Neueste Informationen im Agilent HPLC und Applikationsbereich

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1 Neueste Informationen im Agilent HPLC und Applikationsbereich Natascha Roos Produktspezialistin LC/LCMS, CAG Sebastian Krahe Produktspezialist LC, LSG Agilent Technologies, Deutschland/Österreich 20. August 2013 Berlin 1

2 The Agilent 1200 Infinity Series The Portfolio and the Systems Page 2

3 NEW 1200 Infinity Series More workflow-automation and application-based solutions Analytical SFC Bio-inert HPLC Nano LC & Cap LC Preparative LC & LC/MS GPC SEC Isocratic LC Multi-method and Method Development High-throughput LC & LC/MS Nanoflow HPLC-Chip MS Page 3

4 Frage Was kann ich mit einer 1290 quaternär Pumpe besser machen als mit einer binären Pumpe? 4 Confidentiality Label August 22, 2013

5 Innovation: 1290 Infinity Quaternary Pump Quaternary Pump with Binary Pump-like Performance 1290 Infinity Quaternary Pump Boost Performance Highest accuracy and precision for composition and flow, with exceptionally low delay volume Save Time Accelerates transfer of existing HPLC methods to UHPLC Reduce Costs Outstanding UHPLC performance at HPLC-like costs of ownership

6 Agilent 1290 Infinity Quaternary Pump Specifications & Benefits Power Range For any kind of analysis Flow Accuracy and Precision < 0.07 % RSD or 0.01 min SD ± 1.0 % or 10 µl High RT precision in isocratic runs Composition Accuracy and Precision < 0.15 % RSD or 0.02 min SD ± 0.4 % (1-99 % Composition B) High RT precision in gradient runs Composition Range 1-99 % Wide analytical range Delay Volume < 350 µl For fast quaternary gradients

7 Ternary Gradients Water/Acetonitrile/Methanol Parabenes Toluene Phenol N,N-diethyl-m-Toluamide Column: Agilent ZORBAX Eclipse Plus C18, 3 x 100 mm, 3.5 µm (p/n ) Mobile phase: A = Water / B = Acetonitrile / C = Methanol Gradient: var. Flow rate: 1.2 ml/min Stop time: 8.5 min Injection vol.: 3 µl Column Temp.: 40 C DAD: 254/10 nm, (ref. 360/ 100 nm), 20 Hz

8 Ternary Gradients Water/Acetonitrile/Methanol mau 100 N,N-diethyl-m-Toluamide Butyl paraben Binary gradient Water/MeOH Ternary gradient Water/ACN/MeOH Binary gradient Water/ACN min

9 Ternary/Quaternary Gradients Blend Assist Blend Assist is a driver-based software tool to allow easy solvent/buffer mixing for quaternary pumps based on 1290 Infinity LC pump technology.

10 Ternary/Quaternary Gradients Blend Assist, different TFA concentrations H 3 C CH 3 N H OH OCH 3 Tramadol (+ impurities) Column: Agilent ZORBAX Eclipse Plus C18, 3 x 100 mm, 3.5 µm (p/n ) Mobile phase: A = Water / B = Acetonitrile / C = Water + 1 % TFA / D = Acetonitrile + 1 % TFA Gradient: var. Flow rate: 1.2 ml/min Stop time: 8.5 min Injection vol.: 3 µl Column Temp.: 40 C DAD: 270/10 nm, (ref. 360/ 100 nm), 20 Hz

11 Ternary/Quaternary Gradients Blend Assist H 3 C CH 3 N H OH OCH 3 Tramadol (+ impurities)

12 Ternary/Quaternary Gradients Blend Assist

13 Agilent 1290 Infinity Quaternary Pump Specifications & Benefits Power Range For any kind of analysis Flow Accuracy and Precision < 0.07 % RSD or 0.01 min SD ± 1.0 % or 10 µl High RT precision in isocratic runs Composition Accuracy and Precision < 0.15 % RSD or 0.02 min SD ± 0.4 % (1-99 % Composition B) High RT precision in gradient runs Composition Range 1-99 % Wide analytical range Delay Volume < 350 µl For fast quaternary gradients

14 Resultat: Nutzen der 1290 quat Pumpe Vereinfachung der Methodenentwicklung durch Blend Assist und quaternäre Gradienten Ultraschnelle Trennungen mit höchster Reproduzierbarkeit Nur wenig langsamer als die binäre 1290 Pumpe bei extrem schnellen Gradienten 14

15 Frage: was mache ich bei hochkomplexen Proben...bei denen mir die beste Säule und die Wahl des Eluenten nicht genug Trennleistung liefert? 15 Confidentiality Label August 22, 2013

16 2D-LC 2. Dimension 1. Dimension One or two 1290 Infinity TCC 1290 Infinity Binary Pump 1290 Infinity Binary Pump 1290 Infinity Autosampler or 1260 HiP Autosampler Optional 1260/1290 Infinity Detector Optional 1260/1290 Infinity Detector 1260 Infinity Capillary Pump 1260 Infinity Binary or Quaternary Pump 1260 Infinity Autosampler For 1st dimension chromatogram and peaktriggering 1260/1290 Infinity detector To monitor waste-line Almost any Agilent pump or autosampler in the 1 st dimension! Almost any detectors are supported! A 1290 Infinity Binary Pump for the 2 nd dimension is required. 16

17 Hardware Valves, uniqueness and flexibility 2. Dimension 1. Dimension 2pos/4port-duo Loop1 waste Loop2 New Agilent-only special 2D-LC-QuickChange valve. Single valve with fully symmetric flow-paths and symmetric fill/flush-out behavior. Only valve that allows countercurrent flush-out of both loops. 17

18 Pharmaceutical drug: investigation of main compound and impurities by 2D-LC (Heart Cut) 18

19 Sampling the main compound only (Heart Cut) ( min) 1st dimension Zorbax Eclipse Plus C18 2nd dimension Zorbax Eclipse Plus Phenyl Hexyl 19

20 Sampling the main compound only (Heart Cut) ( min) 20 Confidentiality Label August 22, 2013

21 Sampling the main compound only (Heart Cut) ( min) with added impurity H 21 Confidentiality Label August 22, 2013

22 22 Comprehensive 2D-LC - components of beer samples Analysis of two different commercially available Japanese beer samples with different separation modes System: 1 st dim Pump: 1260 Infinity Binary Pump AutoSampler: 1290 Ininfity Autosampler 2 nd dim. Pump: 1290 Infinity Binary Pump TCC: Detector: Data acquisition: Data analysis: 1290 Infinity Therm. Column Comp. w. 2DLC valve 1290 Infinity Diode-Array Detector G4212A, OpenLAB ChemStation Edition w. 2D-LC Add-on LC image software from GC image LLC Experiments 1st dimension 2nd dimension 1. SEC RP (C18) 2. IEX RP (C18) 3. RP (C18) RP (Phenyl) From WikiCommons, File:Selection of Japanese beer.jpg

23 Separation by RP (C18) 23 Applications examples - components of beer samples Analysis of two different commercially available beer samples with different separation modes 1 st dimension - SEC 2 nd dimension - Reverse phase (C18) Beer A Beer B Separation by SEC

24 Separation by RP (C18) 24 Applications examples - components of beer samples Analysis of two different commercially available beer samples with different separation modes 1 st dimension - ion exchange 2 nd dimension Reverse phase (C18) Beer A Beer B Separation by ion-exchange

25 Separation by RP (Phenyl) 25 Applications examples - components of beer samples Analysis of two different commercially available beer samples with different separation modes 1 st dimension - Reverse Phase (C18) 2 nd dimension - Reverse Phase (Phenyl) Beer A Beer B Separation by RP (C18)

26 On-line Anreicherung mit dem Agilent 1290 Infinity Flexible Cube Workflow-Automatisierung und Applikationen

27 Agenda Einführung Prinzipieller Aufbau 1290 Infinity Flexible Cube Ventilschaltungen Applikation Zusammenfassung August 2013

28 Analytische Methoden Konzentration PAT > QC > Rückstandsanalytik (ng/l) Analyten Einzel oder Mehrkomponenten? Matrix Wasserlöslich oder nicht? August 2013

29 Festphasenextraktion Solid Phase Extraction SPE die Methode für Probenvorbereitung und Anreicherung des Analyten Der gewünschte Analyt wird auf einer SPE Kartusche angereichert und anschließend mit dem gewünschten Lösungsmittel eluiert (sample enrichment) Ungewünschte Verunreinigung oder Probenmatrix wird entfernt (sample cleanup)

30 Festphasenextraktion Solid Phase Extraction SPE die Methode für Probenvorbereitung und Anreicherung des Analyten Der gewünschte Analyt wird auf einer SPE Kartusche angereichert und anschliessend mit dem gewünschten Lösungsmittel eluiert (sample enrichment) Ungewünschte Verunreinigung oder Probenmatrix wird entfernt (sample cleanup) Effizienz und verbesserter Durchsatz mit online SPE LC Agilent Lösung für die Trinkwasseranalytik

31 Herkömmliche Online Anreicherung durch Säulenschalten August 2013

32 Innovation: ventilbasierende Arbeitsabläufe Online SPE Lösungen Der modulare Aufbau der Agilent 1200 Infinity Series Online SPE Lösung mit dem 1290 Infinity Flexible Cube gibt die Flexibilität die Sie brauchen. Erhöhung der Empfindlichkeit Niedrigste Detektionsgrenzen im ng/l Bereich Zeitersparnis Anpassung des Systems auf eigene Arbeitsabläufe und Anforderungen Kostenreduzierung Probenvorbereitung, LC und MS/MS Analyse in einem System, keine zusätzliche Pumpe notwendig 32 Page August 2013

33 1290 Infinity Flexible Cube Reciprocating single-piston pump for flows up to 4 ml/min (60 bar) up to two Quick-Change valves, according to application Solvent selection valve for up to three solvents August 2013

34 Agilent 1290 Infinity Flexible Cube August 2013

35 Agilent 1200 Infinity Series online SPE Produktaufbau Kombination mit dem Starter Kit... SPE starter set Multi SPE kit Direct inject kit High volume inject kit Flask sampling kit Zweck 1 x For on-line SPE analysis using 1 or 2 SPE cartridges. Allows for alternating SPE loading. Loading via auto sampler, vol. of up to (2x) 900 ul. 1x 1 x For on-line SPE analysis using up to cartridges 1 x 2 x 1 x 1 x 1 x 1 x 1 x 1 x 1 x 1x For on-line SPE analysis using up to 12 (2 x 6) cartridges For direct LC injection as well as on-line SPE (vol. 5 ul up to (2x) 900 ul) For direct LC injection as well as on-line SPE (vol. 5 ul up to (2x) 900 ul plus 5 ml sample loop) For SPE loading from auto sampler (up to (2x) 900 ul) as well as sample loading from flasks (ml range)

36 1200 Infinity Series Online SPE SPE starter set G4742A Position 1: Load sample 1 to SPE 1 Features Online SPE with 2 SPE cartridges Injection vol. up to 1800 µl (2 x 900 µl) 1260 Infinity Standard ALS G1329B Page 37

37 1200 Infinity Series Online SPE SPE starter set G4742A Position 2: Elute sample 1 from SPE1 to LC column Load sample 2 on SPE2 Page 38

38 Anwendung der Online SPE: Nachweis von pharmazeutischen Wirkstoffen in Trinkwasser Application Note: Detection of Trace Level Pharmaceuticals in Drinking Water by Online SPE Enrichment with the Agilent 1200 Infinity Series Online-SPE Solution Edgar Nägele P/N EN HPLC Method Agilent 1260 Infinity Quaternary Pump: Solvent A: Water, 5 mm ammonium formiate + 0.1% formic acid Solvent B: ACN + 5% water, 5 mm ammonium formiate + 0.1% formic acid Flow rate: 0.4 ml/min Gradient: 0 minutes 5% B, 5 minutes 5% B, 20 minutes 98% B. Stop time: 25 minutes Post time: 10 minutes Agilent 1290 Infinity Thermostated Column Compartment: Column temperature: 40 C Agilent 1290 Infinity Flexible Cube: Right valve: 2-position/10-port QuickChange valve head Pump: 1.5 ml/min Solvent: A1: Water, B1: ACN 0 minutes Pump 300 s, Solvent A1 5 minutes right valve change position 7 minutes Pump 180 s, Solvent B1 11 minutes Pump 300 s, Solvent A1 Agilent 1260 Infinity Standard Autosampler Injection volume: 1,800 μl (automated multidraw of 2 times 900 μl) Needle wash in vial (MeOH) Draw and eject speed: 1,000 μl/min. Sample temperature: 10 C. 2 halftrays for 15 6 ml vials G mL screw cap vials (glass, p/n ), screw caps (p/n ), pre-slit septa for 6-mL screw cap vials (p/n ) Triple Quadruple MS method Agilent Jet Stream thermal gradient focusing technology: Gas temperature: 325 C Gas fl ow: 9 L/min Nebulizer: 35 psi Sheath gas temperature: 350 C Sheath gas fl ow: 12 L Capillary: 4,000 Volt Nozzle: 0 Volt The MRM and dynamic MRM triple quadrupole MS method was developed by means of the MassHunter optimizer software and direct injections of individual pesticide standards (10 ng/μl) by flow injection into the mass spectrometer. The optimization was done to find the optimum fragmentor voltage for each individual compound and the optimum collision energies for the fragmentation to the quantifier and qualifier ions (Table 3) August 2013

39 On-line SPE Aufbau Figure 1) Setup of the Agilent 1200 Infinity Series Online-SPE solution with MS detector (the solvent bottles in the center are for SPE loading, rinsing and conditioning) August 2013

40 R e s p o n s e s R e s p o n s e s R e s p o n s e s R e s p o n s e s Results and Discussion Figure 4. Calibration curves for selected antibiotics. All compounds were measure from 100 ppt to 1 ppt with seven levels. Depending on the molecule, the LOQ was taken at S/N = 10 and levels adjusted accordingly. x10 2 Vancomycin - 7 Levels, 5 Levels Used, 20 Points, 20 Points Used 9 y = * x R^2 = Type:Linear, Origin:Ignore, Weight:None 5 x10 2 Flumequine - 7 Levels, 6 Levels Used, 12 Points, 12 Points Used y = * x R^2 = Type:Linear, Origin:Ignore, Weight:None 5 HO H 3 C NH 2 HO OH OH O HO HO HN H 3 C HO N H O O O OH OH O O Cl H N O O O N H O O O NH 2 Cl H N O OH O NH H 3 C CH 3 NH CH F N O CH 3 OH O Concentration (ng/l) x10 5 Erythromycin - 7 Levels, 7 Levels Used, 28 Points, 28 Points Used 1.0 y = * x R^2 = Type:Linear, Origin:Ignore, Weight:None OH H 3 C CH 3 O H 3 C O Concentration (ng/l) H 3 C O H 3 C OH CH 3 O CH 3 OH H 3 C CHH 3 O N CH 3 O OH O O O CH 3 CH 3 CH 3 4 x Concentration (ng/l) Ornidazole - 7 Levels, 6 Levels Used, 24 Points, 24 Points Used y = * x R^2 = Type:Linear, Origin:Ignore, Weight:None Concentration (ng/l) O O - N + N N CH 3 OH Cl August 2013

41 Results and Discussion Tab. 5 Overview of calibration data (LOD and LOQ), recovery of the trapping process as well as statistical evaluation (retention time RSD and peak area RSD) of antibiotics used in this study August 2013

42 Results and Discussion Figure 6. Selected antibiotics from a spiking experiment in Rhine river water, tap water, and spring water. The spiked concentration level of all antibiotics was 20 ppt. 3 x10 3 Trimethoprim Ormethoprim Nalidixic acid Rheinwasser Trinkwasser Quellwasser 2 Flumequine Erythromycin 1 Oxolinic acid Ornidazole Sulfamethoxazole Counts vs. Acquisition Time (min) August 2013

43 Zusammenfassung Agilent 1200 Infinity Series Online- SPE Erhöhung der Empfindlichkeit Anreicherung mit online SPE erlaubt die Spurenanalytik (im unteren ng/l Bereich) unter Verwendung des Agilent 6460 Triple Quad MS Kosteneffiziente Lösung basierend auf 1290 Infinity Flexible Cube Einkolbenpumpe - keine zusätzliche zweite LC Pumpe notwendig Enthält Lösungsmittelauswahlventil (3 Eluenten) Einfacher Einbau in das bestehende LC System - Investitionsschutz 2 Ventile im Flexible Cube - kurze Kapillarwege Komplette Steuerung über die Software möglich (OL Chemstation, MassHunter) Sehr flexible Lösung für Labore mit mittlerem Probendurchsatz Online SPE und direkte Injektion, Multi SPE, Methodenentwicklung Einfache Aufrüstung möglich Weitere SPE Kit sind geplant The Jumpstation URL 46 Page August 2013

44 Appendix 47 Page August 2013

45 Agilent 1200 Valves SPE Starter Set pos August

46 Agilent 1200 Valves SPE Starter Set pos August

47 Agilent 1200 Valves SPE Multikit pos August

48 Agilent 1200 Valves SPE Multikit pos August

49 Agilent SPE high volume Injection pos August

50 Agilent SPE high volume Injection pos August

51 Agilent SPE high volume Injection pos August

52 Agilent SPE high volume Injection pos August

53 Agilent Valves SPE Flask Sampling pos August

54 Agilent Valves SPE Flask Sampling pos August

55 Agilent Valves SPE Flask Sampling pos August

56 Agilent Valves SPE Flask Sampling pos August

57 Agilent Auto 1200 Valves SPE direct Injection pos August

58 Agilent Auto 1200 Valves SPE direct Injection pos August

59 Agilent Auto 1200 Valves SPE direct Injection pos August

60 VIELEN DANK FÜR IHRE AUFMERKSAMKEIT!! 63