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Microliter-Volume NMR The Wellplate and Microvial as an Efficient Medium of Sample Transport Among Automated Methods of Analysis.

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Presentation on theme: "Microliter-Volume NMR The Wellplate and Microvial as an Efficient Medium of Sample Transport Among Automated Methods of Analysis."— Presentation transcript:

1 Microliter-Volume NMR The Wellplate and Microvial as an Efficient Medium of Sample Transport Among Automated Methods of Analysis

2 Workflow Diagram for the Natural Product/Metabolite/Medicinal Chemist LC vacuum centrifuge MS DAD/ELSD or bioactivity NMR 2

3 Workflow Diagram for the Natural Product/Metabolite/Discovery Chemist LC vacuum centrifuge MS DAD/ELSD or bioactivity NMR Purification Fraction Collection or Purified Samples Prep Stage Report Crude Samples/Mixtures Low Sensitivity Detection High Sensitivity Detection 3

4 X NMR Measurement is Aided by Good Chromatography! What we care about What we don’t care about Mixture Pure Component LC Chromatogram LC Fraction

5 ELSD FT-IR MS DAD ICP NMR UV-VIS GC LC Analytical Measurement Challenges Budget Management: Mass, Money, Space, Time Copyright © 2006 by Protasis Corporation, All Rights Reserved

6 Wellplate-Inclusive Workflow Budget-Effective Utilization of Analytical Instruments ELSD ICP UV-VIS MS FT-IR GC NMR LC 6 Copyright © 2006 by Protasis Corporation, All Rights Reserved

7 The Advantages of Going Tubeless… No shimming between samples No washing tubes Minimal sample transfer Efficient storage Disposable Economical Incredible Time Savings Plates Make It Easy 7 Copyright © 2006 by Protasis Corporation, All Rights Reserved

8 Protasis One Minute NMR Automation Sample Return Stacked Sample Drawers Wash Bays Microliter Precision Near Zero Dead Volume Easy Parameter Setup Solvent Conditioning Wellplate Web-based Software 8 Copyright © 2006 by Protasis Corporation, All Rights Reserved

9 Principles of Injection NMR Sample Vials Sample is easily dispensed from syringe to probe Easy, Effective, Efficient Simple Plumbing To learn more about Direction Injection Loading, see the Protasis/MRM Technical Bulletin G0020 in the Tech Support Knowledge Base at

10 Solvent Microfilter Vial Spatula MicrofilterSample Step 1: Add sample (5 – 500  g) Step 2: Add solvent (5 – 15  L) Step 3: Spin down (~ 1 min.) Step 4: Discard microfilter Step 5: Place vial on OMNMR Quality Results Start with a Quality Sample 10 Copyright © 2006 by Protasis Corporation, All Rights Reserved

11 Injection 5 µl Push volume 8 µl Courtesy J.-L. Wolfender and A. Thiocone, Laboratory of Pharmacognosy and Phytochemistry School of Pharmaceutical Sciences, University of Geneva, Switzerland 11

12 Dissolution with 6.5 µl of deuterated solvent Samples drying by speed-vac Courtesy J.-L. Wolfender and A. Thiocone Laboratory of Pharmacognosy and Phytochemistry School of Pharmaceutical Sciences University of Geneva, Switzerland 12

13 1) SAMPLE INJECTION 2) PUSH VOLUME 3) NMR MEASUREMENT 4) RECOVERY MeODHOD Courtesy J.-L. Wolfender and A. Thiocone Laboratory of Pharmacognosy and Phytochemistry School of Pharmaceutical Sciences University of Geneva, Switzerland Sample Solvent 13

14 1) SAMPLE INJECTION 2) PUSH VOLUME 3) NMR MEASUREMENT 4) RECOVERY MeODHOD Courtesy J.-L. Wolfender and A. Thiocone Laboratory of Pharmacognosy and Phytochemistry School of Pharmaceutical Sciences University of Geneva, Switzerland Sample Solvent 14

15 1) SAMPLE INJECTION 2) PUSH VOLUME 3) NMR MEASUREMENT 4) RECOVERY MeODHOD Courtesy J.-L. Wolfender and A. Thiocone Laboratory of Pharmacognosy and Phytochemistry School of Pharmaceutical Sciences University of Geneva, Switzerland Sample Solvent 15

16 1) SAMPLE INJECTION 2) PUSH VOLUME 3) NMR MEASUREMENT 4) RECOVERY MeODHOD AnalyteSignals Courtesy J.-L. Wolfender and A. Thiocone Laboratory of Pharmacognosy and Phytochemistry School of Pharmaceutical Sciences University of Geneva, Switzerland Sample Solvent 16

17 1) SAMPLE INJECTION 2) PUSH VOLUME 3) NMR MEASUREMENT 4) RECOVERY MeODHOD AnalyteSignals Courtesy J.-L. Wolfender and A. Thiocone Laboratory of Pharmacognosy and Phytochemistry School of Pharmaceutical Sciences University of Geneva, Switzerland Sample Solvent 17

18 Sample recovery 1) SAMPLE INJECTION 2) PUSH VOLUME 3) NMR MEASUREMENT 4) RECOVERY MeODHOD Courtesy J.-L. Wolfender and A. Thiocone Laboratory of Pharmacognosy and Phytochemistry School of Pharmaceutical Sciences University of Geneva, Switzerland Sample Solvent 18

19 MicroFlow NMR Performance Advantages of Size Scale Enhanced Mass Sensitivity Simple to Use, Easy to Shim! Teflon FEP Flowpaths Reliable Robotics Superior Fluidics Sensitivity EconomySimplicity Efficient Sample & Solvent Utilization Use Less: Dispose of Less  $$ Faster… 19 Copyright © 2006 by Protasis Corporation, All Rights Reserved

20 Data Courtesy of Mike Frey, JEOL USA, Peabody, MA USA CapNMR Performance Get an independent assessment of performance from a major NMR vendor who has used our probe - Download 30-page specifications document at cap_probe.html 20 Copyright © 2006 by Protasis Corporation, All Rights Reserved

21 Great Sensitivity, Easy Operation, Excellent Spectral Quality 600 MHz 5 min 2 hr 6 hr Sensitivity EconomySimplicity 21 Copyright © 2006 by Protasis Corporation, All Rights Reserved

22 Metabolite Identification Novatia, LLC, Princeton, NJ HPLC DAD ELSD OMNMR Mass Spec Michrom HPLC or SepNMR Labconco Centrivap 22

23 Buspirone Metabolites Metabolite Production –1.4 mg of Buspirone in 3mL with HLM –Reduce volume (vacuum centrifuge) to 400 uL Method –Column: Waters SymmetryShield RP8, 4.6X150 mm –Mobile Phases: A: 0.1% TFA in water, B:0.1% TFA in ACN –Method (1 mL/min) 0-13min at 4-63%B min 88%B 14-20min equilibrate –Collect fraction every 30 seconds (500 ul), or SepNMR 23 Copyright © 2006 by Protasis Corporation, All Rights Reserved

24 Buspirone Metabolites: Single Injection MW 385 MW Parent Absorption (240 nm) 24 Copyright © 2006 by Protasis Corporation, All Rights Reserved

25 Buspirone Metabolite MS-MS 25 Copyright © 2006 by Protasis Corporation, All Rights Reserved

26 Buspirone Metabolite: 1H NMR 26 Copyright © 2006 by Protasis Corporation, All Rights Reserved

27 Metabolomics University of Geneva, Switzerland Microfractionation in 96 well plates Micro- fractionation Column 250 x 9mm i.d. CAP-NMR Low sensitivity 1D & 2D NMR HPLC Bioassays Complementary targeted MS experiments Semi-prep Column MS split DAD UV Microfractionation Triggered by MS High sensitivity Split 1:100 Large injection 5-50 mg

28 Example of a CAP-NMR 1 H spectrum NT=4 Spectrum recorded in CD 3 OD No solvent suppression Line width <1Hz Solvent consumption (60 µl) rutin (80 µg) H-6’ H-2’ H-3’ H-8 H-6 H-1’’ H-1’’’CH 3 -6’’’ H-2’’ – H-6’’ H-2’’’ – H-5’’’ 6 8 2’ 3’ 6’ 1’’ 1’’’ 2’’3’’ 4’’ 6’’ 2’’’ 3’’’ 4’’’ 6’’’

29 On-flow LC/NMR sensitivity 10 µg 20 µg 40 µg 60 µg 80 µg 100 µg 200 µg 150 µg 20 µg 80 µg 150 µg transients Swertiamarin (MW 374) 60  l flow probe 500 MHz

30 Proton sensitivity of CAP-NMR On flow LC/NMR 20  g NT 16 MeCN:D 2 O Swertiamarin (MW 374) CAP-NMR 20  g NT 16 CD 3 OD S/N improved by a factor of 8 same S/N 60 times less NT

31 Solvent suppression in CAP-NMR CAP-NMR 2  g NT 128 WET SOLVENT SUPPRESSION 2  g NT Chemical Shift (ppm) Swertiamarin (MW 374) methanol water

32 COSY 1 x min 2D CAP-NMR capabilities Sweroside 100  g (MW 358) TOTAL ANALSIS TIME 8 HOURS COSY G-HSQC 8 x 256 1H30 min *10 1 1’ 3’ 5’’ 2’ 4’ *6’ *7 9 5 *6 solvent 1,8 10 1’ 7 6a 6b 5, 2’-5’ 6 9 G-HMBC 64 x 256 6H30 min 3 8 1’ ’-5’ ,8 10 1’ 7 6a 6b 5, 2’-5’ 9

33 The model plant: Arabidopsis thaliana Genome Proteome Metabolome Transcriptome

34 HPLC microfractionation of the enriched flavonoid extract of A. thaliana Injection 10 mg Column phenyl (250 x 9 mm i.d.) Flow 2 ml/min Fractions 1 ml/min C44C45C46C47C48

35 LC/UV/MS of the chromatogram used for microfractionation exfl10mg # RT:22,87-23,34AV:15SB:7721,55-22,66, 23,61-25,02NL:5,92E5 F:- c ESI Full ms [ 140, ,02] m/z Relative Abundance [M-H] - 739,3 477,5 445,6 740,8 421,6 216,2 741,8 506,6 446,7 921,7 313,3 775,8609,8 281,3 863,8593,7 922,9 738,8 253,3567,7 404,9387,6 776,8 468,5 364,9 957,7742,9 670,4 515,2650,8 832,2 902,0 997,5956,1 m/z 739 TIC UV (254nm)

36 1 H Cap-NMR of adjacent microfractions C44 C45 C46 C47 Each fraction 1ml = 30 sec of peak elution RT:21, ,16SM:3B 21,522,022,523,023,524,024,525,0 Time (min) uAU m/z 755 m/z 477 m/z 739 m/z 445 m/z 369 UV trace Single ion traces (LC/ES-MS neg. ions)

37 On LC/NMR vs CAP-NMR On flow NMR NT=512 CAP-NMR of fraction C47 NT=16 MW 740 -> agl 286

38 Cap-NMR gCOSY spectrum of fraction C47 NT=1 NI=512 Time=12 min

39 Cap-NMR TOCSY spectrum of fraction C47 NT=2 NI=512 Time=24 min

40 Cap-NMR gHSQC spectrum of fraction C47 NT=32 NI=256 Time= 5H00 2’ 3’ 4’ 5’ 6’ 1’ H2’ H3’ H8 H6 H1’’ 1’’ 1’’’ 1’’’’ H1’’’ H1’’’’ 6’’’’ 6’’’ 2’ 6’ 8 6 1’’’ 1’’’’ *6’’ solvent 6’’’’ 6’’’ 6’’’’ 6’’ Sugars H2’- H5’

41 Cap-NMR gHMBC of fraction C47 NT=256 NI=256 Time= 24H00 1’ H2’ H3’ H8 H6 H1’’ H1’’’ H1’’’’ 6’’’’ 6’’’ 7 1’’’ 6’’’’ 6’’’ ’ 3’ 2’ 3’ 4’ 5’ 6’ 1’ ’’ 1’’’ 1’’’’ 6’’’ 6’’’’ 6’’ 4’’’’ 5’’’’ 5’’’ 4’’’ 6’’ Sugars H2-H5

42 Some representative constituents of A. thaliana identified at line by MS and CAP-NMR

43 Search for stress induced metabolites that triggers defense gene expression Clustered Display of cDNA Microarray Analysis of Gene Expression after Mechanical Wounding Reymond, P., et al. Plant Cell 2000;12: Presence of unknown low molecular mass regulators ?

44 CONTROL WOUNDED PCA of total crude extract MS spectra spiked Total crude extract TOF MS spectra RANKED LIST OF INDUCED IONS Total crude extract TOF MS spectra CONTROL WOUNDED C 12 H 17 O

45 UPLC-TOF-ES-MS of A. thaliana Induced by wounding m/z (+/- 10 ppm) m/z (+/- 0.5 Da) CAP-NMR Multiple collection triggered by MS

46 CAP-NMR of ion m/z 209 CAP-NMR ca 1  g NT= ,2,811,5412 Standard 50  g

47 Recently Published: Lambert et al., Anal. Chem. 2007, 79, pp

48 Accelerate Data Acquisition Cornell University < 1 mg crude extract ~ 200 µg/vial 11 Semi-purified fractions ~ 40 nmol/cmpd 50 fireflies Mass Spec 48 Copyright © 2006 by Protasis Corporation, All Rights Reserved

49 Natural Products Firefly Steroids dqf-COSYHMQCHMBCNOESY NMR spectral analysis allowed for complete structural characterization of all major components in each LC fraction, leading to the identification of 13 new steroids

50 Recent Publications Cornell University M. Gronquist, J. Meinwald, F. C. Schroeder, JACS 2005, 127, pp F.C. Schroeder and M. Gronquist, Angewande Chem. Int. Ed. 2006, 45, pp Copyright © 2006 by Protasis Corporation, All Rights Reserved

51 Dionex HPLC Foxy Jr DAD ELSD u Dionex HPLC u ISCO Foxy Jr. Fraction Collector u C18 column with d-ACN/D20 gradient with flow of 1 mL/min u Eluant collected in 96-well conical bottom polypropylene plate  Receiving wells changed every 15 seconds (250  L/well) u Daughter plates assayed for activity Courtesy John Blunt, U. Canterbury, and Kirk Gustafson, NCI (see March 2006 Gordon Conf. On Natural Products Presentations at 100’s µg crude extract ~ 5 µg/well Wellplate-Centric Workflow An Example from the University of Canterbury

52 Workflow Example: Natural Products Dereplication u Evaporation using Jouan RC10-22 centrifugal vacuum evaporator u Plate enclosed with a piercible silicone/Teflon cover ~ 5 µg/well  < 10  L/well solvent u Trace amount injected into Mass Spec u Remainder available for additional analyses Mass Spec NMR 52

53 C 18 HPLC of extract from marine-derived Cephalosporium sp. DAD detection Bioactivity profile Workflow Example: Natural Products Dereplication Active Peak(s) 53 Copyright © 2006 by Protasis Corporation, All Rights Reserved

54 500 MHz 8  g in 6  L CD 3 OD PRESAT 1.5 min Recognisable features: 5 CH 3 groups, of which 1 methoxyl, 3 singlet CH 3 -C, 1 doublet CH 3 -C; 1 aldehyde 54 Copyright © 2006 by Protasis Corporation, All Rights Reserved

55 only 1 hit

56 500 MHz (2 min) 30  g in 6  L CD 3 OD Unknown Peptide, MW=684 TOCSY ? u Rapid 1D NMR enables real-time decision-making u Potentially novel compounds can be further analyzed using 2D experiments Workflow Example: Natural Products Dereplication Informed Decision-Making 56 Copyright © 2006 by Protasis Corporation, All Rights Reserved

57 57 Copyright © 2006 by Protasis Corporation, All Rights Reserved

58 Protasis Capillary NMR Enhanced productivity and Accelerated Decision-Making Bring more “axes of information” to decision-making up-front Incorporate NMR earlier, maximize information-per-unit-time Answer NMR MS ELSD UV FT-IR vs. 58 Copyright © 2006 by Protasis Corporation, All Rights Reserved

59 Enhancing Your NMR Investment Get the most from your instruments Easy Open Access Web login for vials and plates reduces time spent in the lab. Automated System Suitability and Maintenance provides assurance of good spectra. Microgram Samples avoids return trips to the lab and enables scale-down upstream in your workflow. Plate and Vial Submission simplifies reformatting and sample preparation. Closed-Loop Instrument Control enhances NMR error reporting Integrated ACD/Labs and One-Moon Scientific back-end software eliminates manual processing steps. 59 Copyright © 2006 by Protasis Corporation, All Rights Reserved

60 What is it? Everyone wants a magic wand! Leap/CTC PAL Liquid Handler CapNMR Probe NMR Spectrometer ACD/Automation Server Or Web Site You Simply Log In Samples One-Minute NMR Manages The Details ACD/Web Librarian Did I make it? Yield? Copyright © 2006 by Protasis Corporation, All Rights Reserved 60

61 Use CapNMR Sample Log-in

62 Walk away… Let One-Minute NMR run your sample

63 Click … Seconds Later… In your InBox Hot-linked report Integrated ACD/Web Librarian Copyright © 2006 by Protasis Corporation, All Rights Reserved 63

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67 System Maintenance 67 Certified and Sealed standard solutions in your solvent Run before every Nth sample or at startup Checks Signal / Noise and Line Shape to assure good results. Stops if out of spec. System Suitability Check Copyright © 2006 by Protasis Corporation, All Rights Reserved

68 System Maintenance 68 Can be executed before, after, or during a scheduled run. Injects a designated cleaning solution to ensure that flowpath walls are clear. Programmable to accommodate a wide range of sample conditions. Probe Clean Similar to Probe Clean, system can be programmed to inject additional solvent for flowpath rinsing when using problematic samples. Solvent Rinse Copyright © 2006 by Protasis Corporation, All Rights Reserved

69 System Maintenance 69 Provides broad filter face for elimination of particulates Collects particulates during injection Eliminates particulates during reverse rinse Reverse Rinse ……….. injection Injection port Particulates collect on filter face Injection needle filtered sample raw sample …. reverse rinse Particulates removed from filter face Clean solvent …. Particulates flushed to waste Copyright © 2006 by Protasis Corporation, All Rights Reserved

70 70 Sensor monitors backpressure during sample injection Provides indication of proper system function Identifies leaks quickly Identifies blockages and shuts system down safely Indicates advance need for filter change Pressure Monitoring / Pressure History System Maintenance High Pressure Transducer Pressure vs. Time Trace Copyright © 2006 by Protasis Corporation, All Rights Reserved

71 System Maintenance 71 Solvent Management 71 Vacuum Degasser Helium Sparging / Blanketing Protects valuable deuterated solvents from atmospheric contamination Copyright © 2006 by Protasis Corporation, All Rights Reserved

72 72 System Maintenance 72 Temperature -controlled drawer stacks can handle shallow or deep- well plates or vials Plate covers and vial caps maintain dry samples that are free from atmospheric contamination Sample Management Copyright © 2006 by Protasis Corporation, All Rights Reserved

73 Current One-Minute NMR Installations 22 Systems Total 17 Varian 5 Bruker 1 Custom Automated Structure Confirmation Systems (VNMR) 2 Turn-Key Automated Structure Confirmation Systems (VNMR/VNMRJ) 3 Walkup Open Access Systems Copyright © 2006 by Protasis Corporation, All Rights Reserved 73

74 600MHz capillary NMR, ns,64 10  g/10  L Protasis Corporation Protasis technology has been advanced though the support of: NIH RR14392, RR11755, HL56546, RR14661, RR16387, EB , RR15384, RR020239, NSF DMI For More Information, Visit: (product details)www.protasis.com (technical support) Sensitivity EconomySimplicity Graphics on this page complements of: Mark O’Neil-Johnson, Sequoia Sciences Wolfgang Peti, Brown University Brad Bendiak, U. Colorado Health Sciences Center Dave Detlefsen, Novatia Jane Strouse, UCLA Kirk Gustafson, NCI and John Blunt, U. Canterbury


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