL/min Rs (2,3) = 2.32 2.5 µm – 75 mm Injection = 2.5 µL Flow rate = 0.5 mL/min Rs (2,3) = 2.34 1.7 µm – 50 mm Injection = 1.7 µL Flow rate = 0.6 mL/min Rs (2,3) = 2.29 ©2007 Waters Corporation
14
Speed Increases Constant L/dp
Efficiency, N, is directly proportional to column length, L, and inversely proportional to particle size, dp:
L N∝ dp For same N and, therefore, same Rs
dp ↓ 3X,
L ↓ 3X,
N = 1X, Rs = 1X,
(e.g., 5 μm to 1.7 μm) (e.g., 150 mm to 50 mm)
F ↑ 3X,
T ↓ 9X (i.e., F increases 3X, L decreases 3X)
Efficiency & Resolution Remain Unchanged ©2007 Waters Corporation
15
Sensitivity Increases
Constant L/dp
Assuming same efficiency, Peak Height is inversely proportional to column length, L:
1 Height = L For same efficiency, column length, L is decreased proportionally to particle size, dp (constant L/dp)
dp ↓ 3X,
L ↓ 3X,
N = 1X, Rs = 1X,
(e.g., 5 μm to 1.7 μm) (e.g., 150 mm to 50 mm)
T ↓ 9X, Sensitivity ↑ 3X (Increased optimal flow rate & shorter column)
(Peak height increases as peak width and column length decreases)
Efficiency & Resolution Remain Unchanged ©2007 Waters Corporation
16
Backpressure Increases Constant L/dp
Backpressure (P) is proportional to Column Length, L:
P ∝L For constant L/dp, Backpressure is inversely proportional to the square of Particle Size, dp:
1 P∝ 2 dp
dp ↓ 3X,
L ↓ 3X,
P ↑ 9X
(e.g., 5 μm to 1.7 μm) (e.g., 150 mm to 50 mm)
©2007 Waters Corporation
17
Length Proportional to Particle Size Similar L/dp 0.06
1.7 µm, 30 mm, 0.6 mL/min
AU
0.04
0.02
0.00 0.00 0.06
AU
0.04
1.00
2.00
3.00
4.00
Minutes
4.8 µm, 100 mm, 0.2 mL/min
0.02
Reality Same Resolution 8X Faster 2.5X Sensitivity 11X Back Pressure Theory Same Resolution 9X Faster 3X Sensitivity 9X Back Pressure
0.00 0.00
5.00
10.00
15.00
20.00
Minutes
2.1 mm ID columns
25.00
30.00
Manageable Backpressure Increase ©2007 Waters Corporation
18
UPLC® Technology & Gradient Peak Capacity (Resolution) Peak capacity is a measure of the separation power of a gradient on a particular column Pc = # Peaks separated per gradient duration time 0.035
tg Pc = 1 + w ↓, Pc ↑ w
0.030 0.025
Gradient Duration
0.020
tg
0.015 0.010 0.005 0.000 -0.005 0.00
w
w 0.10
0.20
0.30
0.40
0.50
0.60
0.70
w
w 0.80
w
Peak Width 0.90
1.00
1.10
1.20
1.30
1.40
1.50 ©2007 Waters Corporation
19
UPLC® Technology & Gradient Peak Capacity (Resolution)
Peak capacity is affected by: — Gradient duration (tg) — Flow rates (F) — Column length (L)
Influences peak width
— Particle size (dp)
Remember, the flow rate, column length and particle size all influence the plate count (N in isocratic separations) By optimizing these parameters, peak capacities can be maximized
©2007 Waters Corporation
20
Dependence of Peak Capacity on Operating Conditions Start with the simple equation for peak capacity
tg Pc = 1 + w
Make a few substitutions L dp2 L t0 ⋅ a ⋅ dp + b ⋅ DM ⋅ + c ⋅ L DM t0 P =1+ ⋅ 4
B ⋅ Δc t B ⋅ Δc ⋅ 0 + 1 tg
We can now generate a 3-dimensional plot to examine how the gradient time and flow rate effect the peak capacity of a separation. Neue, U. D., Mazzeo, J. R. J. Sep. Sci. 2001, 24, 921-929. Cheng, Y-F., Lu, Z., Neue, U. Rapid Commun. Mass Spectrom. 2001, 15, 141-151. ©2007 Waters Corporation
21
Effect of Particle Size on Peak Capacity for UPLC® Separations Pmax = ~11,000 psi
1.0 x 50 mm Columns 1.7 μm
3.5 μm
5 μm
250
250
100
200
Peak Capacity
200
150
150 100
108
150
100
100
50
8
Gradient Duration (min) 1 4 16 32
Flow Rate (mL/min) 0.249 0.249 0.124 0.088
4 1 0.249
0.176
2 0.088 0.124
0.003 0.004 0.005 0.008 0.011 0.016 0.022 0.031 0.044 0.062
0
0
Flow Ra te (mL/ min)
Pressure Max Peak (psi) Capacity 10852 10852 5426 3837
64 32 16 8
108 172 216 231
Gradient Duration (min) 1 4 16 32
Flow Rate (mL/min) 0.352 0.124 0.088 0.062
4
2
1
Pressure Max Peak (psi) Capacity 3621 1280 905 640
50
Gr ad ie nt (m Du in rat ) io n
64 32 16
0.003 0.004 0.005 0.008 0.011 0.016 0.022 0.031 0.044 0.062 0.088 0.124 0.176 0.249 0.352 0.498 0.704 0.995
50
107
200
63 100 136 151
0 0.003 0.004 0.005 0.008 0.011 0.016 0.022 0.031 0.044 0.062 0.088 0.124 0.176 0.249 0.352 0.498 0.704 0.995 1.407 1.990
250
Gradient Duration (min) 1 4 16 32
Flow Rate (mL/min) 0.35 0.124 0.062 0.044
1
2
64 32 16 8 4
Pressure Max Peak (psi) Capacity 1774 627 314 222
46 76 107 121
ACQUITY UPLC® Columns can provide better Peak Capacity in 1 minute, than a 5 μm column in 16 minutes!! ©2007 Waters Corporation
22
High Resolution Peptide Mapping: Influence of Particle Size 0.08
HPLC Gradient 5 µm Peaks = 70 Pc = 143
AU
0.06
0.04
0.02
0.00 0.08
UPLC® Gradient 1.7 µm Peaks = 168 Pc = 360
AU
0.06
0.04
0.02
2.5X Increase
0.00 0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
45.00
50.00
55.00
60.00
Minutes
More information in the same amount of time ©2007 Waters Corporation
23
Summary:
UPLC® Technology: What is it?
UPLC® Technology is based on chromatographic theory (not marketing) and utilizes: — Small, pressure-tolerant particles that are efficiently packed into short (fast) or long (high resolution) columns — An LC system that can operate at the optimal linear velocity (and resulting pressure) for these particles and possesses minimal system volume and fast, responsive detectors that do not negatively affect efficiency
UPLC® Technology provides information in less time and, hence, at a lower cost
©2007 Waters Corporation
24
Agenda
Introduction: What is UPLC® Technology? Migrating an HPLC Method to a UPLC® Method Efficient UPLC® Method Development and Validation Conclusion
©2007 Waters Corporation
25
Example: Migrating an HPLC Method to a UPLC® Method HPLC Method Pc = 94
0.40
AU
0.30
0.20
0.10
0.00 0.00
5.00
10.00
15.00 20.00 Minutes
0.40
25.00
30.00
35.00 UPLC® Method Pc = 85
AU
0.30
0.20
0.10
0.00 0.00
0.50
1.00
1.50
2.00 Minutes
2.50
3.00
3.50
4.00 ©2007 Waters Corporation
26
Method Migration/Conversion: HPLC to UPLC® Technology
Why Convert HPLC Methods to UPLC® Technology? — Acquire results in less time and/or with more resolution o More information - faster o More robust methods – greater confidence o Better situational response time (stat samples faster, research decisions with more information, process monitoring, product release) o More samples analyzed per system, per scientist
©2007 Waters Corporation
27
USP HPLC Separation of Simvastatin
0.20
0.18
0.16
9.281
0.22
USP Method Requirements k’ > 3.0 N > 4,500 Tf < 2.0
Plates = 12,112
HO
O O
O H3C
H3C
O CH3 H
CH3
0.14
AU
0.12
0.10
0.08
0.06
HPLC: RT = 9.28 min k’ = 4.1 N = 12,112 Tf = 1.1
H3C
0.04
0.02
0.00 2.00
4.00
6.00
8.00
10.00 Minutes
12.00
14.00
16.00
Channel: W2996 238.0nm-1.2; Processed Channel: W2996 PDA 238.0 nm at 1.2; Injection: 3; 10/5/2006 10:12:29 AM EDT; Result Id: 1318; Processing Method: Simvastatin BEH 4_6 x 250
18.00
20.00
Date Acquired:
©2007 Waters Corporation
28
UPLC® Method Conversion Choices
0.21
0.14
AU
AU
0.14
HPLC: RT = 9.28 min k’ = 4.1 N = 12,112 Tf = 1.1
0.00
0.00 0.00
0.50
1.00
1.50 Minutes
Efficiency = 17685
2.00
2.50
0.00
USP Req. k’ > 3.0 N > 4,500 Tf < 2.0 0.50
0.06
0.03
0.07 0.07
0.09
AU
S im v a s t a t in - 1 . 9 2 1
0.21
UPLC®: RT = 1.41 min k’ = 4.9 N = 12,874 Tf = 1.1
Fastest Analysis S im v a s t a t in - 0 . 2 3 4
Equal Efficiency S im v a s t a t in - 1 .4 1 2
Maximum Efficiency
0.00
1.00
1.50 Minutes
Efficiency = 12874
0.00
0.50
1.00
1.50 Minutes
Efficiency = 977 ©2007 Waters Corporation
29
Critical Caveat of Method Conversion The new method will be different from the original method — Operating conditions, e.g., flow rate — Run time — Appearance — Of course, the objective was an improved method
The new method must preserve critical parameters — Complete resolution of all relevant analytes — Peak homogeneity/purity — Certainty of peak identification — Quantitative accuracy and precision ©2007 Waters Corporation
30
Method Conversion Process Steps for Success
1. Gather information about existing method and results 2. Select new or target column — Chemistry — Dimensions
3. Compare instruments 4. Calculate method conversion conditions 5. Evaluate results of transfer 6. Optimize as required
©2007 Waters Corporation
31
1. Gather Required Information Original Method & Results Column
— Chemistry (ligand, brand, particle size) — Dimensions
Conditions
— Mobile phase — Flow rate — Gradient profile, including regeneration and reequilibration — Temperature
Sample
— Diluent — Concentration — Molecular weight(s) — Injection volume
Chromatogram — Number of peaks — Retention — Resolution (critical pairs)
Quantitation — Limit of detection — Limit of quantitation — Linear dynamic range — Accuracy — Precision
©2007 Waters Corporation
32
2. Select Target Column
ACQUITY UPLC® Column Chemistries UPLC® Column Chemistries — ACQUITY — ACQUITY — ACQUITY — ACQUITY — ACQUITY — ACQUITY — ACQUITY — ACQUITY
UPLC® UPLC® UPLC® UPLC® UPLC® UPLC® UPLC® UPLC®
BEH BEH BEH BEH BEH HSS HSS HSS
C18 Shield RP18 C8 Phenyl HILIC T3 C18 C18 SB
Check Column Selection Chart for closest match to original — Chromatographic test at pH 7 — Provides an assessment of a column’s hydrophobicity and base/neutral selectivity — Can be used to select “equivalent” columns for methods transfer
©2007 Waters Corporation
33
Waters Reversed-Phase Column Selectivity Chart 3.6
Waters Spherisorb® S5 P
(ln [α] amitriptyline/acenaphthene)
3.3 3 2.7
ACQUITY UPLC® BEH XBridge™ Phenyl
Waters Spherisorb® S5CN
ACQUITY UPLC® HSS C18 SB
Nova-Pak® CN HP
2.4
Inertsil® Ph-3 ACQUITY UPLC® BEH Hypersil® Phenyl
Waters Spherisorb® ODS1 Resolve® C18
XBridge™ C18
2.1
µBondapak™ C18
1.8 Hypersil® CPS Cyano
1.5
Inertsil® CN-3
YMC-Pack™ Phenyl
Waters Spherisorb® ODS2
ACQUITY UPLC® HSS T3
YMC J'sphere™ ODS–L80
Nucleosil® C18
Nova-Pak® Phenyl
YMC J'sphere™ ODS–M80 YMC J'sphere™ ODS–H80 Nova-Pak® XTerra ® YMC-Pack™ ODS–AQ™ C18 RP-18 Phenyl Nova-Pak® Luna ™ ® Atlantis dC18 YMC-Pack™ Pro C4™ YMC-Pack™ CN Phenyl Hexyl Zorbax® XDB C18 C8 Atlantis® T3 YMC-Pack™ Pro C8™ ACQUITY UPLC® BEH YMC-Pack™ ODS-A™ ACT Ace® C18 Symmetry® C8 XBridge™ Luna ® C18 (2) XTerra® MS C8 Luna® YMC-Pack™ C8 (2) C8 Inertsil® ODS-3 Pro C18™ SunFire ™ C18
1.2
Hypersil® BDS Phenyl
0.9 0.6 0.3
YMCbasic™
ChromolithTM
SunFire ™ C8 XTerra® MS C18
SymmetryShield™ RP8
0 ACQUITY UPLC® BEH
XBridge™
-0.3
Shield RP18
Zorbax® SB C18 SymmetryShield™ RP18
XTerra® RP18 XTerra® RP8
-0.6
Symmetry® C18
ACQUITY UPLC® HSS C18 YMC-Pack™ PolymerC18™
-1.5
-0.5 102007
0.5
1.5
(ln [k] acenaphthene)
2.5
3.5 ©2007 Waters Corporation
34
3. Required Information Original Instrument
Mode of gradient generation — Single pump with gradient proportioning valve (GPV) — Dual pump — Brand and model number
System volume (dwell volume or delay volume) — Value and method used to measure
Injection mechanism Mode of detection ©2007 Waters Corporation
35
3. System Volumes of Pumping Systems Multi-Pump (High Pressure) Smaller System Volume = Smaller Dwell volume
Pump 1
Volume: From where mobile phase is mixed, to where it enters the column.
Mixer
Pump 2
Injector
Column
Detector
Single Pump (Low Pressure) Larger System Volume = Larger Dwell volume
A
B D
C
Gradient Proportioning Valve
Solvent delivery
Injector
Column
Detector
©2007 Waters Corporation
36
Compensating for System Volume Differences
Compare system volumes — This volume should be converted to column volumes for the best comparison
If target system gives smaller isocratic segment — ADD an initial hold to the gradient table to give the identical hold
If target system gives larger isocratic segment — No exact compensation is possible — Chromatographic effect of extra isocratic hold usually small
ACQUITY UPLC® Columns Calculator handles this compensation ©2007 Waters Corporation
37
3. Instrument Comparison Injection
We assume — The specified volume of sample is delivered to the column — The sample composition is not altered — There is no carryover
System differences affect — Volume of sample required — Absolute amount injected — Sample carryover
©2007 Waters Corporation
38
ACQUITY UPLC® System Sample Manager
Minimize sample dispersion during injection Reduce cycle time Preserve — Accuracy — Precision — Low carryover — Sample format flexibility
Dual wash system with a strong wash followed by a weak wash
©2007 Waters Corporation
39
3. Instrument Comparison Detection
We assume — Response is specific — Response is linear with concentration — Detector does not alter peak shape
System differences affect — Specificity — Limit of detection (LOD) — Limit of quantitation (LOQ) — Linear dynamic range — Band-broadening ©2007 Waters Corporation
40
Detection with UPLC® Separations UV, ELSD & FLR
Only Waters ACQUITY UPLC® TUV or PDA — Minimize band-broadening — Provide adequate sampling rate (S/N) — Accurate wavelength
UPLC® ELSD and FLR detectors also available Sensitivity and dynamic range are affected by reduced peak volume and by improved resolution
©2007 Waters Corporation
41
Data Acquisition Rates Impact on UV Data
Peaks are ~ 1-2 seconds wide 1 pt/s 2 pt/s 5 pt/s 10 pt/s
20 pt/s 40 pt/s
How many Data Points is enough? Simple Rule: 15-20 Points per Peak The RATE the points are collected is determined by how wide the peak is in TIME at the baseline. If a peak is only 1 second wide, then you need to collect 20 points in 1 second (20 Hz)
0.50 0.52 0.54 0.56 0.58 0.60 0.62 0.64 0.66 0.68 0.70 0.72 0.74
Minutes
©2007 Waters Corporation
42
Detection with UPLC® Separations MS and MS/MS
Waters MS systems designed for UPLC® separations — Z-spray source minimizes band-broadening — Quattro Premier XE, LCT Premier XE and Q-Tof Premier — SQD & TQD designed specifically for ACQUITY UPLC® system and separations — ZQ single quadrupole data acquisition is compatible with UPLC® separations
Other Waters MS detectors can be operated with compatible sampling rates ©2007 Waters Corporation
43
Ready to Migrate: Target Conditions Mobile Phase and Injection Parameters
Use exactly the same mobile phase — Alter only after evaluating transfer if optimization is required
Use exactly the same sample — Same concentration — Same diluents
ACQUITY UPLC® System needle wash — Use final gradient conditions as strong needle wash (200 µL) — Use initial gradient conditions as weak needle wash (600 µL)
©2007 Waters Corporation
44
4. Example: Original HPLC Method: Caffeic Acid Derivatives of Echinacea Purpurea
5
0.40
1 2
3
AU
0.30
Pc = 94
4
0.20
0.10
0.00 0.00
5.00
10.00
Name 1. Caftaric acid 2. Chlorogenic acid 3. Cynarin 4. Echinacoside 5. Cichoic acid
15.00 20.00 Minutes
Retention Time 5.71 7.07 13.96 16.54 20.32
25.00
30.00
35.00
USP Resolution 4.20 21.19 10.16 17.14 ©2007 Waters Corporation
45
Enter Existing HPLC Gradient Conditions
1. Enter column, system & analyte information
HPLC results given here
2. Enter HPLC gradient Time & %B 3. Select Pmax for UPLC® separation
Calculated for you
4. Press Calculate ©2007 Waters Corporation
46
Five UPLC® Gradient Choices Given Five Gradient Choices: Geometrically Scaled
1. HPLC Linear Velocity 2. UPLC® Linear Velocity 3. User Defined
or Optimally Scaled 4. Maximum Peak Capacity 5. Shortest Analysis Time
Select View for Detailed Gradient Profiles ©2007 Waters Corporation
47
UPLC® Method Gradient Profiles Choices Original HPLC Gradient Method
Maximum Peak Capacity Equivalent Analysis Time
Geometrically Scaled HPLC Linear Velocity
Equivalent Peak Capacity Shortest Analysis Time
Geometrically Scaled UPLC® Linear Velocity
Geometrically Scaled User Defined Flow Rate
©2007 Waters Corporation
48
Calculator Choice 1 (default):
Geometrically Scaled Gradient at HPLC Linear Velocity
Original HPLC Gradient
UPLC® Gradient: HPLC Linear Velocity
Geometrically Scaled UPLC® Gradient at HPLC Linear Velocity: Identical Column Volumes per Time Segment Flow rate scaled for column ONLY
©2007 Waters Corporation
49
Calculator Choice 1 (default):
Geometrically Scaled Gradient at HPLC Linear Velocity HPLC Method Name Pc = 94
0.40
caftaric acid chlorogenic acid cynarin echinacoside cichoic acid
AU
0.30
0.20
0.10
0.00 0.00
5.00
10.00
15.00 20.00 Minutes
0.40
0.30
Retention Time
5.71 7.07 13.96 16.54 20.32
Similar 35.00 Resolution UPLC® Method Retention Choice 1 Name Time Pc = 99 caftaric acid 1.99 25.00
USP Resolution
4.20 21.19 10.16 17.14
30.00
AU
chlorogenic acid cynarin echinacoside cichoric acid
0.20
0.10
2.38 4.85 5.93 7.11
USP Resolution
3.97 25.00 15.09 19.62
0.00 0.00
2.00
4.00
6.00 Minutes
8.00
10.00
12.00 ©2007 Waters Corporation
50
Calculator Choice 2:
Geometrically Scaled Gradient at UPLC® Linear Velocity
Original HPLC Gradient
UPLC® Gradient: UPLC® Linear Velocity
Geometrically Scaled UPLC® Gradient at UPLC® Linear Velocity:
Identical column volumes per Time Segment Flow rate scaled for column AND particle size ©2007 Waters Corporation
51
Calculator Choice 2:
Geometrically Scaled Gradient at UPLC® Linear Velocity HPLC Method Pc = 94
0.40
AU
0.30
0.20
0.10
0.00 0.00
5.00
10.00
15.00
20.00 Minutes
0.40
0.30
Retention USP Time Resolution
Name caftaric acid 5.71 chlorogenic acid 7.07 cynarin 13.96 echinacoside 16.54 cichoic acid 20.32
Similar 35.00 Resolution UPLC® Method Retention Choice 2 Name Time Pc = 85 25.00
4.20 21.19 10.16 17.14
30.00
AU
caftaric acid chlorogenic acid cynarin echinacoside cichoric acid
0.20
0.10
0.71 0.87 1.72 2.06 2.44
USP Resolution
3.96 22.12 11.40 14.28
0.00 0.00
0.50
1.00
1.50
2.00 Minutes
2.50
3.00
3.50
4.00 ©2007 Waters Corporation
52
UPLC® Method Gradient Profiles Choices 0.40
AU
0.30
0.20
Original HPLC Gradient Method
0.10
0.00 0.00
2.00
4.00
6.00 Minutes
8.00
10.00
Geometrically Scaled HPLC Linear Velocity
12.00
0.00
5.00
15.00
20.00
25.00
30.00
35.00
Minutes
Maximum Peak Capacity Equivalent Analysis Time
0.40
0.30
Different Selectivities
AU
Similar Selectivities
10.00
0.20
0.10
0.00
Geometrically Scaled UPLC® Linear Velocity
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
Minutes
Equivalent Peak Capacity Shortest Analysis Time
0.40
AU
0.30
0.20
0.10
0.00 0.00
0.50
1.00
1.50
2.00 Minutes
2.50
3.00
3.50
4.00
0.00
0.50
1.00
1.50
2.00 Minutes
2.50
3.00
3.50
©2007 Waters Corporation
4.00
53
Summary
HPLC to UPLC® Method Conversion
Methods can be moved directly from HPLC to ACQUITY UPLC® technology for — Improved resolution — Improved speed — Improved detectability
Attention to detail leads to success ACQUITY UPLC® Columns Calculator facilitates process Converting to UPLC® technology/methodology increases profitability by lowering cost of sample analysis and various connected operating costs
©2007 Waters Corporation
54
Agenda
Introduction: What is UPLC® Technology? Migrating an HPLC Method to a UPLC® Method Efficient UPLC® Method Development and Validation Summary
©2007 Waters Corporation
55
Pharmaceutical Application Areas
Forced Degradation Studies Stability Indicating Studies Impurity Profiling and Identification Quantitative Bioanalysis Batch Scale Up Studies Batch Comparison Raw Material Acceptance Finished Product Release Testing Cleaning Validation Patent Protection ©2007 Waters Corporation
56
Why Develop Methods with UPLC® Technology? Time Savings Versus 5 µm HPLC Column UPLC Methods Development Protocol 2.1 x 50 mm, 1.7 µm, 0.5 mL/min pH 3/ acetonitrile Flow ramp Column conditioning (2 blank gradients) Sample injection (2 replicates) pH 3/ methanol Flow ramp Column conditioning (2 blank gradients) Sample injection (2 replicates) Column purge pH 10/ acetonitrile Flow ramp Column conditioning (2 blank gradients) Sample injection (2 replicates) pH 10/ methanol Flow ramp Column conditioning (2 blank gradients) Sample injection (2 replicates) Column purge SCREENING TIME
Time 5 min 11 min 11 min 5 min 11 min 11 min 6 min 5 min 11 min 12 min
5 min 11 min 11 min 6 min 120 min 2 Hours/ Hybrid column x 3 columns 1 Hour/ Silica column x 1 column
TOTAL SCREENING TIME
7 HOURS
EQUIVALENT HPLC Methods Development Protocol, 5 µm 4.6 x 150 mm, 5 µm, 1.0 mL/min pH 3/ acetonitrile Time Flow ramp 5 min Column conditioning (2 blank gradients) 79.2 min Sample injection (2 replicates) 79.2 min pH 3/ methanol Flow ramp 5 min Column conditioning (2 blank gradients) 79.2 min Sample injection (2 replicates) 79.2 min Column purge 43.2 min pH 10/ acetonitrile Flow ramp 5 min Column conditioning (2 blank gradients) 79.2 min Sample injection (2 replicates) 79.2 min pH 10/ methanol Flow ramp 5 min Column conditioning (2 blank gradients) 79.2 min Sample injection (2 replicates) 79.2 min Column purge 43.2 min 740 min SCREENING TIME 12.3 Hours/ Hybrid column x 3 columns 6.15 Hours/ Silica column x 1 column
TOTAL SCREENING TIME
43 HOURS
UPLC® Screening: 6.1X faster than 5.0 µm HPLC Column ©2007 Waters Corporation
57
Automated Method Development and Validation
Automated Method Development — ACQUITY UPLC® Column Manager, 4 column selection device — ACQUITY UPLC® Binary Solvent Manager, solvent select valves
Automated Method Validation — Empower® 2 Method Validation Manager (MVM) streamlines method validation process ©2007 Waters Corporation
58
Selectivity Tools
Solvent
pH
α Selectivity
Column Chemistry ©2007 Waters Corporation
59
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
©2007 Waters Corporation
60
Reversed-Phase Retention Map: The Importance of Mobile Phase pH 40
Note: Retention of neutral analytes not affected by pH 35
Retention Factor (k)
Neutral
Acid
30 25
Increased acid retention
20
Increased base retention
15 10 5
Base
0 0
HSS
2
4
6 pH
8
10
12
Silica pH Range
BEH
Hybrid Particle pH Range
Neue et. al. American Laboratory 1999 (22) 36-39. ©2007 Waters Corporation
61
Selectivity Tools
Solvent
pH
α Selectivity
Column Chemistry Ligand & Base particle ©2007 Waters Corporation
62
Waters UPLC® Particles Overview
Ethylene Bridged Hybrid (BEH) Particles — Wide pH range (1-12) — Five chemistries — Seamless HPLC → UPLC® method migration – with same selectivity as XBridgeTM HPLC columns — 130Å and 300Å pore diameters
High Strength Silica (HSS) Particles — ONLY UPLC®-certified 100% silica particle — Three C18 chemistries — Developed specifically for UPLC® applications — Packed, tested and guaranteed compatibility with at pressures up to 15,000 psi (1000 bar)
©2007 Waters Corporation
63
The Chemistries of UPLC® Technology
Launch Date
Mar 2004
Mar 2005
Mar 2005
Mar Dec 2005 2005
Sep 2006
Jun 2007
Dec 2007
©2007 Waters Corporation
64
Selectivity Choices
Separations of Nitroaromatics 3
1 Conditions : Columns: ACQUITY UPLC® BEH 2.1 x 100 mm, 1.7 µm ACQUITY UPLC® HSS 2.1 x 100 mm, 1.8 µm Mobile Phase A: H2O Mobile Phase B: MeOH Flow Rate: 0.5 mL/min Isocratic: 28% MeOH Injection Volume: 5.0 µL Sample Concentration: 10 µg/mL Temperature: 50 oC Detection: UV @ 254 nm Sampling rate: 20 pts/sec Time Constant: 0.1 Instrument: ACQUITY UPLC®with ACQUITY UPLC® PDA
4
2
10 6 789 11
5
3
1
4 5
2
7 10 68 911 1213 14
BEH C8
4
3 2
1
BEH C18 12 13 14
5
7
6
8,9,13
10
11 12
BEH Shield RP18
14
4 1
Compounds 1. HMX 2. RDX 3. 1,3,5-TNB 4. 1,3-DNB 5. NB 6. Tetryl 7. TNT 8. 2-Am-4,6-DNT 9. 4-Am-2,6 DNT 10. 2,4-DNT 11. 2,6-DNT 12. 2-NT 13. 4-NT 14. 3-NT
1
12 1314 4
3 2
1
1
0.00
2.00
6
BEH Phenyl
7
6
HSS T3 10 9
11
12
13
14
4
3
3
9
8
7,8 5
2
2
10,11
3
5
2
5
4
5,6 7
4.00
6 7
10 8 9
8 9 10
11
HSS C18 12
13
14
11 12
6.00
13 14
8.00 Minutes
HSS C18 SB 10.00
12.00
14.00
©2007 Waters Corporation
65
Different Ligands: Different Selectivities
Changes in hydrophobicity — Longer alkyl chain will provide greater retention
Changes in silanol activity — Affect peak asymmetry and influences secondary interactions
Changes in hydrolytic stability — Longer column lifetimes with greater number of ligand attachment points to the particle surface
Changes in ligand density — Influences sample loadability
©2007 Waters Corporation
66
Low pH Selectivity Differences
35%ACN/65% 15.4 mM HCOONH4, pH 3.0 Material
k (Tol)
BEH C18
13.01
0.365
BEH C8
8.27
0.469
2.11
-0.76
BEH Shield RP18
11.19
0.256
2.42
-1.36
BEH Phenyl
6.60
0.613
1.89
-0.49
BEH 300 C18
5.97
0.409
1.79
-0.89
HSS C18
20.45
0.299
3.02
-1.21
HSS T3
17.91
0.393
2.89
-0.93
HSS C18 SB
9.23
0.842
2.22
-0.17
ln K (toluene)
3.5
HSS C18
3.0
HSS T3
BEH C18
2.5 2.0
α (Ami/Tol)
Y-AXIS X-AXIS ln k (Tol) ln α (Ami/Tol) 2.57 -1.01
Shield RP18
HSS C18 SB
BEH C8
1.5
BEH 300 C18
1.0 -1.75
-1.25
BEH Phenyl
-0.75
-0.25
0.25
ln α (ami/tol) pH 3.0 ©2007 Waters Corporation
67
ACQUITY UPLC® USP “L” Designation – 2007/2008 August 2007: ACQUITY UPLC® Columns meet USP requirements are now officially considered “L” columns
©2007 Waters Corporation
68
Selectivity Tools
pH
Solvent
α Selectivity
Column Chemistry ©2007 Waters Corporation
69
Solvent Properties
Methanol — Weaker eluent — H-bonding solvent
Acetonitrile — Aprotic solvent — Stronger eluent — Lower viscosity
©2007 Waters Corporation
70
Automated Method Development and Validation Example: Paroxetine and Related Compounds
Method Development — Use systematic screening protocol — Paroxetine (API) concentration: 0.2 mg/mL in 50:50 MeOH:H2O — Related compounds at 10% concentration of API for easy identification during scouting
Method Optimization — Related compounds at 0.1% concentration of API
Paroxetine m.w. 374.8
©2007 Waters Corporation
71
Paroxetine Related Compounds
Paroxetine HCl — (-)-trans-4R-(4'-fluorophenyl)-3S-((3',4‘ methylenedioxyphenoxy)methyl)piperidine
Paroxetine related compound B — (trans-4-phenyl-3-[(3,4-methylenedioxy)phenoxymethyl]-piperidine HCl
Paroxetine related compound D — (cis)-paroxetine HCl
Paroxetine related compound F — Trans (-)-1-methyl-3-[(1,3-benzodioxol-5-yloxy)methyl]-4-(4fluorophenyl)piperidine
Paroxetine related compound G — [(+/-)Trans-3-[(1,3-benzodioxol-5-yloxyl)methyl]-4-(4”-fluorophenyl4’phenyl)piperidine HCl
©2007 Waters Corporation
72
Systematic Screening:
Combining Chemical Factors
3. Solvent
1. pH
α Selectivity
2. Column Chemistry ©2007 Waters Corporation
73
Stationary Phase Selectivity:
0.40
Paroxetine
Paroxetine
ACQUITY UPLC® BEH C18
AU
0.30
B D
0.20 0.10
Methanol pH 3.0 F
G
0.00
0.40
AU
0.30
0.50
1.00
1.50
2.00
2.50
3.00
3.50
ACQUITY UPLC® BEH Shield RP18
0.20
D
0.10
B
4.00
Paroxetine
0.00
4.50
5.00
5.50
6.00
Observation:
Poor resolution of paroxetine and Related Compounds (RC)
F
G
0.00
AU
0.30
1.00
1.50
2.00
2.50
3.00
3.50
ACQUITY UPLC® BEH Phenyl
0.20
B
0.10 0.00 0.00 0.40
0.50
1.00
1.50
2.00
2.50
3.00
AU
0.30
B D
0.10
4.50
5.00
5.50
6.00
5.00
5.50
6.00
F
G D
3.50
ACQUITY UPLC® HSS T3
0.20
4.00
Paroxetine
0.40
0.50
Action: 4.00
4.50
Investigate high pH
Paroxetine
0.00
F
G
0.00 0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
CM, ESG
©2007 Waters Corporation
74
pH Selectivity: Paroxetine 0.40
Methanol
Paroxetine
0.30 0.25 AU
Observation:
pH 3.0
0.35
0.20
Better retention and resolution of API from RC due to neutral charge state of analytes at alkaline pH
0.15
B D G
0.10
F
0.05 0.00 0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
0.40
0.30
AU
0.25
5.50
6.00
Actions:
pH 10
Paroxetine
0.35
5.00
Select pH 10 due to better separation
Methanol
0.20
Compare stationary phase selectivity with pH 10 buffer
0.15 0.10
B
D G
0.05
F
0.00 0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
CM, ESG
©2007 Waters Corporation
75
Stationary Phase Selectivity:
0.40
Paroxetine
Paroxetine
ACQUITY UPLC® BEH C18
AU
0.30
0.20
Methanol pH 10.0 Observation:
0.10
B D G
F
0.00
0.40
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
ACQUITY UPLC® BEH Shield RP18
AU
0.30
0.20
B
0.10
4.50
5.00
5.50
6.00
Paroxetine
0.00
D G
Any column may provide successful separation
Actions:
F
0.00
0.40
0.50
1.00
ACQUITY
1.50
UPLC®
2.00
2.50
3.00
3.50
4.00
BEH Phenyl
0.30 AU
4.50
5.00
5.50
6.00
Paroxetine
0.00
0.20
B D G
0.10
Compare selectivity between organic modifiers
F
0.00 0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
Select ACQUITY UPLC® BEH C18
5.50
6.00
CM, ESG
©2007 Waters Corporation
76
Solvent Selectivity: Paroxetine
ACQUITY UPLC® BEH C18
0.35 0.30
Observations:
Acetonitrile pH 10.0
0.25 AU
Methanol is weaker elution solvent resulting in greater retention
Paroxetine
0.40
0.20 0.15
G
0.10
B
0.05
F
D
0.00
0.40
0.50
1.00
1.50
0.35
Methanol
0.30
pH 10.0
2.00
2.50
3.00
3.50
4.00
0.25 AU
4.50
5.00
5.50
6.00
Paroxetine
0.00
Better resolution exhibited with acetonitrile as organic modifier Actions:
Select acetonitrile as organic modifier
0.20 0.15 0.10
B D
G
F
0.05 0.00 0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
Optimize separation using appropriate concentration of RC CM, ESG ©2007 Waters Corporation
77
Related Compounds at 0.1% Concentration of Paroxetine Paroxetine
0.10
0.08
B
Observation:
Related Compounds at 10%
Inadequate resolution among paroxetine and related compounds B and D due to disparate levels of concentration
G
AU
0.06
F D
0.04
0.02
0.00 2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40 3.60 Minutes
3.80
4.00
4.20
4.40
4.60
4.80
5.00
0.005
Paroxetine
0.004 0.003 0.002 AU
Related Compounds at 0.1%
Action:
Change gradient slope
B
0.001 0.000
G D
-0.001
F
-0.002 -0.003 2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40 3.60 Minutes
3.80
4.00
4.20
4.40
4.60
4.80
5.00
©2007 Waters Corporation
78
Method Optimization: Gradient Slope
ACQUITY UPLC® BEH C18 0.005
0.003 0.002 AU
5 Minute Gradient
Paroxetine
0.004
Acetonitrile pH 10.0 30oC
5%-90%
Observations:
0.001
G
0.000
B
D F
-0.001 -0.002 -0.003 2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40 3.60 Minutes
3.80
4.00
4.20
4.40
4.60
4.80
5.00
Paroxetine
0.008
0.007
0.006
5 Minute Gradient
20%-90% Action:
AU
G 0.005
B
D
F
Alter gradient endpoint to produce shallower slope
0.004
0.003
0.002 2.00
2.20
2.40
2.60
Marginal improvement in separation of impurities from parent compound with shallow gradient slope
2.80
3.00
3.20
3.40 3.60 Minutes
3.80
4.00
4.20
4.40
4.60
4.80
5.00
CM, ESG
©2007 Waters Corporation
79
Method Optimization: Gradient Slope Paroxetine
0.008 0.007 0.006 0.005 AU
B
5 Minute Gradient
Acetonitrile pH 10.0 30oC
20%-90% G F
D
Observations:
0.004
Resolution remains inadequate with shallow gradient slope
0.003 0.002 0.001 2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40 3.60 Minutes
3.80
0.008
0.006
4.20
4.40
4.60
4.80
5.00
5 Minute Gradient
Paroxetine
0.007
4.00
20%-65%
B
AU
0.005
G
D
0.004
Action:
F
Investigate column temperature
0.003 0.002 0.001 2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40 3.60 Minutes
3.80
4.00
4.20
4.40
4.60
4.80
5.00
CM, ESG
©2007 Waters Corporation
80
Method Optimization: Column Temperature
Paroxetine
0.008
AU
0.006
B
0.004
30 oC G
D
F
Observations:
0.002 2.00
2.20
2.40
2.60
2.80
3.00
3.20
Paroxetine
0.008
0.006 AU
B
3.40 3.60 Minutes
3.80
4.00
4.20
4.40
4.60
4.80
5.00
45 oC D
G
F
0.002 2.20
2.40
2.60
2.80
3.00
0.006 AU
B
3.20
Paroxetine
2.00
3.40 3.60 Minutes
3.80
4.00
4.20
4.40
4.60
4.80
5.00
60 oC G
D
F
0.002 2.20
2.40
2.60
2.80
3.00
3.20
3.40 3.60 Minutes
3.80
4.00
4.20
4.40
Action:
Select 60 oC for best resolution and peak shape
0.004
2.00
Higher temperature improves separation of RC from paroxetine Peak shape improves as temperature increases
0.004
0.008
Acetonitrile pH 10.0
4.60
4.80
5.00
CM, ESG
©2007 Waters Corporation
81
Final Method
Paroxetine and Related Compounds at 0.1% 0.010
Paroxetine
0.50
0.008
0.40 AU
0.009
0.30 0.20 0.10
0.007
0.00 0.00
1.00
2.00
3.00 Minutes
4.00
5.00
6.00
AU
0.006
0.005
G
D
B
F
0.004
0.003
0.002
0.001 2.00
2.20
2.40
2.60
2.80
Compound Related compound B Paroxetine Related compound D Related compound G Related compound F
3.00
3.20
3.40 3.60 Minutes
USP Rs 1.95 3.07 13.00 6.74
3.80
4.00
4.20
4.40
4.60
4.80
5.00
ACQUITY UPLC® BEH C18,, 2.1 x 50 mm, 1.7 µm Mobile phase A: 20 mM NH4HCOO3, pH 10 Temperature: 60 oC 5 Min Gradient: 20%-65% ACN Flow rate: 0.5 mL/min Injection Volume: 4 µL Detection: UV @ 295 nm ©2007 Waters Corporation
82
Why Validate?
Ensures that analytical methodology is accurate, reproducible and robust over the specific range that an analyte will be analyzed Provides assurance of reliability FDA Compliance Good Science!!
"The process of providing documented evidence demonstrating that something (the method or procedure) does what it is intended to do; is suitable for its intended purpose." ©2007 Waters Corporation
83
Method Validation
Which Validation Steps need to be performed after a method has been converted to UPLC® technology? Waters cannot define that for you You must define or follow your own corporate policies However, there are some strategies colleagues are using today — Full Blown Validations (conservative approach) — Cross validations o SLAP technique– perform selectivity, linearity, area, and precision tests
Relate specifications to critical quality attributes — Propose acceptance criteria based on scientific rationale — Relationships established from DOE and prior knowledge Automation tools can be used to facilitate the process ©2007 Waters Corporation
84
New Method Validation Manager Option For Empower 2
"The industry is very much in need of a workflow-based, configurable system that allows users to implement an organization’s method validation practices. An approach that seamlessly implements method validation requirements but is inherently flexible and manages the data, effectively standardizes an often tedious and time-consuming process“ — James Morgado, Pfizer Global R&D
Method Validation Manager option automates the laborious method validation process within Empower 2 Software Realize time savings of 50% – 80% in this formerly manual and error-prone iterative process
©2007 Waters Corporation
85
Analytical Method Validation Process with Method Validation Manager
Calculation Statistical Results Create Sample Sequence
Data Acquisition & Processing
Reports Compiled
Prepare Standards & Samples
Corporate Method Method Validation Validation SOP
Manager
Time consuming, repetitive tasks consisting of Faster Easiersteps Method Validation severaland sequential
©2007 Waters Corporation Data Management
86
Automated Method Validation Manager: Paroxetine Validation Results Parameter
Acceptance Criteria
Reported Value
Pass/Fail
Linearity
R2 > 0.995
0.9999
pass
Residuals < 2.0% RSD
1.74% RSD
pass
Accuracy
80 – 120 %
97 – 102 %
pass
Intermediate Precision
Variance Component < 10 %RSD Peak Area
Analyst 1.33% RSD
pass
(Analyst, Instrument, Column)
Instrument 7.72% RSD
pass
Column 0.00% RSD
pass
LOD of impurities
Impurities 0.1% of active at 0.2 mg/mL
0.05% of active at 0.2 mg/mL (s/n 2.2 – 6.23)
pass
Method Robustness
Variance Component < 2 %RSD Peak Area
0.06 – 1.64% RSD
pass
Peak Area
(Buffer strength, Additive Conc., Column Temperature)
Method Robustness
Variance Component < 5 %RSD Retention Time
0.00 – 2.57% RSD
pass
Retention Time
(Buffer strength, Additive Conc., Column Temperature, Flow Rate, Injection Volume)
©2007 Waters Corporation
87
Methods Development and Validation Timeline: Instrument Time
UPLC® Methods Development and Validation Timeline 2.1 x 50 mm, 1.7 µm, 0.5 mL/min Screening 4 Columns, 2 Organics, 2 pH’s
Time 7.0 hours
Optimization Gradient Slope and Temperature
1.7 hours
Validation Accuracy, linearity, repeatability, Reproducibility, LOD/LOQ, Intermediate precision, robustness
21.1 hours
TOTAL TIME
29.8 HOURS ©2007 Waters Corporation
88
Method Validation Manager Benefits
Save time over entire process and less error prone — Data management is handled by Empower 2, not by user — Automatic data checks performed at each step of the workflow — Data approvals can be configured at each step of the workflow — Calculations done in Empower 2 o No transfer to spreadsheets or other software o No transcription error / No need to check data transfer o No need to validate spreadsheet functions o Multi-component analysis and batch processing of validation results — Report templates can be used to standardize the report format o Automatic report generation o Ease of Review ©2007 Waters Corporation
89
Summary:
Efficient UPLC® Method Development and Validation
Achieve more resolution, faster by utilizing sub-2 µm UPLC® columns at optimal linear velocities with full pressure capabilities up to 15,000 PSI Principles of methods development remain the same UPLC® column chemistries provide a broad range of selectivity to successfully develop methods efficiently UPLC® Technology allows for faster methods development and validation UPLC® Technology, Empower® 2 and Method Validation Manager software can significantly improve laboratory productivity and compliance
©2007 Waters Corporation
90
Agenda
Introduction: What is UPLC® Technology? Migrating an HPLC Method to a UPLC® Method Efficient UPLC® Method Development and Validation Conclusion
©2007 Waters Corporation
91
Conclusion
UPLC® Technology provides more reliable information FASTER and at a LOWER cost per analysis UPLC® Technology is NOT: — Just fast LC (speed WITHOUT resolution) — 2.X µm HPLC particles packed into short (≤100 mm) HPLC column hardware that are run on an HPLC system under HPLC operating pressures (beware of these claims)
UPLC® Technology and Empower 2 software can improve productivity and compliance by streamlining method development and validation protocols
©2007 Waters Corporation
92
THANK YOU
©2007 Waters Corporation
Presentation pdf files available on www.waters.com/slides
©2007 Waters Corporation
94
Please complete your seminar evaluation form to receive a free copy of the application book of your choice
©2007 Waters Corporation
95
