![49
Prelable derivatization, internal standard addition, sample dilution, etc.
0.0 1.0 2.0 3.0 4.0 5.0 min
Asp
Ser
Gly
Thr
Arg
His
Ala
Tyr
Val
Met
Ile
Phe
Leu
Lys
Glu
Arg
Val
Ile
Leu
Amino acid standard
Analysis of BCAA in
supplement
[Conditions]
Column : ODS column (3.0mm i.d. x 50mm, 2.2μm)
Mobile phase : A :20mmol/L Potassium Phosphate Buffer
: B :aceotnitrile
Flow rate : 1.2mL/min
Column temp. : 40 ℃
Detection : Fluorescence detection
(Ex. 350nm, Em 450nm)
Auto Sample Pretreatment by SIL-30AC
Analysis of OPA derivatized amino acids](https://image.slidesharecdn.com/nexeraintroductionpresentationapr19-231019114909-14315f06/85/Nexera-Introduction-presentation-Apr19-ppt-49-320.jpg)
Nexera Introduction presentation Apr19.ppt
- 2. Background of Development How maximize sample throughput ? Rethinking of efficiency and productivity Consideration of efficiency/cost Many UHPLCs have been commercialized during this 6 years. Ultra-high pressure delivery and smaller column particles are the key for speed-up of analysis. All focus on pressure. The performance is compromised in exchange for pressure ? Flow rate limitation Slow injection speed Poor reproducibility Poor durability Limited expandability Etc….. ?
- 3. Development Concept of Nexera Expandability Performance UHPLC UFLC HPLC Maximizing Throughput Maximizing Expandability Maximizing Performance •Highest pressure range with widest flow rate range. •Fastest cycle time for maximization of sample throughput •Solvent consumption reduction for environment To produce next generation UHPLC without compromise of performance •Highest accuracy and precision in ultra fast analysis •Fastest injection with minimized carryover •Versatile system configuration •Pretreatment, multi-dimensional LC with valve control, etc.
- 4. CTO-30A • Minimized temperature distribution in the column -Usable up to 150℃ LC-30AD • World’s highest pressure Specification 130MPa • Ultra Low volume high efficiency Mixer（20μL） SIL-30AC • 130MPa Max pressure • 4 liquid rinsing • AutoPretreatment • Loop injection （optional） Rack Changer II • High sample capacity (4600 samples ) • front window to support easier maintenance. Nexera New Component Detectors • Shimadzu high-sensitivity detector lineup, UV, PDA, RF, MS spec….. • High speed sampling
- 5. Nexera Features, Maximizing Throughput Ultra-high Speed/Ultra-high Resolution Analysis •World’s highst pressure 130MPa •Minimized system delay volume Ultra-Fast Injection •World’s fastest autosampler 10sec/ injection •Minimized injection time by overlap injection Maximized Sample Capacity •Nexera Rack changer 4600 samples •Multiplexed system
- 6. Nexera Features, Maximizing Throughput Ultra-high Speed/Ultra-high Resolution Analysis •World’s highst pressure 130MPa •Minimized system delay volume Ultra-Fast Injection •World’s fastest autosampler 10sec/ injection •Minimized injection time by overlap injection Maximized Sample Capacity •Nexera Rack changer 4600 samples •Multiplexed system
- 7. S4-Plunger surface(x50) World’s Highest Pressure 130MPa S4–Plunger （Super Smooth Surface Sapphire） Minimized frictional heat on plunger surface Optimized material for UHPLC Plunger, Plunger seal, Needle seal, High-pressure valve, etc. New high pressure valve design High-strength seal material S4-Plunger High-pressure valve
- 8. Vendor W Vendor A Vendor T Vendor D Vendor A’ MPa bar 1200 1000 800 600 400 200 mL/min 1400 Shimadzu Nexera Power Range
- 9. Nexera Ultra-High Speed High Speed and High Resolution Ultra-High Resolution Enhanced Balance between Speed and Resolution Nexera meets customers’ diverse needs in speed/resolution thanks to the widest pressure range .
- 10. Ultra-high Speed Analysis 0.0 2.5 5.0 7.5 10.0 12.5 15.0 min 0 25 50 75 100 125 mAU Shim-pack VP-ODS (4.6mmi.d.150mm, 5um) 1.0 mL/min Water/acetonitrile = 45/55 40℃ 245nm 5 um 1.0 mL /min 5.4 MPa TPN at peak 5 = 11352 Rs (4,5 = 10.849) Zorbax eclipese plus C18 ODS (2.1mmi.d. 50mm, 1.8 um) 0.6 mL/min Water/acn = 45/55 40C 245nm 1. Acetophenone 2. Propiophenone 3. Butyrophenone 4. Valerophenone 5. Hexanophenone 25 times faster 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 min 1050 1075 1100 1125 1150 1175 mAU 45.0 46.0 47.0 48.0 49.0 50.0 51.0 52.0 MPa 1.8 um, 50mm 1.8mL /min 105 MPa TPN at peak 5 = 6634 Rs (4,5 = 8.40)
- 11. Ultra-High Resolution 0.0 1.0 2.0 3.0 4.0 5.0 min 0 25 50 75 100 125 mAU Shim-pack VP-ODS (4.6mmi.d. 150mm, 5 um) 1.0 mL/min Water/acn = 30/70 40C 245nm 1.8 um, 300 mmL 115 MPa TPN at peak 7 = 69325 * 130 MPa allows to use longer columns with sub-2 um particles. In this example, two 150mm columns with sub 1.8 um were connected in series. 0.0 2.5 5.0 7.5 10.0 12.5 min 1 2 3 4 5 6 7 5 um, 150 mmL 4.7 MPa TPN at peak 7 = 14800 Zorbax eclipese plus C18 ODS (2.1mmi.d. 300mm, 1.8 um) 0.5 mL/min Water/acn = 20/80 50C 245nm 5 times higher separation efficiency 1. Acetophenone 2. Propiophenone 3. Butyrophenone 4. Valerophenone 5. Hexanophenone 6. Heptanophenone 7. Octanophenone
- 12. Speed and Resolution 0.0 2.5 5.0 7.5 10.0 12.5 15.0 min 0 25 50 75 100 125 mAU Shim-pack VP-ODS (4.6mmi.d.150mm, 5um) 1.0 mL/min Water/acetonitrile = 45/55 40℃ 245nm 5 um, 150mm 1.0 mL /min 5.4 MPa TPN at peak 5 = 11352 Rs (4,5 = 10.849) Zorbax eclipese plus C18 ODS (2.1mmi.d. 50mm, 1.8 um) 0.6mL/min Water/acn = 45/55 40C 245nm 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 min 0 25 50 75 100 125 150 175 mAU 14 times faster 1.5 times higher separation efficiency 1.8 um, 100mm 1.4 mL /min 100 MPa TPN at peak 5 = 16510 Rs (4,5 = 12.21) 1. Acetophenone 2. Propiophenone 3. Butyrophenone 4. Valerophenone 5. Hexanophenone
- 13. Area %RSD(n=6) Peak1 0.29 peak2 0.05 peak3 0.15 Peak4 0.15 Peak5 0.26 0.0 0.1 0.2 0.3 0.4 0.5m in 0.0 1.0 2.0 3.0 4.0 5.0 6.0 m V(x100) 25.0 50.0 75.0 % B.Conc.(Method) Detector A:254nm t0 1 2 3 4 5 Maximized Peak Capacity Peak capacity = W tg +1 tg : gradient time W : peak width at 50 % of peak height Peak capacity at peak 4 : 190 in 24 sec Peak width at t0 : 80 msec Reproducibility Phenomenex Kinetex 2.1mm 30mm (2.6 um) Column Temp : 70C Flow : 3.0 mL/min A: water, B : acetornitrile GE : 2%(0min) 98% (0.40 min) UV 254nm t0 :Uracil Peak 1 :Methylparaben Peak 2 :acetophenone Peak 3: Propyl paraben Peak 4 :Butyl paraben Peak 5 : Naphthalene
- 14. Peak capacity : 244 in 8 minutes Ultra High Resolution Application For improvement of separation efficiency in proteomic, food, pharama and various application fields 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 min 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 mAU BSA triptic digest Column : Zorbax Eclipse RRHD C18 2.1mm, 100mm, 1.8um Mobile phase : A : 0.03 % TFA in water B : 0.03 % TFA in acetonitrile Gradient : B 5% → 40 % (8 min) Mixer: 180 uL Flow rate : 0.9 mL/min Column temp.: 40C Detection : 214 nm Sample : Trypsin digested BSA (total of 1 pmol / uL)
- 15. Minimized Delay Volume MiRC Mixer incorporating micro reactor technology (20 uL) Loop injection (delay volume: 18 uL) Low volume injection valve and injection port High-pressure valve MiRC mixer Injection port
- 16. System Delay Volumes Nexera Nexera with 5uL fixed loop injection ProminenceXR Inlet tube OD1.6xID0.3 x400mm 28.27 OD0.8xID0.1x 600mm 4.71 OD1.6xID0.3 x400mm 28.27 Auto-sampler Sample loop (50uL) 84.82 Sample loop (5uL) 5.00 Sample loop (50uL) 84.82 Others 14.84 Others 1.57 Others 14.92 Outlet tube OD0.8xID0.1 x800mm 6.28 OD0.8xID0.1 X800mm 6.28 OD0.8xID0.1 x600mm 4.71 Total 134.21 17.56 132.72 Unit: mL *Plus the selected mixer volume
- 17. Incorporating micro reactor technology (MiRC) Split / merge flow mechanism Multi layer structure Lowest delay volume standard : 20 mL TFA applications : 180 mL 17 A A+B A A B B MiRC Gradient Mixer
- 18. ① ④ ① ② ③ ④ ⑤ ⑥ ⑦ ⑥ ⑤ ③ ② Loop injection A B C D E ① ④ ① ② ③ ④ ⑤ ⑥ ⑦ ⑥ ⑤ ③ ② Direct injection B C D E Tube B LPV① - HPV② Tube C Pump - HPV⑥ Tube D HPV⑤ - column Tube E HPV③ - drain valve Tube A Sample loop (1.5uL/5uL/20uL) Tube B LPV① - sample loop Tube C Pump – HPV② Tube D HPV① - column Tube E HPV⑤ - drain valve Flow Diagram for Fixed Loop Injection HPV LPV Drain valve Column Pump HPV LPV Drain valve Column Pump
- 19. Column :Kinetex 2.1mm, 50mm, 2.6um Mobile phase :A: water :B: acetonitrile Gradient :0min (B.15%), 1min (B 80%), 1.01min(B15%), Flow rate :0.8mL/min Methylparaben:50ppm, Acetophenone :50ppm Propylparaben:50ppm, Butylparaben :50ppm 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 min -25000 0 25000 50000 75000 100000 125000 150000 175000 200000 225000 250000 275000 300000 325000 uV Direct inj Fixed loop inj Minimized Gradient Delay Minimized internal volume in mobile phase path 254nm
- 20. Column :Kinetix 2.1mm, 50mm, 2.6um Mobile phase :A: water :B: acetonitrile, A/B =7/3 Flow rate :0.8mL/min Methylparaben:50ppm、Acetophenone:50ppm Propylparaben :50ppm、Butylparaben:50ppm When isocratic conditions are used, width and theoretical plates # of early eluted peaks are improved. Rt k' Peak width50% (sec) Theoretical plates Direct inj Loop inj Direct inj Loop inj uracil 0.15min - 0.610 0.495 - - methy paraben 0.42min 1.8 0.777 0.649 5073 7006 acetophenone 0.61min 3 0.966 0.865 7346 8880 propylparaben 1.32min 7.8 2.033 1.926 8173 9036 butylparaben 2.75min 17.2 4.237 4.150 8808 9232 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 min 0 25000 50000 75000 100000 125000 150000 uV 0.30 0.35 0.40 0.45 min 0 25000 50000 75000 100000 125000 150000 175000 uV Total inj Loop inj Loop inj Total inj Methl Paraben 254nm Improved Peak Width
- 21. Nexera Features, Maximizing Throughput Ultra-high Speed/Ultra-high Resolution Analysis •World’s highst pressure 130MPa •Minimized system delay volume Ultra-Fast Injection •World’s fastest autosampler 10sec/ injection •Minimized injection time by overlap injection Maximized Sample Capacity •Nexera Rack changer 4600 samples •Multiplexed system
- 22. World Fastest Autosampler Reliable X-Y-Z needle carrying mechanism to enable high speed motion Ultra-low carryover design eliminates extra rinse time.
- 23. Overlap Injections Injection Analysis Injection Analysis Injection Analysis Start 0 min 0 sec Injection Analysis Injection Analysis Injection Analysis Other UHPLC 30sec for injection Simadzu UFLC 10sec for injection Injection Analysis Injection Analysis Injection Analysis Finish 3 min 0 sec Finish 2 min 0 sec Finish 1 min 40 sec 33% Faster 44% Faster Nexera with overlapping injection
- 24. 24 Example of Overlap Injections 30 % cycle time reduction (in case of 0.7 min/1 cycle) 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 min 0 10 20 30 40 50 60 70 80 90 mV 0 50 100 150 200 250 300 350 400 450 kgf/cm2 ŒŸ oŠíA 245nm 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 min 0 10 20 30 40 50 60 70 80 90 mV 0 50 100 150 200 250 300 350 400 450 kgf/cm2 ŒŸ oŠíA 245nm
- 25. 25 Mobile phase Flow amount (L) 100MPa 130MPa Water 176.4 25.2 100 mM phosphate buffer 80.6 20.2 Methanol 156.2 22.0 Acetonitrile 141.1 25.2 Flow rate : 3.5 mL/min Longer seal life supports UHPLC analysis. At even 100 MPa, seal life of about half year
- 26. Nexera Features, Maximizing Throughput Ultra-high Speed/Ultra-high Resolution Analysis •World’s highst pressure 130MPa •Minimized system delay volume Ultra-Fast Injection •World’s fastest autosampler 10sec/ injection •Minimized injection time by Overlap injection Maximized Sample Capacity •Nexera Rack changer 4600 samples •Multiplexed system
- 27. Rack Changer for More Sample Capacity Provides over 4600 consecutive sample analysis 96 well, 384 well, and vial plates coolable rack changer (4 to 40 ºC)
- 28. Multiplexed System for Higher Throughput The multiplexed system (MPX) is a dual stream multiplexing solution to increase LCMS/MS throughput. The samples are alternately injected from the two UPLC lines without dead time.
- 29. Nexera Features, Maximizing Performance Minimized carry over for trace analysis •Flow-through needle with minimized contact area •Thorough rinse of sample path and needle surface by multiple solvents (up to 4) Precise and accurate injection at lower volume •Direct injection with high-resolution metering pump •Metering pump isolated from flow path Highly-reproducible gradient at ultra high speed conditions •High-resolution gradient control •Efficient mixing mechanism by MiRC mixer Uniform column heating for UHPLC •Minimized temperature distribution across column by IHB •Efficient preheating by small volume preheater
- 30. Nexera Features, Maximizing Performance Minimized carry over for trace analysis •Flow-through needle with minimized contact area •Thorough rinse of sample path and needle surface by multiple solvents (up to 4) Precise and accurate injection at lower volume •Direct injection with high-resolution metering pump •Metering pump isolated from flow path Highly-reproducible gradient at ultra high speed conditions •High-resolution gradient control •Efficient mixing mechanism by MiRC mixer Uniform column heating for UHPLC •Minimized temperature distriution across column by IHB •Efficient preheating by small volume preheater
- 31. 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
- 32. Carryover of neutral compounds (Caffeine ) @ 100 MPa Specification : < 0.0015% Results : 0.0004 % Without injection port rinse, needle internal rinse and needle outer surface rinse Carryover Caffeine 2000 ng/mL － Post-blank 1 0.0004% Post-blank 2 N.D. Column : ODS (2.0 mmI.D.×100 mmL., 1.8 um) Mobile Phase: : Methanol, Water= 2/8 Flow rate : 0.4 mL/min Temperature : 40 ℃ Injection volume : 5 mL Carryover - Caffeine - Caffeine (4 g/L) 0 1.0 2.0 3.0 4.0 5.0 min 0.0 1.0 2.0 3.0 4.0 5.0 mAU Blank 0.0004%
- 33. Carryover Chlorhexidine 2,000 ng/uL － Post-blank 1 0.0006% Post-blank 2 0.0003% Post-blank 3 0.0004% Carryover of chlorohexidine @ 100 MPa Specification : < 0.0015% 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= 2/8 Flow rate : 0.4 mL/min Temperature : 30 ℃ Injection volume : 5 mL Rinse solution : 0.05 % formic acid in methanol Needle 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 4.0mV 50 60 70 80 90 100 110 MPa Detection :260nm Carryover - Chlorohexidine -
- 34. Carryover Chlorhexidine 2,000 ng/uL － Post-blank 1 N.D. Carryover Carryover of chlorohexidine @ 100 MPa Specification : < 0.0015%, Results : Not detected With injection port rinse, needle internal and 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 MPa Detection :260nm Column : ODS (2.0 mmI.D.×100 mmL., 1.8 um) Mobile Phase: : Methanol, Water= 2/8 Flow rate : 0.4 mL/min Temperature : 30 ℃ Injection volume : 5 mL Rinse solution : 0.05 % formic acid in methanol Needle wash : outer surface flush by rinse pump (1 second flush) : needle soak wash (0 second) : needle internal rinse Injection port rinse : performed Carryover - Chlorohexidine -
- 35. Nexera Features, Maximizing Performance Minimized carry over for trace analysis •Flow-through needle with minimized contact area •Thorough rinse of sample path and needle surface by multiple solvents (up to 4) Precise and accurate injection at lower volume •Direct injection with high-resolution metering pump •Metering pump isolated from flow path Highly-reproducible gradient at ultra high speed conditions •High-resolution gradient control •Efficient mixing mechanism by MiRC mixer Uniform column heating for UHPLC •Minimized temperature distriuition across column by IHB •Efficient preheating by small volume preheater
- 36. Injection volume precision & linearity in the range of 1 to 50 mL @ 100 MPa N=6 x 5set %RSD (Area) Spec. 1 mL x 6 0.17 % 2 mLx 6 0.23 % － 5 mLx 6 0.06 % < 0.25 % 10 mLx 6 0.11 % － 20 mLx 6 0.12 % － 50 mLx 6 0.10 % － Injection volume Peak Area (n=6) Linearity 1 mL 63857 3.4 % 2 mL 129668 1.9 % 5 mL 329103 0.4 % 10 mL 664574 - 0.5 % 20 mL 1328747 - 0.5 % 50 mL 3305754 ±0.0 % Correlation Coefficient 0.999996 > 0.999 Sample : Caffeine 20 mg/L Precision Linearity Injection Precision and Linearity in Direct mode
- 37. Nexera Features, Maximizing Performance Minimized carry over for trace analysis •Flow-through needle with minimized contact area •Through rinse of sample path and needle surface by multiple solvents (up to 4) Precise and accurate injection at lower volume •Direct injection with high-resolution metering pump •Metering pump isolated from flow path Highly-reproducible gradient at ultra high speed conditions •High-resolution gradient control •Efficient mixing mechanism by MiRC mixer Uniform column heating for UHPLC •Minimized temperature distribution across column by IHB •Efficient preheating by small volume preheater
- 38. Time %B High resolution gradient control Conventional gradient control 1/6 High Resolution Gradient Control
- 39. Gradient Reproducibility- Zorbax Eclipse plus C18, 2.1mm, 50 mm, Water / Acetonitrile 15 → 95% (0.80min) 1.2 mL/min 20 uL mixer 40C 245nm 2 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 0.033 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 min 0 25000 50000 75000 100000 125000 150000 175000 200000 225000 uV N=6 (overlay) Excellent gradient reproducibility even in the ultra fast analysis.
- 40. Nexera Features, Maximizing Performance Minimized carry over for trace analysis •Flow-through needle with minimized contact area •Through rinse of sample path and needle surface by multiple solvents (up to 4) Precise and accurate injection at lower volume •Direct injection with high-resolution metering pump •Metering pump isolated from flow path Highly-reproducible gradient at ultra high speed conditions •High-resolution gradient control •Efficient mixing mechanism by MiRC mixer Uniform column heating for UHPLC •Minimized temperature distribution across column by IHB •Efficient preheating by small volume preheater
- 41. Uniformed Column Heating IHB (Intelligent Heat Balancer ) Independently controllable two heaters to minimized temperature distribution across column Low volume preheater (< 3uL)
- 42. Nexera Features, Maximizing Expandability Scalable modular UHPLC design •Wide range-configurable system by combination of conventional HPLC units Auto sample pretreatment by autosampler •Precolumn derivatization •Internal standard addition, sample dilution High temperature analysis150℃ Column oven •Wide temperature range control by Column oven CTO-30A (up to 150 ℃) •Use of new technology area such as green chromatography High speed LCMS analysis •Combination with ultra fast LCMS, LCMS-2020
- 43. Nexera Features, Maximizing Expandability Scalable modular UHPLC design •Wide range-configurable system by combination of conventional HPLC units Auto sample pretreatment by autosampler •Precolumn derivatization •Internal standard addition, sample dilution High temperature analysis150℃ Column oven •Wide temperature range control by Column oven CTO-30A (up to 150 ℃) •Use of new technology area such as green chromatography High speed LCMS analysis •Combination with ultra fast LCMS, LCMS-2020
- 44. Multi Dimensional Configuration with valve control
- 45. 1ppmX1μL (n=6) 1 2 3 4 5 6 7 8 9 10 11 1.Norfloxacin 2.Ofloxacin 3.Ciproploxacin 4.Danofloxacin 5.Enrofloxacin 6.Orbifloxacin 7.Sarafloxacin 8.Difloxacin 9.Oxolinic acid 10.Nalidixic acid 11.Flumequine Use of High Sensitive RF Detector Ultra-high sensitive RF detector, RF-20A/RF-20AX water/raman S/N ratio : >2000 (RF-20AXS) Stable analysis by temperature controlled cell Quinolones analysis by Nexera-RF Ex.295nm Em.455nm (0-1.05min) Ex.325nm Em.365nm, (1.05-3min)
- 46. Scalable Pump Configuration Isocratic, binary and ternary gradient are available. MiRC mixer allows gradient elution by combination of various mobile phases. Analysis of DNPH-aldehydes/ketones by ternary GE 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 min -5000 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 55000 60000 65000 70000 75000 80000 uV 1 2 3 4 5 6 7 8 9 10 11 12 13 1. Formaldehyde 2. Acetaldehyde 3. Acetone 4. Acrolein 5. Propionaldehyde 6. Crotonaldehyde 7. 2-Butanone 8. Methacrolein 9. N-Butylaldehyde 10. Benzaldehyde 11. Valeraldehyde 12. m-Tolualdehyde 13. Hexaldehyde Column : Phenomenex Kinetex , 2.1mm, 50mm, 2.6 um Mobile phase : A: water / B :acetonitrile / C : THF (Ternary gradient) Flow rate : 0.9mL/min Column temp. : 50C Detection : 360 nm
- 47. 0.0 0.5 1.0 1.5 2.0 2.5 min 0.0 2.5 5.0 7.5 10.0 12.5 mAU Higher pressure capability allows alternate solvent usage for selectivity purpose. Analysis of cortisone and cortisol in Urine (each 5 ppm, spiked; UV @ 254nm; 40C) Mobile phase : A :0.1 % formic acid in water : B : acetonitrile Mobile phase : A : 0.1 % formic acid in water : B : methanol Cortisone Cortisol 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 min 0 5 10 15 20 25 30 mAU Cortisol Cortisone Methanol ACN : 69 MPa Methanol : 108 MPa Use of Alternate Solvents for Separation
- 48. Nexera Features, Maximizing Expandability Scalable modular UHPLC design •Wide range-configurable system by combination of conventional HPLC units Auto sample pretreatment by autosampler •Precolumn derivatization •Internal standard addition, sample dilution High temperature analysis150℃ Column oven •Wide temperature range control by Column oven CTO-30A (up to 150 ℃) •Use of new technology area such as green chromatography High speed LCMS analysis •Combination with ultra fast LCMS, LCMS-2020
- 49. 49 Prelable derivatization, internal standard addition, sample dilution, etc. 0.0 1.0 2.0 3.0 4.0 5.0 min Asp Ser Gly Thr Arg His Ala Tyr Val Met Ile Phe Leu Lys Glu Arg Val Ile Leu Amino acid standard Analysis of BCAA in supplement [Conditions] Column : ODS column (3.0mm i.d. x 50mm, 2.2μm) Mobile phase : A :20mmol/L Potassium Phosphate Buffer : B :aceotnitrile Flow rate : 1.2mL/min Column temp. : 40 ℃ Detection : Fluorescence detection (Ex. 350nm, Em 450nm) Auto Sample Pretreatment by SIL-30AC Analysis of OPA derivatized amino acids
- 50. Nexera Features, Maximizing Expandability Scalable modular UHPLC design •Wide range-configurable system by combination of conventional HPLC units Auto sample pretreatment by autosampler •Precolumn derivatization •Internal standard addition, sample dilution High temperature analysis150℃ Column oven •Wide temperature range control by Column oven CTO-30A (up to 150 ℃) •Use of new technology area such as green chromatography High speed LCMS analysis •Combination with ultra fast LCMS, LCMS-2020
- 51. Uniform temperature control at high temperature conditions by CTO-30A oven Optional post-column cooler minimize noise increase at high temperature conditions. Reliable High-Temperature Control
- 52. Mobile phase: water/acetonitrile (3/7, v/v), Detection: absorbance at 245 nm. Peaks; 1: actophenone 2: propiophenone 3: butyrophenone 4: balenophenone 5: hexanophenone 6: heptanophenone 7: octanophenone Improved Column Efficiency by Elevated Temperature Column efficiency improves as column temperature increases. Increased theoretical plate number Decreased column pressure 4 5 6 7 0.00 0.25 0.50 0.75 1.00 1.25 min -300 -250 -200 -150 -100 -50 0 50 100 150 mAU 0.00 0.25 0.50 0.75 1.00 1.25 min -150 -100 -50 0 50 100 150 200 250 300 350 mAU 0.00 0.25 0.50 0.75 1.00 1.25 min 0 50 100 150 200 250 300 350 400 450 500 mAU 1 2 3 Shim-pack XR-ODS (3.0 mmi.d. ´ 50 mm , 2.2mm) Flow rate : 1.5mL/min Temperature : 80° C (Pressure : 11.8MPa) N for Peak 7 : 9,886 Temperature : 60° C (Pressure : 14.0MPa) N for Peak 7 : 9,426 Temperature : 40° C (Pressure : 17.4MPa) N for Peak 7 : 9,154 4 5 6 7 0.00 0.25 0.50 0.75 1.00 1.25 min -300 -250 -200 -150 -100 -50 0 50 100 150 mAU 0.00 0.25 0.50 0.75 1.00 1.25 min -150 -100 -50 0 50 100 150 200 250 300 350 mAU 0.00 0.25 0.50 0.75 1.00 1.25 min 0 50 100 150 200 250 300 350 400 450 500 mAU 1 2 3 Shim-pack XR-ODS (3.0mm i.d., 50mmL, 2.2 um) Flow rate : 1.5 mL/min Temperature : 80℃ (Pressure : 11 MPa) N for Peak 7 : 9,886 Temperature : 60℃ (Pressure : 14 MPa) N for Peak 7 : 9,426 Temperature : 40℃ (Pressure : 17 MPa) N for Peak 7 : 9,154
- 53. High temperature analysis (mobile phase : only water) Green HPLC 1.0 2.0 3.0 4.0 5.0 6.0 min Conventional LC Green LC 1 : Theophylline 2 : Caffeine 1 2 1 2 150℃ Water only 40℃ 30% methanol Green LC Column : Shodex ET-RP1 (3.0mm,150mm) Mobile phase : water Flow rate : 0.5 mL/min Column temp. : 150C Conventional LC Column : ODS (4.6mm, 150mm, 5 um) Mobile phase : methanol / water = 3/7 Flow rate : 1.0 mL/min Column temp. : 40C Green HPLC
- 54. Nexera Features, Maximizing Expandability Scalable modular UHPLC design •Wide range-configurable system by combination of conventional HPLC unit Auto sample pretreatment by autosampler •Precolumn derivatization •Internal standard addition, sample dilution High temperature analysis150℃ Column oven •Wide temperature range control by Column oven CTO-30A (up to 150 ℃) •Use of new technology area such as green chromatogaphy High speed LCMS analysis •Combination with ultra fast LCMS, LCMS-2020
- 55. UF scanning technology Fastest scan speed of 15,000 u/sec UF switching technology High-speed polarity switching between +/- ion modes -15 msec interval - UF sensitivity Superior sensitivity for ultra fast analysis UF switching UF sensitivity UF scanning LCMS-2020
- 56. Positive 1. Atenolol (267) 2. Procaine (237) 3. Lidocaine (237) 4. Atropine (290) 5. Yohimbine (355) 6. Chlorpheniramine (275) 7. Propranolol (260) 8. Alprenolol (250) 9. Tetracaine (265) 10. Diphenhydramine (256) 11. Doxepin (280) 12. Dipyridamol (505) 13. Desipramine (267) 14. Imipramine (281) 15. Nortriptyline (264) 16. Amitriptyline (278) 17. Dibucaine (344) 18. Verapamil (455) 19. Reserpine (609) 20. Carbamazepine (237) 21. Isopropylantipyrine (231) 22. Alprazolam (309) 23. Trizolam (343) 24. Cilostazol (370) 25. Nifedipine (347) 26. Diazepam (285) 27. Warfarin (309) Negative 1. Cefuroxime (423) 2. Chloramphenicol (321) 3. Nitrendipine (359) Column : Zorbax Eclipse RRHD C18 2.1mm, 50mm, 1.8um Mobile phase : A : 0.1 % formic acid in water / B : 0.1 % formic acid in acetonitrile Gradient : B 3% → 95 % (0.50 min) Detection : ESI (+/-), LCMS-2020 , Scan speed : 15000 u/sec Ultra-Fast Analysis by Nexera-LCMS-2020 Nexera-LCMS-2020 Analysis of 30 pharmaceuticals by high-speed polarity switching (15 msec) and high-speed scanning technology (15000 u/sec)
- 57. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 min 0 25000 50000 75000 100000 125000 150000 175000 200000 225000 250000 275000 300000 0.0 0.1 0.2 0.3 0.4 0.5 0.6 min 0 25000 50000 75000 100000 125000 150000 175000 200000 225000 250000 275000 300000 Injection 1 Injection 300 Highly-Reproducibe High Speed Scan LCMS-2020 offers stable high-speed scan and catches any UHPLC peaks. The reproducible high speed scan provided very reproducible LCMS chromatogram during 300 injections.
- 58. Minimizing Operation Cost •Solvent Saving ・Flow Rate : 3/5 reduction x Analysis time : 1/25 reduction = Mobile phase consumption/analysis is reduced to 1/40 •Cost for power ・Analysis Time : 1/25 reduction = Power consumption is reduced to 1/25 •Cost for performance ・Initial cost + 5 years maintenance cost > up to x 1.3 only ・Sample / System > up to x 5 = Cost / performance is reduced to 1/15 •Cost saving for method migration ・Method migration （conventional LC ↔ UHPLC) tool available
- 59. • Maximized Throughput ⇒ 25 times faster analysis cycle, 10 sec injection speed , 2300 samples/day ⇒ Solvent consumption reduced to 1/40 – Cost saving and environmentally-friendly UHPLC • Maximized Performance ⇒ 5 times higher separation efficiency ⇒ Carryover reduced to one/third • Maximized Expandability ⇒ Expanded configurations by more than 100 optional units ⇒ World’s highest sensitivity detectors – PDA : 6x10-6Au, RF-20AXS : water/raman S/N>2000 Nexera will improve lab productivity by,