920-lc analytical hplc 940-lc analytical to prep hplc ihplc
TRANSCRIPT
920-LC Analytical HPLC940-LC Analytical to Prep HPLC
iHPLC
Varian 900-LC Series iHPLC
3
Agenda
• Introduction• Introduction to the 9X0-LC series HPLC
• Total market size by technique and market segment
• An overview of the 920-LC Analytical HPLC• Features and benefits
• Positioning, competition and target markets
• Applications
• An overview of the 940-LC Analytical to Prep and Prep HPLC• Features and benefits
• Positioning, competition and target markets
• Applications
4
What is the iHPLC?
• 2 models 920-LC and 940-LC
• Compact, stylish, automated analytical and semi prep HPLC
• Pre-plumbed and factory tested for the highest confidence
• Easy to order, install and use
– Single Ethernet cable connection
• Focused on improved productivity through automation
• Updated premium performance hardware
• Autosampler, Fraction Collector and Scale up Module for semi-prep
• Quaternary pump, precision Column Heater,RI, Fluor, ELSD and autosampler for analytical
• Updated Integrated Instrument Software Interface
• Eliminate repetition, modernise UI
• Consistent
• Easy to use with added functionality
• Adds to modular range
5
HPLC DemandSDI 2007
Service16%
Initial Systems
38%
Aftermarket46%
Fast LC10%
GPC4%
Other9%
Prep14% Conventional
HPLC63%
• Overall LC is $3.2B @ 6% growth
• 23000 systems sold in 2006
• New HPLC systems sales (initial)
• $1220M
– $764M Conventional analytical
– $180M Prep
– $113M Fast LC
6
Current Status of Varian HPLC
• Installation/setup difficulties• No supporting materials• Perceptions• Ease of ordering• Competitive performance• Lack of competitive specifications• Ease of use• Reliability• Productivity• Validation and Compliance• Flexibility/expansibility (especially in semi-prep)• Competitive pricing• Wide product range – hardware, columns, consumables
7
Varian 920-LC Integrated Analytical HPLC
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Varian 920-LC Silver Bullets
• Premium Analytical Pump Performance and reliability
• Superb Fast LC Performance optimised for sub 3 um columns– Improved throughput without compromising robustness
• High sensitivity/Top Line Performance detection systems across UV/PDA/RI/Fluor and ELSD – Unparalleled strength
• Unbeatable Injection performance and carryover statistics with 2008 Loopless Technology Autosampler
• Full system level computer controlled AIQ (validation)
• Updated UI for Enhanced User Experience
9
Varian 920-LC Analytical HPLC Goals
• Aim
• Become credible player in this $800M segment by doubling market share and revenue
• Markets
• Pharma / BioPharma
• Environmental
• Industrial
• Strategy
• Leverage Varian distribution channel to deliver competitive HPLC hardware, consumables, software and services
10
Varian 920-LC What Is It?
• Compact, stylish automated analytical HPLC
– Pre-plumbed and factory tested for the highest confidence
– Single cable communications
• Virtually pulse-free dual piston pump
– Does not require a pulse damper
• State-of-the-art low pressure quaternary gradient module with built-in degasser
• Autosampler with cooling option
– Synchronised injection for outstanding retention time reproducibility
– Compatible with 200 x 1.6 ml HPLC vials or 3 microplates
11
Varian 920-LC What Is It?
• Either dual UV-Vis or PDA detector
– Optional second detector - Fluorescence or RI
• High precision (<0.15oC) dual column heater available
– Low delay solvent pre-heater for improved heat transfer
– Optional column switching and electric injection valve
• NEW improved instrument interface on GalaxieTM software
• External CVM, ELSD or 356-LC RI can be plugged directly into the back of the unit.
12
920-LC for Standard HPLC to Fast HPLC
25cm x 4.6mm Pursuit XRS 5um 1.0ml/min
25cm x 4.6mm Pursuit XRS 5um 2.2ml/min
15cm x 4.6mm Pursuit XRS 5um 1.0ml/min
15cm x 4.6mm Pursuit XRS 5um 3.75ml/min
10cm x 2.0mm Pursuit XRS 2.8um Ultra 0.8ml/min
5cm x 2.0mm Pursuit XRS 2.8um Ultra 0.4ml/min
5cm x 2.0mm 2.4um 1.3ml/min16 x Speed Enhancement
13
920-LC for Standard HPLC to Fast HPLC
25cm x 4.6mm Pursuit XRS 5um 1.0ml/min
25cm x 4.6mm Pursuit XRS 5um 2.2ml/min
15cm x 4.6mm Pursuit XRS 5um 1.0ml/min
15cm x 4.6mm Pursuit XRS 5um 3.75ml/min
10cm x 2.0mm Pursuit XRS 2.8um Ultra 0.8ml/min
5cm x 2.0mm Pursuit XRS 2.8um Ultra 0.4ml/min
5cm x 2.0mm 2.4um 1.3ml/min16 x Speed Enhancement
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Varian 920-LC Fast LC PAHs
Varian 920-LC6 minute Analysis
Waters Acquity7 Minute Analysis
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Varian 920-LC Pump
• Dual piston quaternary pump produces fast gradients – even at 200 l/min
• Virtually pulse-free pumping without pulse damper
• Outstanding gradient compositional accuracy give retention time precision at the <1% level
• Built-in vacuum degas maintains pump prime and enhanced injection reliability
• Accurate flow from 0.1 to 10 mL/min for 2 to 4.6 mm columns
• Overlay of 10 chromatograms
• 10% steps at 1ml/min
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Varian 920-LC Pump - FAB
Feature Advantage Benefit
Dual Piston Quaternary Capability
Market leading precision and accuracy
Performance benchmark
Built in Degasser and Pulse Free Flow
Enhanced run to run reproducibilty
Dependable
Confidence in results
Optional Semi-Micro
Kit
Fast LC capability Performance with Reliability
17
Varian 920-LC Autosampler
• Needle-in-flow path geometry for low carry-over (<0.01%)
• Typical inj precision (< 0.3% at 10 l)
• Optimized flow paths for fast injection turn-around
• Synchronised injection for best RT reproducibility
• Programmable wash setting for low carry-over
• Injection volumes from 1ul to 50L
• Cooled option available
18
Varian 920-LC Washout Performance
Blank injection <0.01% carryover
125 ug/ml Caffeine
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920-LC Autosampler How Does it Work?
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Varian 920-LC Injection Synchronization
-20%
0%
20%
40%
60%
80%
100%
120%
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2
21
Varian 920-LC Autosampler - FAB
Feature Advantage Benefit
Needle in Path
Design
No need for Injection loops
No Carryover
Versatility
Performance
Synchronised injections with Pump Stroke cycle
Enhanced retention time reproducibilty
Performance
Accuracy
One Autosampler, all rack formats
No need for multiple injector options
Versatility
Simplicity
22
Sensitive UV-Vis Detectors
Typical Agilent VWD S/N ~ 25
Typical Varian 920-LC UV Vis S/N ~ 38
23
Varian 920-LC UV/Vis - PDA Detectors - FAB
Feature Advantage Benefit
Best signal to Noise Enhanced sensitivity and detection limits
Performance
Tool Free maintenance
Easy to change lamps and flow cells
Simplicity
Range of cells for ul/min to Litres/min
Fits all applications from LCMS to Benchtop Prep
Versatility and Performance
24
Varian 920-LC Fluorescence Detector
• Sensitive Fluorescence detector (S/N > 600 Raman H2O peak baseline method)
• Time programmable for increase sensitivity• Automatic validation with built-in Hg lamp• Low 12 l dead volume flowcell for improved
chromatography
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Varian 920-LC Fluorescence Detector - FAB
Feature Advantage Benefit
Improved Sensitivity
(S/N > 600 Raman)
Lower limits of detection
Performance
Automatic calibration
( Hg lamp)
Greater accuracy of analysis
Confidence in results; Dependable
Low dead volume cell ( 12ul volume)
Sharper peaks, enhanced resolution
Performance
26
Varian 920-LC Refractive Index Detector
• Same as 356-LC Refractive Index Standalone
• Low Volume, flow efficient cell for Superior Peak Shape
• Stable, Long Life and High Intensity LED
• Temperature Controlled Optics for minimised baseline drift and outstanding detection limits
• Complete automation thru Galaxie Software
27
Varian 920-LC RI Detector - FAB
Feature Advantage Benefit
Low Volume Cell Minimal Peak Dispersion giving enhanced resolution
Performance and Accuracy
High Intensity, long life LED
Maintenance Free for consistant long term performance
Dependability, Durability and Confidence in results
Temp Controlled optics
Minimises baseline drift for lower settling times, better sensitivity
Performance and Ecconomy – saves the analyst precious time
28
Varian 380-LC and 385-LC ELSD
• Varian Product numbers
• 2 to 3 Times More Sensitive
• NEW Glass Nebuliser for Improved Reproducibility
• 40Hz Data Rate for Fast LC
• Onboard timetable program to enable parameter change thru a run
29
Varian 380-LC and 385-LC ELSD - FAB
Feature Advantage Benefit
Improved Sensitivity Lower limits of detection
Performance
NEW Glass Nebuliser
Greater run to run reproducibility
Confidence in results; Performance
40Hz Data Rate Suitable for Fast LC Performance coupled with 920-LC in Fast LC operation
30
Heated Column Compartment
• Temperature range from ambient + 10oC up to 65oC
– Temperature reproducibility +0.15oC
– Ensures excellent analytical reproducibility
– Uses a low delay volume pre-heater for efficient heat transfer to mobile phase
– Spacious column compartment easily accommodates 2 x 250mm columns
– Option with electrically actuated column switching valve and injection valve
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Outstanding Stability
20
22
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32
34
36
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40
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11:4
6:00
12:2
2:00
12:5
8:00
13:3
4:00
14:1
0:00
14:4
6:00
15:2
2:00
15:5
8:00
16:3
4:00
17:1
0:00
17:4
6:00
18:2
2:00
18:5
8:00
19:3
4:00
20:1
0:00
20:4
6:00
21:2
2:00
21:5
8:00
22:3
4:00
23:1
0:00
23:4
6:00
0:22
:00
0:58
:00
1:34
:00
2:10
:00
2:46
:00
3:22
:00
3:58
:00
4:34
:00
5:10
:00
5:46
:00
6:22
:00
6:58
:00
7:34
:00
8:10
:00
8:46
:00
9:22
:00
Time
Te
mp
era
ture
(d
eg
ree
s C
)
Mobile phase input Temp
Mobile phase temp inside column
Oven Temp
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Reproducibility of 50 Injections
Reproducibility <0.8% over 50 injections and 12 hours
33
Varian 920-LC Column Heater - FAB
Feature Advantage Benefit
Low Volume Solvent Pre-Heater
Eliminates thermal column effects and enhances resolution/method transfer
Performance
Outstanding Stability and Accuracy
Provides greater method robustness
Dependable, Confidence in results
Accurate
Spacious compartment with easy access
Fast column changes, room to work
Simple and Convenient
34
Varian 920-LC Software Usability
• User friendly GalaxieTM software
• Simplified, modern, consistent Instrument interface
• Modular 11 pages
• 920-LC 4 pages!!
• One page real-time status and chromatogram
• Advanced integration and calibration
• New IMS capabilities
• Automated instrument validation
• Reduces downtime and operating costs
• Fusion AE
35
Varian 920-LC AIQ (Validation)
AIQ (Analytical Instrument Qualification)• Fully PC controlled IQ and OQ (not PQ)
• Puts the Varian 920-LC at the forefront of regulated capabilities
• Consistent, reproducible methodology• Includes pumps, autosampler, detectors, column
heater
• Chargeable option
• Fully documented
36
Varian 920-LC User interface - FAB
Feature Advantage Benefit
Modern, User friendly UI
( 11 pages to 4!)
Simple to use and an enjoyable experience
Simplicity coupled with Stylish Appearance
Shockwave Graphics for 2008 Software Technology
Visually pleasing with Flashing instrument status
Stylish appearance
Instrument maintenance Software
Automated instrument validation/testing
Ensures instrument Accuracy and Performance
37
Varian 920-LC Concept - FAB
Feature Advantage Benefit
1st Fully Integrated
iHPLC System
Unique and Interesting concept
Prestige resulting in elevated HPLC interest/prospects
Easy to Use, Install and Maintain
Creates a new level of customer satisfaction
Product Confidence and enhanced repeat business
Compact and Stylish High level of instrument capability in Unique footprint
Desirable to own, Convenient to use
38
Varian 920-LC Concept - FAB
Feature Advantage Benefit
Single Part Numbers Easy to quote Much simpler to sell, and easier experience
One Power, Communications cable and “On” Button
Easy to configure, clean and tidy look
Simplicity with enhanced Appearance
Pre-Plumbed and “Ready” out of the Box
Fast installation so you are quickly able to use the instrument
Convenient and Simple
39
Varian 920-LC Concept - FAB
Feature Advantage Benefit
Sample tray access without opening the main doors
System looks good at all times without exposing internals
Clean Appearance
Door trays and holders for columns and accessories
Provides space to tidily store the many parts used daily
Clean Appearance
Tray Storage Provides additional storage space for Software CD, tools, columns etc…
Clean Appearance and Versatility
40
Varian 920-LC Concept - FAB
Feature Advantage Benefit
Solvent Tray capacity
Shape of instrument provides ample space for solvents
Convenience and Safety
Ski Slopes for easy tray insertion/removal
Eliminates tray location errors
Provides Autosampler Confidence and Durability
Single Centralised Waste port
No need for multiple waste vessels positioned around the instrument
Clean Appearance and Simplicity.
Only 1 waste to empty
41
Varian 920-LC Concept - FAB
Feature Advantage Benefit
Leak Detector Ensures the system is “leak free” and performing to the max
Performance and Confidence
Modern Manual Injectors
Push button valve positioning with automatic return, eliminate operator error
Even Simpler to use, eliminates user error improving Confidence in results
Bottle Management Ability to fill reservoirs and empty waste
Provides a Safety mechanism for Confidence in overnight runs
42
Varian 920-LC Concept - FAB
Feature Advantage Benefit
Tool Tips, Help and Videos
Extensive library of Mouse tool tips and Online help
Makes the instrument Simple to use and Convenient to access
New Brochures Physical material to present to customers
Convenient and Powerful messages to explain to customer
Updated Data Sheets, Price List and App Notes
Supporting materials to leave with the customer
Provides Confidence in product range and Simple to Quote
43
Varian 920-LC Ordering Matrix
pump inject detector Oven Detector 2
option
Options
Modular Isocratic 210-LC 10ml Manual 325 PDA, RI, ELSD
Isocratic
00-100890-00Dual piston pump 10 ml head
Auto Dual UV/Vis
Opt FL, RI ELSD
Gradient
00-100891-00Quat, Grad, Degas Auto Dual
UV/VisOpt FL, RI ELSD opt
High Performance Gradient
00-100892-00
Quat, Grad, Degas Auto cooled Dual UV/Vis
Opt FL, RI ELSD opt
Development
00-100893-00Quat, Grad, Degas Auto PDA Std FL, RI ELSD opt
Research
00-100894-00Quat, Grad, Degas Auto cooled PDA Std FL, RI ELSD opt
44
Varian 920-LC Target Markets
• Strong historical position for Varian in analytical
• Varian has the leading range of analytical technologies: spectroscopy, chromatography including consumables, data and services
• Target Users
• Environmental
• Food/Beverage
• Industrial
• Generic Pharmaceutical *
• Academia and Biotech
• Same (but wider) target groups with a stronger product offering
*40% of Regulated Pharma has significant competitive barriers due to installed software base
45
Varian 920-LC Consumables
• Excellent range of HPLC Columns providing industry leading resolution and robustness
• Pursuit
• Pursuit XRS
• Polymeric Phases - PL
• From 2mm to 2” ID Prepacked
• Other Speciality Phases
• NEW 2.8 Ultra Column AND 2.4um Columns for Fast or High Res LC
• 2.8um Ultra is optimised for Speed
• 2.4um gives further enhancements in Resolution
46
2.8um Vs 2.4 um – High Res or Extra Speed
2.8um 2mm x 5cm for speed
2.4um 3mm x 5cm for RESOLUTION
Varian 920-LC Positioning
UPLC, Fast LC and the Varian 920-LC
49
What is Fast LC?
• Involves using LC columns packed with very small (sub 3 m diameter) particles
• Smaller particles column are more efficient giving greater resolution per unit length
• For the same separation (at the same flow rate) can use a shorter column which reduces run time for improve productivity
• As Van Deemter plot is flatter can operate at higher flow rates efficiently
But smaller particles give greater back pressure!!
50
Van Deemter Plot
3.5 m
5 m
m
HE
TP
(cm
/pla
te)
Linear velocity u cm/sec
0.000
0.005
0.010
0.015
0.020
0.025
0.035
0.030
0.040
0.045
0.0 0.2 0.4 1.61.0 1.2 1.40.6 0.8
51
Fast LC Productivity
25cm x 4.6mm Pursuit XRS 5um 1.0ml/min
25cm x 4.6mm Pursuit XRS 5um 2.2ml/min
15cm x 4.6mm Pursuit XRS 5um 1.0ml/min
15cm x 4.6mm Pursuit XRS 5um 3.75ml/min
10cm x 2.0mm Pursuit XRS 2.8um Ultra 0.8ml/min
5cm x 2.0mm Pursuit XRS 2.8um Ultra 0.4ml/min
5cm x 2.0mm 2.4um 1.3ml/min16 x Speed Enhancement
52
Market Size for Fast LC - 2007
2007 HPLC Demand by Product Type
GPC4%
Other9%
HPLC61%
Prep LC15%
Fast LC11%
Total Systems market Size 2007 = US$1.3b (SDI 2006)
53
Fast LC
• Fast LC market estimated at $160mill growing rapidly• Many names
• Ultrahigh Pressure LC
• Rapid Resolution LC
• High Resolution LC
• Fast LC
54
UPLC• Research Pharma – Drug discovery• QA/QC Pharma – Not as successful as first thought• No.1 use - separation front end for MS
Target Markets for UPLC and Fast LC
55
UPLC or Fast LC• Small particle porous column packing 1.7 – 3.0 m in
diameter• Increased flow rates• Increased pressure• Increased column temperatures
Advantage:• Improved resolution of peaks in complex samples• Improved sensitivity for low concentration or limited
samples• Reduced analysis times
Theory
56
UPLC or Fast LC• Small particle porous column packing 1.7 – 3.0u in dia.• Increased flow rates• Increased pressure• Increased column temperatures
Disadvantage:• Increased back pressure – special hardware requirements
needed, micro leaks become a problem = Poor reproducibility
• Band spreading in the column decreases – the HPLC will need to account for this
• High acquisition rates for detector collection
• Expensive
• Proprietary columns in some instances
Theory
57
• Price – Binary gradient LC with autosampler and detector ~
50-60% of UPLC cost
• Flexibility– Operates as standard HPLC and in Fast LC mode
• Allows all LC applications to be run
• Reliability– Reproducibility of results
Advantages of Fast LC
58
• Speed – Reduced analysis times = cost savings
• Flexibility– Optimum performance as front end for MS
• Performance– Improved sensitivity
Advantages of UPLC
59
Typical Speed Performance of a UPLC Separation
60
Typical Speed Performance of Fast LC
2.4 minutes
Fanta Orange Drink
Sugar Standard
2.72.62.52.42.32.22.121.91.81.71.61.51.41.31.21.110.90.80.70.60.50.40.30.20.1
170
165
160
155
150
145
140
135
130
125
120
115
110
105
100
95
90
85
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70
65
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55
50
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35
30
25
20
15
10
5
0
Ace
K
Sod
ium
Ben
zoat
e
Pot
assi
um S
orba
te
Caf
fein
e
Asp
arta
me
RT [min]
mAU Soft drinks_19.DATA [PDA detector Absorbance Analog Channel 1 ]
Conditions
Mobile Phase: 10mm Sodium Phosphate ph6.0/ACN gradientDetection: UV DetectionTemperature: 40°CFlow-Rate: 1,2mL/minColumn: Varian Ultra 50x2,0mm
61
Waters Acquity• High pressure – quoted to 15,000psi but generally
run at around 10,000psi• Low dispersion• Small particle size columns• Small columns – generally <2mm in dia.• Binary gradient system – 4 solvent selection per
pump• Typical column temp of 30°C to 50°C
Current Systems on the market
62
Agilent 1200 RRLC• High pressure – quoted to 9,000 psi but generally run
at lower pressures• Low dispersion• Small particle size columns• Flow rate maximum of 5 ml/min• Binary gradient system – 4 solvent selection per
pump• Typical column temp of 30°C to 100°C• Can switch between Fast LC and normal LC (variable
dead volume control).
Current Systems on the market
63
Thermo Accela• High pressure – quoted to 15,000psi only with flow
rates of 1-1000 L/min• Low dispersion• Compatible with small particle size columns• Promoted as conventional and Ultrahigh press LC
– max flow rate of 1ml/min limits conventional HPLC
• Quaternary pumping system
Current Systems on the market
64
Others
Shimadzu• Conventional LC that shows 2.2 m particle column
applications – Fast LC approach
Jasco• UPLC system utilising 15000psi pressure capability
and column temperatures to 60°C
Hitachi• 9000psi pressure capability with 90°C temperature
performance. Variation on their L2000 series HPLC
Current Systems on the market
65
Van Deemter Plot
3.5 m
5 m
m
m
HE
TP
(cm
/pla
te)
Linear velocity u cm/sec
0.000
0.005
0.010
0.015
0.020
0.025
0.035
0.030
0.040
0.045
0.0 0.2 0.4 1.61.0 1.2 1.40.6 0.8
66
Trends in Fast LC
• Movement away from sub 2 m particles owing to– Ultrahigh back pressures & reproducibility– High temperatures & column lifetime– Data acquisition rates
• 2.4 m and 2.8 m particles allows– Improved resolution without ultrahigh back pressures– Use of modified conventional HPLC (reduced delayed
volumes)– Use of more moderate conditions e.g. temp & pressure
• Many column and instrument companies developing products– Shimadzu– Phenomenex– Varian
67
16 PAHs by UPLC and Fast LC
Waters Acquity UPLC
Varian 920-LC Fast LC
Mobile Phase: Acetonitrile Water GradientDetection: UV@254nmTemperature: 40°CFlow-Rate: 0,8mL/minColumn: Pursuit XRs C18 3um, 100x2mm
68
Soft Drinks Using 920-LC
2.4 minutes
Fanta Orange Drink
Sugar Standard
2.72.62.52.42.32.22.121.91.81.71.61.51.41.31.21.110.90.80.70.60.50.40.30.20.1
170
165
160
155
150
145
140
135
130
125
120
115
110
105
100
95
90
85
80
75
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65
60
55
50
45
40
35
30
25
20
15
10
5
0
Ac
e K
So
diu
m B
en
zo
ate
Po
tas
siu
m S
orb
ate
Ca
ffe
ine
As
pa
rta
me
RT [min]
mAU Soft drinks_19.DATA [PDA detector Absorbance Analog Channel 1 ]
Conditions
Mobile Phase: 10mm Sodium Phosphate ph6.0/ACN gradientDetection: UV DetectionTemperature: 40°CFlow-Rate: 1,2mL/minColumn: Varian Ultra 50x2,0mm
69
Alkylated Phenones Using 920-LC
1.4 minutes
1.81.751.71.651.61.551.51.451.41.351.31.251.21.151.11.0510.950.90.850.80.750.70.650.60.550.50.450.40.350.30.250.20.150.10.05
110
105
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
Ace
toph
enon
e
Pro
piop
heno
ne
Ben
zoph
enon
e
Val
erop
heno
ne
Hex
anop
heno
ne
Hep
tano
phen
one
Oct
anop
heno
ne
TI
OF
F
RT [min]
mAU Phenones_31.DATA [PDA detector Absorbance Analog Channel 1 ]Conditions
Mobile Phase: Acetonitrile Water GradientDetection: UV DetectionTemperature: 60°CFlow-Rate: 1,6mL/minColumn: Varian Ultra 3um 50x2,0mm
70
Anthocyanadins Using 920-LC
2,221,81,61,41,210,80,60,40,20
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
RT [min]
mAU anthocyanidins mixtureHS_16_3.DATA [PDA detector Absorbance Analog Channel 2 ]
ConditionsMobile Phase:-solvent A Water phosphoric acid 0,3% -Solvent B Acetonitrile -Gradient separation from 88% A to 78% B in 2,5 minDetection: DAD 525 nmTemperature: 55°CFlow-Rate:0.9ml/minColumn:HP 3 C18 CPInjection volume : 1µl sample Delphinidin, Cyanidin, Petunidin, Pelargonidin, Peonidin, Mavidin
71
Fat Soluble Vitamins Using 920-LC
11109876543210
750
700
650
600
550
500
450
400
350
300
250
200
150
100
50
0 RT [min]
liposolubles vitamins C18 10cm_20_2.DATA [PDA detector Absorbance Analog Channel 1 ]mAU
11109876543210
1 600
1 500
1 400
1 300
1 200
1 100
1 000
900
800
700
600
500
400
300
200
100
0 RT [min]
liposolubles vitamins C18 10cm_20_2.DATA [Max. absorbance betw een [210,00 ; 500,00] nm]mAU
Chromatogram 260 nm
Chromatogram MaxPlot
Conditions
Mobile Phase: A Water 0;1% formic acid B CH3CN 0,1% Formic acid gradient t 0min B 80% ; t 1.5 min B80%; t 2min B 100% T 12 min B100%Detection: UV 260 nm & MaxPlotTemperature: 50°CFlow-Rate: 0,7mL/minColumn: Pursuit C18 3 µm 100 x 2,1mmInjection volume 25 µL, sample Retinol, vitamin D2,D3,E, E Ac, K1
72
Fast Sugars Using 920-LC
Fanta
Sprite
32.92.82.72.62.52.42.32.22.121.91.81.71.61.51.41.31.21.110.90.80.70.60.50.40.30.20.10
320
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255
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SP
W 0
.4
0ST
H 1
0.0
0T
I O
FF
SM
A 1
.0
0
TI O
N
Fru
cto
se
Glu
co
se
Su
cro
se
RT [min]
µRIU Fanta_25.DATA [RI detector Channel]
Fanta Orange Drink
32.92.82.72.62.52.42.32.22.121.91.81.71.61.51.41.31.21.110.90.80.70.60.50.40.30.20.10
230220210
200190180170160
150140130120
110100908070
60504030
20100
-10
-20-30-40-50-60
-70-80-90
-100
-110-120
SP
W 0.2
0S
TH
10.00
TI O
FF
SM
A 1.00
TI O
N
Fructose
Glu
cose
Suc
rose
RT [min]
µRIU Sugar Mix_50uL_41.DATA [RI detector Channel]
Sugar Standard
Conditions
Mobile Phase: Acetonitrile Water 75:25Detection: RI DetectionTemperature: 40°CFlow-Rate: 2,5mL/minColumn: Polaris NH2 5um 150x4,6mm
73
Varian 920-LC Conclusion
• UPLC does not currently suit all applications• Restricted column choices
• Demanding H/W are chemistry requirements
• Fast LC with 2.4 m to 3 m particle columns provides flexibility
• Compatible with 920-LC
– Low delay volume kit e.g mixer, flowcell, tubing
• Wide choice of chemistries
• Price competitive (can purchase 2 Fast LC for UPLC)
Varian 920-LC Target Markets
75
SDI 2006 Market by Industry Split
Other, 3%
Petrochem, 3%
Polymers, 4%
Ag/Food, 6%
Pharma, 28%
Indep Test, 10%
Industrial, 10%
Biotech, 11% Gov, 11%
Academic, 14%
76
Varian LC Sales by Industry Type
Academic30%
Pharma22%
Industrial 15%
Food/Ag4%
Enviro9%
R & D3%
Govt9%
other 8%
77
Varian 920-LC Target MarketsEnvironmental
• Build-on Spectroscopy, GC-MS & LC-MS products– Superb gradient pumping system
– Sensitive Fluoro detector for PAH’s
78
Varian 920-LC Target MarketsIndustrial
• Traditionally strong Varian market• Leverage profile of GC, Spectroscopy and Polymer
Lab products• Large range of systems
• Isocratic system with UV, RI, PL columns, standards & Galaxie add-in ideal for low temp GC applications
• 920-LC has a wide range of detection incl:
– UV, PDA, FL, RI, ELSD
Varian 920-LC Competitors
80
Varian 920-LC Market Competitors
Competitors• Shimadzu• Waters• Agilent• Dionex
•The 920-LC presents a significant step forward for the Varian
Varian 920-LC Compared to the Waters Alliance HPLC
82
Waters Alliance
• Consists of 2695 Separations Module• Pump, autosampler and controller
• Options of– 2996 Diode Array Detector
– 2487 Dual wavelength Detector
– 2475 multi-wavelength Detector
– 2414 RI Detector
– 432 Conductivity Detector
– 2420 ELS Detector
– 2465 Electrochemical Detector
83
Varian 920-LC Compared to Waters Alliance
Waters Alliance Varian 920-LC
Maximum flowrate 5 ml/min 10 ml/min
Maximum pressure
Autosampler Capacity
120 HPLC vials 200 HPLC vials
Microplate Capability NA 3 plates
Maximum Column Heater Temp
60oC 65oC
Number of Detectors None Up to 2 built-in
Sample Carryover <0.1% <0.01%
Varian 920-LC compared to the Agilent 1200 Series LC
85
Agilent 1200 Quaternary Pump G1354A
• Dual piston, low pressure quaternary gradient pump• Electromechanical check valve
• Variable piston stroke
• Pulse damper between primary and secondary piston
• Upgrade?? of 1100 series• Requires degasser module
86
Agilent 1200 Autosamplers
• Option of • G1329A standard Autosampler capable of 100 HPLC
vials
• G1367B Enhanced Autosampler compatible with either 2 microplates or 108 HPLC vials
• Standard carryover <0.1% but can be improved to <0.01% using needle wash
• Both have peltier cooled option
87
Agilent 1200 Detectors
• Wide range of detectors• Variable Wavelength Detector
• Multi-Wavelength Detectors
• Diode Array Detector
• Fluorescence Detector
• RI Detector
88
Varian 920-LC Compared to Agilent 1200
Agilent 1200 Varian 920-LC
Pulse Damper Yes Not Required
Microplate compatibility
Not Compatible Up to 3 96 well plates
Autosampler Capacity
100 HPLC vials 200 HPLC vials
Dual UV-Vis Capability
NA Yes
Carryover <0.1% <0.01%
UV-Vis range 190 – 600 D2 only 190 – 900 D2 & QI
UV detector noise + 0.75 x 10-5 Au + 0.5 x 10-5 Au
Applications of the 920-LC
Improved Analysis of 18 Polycyclic Aromatic Hydrocarbons using a Varian 920-LC Analytical
HPLC and Pursuit® 3 PAH Column
By Phuong TruongVarian Australia Pty Ltd
679 Springvale Rd Mulgrave VIC Australia
91
Varian 920-LC Analytical HPLC
92
Introduction
• PAH’s are carcinogenic contaminants produced as a by-product of combustion
– Found in drinking water, soils and sludges
• Can be analysed by GC, GC-MS• Compared to GC, HPLC offers
• Easier sample preparation
• Improved sensitivity with FLD
• Better selectivity
• PAH’s are difficult to analyse because of their structural similarity
• Results in long gradient runs of 25 to 30 minutes
93
Instrumentation
• Varian 920-LC Analytical HPLC (0010089300)• Quaternary gradient pump with 4 channel degasser
• 200 position autosampler with 100 L syringe
• Thermostated column compartment
• Diode Array detector
• Optional Fluorescence detector (99100xxx00)• Varian Pursuit® 3 PAH 100 x 4.6 mm column with
3m packing material (A7001100X046)
94
Instrument Conditions - Gradient Conditions
Time (min) Flow rate
(ml/min)
% Water % Acetonitrile
0.0 1.3 40 60
3.0 1.3 40 60
7.0 1.3 10 90
13.3 1.3 0 100
16.0 1.3 0 100
16.1 1.3 40 60
21.0 1.3 40 60
95
Instrument Conditions - Fluorescence Detector Conditions
Time (min) Excitation
(nm)
Emission
(nm)
0.0 270 323
5.6 250 375
7.1 270 410
8.5 265 380
10.5 280 420
14.7 293 498
96
Results
Component UV LOD
(g/L)
Fluoro LOD
(g/L)
USEPA RDL
(g/L)
Naphthalene 2 0.04 1.8
Acenaphthylene 5 NA 2.3
1-Methylnaphthalene
5 0.05 NR
2-Methylnaphthalene
5 0.06 NR
Acenaphthene 8 0.03 1.8
Fluorene 2 0.003 0.21
Phenanthrene 0.5 0.04 0.64
Anthracene 0.5 0.06 0.66
Fluoranthene 1 0.14 0.21
97
Results
Component UV LOD
(g/L)
Fluoro LOD
(g/L)
USEPA LOD
(g/L)
Pyrene 1 0.02 0.27
Benzo(a)anthracene 1 0.01 0.013
Chrysene 0.5 0.04 0.15
Benzo(b)fluoranthene 1 0.01 0.018
Benzo(k)fluoranthene 1 0.01 0.017
Benzo(a)pyrene 1 0.01 0.023
dibenzo(ah)anthracene
2 0.01 0.03
Benzo(ghi)perylene 2 0.01 0.076
Indenol(123-cd)pyrene 0.5 0.02 0.043
98
18 PAH by Varian 920-LC & Pursuit 3 PAH
99
16 PAH by Dionex HPLC
• Gradient separation at 1.5 ml/min with Fl Detection
100
17 PAH by Grace Vydac 201 TP 5 Column
• Flow rate 1.5 ml/min
101
Conclusion
• The Varian 920-LC and Pursuit 3 PAH column are capable of separating all 18 PAH’s in 15 minutes
• The reproducible gradient delivery and thermostated column compartment ensure excellent retention time reproducibility
• With the optional built-in fluorescence detector, detection limits exceed the requirements of USEPA method 8310
• Offers improvements in throughput compared to alternative HPLC separations (Grace Vydac 201 TP 5 column)
Analysis of Ibuprofen in Tablets According to the US Pharmacopoiea using a Varian 920-LC Analytical HPLC and Pursuit®XRs column
By Phuong TruongVarian Australia Pty Ltd
679 Springvale Rd Mulgrave VIC Australia
103
Varian 920-LC Analytical HPLC
104
Introduction
• Assays of active pharmaceutical ingredients (API) are common in the pharmaceutical industry
• Often used to quantitate the API and contaminants• Ibuprofen is a NSAI in widespread use around the world• 4-isobutylacetophenone (Ibuprofen related compound C) is a
breakdown product of Ibuprofen• It can adversely affect the central nervous system• The USP requires Ibuprofen related compound C to be
determined at < 0.1% per tablet• USP prescribe
• a 250 mm x 4.6 mm ID x 5 m L1 type (C18) column• Valerophenone internal standard
• In this work the recommended column was compared to a 100 x 4.6 x 3 m column
105
Instrumentation
• Varian 920-LC Analytical HPLC (0010089300)• Quaternary gradient pump with 4 channel degasser• 200 position autosampler with 100 L syringe• Thermostated column compartment• Diode Array detector
• Columns• Varian Pursuit®XRs columns feature 100Å silica, high
sample loadability, extended pH range, outstanding resolution and excellent column lifetime
– Varian Pursuit® XRs 100 x 4.6 mm x 3m column (A6011100X046)
– Varian Pursuit® XRs 250 x 4.6 mm x 5m column (A6010250X046)
106
Instrumental Conditions
Mobile Phase 1% chloroacetic acid in water pH 3/Acetonitrile 40:60 v/v
Flow rate 2 ml/min
Wavelength 254 nm
Temperature Ambient
107
Definitions – Resolution and Selectivity ()
'Bk
mAU
rt
mt
'Ak
108
Chromatographic Resolution and Selectivity
• Resolution R is similar to selectivity but takes into account peak width
• Baseline resolution is when R=1.5
• Capacity factor • Selectivity
m
mrA t
ttk
'
BA
ArBr
WW
ttR
])()[(2'
Ak
'
'
B
A
k
k
109
Standards - Pursuit®XRs 250 x 4.6 x 5m Column
• Ibuprofen and Internal Std with 250 mm x 5 m column
110
Standards – Pursuit®XRs 250 x 4.6 x 5m Column
• Internal Std and Ibuprofen related compound C with 250 mm x 5 m column
111
Results – Pursuit®XRs 250 x 4.6 x 5m Column
Resolution
(USP > 2.5)
Assymetry
(USP < 2.5)
Selectivity
Ibuprofen 1.29
I/S in Ibuprofen solution
7.3 1.00 1.29
I/S 1.06
Ibuprofen related C solution
5.1 1.01 1.18
112
Standards – Pursuit®XRs 100 x 4.6 x 3m Column
• Ibuprofen and Internal Std with Pursuit® XRs 100 mm x 3 m column
113
Standards – Pursuit®XRs 100 x 4.6 x 3m Column
• Internal Std and Ibuprofen related compound C with Pursuit® XRs 100 mm x 3 m column
114
Results – Pursuit® XRs 100 x 4.6 x 3m Column
Resolution
(USP > 2.5)
Assymetry
(USP < 2.5)
Selectivity
Ibuprofen 1.27
I/S in Ibuprofen solution
5.3 1.01 1.29
I/S 1.13
Ibuprofen related C solution
4.0 1.10 1.18
115
Ibuprofen Tablet
Chromatogram of Ibuprofen tablet sample spiked with I/S using a Pursuit® XRs 100 x 3m column
116
Spectral Confirmation using PDA Detector
117
Data for Ibuprofen Tablets
% of labelled Concentration of Ibuprofen
Assymetry of Ibuprofen
Assymetry of I/S
Resolution between Ibuprofen and Valerophenone I/S
101.9% 1.27 1.08 5.4
118
Conclusion
• The Varian 920-LC and Pursuit® XRs 3m columns significantly improve throughput of an Ibuprofen assay compared to the USP recommended columns
• The calculated concentration of Ibuprofen in the tablet complies with USP requirements of nominal + 10%
• The sensitivity of 920-LC PDA detector ensures the LOD for the Ibuprofen related C compound is 10 fold lower than USP
• The PDA detector allowed positive confirmation the unknown peak in the tablet was Ibuprofen related compound C (4-isobutylacetophenone)
119
Varian 920-LC All Your HPLC needs
• The 920-LC
– Is a robust high performance automated Analytical HPLC
– Fast LC compatible using 2.4/2.8 m columns
– Has a state-of-art LP quaternary gradient solvent delivery system
– A high capacity, low carry-over autosampler with leading performance specifications
– Features sensitive UV-Vis and PDA detection technology for unmatched performance
– An option of second detector fitted – Fluoro or RI
– Uses highly capable GalaxieTM software with a simpler, consistent instrument interface for real productivity
– Has automated AIQ and maintenance tools
Questions??