Smart HPLC LC800. Ph

Transcription

1 Smart HPLC LC800 Distributed in Australia & New Zealand WINLAB PTY LTD PO Box 5007, Brendale,Winlab Queensland, Australia Pty Ltd 4500 Ph: +61 (0) Web: Ph

2 HPLC GC Compounds of higher decomposition are not analyzed with GC easily. The compounds can be analyzed with HPLC. HPLC analyze pesticides and pharmaceutical products of higher decomposition or non-volatile compounds that cannot be analyzed with GC. Measurable target compounds have been increased.

3 How to select HPLC *Same conditions* Column, Mobile phase Gradient program Which data do you prefer? SMART HPLC LC800 UHPLC

4 Analytical Market Pharmaceutical Efficient method development Samples are increased High Speed Analysis Environmental Reduction of environmental burden Running cost is reduced Food Chemical

5 High Speed Analysis How to do high speed analysis Using small particle column material Benefit? Shorter analytical time High sensitive analysis

6 Comparison between 2μm and 5μm Using smaller particle size or higher linear speed can be the same good separations! Particle size 5μｍ 2μｍ Column length 150ｍｍ 50ｍｍ I.ID size 3.0ｍｍ 3.0ｍｍ Flow rate 0.4mL/min 0.8mL/min Retention time 6.96min 1.19min Peek height 179, ,000 Separation 5 5 Pressure 3.5MPa 19.5MPa Solvent Acetonitrile/Water=65/35(v/v) Column temp. 40 2μm To be 6times faster analytical time! 2.5 times higher Peaks! Wonderful! 5μm Time (min)

7 Current UHPLC problem High Speed High Separation Analysis = Particle size (Sub2μm) = High pressure system (UHPLC) Conventional column 5μm UHPLC column 1.7μm Resolution (Theoretical plate number) is increased but also system pressure increase! By necessity, UHPLC needs expensive system and high temperature oven to decrease the system pressure. ΔP 1/Dp2

8 Problems of High Pressure Analysis Column problems Poor column durability Clogging issue Poor Peak shapes are caused by different temperature of the internal and the outer column. (The cause of high temperature effective for analysis to decrease the high pressure.) System problems Mixer Degradation of Basic performance Pump Poor consumables durability Pre-column filter for clogging issue Pump ( sample diffusion) Preheating tube ( sample diffusion) Detector Auto Sampler Column Oven

9 Line filter UHPLC Pre-heating tube Column Injector Oven ① Line filter to prevent clogging Unnecessary dead volume Very narrow tube mm I.D. Very small particle 1.7 um Clogging issue Poor durability ② Block heating oven Only the outer surface of the column is temperature controlled. A pre-heating tube is needed at the column inlet. Unnecessary dead volume They system cannot make the maximum column performance.

10 LC800の開発コンセプト ジーエルサイエンスが目指す真の高分離 高速分析 超微粒子カラムに頼る高分離 高速分析から システムの最適化による高分離 高速分析へ Inertsil シリーズ (2um 3um 5um) MonoClad C18-HS

11 Smart HPLC LC800 Auto sampler Oven Manual key-pad Pump

12 Sample Injection Needle Needle Wash Port Smart HPLC Sample Tray w/cooling function)) Oven Until now UHPLC From now Smart HPLC Minimized Dead Volume Minimized system volume exception the column (<3.4μL) for maximizing column performances

13 Oven Analytical column Optical fiber Injection valve System volume exception the column after injector Static mixer Only < 3.4μL Pre-heating & coil mixer 重 Temperature change要is decreased when eluent are mixed, temperature distribution in/out the column is decreased. UV Flow cell Capacity: < 1μL Minimized Dead Volume (applied for patent)

14 LC800 Mixing principle Mixing ① Radius direction mixing Diffusion of radius direction against the flow. ② Flow direction mixing Diffusion of concentration distribution to the flow direction LC800 mixing principle ① Static mixer Diffusion by branching interflow Acceleration of mixing to the radius direction + ② Coil mixer Diffusion by 2nd flow Acceleration of flow direction mixing 2nd flow direction The different pressures of the outer tube and the inner tube are generated by centrifuge power, it can make the 2nd flow line which is different than the main flow.

15 LC800 Mixer performance Mobile Phase A 0.1 TFA water solution Mobile Phase B 0.1 TFA in acetonitrile Wave length : 210nm A/B = 85/15 1min 65/35 0.4min 65/ LC800 Standard mixer:150ul AU AU min UV 0.10 Ch Commercial mixer:150ul AU Ripple min AU 0.10

16 Flexible design for any column length up to 300mm 50 mm 150mm

17 Column replacement

18 Smart HPLC Autosampler Until now UHPLC From now Smart HPLC

19 Auto sampler (Fixing loop system) Needle washing port Sample injection needle Sample tray Cooling function as standard)

20 Autosampler

21 Sample capacity of the Autosampler Sample process Cooling function as standard 1.5mL vials x 360 (180vial tray x 2 as standard) 4mL vials x 256 Well plate x 6 (96 shallow/deep 384 shallow/deep) Different type trays can be used at the same time (Ex 1.5mL vials and 96 deep well analysis at once)

22 Smart HPLC Pump Until now UHPLC From now Smart HPLC

23 Pump 2 High pressure gradient pumps Degassing unitト 2CH as standard Mobile phase solvent tray (it can be pulled out) Plunger washing pump

24 Smart HPLC LC800 Features Minimized Dead Volume Minimized system volume exception the column (<3.4μL) for maximizing column performances Analytical Reliability Increase Ultimate stable analytical reproducibility in high speed analysis Strong system design in environmental temperatures Minimized Carry Over Internal polishing of the needle Electrolytic polishing in contacted site) Thorough washing of sample contact area.

25 Smart HPLC LC800 Features Minimized Dead Volume Minimized system volume exception the column (<3.4μL) for maximizing column performances Analytical Reliability Increase Ultimate stable analytical reproducibility in high speed analysis Strong system design in environmental temperatures Minimized Carry Over Internal polishing of the needle Electrolytic polishing in contacted site) Thorough washing of sample contact area.

26 LC800 performance Comparison data of between LC800 and the other UHPLC (1) Standard samples-1 Theoretical plate number of standard samples in isocratic analysis (2) Standard samples-2 Performance comparison between the other UHPLC w/1.7um column vs LC800 w/ Inertsil ODS-4 2um (3) Actual samples Impurity analysis of medicines in gradient analysis (4) LC/MS-MS Sensitivity and Separation comparison data in pesticides analysis

27 (1) Standard sample Performance comparison between LC800 and the others Performance comparison with general standard samples (Theoretical plate number) Same mobile phase Same column Same sample Same analytical conditions Column Flow rate Mobile phase Column temp. Detection Injection volume Sample Inertsil ODS-3 2μm 2.1 mm I.D. 50 mm 0.6 ml/min 65 % Acetonitrile water solution 40 UV254 nm 1μL 1 Acetophenone 2 Benzene 3 Toluene 4 Naphthalene

28 Theoretical plate number comparison data between LC800 and the other UHPLC Peak 1 Acetophenone Peak 2 Benzene Peak 3 Toluene Peak 4 Naphthalene Hitachi Lachrom ultra Thermo Accera Shimadzu UFLCXR Waters UPLC Smart HPLC LC LC800 is the highest system performance in UHPLC htsw)

29 Performance comparison in Isocratic analysis UV UV Ch1 Smart HPLC LC800 A UHPLC N=6300 Twice higher theoretical plate number!! Sharper Peaks! N=1800 N=4900 AU N= N=8100 N=4500 N= N= AU AU 0.8 AU 1.0 Influenced by dead volume of front/back the column min min min 0.5 min

30 (2) Standard sample -2 (1) LC800 Inertsil ODS-4 2um mm (2) UHPLC system ODS1.7um mm (1) (2) LC800 Theoretical plate number? Particle size 2.0 um Low pressure analysis UHPLC Particle size 1.7 um High pressure analysis

31 LC800 Inertsil ODS-4 2um mm D社UHPLC C18 1.7um mm High pressure Low pressure 41 MPa 24 MPa Time (min) Time (min) : Impurity of Naphthalene ①Acetophenone 3400 ①Acetophenone 5100 ②Benzene 5100 ②Benzene 6800 ③Toluene 6600 ③Toluene 7900 ④Naphthalene 7500 ④Naphthalene 8500

32 (3) Actual sample Gradient User Purpose Sample Column Instruments A pharmaceutical company Performance test for each UHPLC 2nd peak (main) and by product (impurity) 2.1mm ID x 50mm Particle size around 2um. LC800 and the other brand UHPLC Which UHPLC is the best performance?

33 The best evaluation LC system by the customers Column C18 2.0μm 2.1mmI.D.x50mm The other UHPLC UV Ch LC AU 0.4 AU Very good separation with the column used for the above 0.0LC min

34 Very good data with LC800 and the other brand columns 1 UV Ch1 004 UV Ch1 004 UV Ch1 004 UV Ch1 004 UV Ch1 004 UV 004 LC800 with the other brand columns Column A Column B Column C Column D Column E Column F Column G Column H Column I min

35 LC800 is coupled with LC/MS/MS! Controlled by Analyst LC800 is controlled by Analyst software of Applied biosystems/mds Analytical Technologies LC Q TRAP

36 Controlled by Analyst Software

37 LC800 Performance (LC/MS-MS) for pesticide residue HPLC MASS Method Mass Detector: LC System: Flow Rate: Injection Volume: Column Temperature: 4000 Q TRAP (AB Sciex) LC800 vs UHPLC 0.5mL/min 5μL 40 MS Conditions Positive ESI 1 Oxamyl Q1/Q3=237/220 2 Methomyl Q1/Q3=163/88 3 Aldicarb Q1/Q3=208/88 4 Bendiocarb Q1/Q3=224/167 5 Methiocarb Q1/Q3=226/107 6 Pirimicarb Q1/Q3=239/182 Column Inertsil ODS-3 2μm mm Mobile Phase H2O : MeOH=50 : 50

38 ﾁｬﾝﾈﾙ A ﾁｬﾝﾈﾙ A ﾁｬﾝﾈﾙ A ﾁｬﾝﾈﾙ A ﾁｬﾝﾈﾙ A ﾁｬﾝﾈﾙ A Smart HPLC LC800 ② Weak retention time compounds can be detected sharply! ﾁｬﾝﾈﾙ A 1.2 ① ④ ③ ⑤ min ﾁｬﾝﾈﾙ A ﾁｬﾝﾈﾙ A ⑥ ② ① ④ ③ ⑤ ① Oxamyl Q1/Q3=237/220 ② Methomyl Q1/Q3=163/88 ③ Aldicarb Q1/Q3=208/88 ④ Bendiocarb Q1/Q3=224/167 ⑤ Methiocarb Q1/Q3=226/107 ⑥ Pirimicarb Q1/Q3=239/ ﾁｬﾝﾈﾙ A Influenced by dead volume of front/back the column ﾁｬﾝﾈﾙ A C UHPLC ⑥ ﾁｬﾝﾈﾙ A min Intensity Intensity Intensity Intensity Sensitivity comparison data in Isocratic analysis by MS

39 Sensitivity comparison in Isocratic analysis by MS ﾁｬﾝﾈﾙ A ﾁｬﾝﾈﾙ A ﾁｬﾝﾈﾙ A ﾁｬﾝﾈﾙ A ﾁｬﾝﾈﾙ A ﾁｬﾝﾈﾙ A Smart HPLC LC800 ﾁｬﾝﾈﾙ A ﾁｬﾝﾈﾙ A ﾁｬﾝﾈﾙ A ﾁｬﾝﾈﾙ A ﾁｬﾝﾈﾙ A ﾁｬﾝﾈﾙ A C UHPLC ② Peaks of the shorter retention time are much influenced even by MS! ① Intensity Intensity Intensity ② sec. 9 sec ① Intensity

40 Smart HPLC LC800 Features Minimized Dead Volume Minimized system volume exception the column (<3.4μL) for maximizing column performances Analytical Reliability Increase Ultimate stable analytical reproducibility in high speed analysis Strong system design in environmental temperatures Minimized Carry Over Internal polishing of the needle Electrolytic polishing in contacted site) Thorough washing of sample contact area.

41 Smart HPLC LC800 Features Minimized Dead Volume Minimized system volume exception the column (<3.4μL) for maximizing column performances Analytical Reliability Increase Ultimate stable analytical reproducibility in high speed analysis Strong system design in environmental temperatures Minimized Carry Over Internal polishing of the needle Electrolytic polishing in contacted site) Thorough washing of sample contact area.

42 Extremely stable analysis reproducibility Isocratic analysis Column Inertsil ODS-3 2μm 2.1mmI.D. 50mm Flow rate 0.6mL/min Mobile phase Acetonitrile / Water = 65/35(v/v) Column temp. 40 Detection UV254nm RSD 0.45 Running time : 1min ①Acetophenone ②Benzene ③Toluene N=5 Overwriting ④Naphthalene 0.35 ① 0.30 Retention time 0.45 ③ ② ④ AU 0.25 AU 0.25 Uracil min

43 Extremely good reproducibility (Gradient analysis) Inertsil ODS-3 2μm 2.1mmI.D. 50mm 0.6mL/min 0.1 TFA water solution 0.1 Acetonitrile including TFA A/B = 85/15 1min 65/35 0.4min 65/35 Column Temperature 40 Detection UV214nm 0.25 UV Ch1 UV Ch1 UV Ch UV Ch1 RSD Running time : 2min ① Gly-Tyr 0.20 AU 0.14 ②Val-Tyr-Val ① 0.15 ③④ ⑤ ② Retention time 0.20 ③Met-Enkephalin 0.07 ④Leu-Enkephalin ⑤AngiotensinⅡ AU Column Flow rate Mobile Phase A Mobile Phase B

44 Pump of maximum resolution 2ul % % AU % 0.01 B 0% % 4% 3% % 8% 7% % Minutes Flow rate 0.2 ml/min Mobile phase A Methanol Mobile phase B Methanol including 0.01g/L n-porpylparaben A/B 100/0-(1%各20min)-90/10 Detection UV254 nm

45 Internal UV flow cell temperature stability Base line fluctuation by room temperature change Flow cell is set in the oven Cell temperature Cell temperature Base line min Base line min Flow rate 0.2 ml/min Solvent Acetonitrile/Water 25/75(v/v)(including 0.05%TFA) Detection UV214 nm AU AU

46 Smart HPLC LC800 Features Minimized Dead Volume Minimized system volume exception the column (<3.4μL) for maximizing column performances Analytical Reliability Increase Ultimate stable analytical reproducibility in high speed analysis Strong system design in environmental temperatures Minimized Carry Over Internal polishing of the needle Electrolytic polishing in contacted site) Thorough washing of sample contact area.

47 Smart HPLC LC800 Features Minimized Dead Volume Minimized system volume exception the column (<3.4μL) for maximizing column performances Analytical Reliability Increase Ultimate stable analytical reproducibility in high speed analysis Strong system design in environmental temperatures Minimized Carry Over Internal polishing of the needle Electrolytic polishing in contacted site) Thorough washing of sample contact area.

48 Necessary minimized carry over for high sensitivity analysis What is Carry over? Carry over is remained samples or contaminations which are detected with the system. The samples should be remained in the HPLC system. (Particularly in the Autosampler unit). The carry over makes poor analysis accuracy and quantitative performance, it also need to re-analyze or consider to countermeasure, so the work efficiency is getting worse. Minimized carry over system merit Quantitative performance increase by high sensitivity analysis Data reliability by high sensitivity analysis Work efficiency increase

49 Cause of carry over It is caused by residual contamination. Residue Residue at connecting parts like joints Adsorption

50 To reduce carry over Improvement of the injection port design ① Injection port ② Sample needle Improvement of the washing methods

51 ① Injection port shape General Injection Port Contamination on the injection port surface

52 ① Injection port shape LC800 Injection Port The sample contact area is much decreased by contact in point and point even if contamination was remained. Especially, in case of the direct injection, the sample contact area is increased by contact in surface and surface since it gets the pressure.

53 ② Needle Surface finishing Imagination 凹凸 rough surface of several um Smoothing If rough surface, sample can be remained on the internal and outer surface. Sample is not remained Easily on the surface. Special polishing Needle surface Special polishing needle

54 ② Needle Monolithic type Sample holding part Sample holding part Sample can be remained at the connections Union Needle General needle Needle Monolithic needle LC800

55 General washing (UHPLC)

56 LC800 Standard wash Before injection ①Sample suction ②Outer needle surface wash ③Sample injection Wash The wash used once is automatically discharged to drain after finish of the needle insert.

57 LC800 Standard Wash After injection ④ Strong wash is flown ⑤ Strong wash is forced ⑥ It substitutes with to discharge weak wash Weak wash is flown from the top Sample Contact at the points Discharge Discharge In the direct injection, only the mobile phase can be flown, but Strong wash is also flown. Sample Strong wash Weak wash

58 LC800 Wash Needle wash port Discharge Injection port Discharge

59 LC800 Custom Wash Needle in/out surface wash Valve wash Injection port wash Strong wash is supplied from the needle Drain Strong wash is flown into the syringe and discharged, strong wash is filled in the wash port to wash the outer surface. Drain Hold switching the valve and wash the rotor seal

60 Carry over test① in Pharmaceutical product (Reserpine Low carry over is achieved by standard wash Standard wash 100 ng/ml Reserpine Blank Carry Over S/N=2.5

61 Carry over test② in Pharmaceutical product (Chrorhexidine Custom wash on analytical conditions by custom wash XIC of +MRM (1 pair): 505.1/335.9 amu 5.5e6 5.0e6 4.5e6 4.0e6 500 ug/ml blank XIC of +MRM (1 pair): 505.1/335.9 amu e6 Carry Over % e6 Intensity, cps Intensity, cps 3.0e6 2.0e6 1.5e6 1.0e e Time, min Time, min 2.5

62 Smart HPLC Complement Until now UHPLC From now Smart HPLC Operability

63 LC800 pump

64 UHPLC pump

65 LC800 pump UV lamp containment

66 UHPLC UV Detector UV leakage

67 Summary Theoretical plate is changeable by tubing capacity and joint quantity. The pressure is changeable by thin of tubing.

68 Summary To obtain high separation Sub 2um particle High pressure analysis 2um particle Mid. pressure analysis

69 Summary To obtain high separation Unnecessary Dead volume Max. column performance Sub 2um High pressure analysis 2um Lower pressure analysis

70 Summary To obtain high separation Clogging Unnecesasry Dead volume Sub 2um Analytical stability Column performance to be max! High pressure analysis 2um Lower pressure analysis

71 Summary To obtain high separation Clogging Unnecessary Dead volume Analytical stability Sub 2um Column performance to be max! High pressure analysis 2um Lower pressure analysis

72 Smart HPLC Thank you very much for listening! Distributed in Australia & New Zealand WINLAB PTY LTD PO Box 5007, Brendale, Queensland, Australia 4500 Ph: +61 (0) Web: Winlab Pty Ltd Ph