New Advances in UHPLC-Resolution, Speed & Sensitivity
Enhancing Productivity with Agilent’s Newest Instrumentation
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Patrick CronanLC Applications Scientist
Boston, MA
September 14, 2012
The 1290 Infinity Quaternary LC SystemHow binary pump-like performance can enhance your lab efficiency
1290 Infinity DAD with 60mm cell (& HDR optional)
1290 Infinity TCC with broadest
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September 24, 2012Page 2
1290 Infinity TCC with broadest valve selectivity
1290 Infinity Autosampler (with Flexible Cube for lowest carry-over)
1290 Infinity Quaternary Pump
Innovation: 1290 Infinity Quaternary PumpQuaternary Pump with Binary Pump-like Performance
1290 Infinity Quaternary Pump
• Boost PerformanceHighest accuracy and precision for composition and flow, with exceptionally low delay volume
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• Save TimeAccelerates transfer of existing HPLC methods to UHPLC
• Reduce CostsOutstanding UHPLC performance at HPLC-like costs of ownership
New Inlet-Mixer - based on JetWeaver technology
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Oct. 2011
4
1290 Infinity Quaternary PumpVarious tools enabling the high performance
Multi-Purpose-
Switchable Jet Weaver Mixer
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5
4-channel Degasser
MCGV (Multi-Channel-Gradient-Valve) Inlet-Weaver Pump Head
Multi-Purpose-Valve
In-line Filter
Multipurpose valve functions for highest comfortStandard Application for standard Delay Volume
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Multipurpose valve functions for highest comfortExtra Mixing Volume for lowest baseline ripple (TFA applications)
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Multipurpose valve functions for highest comfortAutomatic Purging
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Multipurpose valve functions for highest comfortAutomatic filter backflush
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Power Range� For any kind of analysis
Composition Accuracy and Precision< 0.15 % RSD or 0.02 min SD
� 0.4 % (1-99 % Composition B)
� High RT precision in gradient runs
Agilent 1290 Infinity Quaternary PumpSpecifications & Benefits
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gradient runsFlow Accuracy and Precision
< 0.07 % RSD or 0.01 min SD
� 1.0 % or 10 µL
� High RT precision in isocratic runs
Composition Range1-99 %
� Wide analytical range
Delay Volume< 350 µL
� For fast quaternary gradients
� Different mixtures (H2O, MeOH, ACN) at � different pressures and � different flow rates
Full application range!
Composition Accuracy and PrecisionStep Gradient Analysis with Tracer
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11 January, 26, 2012
Flow and Composition Accuracy and PrecisionIsocratic analysis of Glucocorticoids
3: Hydrocortisoneacetate
1: Hydrocortisone 2: Beclomethasone-di-proprionate
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Column: Agilent ZORBAX Eclipse Plus C18, 4.6 x 100 mm, 5 µm (p/n 959996-902)
Mobile phase: Water/ Acetonitrile = 30/70Flow rate: 1 mL/min
Stop time: 5 min
Injection vol.: 3 µL (needle wash for 3 sec)
Column Temp.: 30 °C
DAD: 254/10 nm, (ref 400/ 80 nm), 20 Hz, slit 4 nm
Flow and Composition Accuracy and PrecisionIsocratic analysis of Glucocorticoids
mAU
25
30
1
2
RT of compounds very sensitive regarding changes in
mobile phase composition!
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min1 1.5 2 2.5 3 3.5
0
5
10
15
20
Water/ACN 29:71
3
Water/ACN 31:69
Water/ACN 30:70
Accuracy and PrecisionIsocratic analysis of Glucocorticoids
mAU
35
1: Hydrocortisone
Specification (< 0.07 %)
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min0 1 2 3 4
0
5
10
15
20
25
30
35
3: Hydrocortisoneacetate
2: Beclomethasone-di-proprionate
Power Range� For any kind of analysis
Composition Accuracy and Precision< 0.15 % RSD or 0.02 min SD
� 0.4 % (1-99 % Composition B)
� High RT precision in gradient runs
Agilent 1290 Infinity Quaternary PumpSpecifications & Benefits
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gradient runsFlow Accuracy and Precision
< 0.07 % RSD or 0.01 min SD
� 1.0 % or 10 µL
� High RT precision in isocratic runs
Composition Range1-99 %
� Wide analytical range
Composition RangeShallow gradient at low organic concentration
1: Theophylline
N
N
N
N
CH3
O
CH3
O
H
N
N
N
N
CH3
O
CH3
O
CH3
2: Caffeine
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Column: Agilent Poroshell 120 EC-C18, 4.6 x 50 mm, 2.7 µm (p/n 699975-902)
Mobile phase: A = Water / B = Acetonitrile
Gradient: 2 – 12 % B in 5minFlow rate: 2 mL/min
Stop time: 5 min
Injection vol.: 5 µL
Column Temp.: 40 °C
DAD: 254/10 nm, (ref. 360/ 100 nm), 20 Hz
Composition RangeShallow gradient at low organic concentration
mAU
80
100
1: Theophylline
N
N
N
N
CH3
O
CH3
O
H
N
N
N
N
CH3
O
CH3
O
CH3
2: Caffeine
1
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min2 2.5 3 3.5 4 4.5
0
20
40
60
2
Composition RangeShallow gradient at low organic concentration
0.3
0.4
0.5
RS
D R
T (%
)Retention time precision
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0
0.1
0.2
0.3
1290 InfinityQuat. System
RS
D R
T (%
)
Theophylline
Caffeine
Specification (< 0.15 %)
Composition RangeShallow gradient at low organic concentration
0.3
0.4
0.5
RS
D R
T (%
)Retention time precision
2.4
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0
0.1
0.2
0.3
1290 InfinityQuat. System
1260 InfinityQuat. System
Quat. UHPLCOther vendor
RS
D R
T (%
)
Theophylline
Caffeine
Specification (< 0.15 %)
Composition RangeShallow gradient at low organic concentration
mAU
200
250
Step gradient1 % steps from 0 to 10 %
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0 10 20 30 40 50
0
50
100
150
1290 Infinity Quaternary system
Quaternary UHPLC from other vendor
Ternary/Quaternary GradientsBlend Assist
You need different concentrations of modifiers in your analysis, would like to have just one stock-solution and do online dilution to profite from the quaternary mixing capability of your pump? Here is a simple tool – BlendAssist!
Water Water 1% TFA
Desired method conditions - example: 1. 5 to 95% gradient of ACN with 0.1% TFA in
Water and 0.08% TFA in ACN2. 20 – 80% gradient of ACN with 0.5% TFA in
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1% TFA
ACNACN
1% TFA
Water and 0.4% TFA in ACN
Without BlendAssist you need to either pre-mix the required solvents or by using stock-solutions of TFA in Water and ACN to program complex gradients (%A, B, C, D).
With BlendAssist: just program your binary organic/aqueous gradient and define the dilution factor!
Ternary/Quaternary GradientsBlend Assist - simple Tool for Online-Dilution of Modifiers and Gradient Set-Up
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BlendAssist - Solvent/modifier channel definition
Buffer Advisor SoftwareQuaternary mixing
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September 24, 201223
1260 Infinity Bio-inert HPLC
What is similar compared to standard1260 Infinity Quaternary LC?
� Pumping performance, gradient performance
� Pumping Specifications� Detector, Autosampler Performance and
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� Detector, Autosampler Performance and Specs
� Overall System Robustness� TCC specs
600 bar also for fast or high resolution Bioseparations
Modular flexibility maintained
September 24, 201224
1260 Infinity Bio-inert HPLC
What is different compared to standard 1260 Infinity Quaternary LC
� Pump titanium based, no SST �Autosampler metal-free�Capillaries and connections metal-free�Detector Flow Cell metal-free
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�Detector Flow Cell metal-free�pH-range 1-13, short term pH 14� Active Seal wash included in pump� Low pressure is possible
•Wide pH range•High salt tolerance•Lowest surface activity� Bio-Inertness
September 24, 201225
New Capillary Design Ensures Bio-inertnessCapillaries:
Metal cladded PEEK capillary
New capillary technolgy enables 600 bar
AND is completely metal free !!
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September 24, 201226
Needle:
SST housingfor robustness
PEEK and ceramic for metal free sample path
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Buffer Advisor SoftwareWorkflow
1: Select Buffer and Gradient Solution
2: Enter Gradient Timetable
3: Review Composition
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September 24, 201229
3: Review Composition of Stock Solutions and Adjust
4: Gradient Display Section: Process and Review Gradient
Experimental Situation
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Buffer Advisor SoftwareWorkflow
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Buffer Advisor SoftwareTimetable import into Chemstation
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Buffer Advisor SoftwarepH scouting
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Buffer Advisor Softwarestable pH
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30x wider linear UV range -Quantification of widely different concentration levels in one single run
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Agilent 1200 Infinity High Dynamic Range (HDR) Solution
History of DAD Linearity Gain The last 30 years
> 30
20.0
25.0
30.0
35.0
40.0
45.0
Line
arity
Gai
n
1200 Infinity HDR-DAD
With HDR
>30X Linearity
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Detector Model (Intro Year)
1.4 1.7 2.0 1.4 2.9
0.0
5.0
10.0
15.0
20.0
1040 to 1090 1090 to 1050 1050 to 1100 1110 to 1200 1200 to 1290/60 1200 to HDR
Line
arity
Gai
n
(1988)(1986) (1995) (2006) (2010) (2011)
September 24, 201236
Programmable
GratingMirror
Optofluidic Waveguides: Max-Light Flow Cells Total-internal reflection in a non-coated fused silica fiber
1260 / 1290 Infinity DAD
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Deuterium Lamp
Max-Light Cartridge Cell10 mm or 60 mm path length
Programmableor fixed slit
1024 elementdiode-array
September 24, 201237
Effects of path length increase
Peak Dispersion
60 mm pathlengthlarge geom. cell volume (78 µL)
- Loss of resolution- Loss of signal height
10 mm pathlengthsmall geom. cell volume (13 µL)
high light transmission
Conventional flow cells:
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large geom. cell volume (78 µL)lower light transmission
60 mm pathlength4 µL �V dispersion volume
Max-Light High Sensitivity cell:
Optofluidic waveguides (total internal reflection)
September 24, 201238
10x Higher Sensitivity 1290/1260 Infinity DAD compare to 1200 Series DAD SL
mAU
20
25
30
Columns: 150 x 4.6mm Zorbax SB C18, 5µm
Sample: Anthracene: 835 pg/µL
Mobile phase: A: Water, B: Acetonitrile
Elution: isocratic 80 % B
Injection volume: 5 µL
Flow: 1.5 mL/min
DAD: 251/4nm, Ref= 450/80nm, 2.5Hz, slit width 4nm
1260/1290 Infinity DAD
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1260/1290 DAD60 mm
1200 DAD SL 10mm
Height [mAU] 28.876 4.938
Noise [mAU] 0.0098 0.0190
Signal/ noise 2944 259
Increase 11.4 xmin0 1 2 3 4 5
0
5
10
15
20 DAD: 251/4nm, Ref= 450/80nm, 2.5Hz, slit width 4nm
1200 Series DAD / DAD SL
September 24, 201239
30x Wider Linear Range with HDR-DAD3.7 mm and 60 mm and Max-Light flow cell
3.7 mm path-length
Detector Signal[AU/cm]
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60 mm path-length
Computing the signals from60 mm path-length for the low concentration3.7 mm path-length for the high concentration
Conc [µg/mL]
September 24, 201240
1290/1260 High Dynamic Range – HDR Increasing Linear UV Range by >30x
Technology
� Cluster of two DADs
� DAD 1: 60 mm Cell – for low concentrations
� DAD 2: 3.7 mm Cell – for high concentrations
� Output: One, combined signal, normalized to 10 mm path length (HDR range: 0.6 x 10-6 to 6.7 AU/cm) vs for 1200 Series: 7 x 10-6 to 2 AU/cm)
60mmDAD 1:
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Control / Usability
� Like standard 1290/1260 DAD: Control, data analysis, reporting
Investment Protection
� Existing 1290/1260 DAD can be upgraded (by 2nd DAD)
� Existing 1100/1200 Systems can be upgraded (by two 1290 or 1260 DAD)3.7mm
Clustered DADDAD 2:
September 24, 201241
Fixed Dose CombinationsFrom Roche Switzerland
Fixed dose combinations:
A combination of two or more
APIs in a fixed ratio of dosesAgilent 1200 (black): API 2: 10 µl injected
Agilent 1200 (red): API 1: 100 µl injected
Infinity HDR-DAD (blue): API 1 and 2 simultaneously: 70 µl injected
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API 1
API 2
September 24, 201242
Fixed Dose CombinationsFrom Roche Switzerland
“With the Agilent 1200 Infinity HDR-DAD we significantly decreased our turnaround time. We also get lower
LODs and better area precision”
Dr. Rose-Marie Meier F. Hoffmann - La Roche AG
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Infinity HDR-DAD
Agilent 1200 DAD
September 24, 201243
2 Injections needed if a conventional DAD is used
1µl injection to determine the first 3 peaks
min0 1 2 3 4 5
mAU
0
200
400
600
800
1000 Vitamin C
Paracetamol
Caffeine Chlorphenamine ?
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September 24, 201244
Vitamin CParacetamol
Caffeine
Chlorphenamine
5µl injection to determine the last peak and further impuritiesThe first 2 peaks are out of linear range
min0 1 2 3 4 5
mAU
0
1000
2000
3000
3800Detector saturationOutside the linear range
2 Injections needed if a conventional DAD is used
ChlorphenaminemAU
15
17.5
20
DAD1 A, Sig=254,20 Ref=380,80 (SOP 98 GRI...0FEB\HDR_GRIPPO_OLD_20FEB 2012-02-20 10-40-57\GRIPPO_1UL09.D)
DAD1 A, Sig=254,20 Ref=380,80 (SOP 98 GRI...0FEB\HDR_GRIPPO_OLD_20FEB 2012-02-20 10-40-57\GRIPPO_5UL10.D)
mAU
0
0.2
0.4
0.6
0.8
1
Chlorphenamine
Imp 3
Imp 4
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September 24, 2012Confidentiality Label
45
Blue: 1µl injection with 3.7mAU peak height for Chlorphenamine
Red: 5µl injection with 21.8mAU peak height for Chlorphenamine
min4 4.5 5 5.5 6
0
2.5
5
7.5
10
12.5
15
Imp 3 Imp 4
min4.6 4.7 4.8 4.9 5 5.1 5.2 5.3
-0.2
0
Comparison of conventional DAD vs. HDR DAD, 5µl injection
mAU
2500
3000
3500
4000
Red conventional DAD: 2 peaks out of linear range with 5µl injection
Green HDR-DAD: All peaks within linear range
Paracetamol
Vitamin CDetector saturationOutside the linear range
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September 24, 201246
min0 1 2 3 4 5
0
500
1000
1500
2000All peaks within linear range (up to 6AU)
Caffeine
Chlorphenamine
Comparison of LOD
mAU
0.8
0.9
1
1.1
1.2
1.3
HDR DAD
Conventional DAD
More reliable automated peak integration
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September 24, 201247
Compound LOD with S/N=3 for Conventional DAD
LOD with S/N=3 for HDR- DAD solution
Chlorphenamine ~1ng ~0.1ng
min3.9 4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.80.5
0.6
0.7
0.8
Comparison of conventional DAD and HDR DAD, 5µl injection
mAU
15
20
DAD1 K, Sig=254,20 Ref=380,80 (SOP 98 GRI...B\HDR_GRIPPO_HDR_21FEB 2012-02-21 07-51-02\GRIPPO_HDR5UL09.D)DAD1 A, Sig=254,20 Ref=380,80 (SOP 98 GRI...0FEB\HDR_GRIPPO_OLD_20FEB 2012-02-20 10-40-57\GRIPPO_5UL09.D)
Red conventional DAD: determination of impurities and chlorphenamine in a second run
Blue HDR DAD: dermination of all peaks in one run
ParacetamolCaffeine
ChlorphenamineVitamin C
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48
min1 2 3 4 5
0
5
10
Imp 1 Imp 2 Imp 3 Imp 4
Comparison of area precision
14.908 13.013 5.37 5.723
1
1.5
2
2.5
3
RS
D A
rea
(%)
Comparison of Area Precision
HDR Signal
Conv.DAD signal (1µl)
Conv.DAD (5µl)
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September 24, 201249
HDR DAD 5µl injection volume for detemination of all compounds (blue) Conventional DAD: 1µl injection volume for determination of Vit C, paracetamol and Caffeine (orange)
5µl injection volume for determination of Chlorphenamine and further impurities (green)
0
0.5
Intelligent System Emulation Technology
(ISET)
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September 24, 2012Page 50
What is ISET?- Two components
A. Best in class performance of the 1290 Infinity LCBroad power range, ultra-low delay volume, superior sensitivity, unmatched flow and composition accuracy
Peak 3Overlay of 10 runs
RSD = 0.005 %
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B. The revolutionary ISET emulationalgorithmFull characterization of target LC systemsCreation of emulation function
FA
Method Transferability: 1290 Infinity LC- System Emulation Technology
Concept
EmulatedSystem A
FA
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Select System to be emulated by a simple mouse click:
Agilent 1100 Series
Agilent 1200 Series LC
Agilent 1260Infinity LC
Agilent 1220Infinity LC
Alliance(2012)
Intelligent System emulation technology- How it works (1290 to 1200)
250
300
350
400
mAU
Injection
Same gradient conditions by moving the gradient and by emulating the gradient mixing behavior.
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Programmed gradient
1290 gradient
1200 gradient
0 0.5 1 1.5 2 2.5 3 3.5
0
50
100
150
200
min
gradient mixing behavior.
Select pump
Intelligent System Emulation Technology ISETImplementation
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Select sampler
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PesticidesColumn: 3 x 50 mm, 2.7 µm Poroshell Run time: 3.5 min
mAU
500
600
700
800
Sim
azin
e
Chl
orid
azon
e
Chl
orto
luro
n
Diu
ron
Pro
pazi
neTe
rbut
ylaz
ine
Pro
met
ryn
1290 System with iSETM
etam
itron
Cya
nizi
ne
Rs= 1.87
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55
min1 1.5 2 2.5 3 3.5
0
100
200
300
400
1290 System
Rs= 1.90
1100 Binary System
Rs= 1.15
PesticidesColumn: 3 x 50 mm, 2.7 µm Poroshell Run time: 3.5 min
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PesticidesColumn: 3 x 50 mm, 2.7 µm Poroshell Run time: 3.5 min
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Generic emulation- Limitations
Example: 1290 to Vendor X
Injection
250
300
350
Generic emulation: Move the gradient
mAU
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0 0.5 1 1.5 2 2.5 3 3.5
0
50
100
150
200
min
Programmed gradient
1290 gradient
Vendor X
Generic emulation: Move the gradient
Generic emulation- Limitations
Example: 1290 to Vendor X
Injection
250
300
350
mAU
Generic emulation: Move the gradient
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Programmed gradient
1290 gradient
Vendor x
0 0.5 1 1.5 2 2.5 3 3.5
0
50
100
150
200
min
Does not consider the gradient mixing behavior (different curves) ordelay volume changes because of pressure changes for pumps with damper.
Method Transfer Possibilities Today- A) Adding physical volume (plumbing solution)
1290 + 1000 �L dwell vol.
Example: 1290 Infinity LC
1100/1200 Quat Pump
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Advantages
• Works well for generic, slower analysis• No Method change
Disadvantages
• Requires hardware change, no flexibility
• Hardware change difficult in validated environment
• Requires manual determination of the dwell volume (Pumps with damper the dwell volume is not constant and depends on pressure)
Isocratic hold: move the gradient
Method Transfer Possibilities Today- B) Isocratic Hold
Example: 1290 to 1200
Injection
250
300
350
mAU
Isocratic hold: move the gradient
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Programmed gradient
1290 gradient
1200 gradient
0 0.5 1 1.5 2 2.5 3 3.5
0
50
100
150
200
min
Does not consider the gradient mixing behavior (different curves).
Method Transfer Possibilities Today- B) Isocratic Hold
Example: 1290 to 1200
1200 Series LCBinary Pump
1290 Infinity LC+ 860 µl “Delay Volume”
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1290 Infinity LC+ 900 µl “Delay Volume”
1290 Infinity LC+ 940 µl “Delay Volume”
Method Transfer Possibilities Today- B) Isocratic Hold
Example: 1290 to 1200
1260 Infinity LC
1290 Infinity LC+ 900 µl hold
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Advantages
• Flexibile, Isocratic Hold is part of the method
Disadvantages
• Works not in all cases• Requires manual determination of the dwell volume/
isocratic hold (Pumps with damper the dwell volume is not constant and depends on pressure)
• Requires modification of the methods (should be avoided in validated environment,but doesn’t require revalidation USP Chapter <621>)
Method transfer, pesticide example2.1x100 mm Zorbax Eclipse Plus, 1.8 µm column, flow: 0,8 mL/min
(A)1100
Quaternary400 bar
(B)
1 2,3 4 5 6,7 8 9 10,11,12 13 14 15 16
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September 24, 2012Page 64
(C)1290 Infinity
1200 barwith ISET
(B)1290 Infinity
1200 bar
Summary1290 Quaternary Pump:� Full 1290 Infinity LC Power range
� Highest composition precision and accuracy across pressure and flow rate range
� Full application range with binary-like pump performance! 1290 Systems using ISET:• Achieves higher productivity for method development • Simplify your instrument to instrument method transfer while minimizing costs
• Run your legacy methods with ISET and use UHPLC speed for faster separations
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• Run your legacy methods with ISET and use UHPLC speed for faster separations
HDR DAD Solution:• Determination of low and high dose drugs is possible in one run• Improved Precision and Lower Detection Limits as compared to convention DAD
Using Buffer Advisor with BioInert Quat System:• Shortens and simplifies the workflow for pH scouting by using pre-validated
buffer systems and dynamically mixed gradients• Excellent tool using for automated pH scouting and accurate ion exchange
chromatography
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