Next Generation of HPLC
Curtis R. Campbell, Ph.D.Shimadzu Scientific Instruments, Inc.
The BasisProminence HPLC (2005)– World’s 1st Web Enabled Controller
– World’s Fastest Autosampler
– World’s Cleanest Autosampler
The ContinuationProminence UFLCXR (2008)
– Enhanced Pressure Tolerance
– Reduced delay volume
– Advanced Materials
– NO compromise in basic performance
The Next GenerationNexera UHPLC (2010)– Widest Pressure and Flow Range
– Enhanced Autosampler Performance
– Unique Engineering and Materials
– NO compromise in basic performance
A rose by any other name…
• Chromatography– Regardless of the acronyms thrown about, that is what we are talking about here.
– The ability to reliably and reproducibly separate, detect, and quantitate a liquid mixture
– The results of which are used to make innumerable decisions
• Crucial not to sacrifice basic performance in exchange for pressure tolerance
Qualities of a Good System
• Speed– Cycle Time
– Carryover
Regardless of System Pressure
Cycle Time
Injection Analysis Injection Analysis Injection Analysis
Start0 min 0 sec
Injection Analysis Injection Analysis Injection Analysis
Other UHPLC30sec for injection
Simadzu UFLC10sec for injection
Injection Analysis
Injection Analysis
Injection Analysis
Finish3 min 0 sec
Finish2 min 0 sec
Finish1 min 40 sec
33% Faster
44% FasterNexera with overlapping injection
Carryover
• Two ways to attack carryover– Innovative engineering and proper material choices
– Provide effective countermeasures
Minimized Carryover
Flow‐through needle design to prevent carryover
Thorough rinse of sample path by multiple rinse
Dip wash and active wash of needle surface
HighHigh‐‐strength seal strength seal materialmaterial
To achieve zero carryover and high‐pressure capability •Fine‐tipped needle•Reduced sealing area•Smooth inner surface of needle•High‐strength seal material
Newly Designed Needle / Injection Port
‐‐ 60%60%
SIL‐20AXR SIL‐30AReduced Reduced Sealing areaSealing area
Qualities of a Good System
• Speed– Cycle Time
– Carryover
• Precision– Gradient Reproducibility
– Injection Accuracy and Reproducibility
Regardless of System Pressure
Gradient Reproducibility‐
Zorbax Eclipse plus C18, 2.1mm, 50 mm, Water / Acetonitrile 15 → 95% (0.80min) 1.2 mL/min20 uL Shimadzu MIRC mixer40C245nm2 uL injection, 25ppm
R.T.
%RSD
Area R.T.
Acetophenone 0.51 min 0.156 0.068
Propiophenone 0.62 min 0.278 0.039
Butyrophenone 0.70 min 0.108 0.034
Valerophenone 0.77 min 0.259 0.045
Hexanophenone 0.84 min 0.196 0.042
Heptanophenone 0.90 min 0.260 0.034
Octanophenone 0.96 min 0.220 0.0330.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00min
0
25000
50000
75000
100000
125000
150000
175000
200000
225000
uV
N=6 (overlay)
Excellent gradient reproducibility even in the ultra fast analysis.
Injection volume precision & linearity in the range of 1 to 50 μL @ 100 MPa
N=6 x 5set %RSD (Area) Spec.
1 μL x 6 0.17 %
2 μLx 6 0.23 %
5 μLx 6 0.06 % < 0.25 %
10 μLx 6 0.11 %
20 μLx 6 0.12 %
50 μLx 6 0.10 %
Injection volume
Peak Area (n=6) Linearity
1 μL 63857 3.4 %
2 μL 129668 1.9 %
5 μL 329103 0.4 %
10 μL 664574 - 0.5 %
20 μL 1328747 - 0.5 %
50 μL 3305754 ±0.0 %
Correlation Coefficient 0.9999960.999996 > 0.999
Sample : Caffeine 20 mg/L
Precision Linearity
Injection Precision and Linearity
Qualities of a Good System
• Speed– Cycle Time
– Carryover
• Precision– Gradient Reproducibility
– Injection Accuracy and Reproducibility
• Reliability
Regardless of System Pressure
Ω shaped rotor
New High Pressure Valve DesignTo withstand operation @ 130 MPa
•Minimized friction by specially coated stator•Ω‐shaped rotor seal (Patent pending) → to equalize pressure
Distribution of force eliminates rotor tilt →longer consumable life
High Pressure area @High Pressure area @ Run and Load/INJ modeRun and Load/INJ mode
Low pressure area @ Load modeLow pressure area @ Load mode
Qualities of a Good System
• Speed– Cycle Time
– Carryover
• Precision– Gradient Reproducibility
– Injection Accuracy and Reproducibility
• Reliability
Regardless of System Pressure
••System FlexibilitySystem Flexibility
Unsurpassed Flexibility
•First and foremost the system has to be able to run current methods•Must have the ability to adapt for system variations•Capable of growing with the application demands
Pumping Configuration
• Two ways to form a gradient– Binary High Pressure Mixing
– Low Pressure Gradient Formation (LPGE)
Pumping Configuration
AbsorbanceDetector
LC/MS
• Neither is “correct”
• Combination of the two is optimal
• Modular system allows the ability to do even more– 2 Dimensional system
– Multi‐plex system
Multiplexed System for Higher Throughput
The MPX‐2 is a dual stream multiplexing solution to increase LCMS/MS throughput. The samples are alternately injected from the two UPLC lines without dead time.
Injector Capabilities
• Minimized carryover
• Multiple injection modes
• Flexible format– Different sample vial types
– Expandability for high throughput labs
Loop injection
A
B
C
D
E
Direct injection
B
CD
E
Tube B LPV ‐ HPVTube C Pump ‐ HPVTube D HPV ‐ column
Tube E HPV ‐ drain valve
Tube A Sample loop (1.5uL/5uL/20uL)
Tube B LPV ‐ sample loop
Tube C Pump – HPVTube D HPV ‐ column
Tube E HPV ‐ drain valve
Flow Diagram for Fixed Loop Injection
HPV
LPV
Drainvalve
Column
Pump
HPV
LPV
Drainvalve
Column
Pump
134 uL 18 uL
Rack Changer for More Sample Capacity
Provides over 4600 consecutive sample analysis96 well, 384 well, and vial platescoolable rack changer (4 to 40 ºC)
Summary
• Prominence HPLC– Great All Round HPLC
• ProminenceXR– Fantastic All Round Enhanced HPLC
• Nexera UHPLC– The next era of HPLC– Widest Pumping Range– Fastest, Cleanest Autosampler– Most Flexible
Summary• Wide Range of S/W Control
– Shimadzu Lab Solutions– Atlas by ThermoFisher– Chromeleon by Dionex– Clarity by Data Apex– Empower by Waters– EZChrom Elite by Agilent
• MS S/W Control– Analyst by AB Sciex– Xcalibur by ThermoFisher– Hystar by Bruker
THANK YOU
I look forward to the discussion and your questions.
For more go to Keyword: Nexera
Carryover
Chlorhexidine 2,000 ng/uL
Post-blank 1 0.0006%0.0006%Post-blank 2 0.0003%
Post-blank 3 0.0004%
Carryover of chlorohexidine @ 100 MPa
Results : 0.0006 % Without injection port rinse, needle internal rinse
With needle outer surface rinse
Carryover
Column : ODS (2.0 mmI.D.×100 mmL., 1.8 um)Mobile Phase: : Methanol, Water= 2/8Flow rate : 0.4 mL/minTemperature : 30 Injection volume : 5 μLRinse solution : 0.05 % formic acid in methanolNeedle wash : outer surface flush by rinse pump (1 second flush)
: needle soak wash (0 second)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0 mV
50
60
70
80
90
100
110
MPa
Detection :260nm
Carryover
Chlorhexidine 2,000 ng/uL
Post-blank 1 N.D.N.D.
Carryover Carryover of chlorohexidine @ 100 MPa
Specification : < 0.0015< 0.0015%,
Results : Not detected With injection port rinse, needle internal rinse and needle outer surface rinse
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min-1
0
1
2
3
4mV
50
60
70
80
90
100
110
MPaDetection :260nm
Column : ODS (2.0 mmI.D.×100 mmL., 1.8 um)Mobile Phase: : Methanol, Water= 2/8Flow rate : 0.4 mL/minTemperature : 30 Injection volume : 5 μLRinse solution : 0.05 % formic acid in methanolNeedle wash : outer surface flush by rinse pump (1 second flush)
: needle soak wash (0 second): needle internal rinse
Injection port rinse : performed
Rinse Function Test 1 Test 2 Test 3 Test 4
Injection Port Rinse New
Needle Internal Rinse New
Needle Internal Flush–Back & Forth Rinse- New
Needle Outer surface rinse-Needle Dip-
Needle Outer surface rinse-Flush by Rinse pump- 1sec
Carryover ( % ) 0.0106 %0.0106 % 0.0006 %0.0006 % 0.0015 %0.0015 % N.D.N.D.
Carryover Summary ‐Chlorohexidine‐
Chlorohexidine 2000 ng/μL, 5 μL injection
Binary Pumping system