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Agenda

Hyphenation of Chromatography to NMR and MS: Technical Realization and Applications Markus Godejohann Bruker Biospin Germany. Agenda. Onflow HPLC-NMR, stop-flow HPLC-NMR Loops vs. SPE Probes for flow-applications Integrating an MS into LC-(SPE)-NMR LC-SPE-NMR multi-trapping

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Agenda

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  1. Hyphenation of Chromatography to NMR and MS: Technical Realization and ApplicationsMarkus GodejohannBruker Biospin Germany

  2. Agenda Onflow HPLC-NMR, stop-flow HPLC-NMR Loops vs. SPE Probes for flow-applications Integrating an MS into LC-(SPE)-NMR LC-SPE-NMR multi-trapping LC-SPE-NMR with NMR tubes Applications

  3. Onflow HPLC-NMRstop-flow HPLC-NMR

  4.  Measurements - Schematic view Spectrometer Sample typically a mixture On-flow LC-NMR Interface Direct . Intermediate storage Chromatographic System separation  compounds  Stop-flow HPLC Detector UV MS Storage/transfer

  5. On-flow experiment on AVIII 0.25ml/min 0min 80% D2O Step to 50% D2O Gradient to 5% D2O

  6. On-flow experiment on AVIII

  7. PHBA-methyl-ester 1: 2.07min, 28.31% 2: 2.91min, 26.26% PHBA-ethyl-ester PHBA-n-propyl-ester PHBA-methyl-ester 3: 3.85min, 23.80% nmr+recover RT(2.03) nmr+recover RT(2.92) nmr+recover RT(3.89) nmr+recover RT(4.77) PHBA-n-butyl-ester 4: 4.73min, 21.63% PHBA-ethyl-ester PHBA-n-propyl-ester PHBA-n-butyl-ester pump pressure pump pressure stop for RT 2.92min stop for RT 2.03min stop for RT 3.89min stop for RT 4.47min 0 0 1 2 3 4 5 Time [min] 0 1 2 3 4 5 Time [min] Chromatography: Stop-flow Effect • System stands approx. 15min per stop. • Slight distortions due to startup visible

  8. nmr RT(12.33) nmr RT(14.71) nmr RT(14.95) nmr RT(15.36) nmr RT(17.40) nmr RT(20.29) 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 Time[min] 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 Stop-flow with multiple Peaks       15min 84% 100% • ReferenceChromatogram • No interruption • Stop-flow Chromatogram. For each peak … • Stop chromatography • Pause 15min to acquire NMR • Restart chromatography • Check the peak @ 20.3min … • Experienced 5 start/stopoperations • Remained 1,5hon the column. • Still shows 84% intensitycompared with peakfrom referencechromatogram.

  9. Flowrate: 1 ml/minProbehead: 120 µl Distance: 0.35 min = 350µl Stop-flow  Loop-Transfer

  10. Stop-flow  Loop-Transfer Direct stop-flowcross contamination Loop Transferno cross contamination

  11. Bruker Stop Flow Unit- High Performance. Holds up to 80kg on top Fits into Agilent Tower Fits below LC-SPE-Interface Includes Manual injector Includes Column Holder capillaries and valvesi.d. 0.13mm Position indicationfor stop-flow valve. WEB-interface forfuture applications LC-NMR Interfaces – BSFU-HP

  12. BPSU-36/2 • Interface for all LC-NMR workingmodes. • Chromatography(no loops, no NMR) • On-flow LC-NMR. • Stop-flow LC-NMR. • Loopstorage basedon UV or MS. • Automatic switch from loop storagetotransfer. • Looptransfer into NMR.

  13. Loops versus SPE

  14. LC-NMR Limitations/Disadvantages • Amount of sample is limited by chromatography. • Amount from one injection is available for the NMR. • Only a fraction of a broad peak is positioned inside the NMR volume (approx. 120µl). • Non deuterated chromatography solvents cause background signals in the NMR. • Deuterated solvents needed (D2O), increase price, prevent observation of exchangeable protons and cause problems with MS detection

  15. Pass the raw samplesolution through aSPE cartridge „Strong“Organicsolvent NMR Sampleelutes insmallelution band SampleoncartridgeSolvent LC ConcentratedSample Solutionin organic solvent SPE – Solid Phase Extraction AqueousSampleSolution

  16. Mixture of Water andorganic solvent Water addition 2. Drywith N2 Separationin HPLC SPE –Collector for the NMR Transfer directly intoflow probe withdeuterated solvent 1. WashH2O Sample( Mixture ) Components onindividual cartridges LC SolventsSalts, Buffer

  17. Make up (water) 1st UV HPLC column SPE cartridge 2nd UV LC-SPE-NMR: retention on SPE cartridges

  18. Bruker/SPARK Prospekt 2 SPE-UNIT • LC-SPE-NMR interface. • Automatically Handles 192 cartridges. • Preparation, Trapping, Drying, Transfer. • Syringe pumps for deuterated Solvents. • Complete Process automated • Preparation, Trapping, Washing/drying • Transfer to NMR/tube

  19. LC-SPE „Consumables“ • SPE Cartridges • 2x10mm or 1x10mm • Approx 40 differentpacking materials(C2, C8, C18, polymer,ion exchange, …) • ~4 €/cartridgeapprox. 15-30x reusable • Capacity several 100µg(depending on sample) • Solvents • Cleaning, Preparation, chromatography = non-deuterated • 300µl/sample deuterated solvent for elution of cartridges

  20. Loop collection: 5µg PHBA Ethylester • Peak stored in BPSU-36 Loop and transferred into NMR • 5µg on column • Solvent CH3CN/D2O 50.6%/49.4% • Signal to Noise 10.4:1 ( 9 - 5.5 ppm ) • 24 scans, 500 MHz, double presat.

  21. Peak trapping: 5µg PHBA Ethylester • Peak trapped on cartridge and transferred into NMR • 5µg on column • Solvent CD3CN 100% • Signal to Noise 23.5:1 2.3x loop storage • 24 scans, 500 MHz,no solv. suppression.

  22. Comparison LC-SPE-NMR vs. LC-NMR Intens. [mAU] 600 400 UV 254 nm 200 UV 370 nm 0 2 4 6 8 10 Time [min] • Compare twodifferentpolarities • Polar: Amino-acid Tryptophan • Medium polar: polyphenol-glycosideRutin • Analysis: • LC-SPE-NMRusing GP resin. • LC-NMR withloop storage/transfer

  23. Medium polar compound Rutin:LC-SPE-NMR 1,5x better than LC-NMR! CH3CN CH3OH HDO FA CH3CN HDO LC-NMR15:1 1xLC-SPE23:1 3xLC-SPE75:1

  24. Polar Compound Tryptophan:LC-NMR wins! CH3CN CH3OH HDO FA CH3CN HDO LC-NMR39:1 LC-SPE2.3:1 3xLC-SPE6.2:1

  25. LC-SPE-NMR on mixed mode strong cation exchange: elution with MeOD / 5% NH4OH HDO CH3CN FA MeOD LC-NMR105:1 LC-SPE476:1 3xLC-SPE1055:1

  26. LC-IX-SPE-NMR: Enrichment and extraction Catch “weak” cations Release “weak” cations No interaction with stationary phase! Positively charged @ pH < 6 (mobile phase: H2O 0.1% HCOOH, pH 2.6) Neutral @ pH > 10 (elution from cartridge with CD3OD 5% NH4OH) Always negatively charged Catch “weak” anions Release “weak” anions No interaction with stationary phase! Negatively charged @ pH > 7 (mobile phase: H2O 0.1% NH4OH, pH 10.8) Always positively charged Neutral @ pH < 1.4 (elution from cartridge with CD3CN 2% DCOOD)

  27. LC-NMR versus LC-SPE-NMR • LC-SPE-NMR: • Lipophilic or ionizable polar compounds that can be trapped under standard conditions. • Broad chromatographic peaks which benefit from the SPE-concentration effect. • For low concentrated samples or less sensitive NMR experiments (1H/13C) that require sample enrichment by multiple injections. • LC-NMR: • Polar compounds that would require method development for successful LC-SPE-NMR trapping. • Unstable compounds which tend to decompose during the drying process LC-NMR may provide better results. • Compounds with sufficient sample amount in one injection and with good “sharp” chromatographic peak shape. • Fast analysis where cartridge drying time is annoying. • Simple analysis, where no choice of trapping material, amount of makeupflow and selection of transferliquid should be done.

  28. Probes for Flow Applications

  29. out in Eluent Probe Design for LC-NMR RF-coils Coils directly attached toflow cell for best fillingfactor and highestsensitivity. Volume typically smaller~120 to 30µl. Notubechange onlysample changes. Effectfor shimming minimized.

  30. Convert an NMR into an LC-NMR • Insert Flow Probe intostandard NMR. • Noother modifications. • No special NMRconfiguration required • standard HR-amplifiers • Two RF-channels • Required timeapprox. 15min. • NMR instrument can beshared for HR- tubeand flow applications. • Available with30, 60 and 120µlNMR active volume.

  31. CryoNMR for Flow Applications • Major Properties of CryoProbes • CryoProbe provides increase of signal to noise of ~4. • CryoFlow NMR??? • General usage of FlowProbes • Change from flow to tube is 15min for RT probes. • Changing a CryoProbe is severalhours. • Price for a (additional) flow CryoProbe would be high. • Solution  CryoFit! • ConvertsCryo Probe from tube into flowprobe within 15min. • One CryoProbe can be used with multipleCryoFits of different geometry (capillaries diameters, cell size).

  32. CryoFit™ - Properties Metal rod with flow cellinside. Inserts from the top into the magnet. Bendable for convenient mountingwith low ceiling height.

  33. New CryoFit – Improved design Simple replacementof flowcell Capillary can bedisconnected Robust PEEK inlet capillary. Simple replacement of inletcapillary possible. Capillary and flow separate parts. On site exchange of flow cellpossible as well.

  34. Integrating an MS intoLC-(SPE)-NMR

  35. Special requirements in LC-NMR/MScompared with LC-MS • MS destroys the sample • Use a split and send only a small fraction of the eluent to the MS. • NMR requires large sample amounts • Use a split and send only a fraction of the sample to the MS to avoid overloading and fast contamination of the spectrometer. • Use a switchingvalve to allow disconnection of the eluent from the MS during chromatography while major peaks elute. • LC-NMR often uses highflowrates ~1ml/min flow • Use a split and send only 10-50ul/min to MS. • Verylowflow rate in the MS flow path (1%=10ul/min) • Add a makeupflow to improve peak shape. • If used as trigger signal UV and MS chromatogram should be synchronized • Add a makeupflow to reduce transfer time between split and MS inlet.

  36. BNMI-HP (Bruker NMR MS Interface) Bruker NMR MS Interface– High Performance Microbore (.15mm) valvefor dis-/connection of MSand selection of workingmodes. Split 1:100 or 5:95 Two highpressurepumps forcontinuous addition ofmakeupflow up to 200ul/min Optional 3rd pump forcalibrant addition(required for micrOTOF).

  37. Chromatography with ACN/D2O (H2O buffer added in BNMI) Chromatography with ACN/H2O: [M(HnDm)+D]+ [M+H]+ 13C-isotope peak LC-NMR/MS with D2O

  38. Intens. Intens. 19.1 16.1 5 6 4 x10 x10 1.5 3 1.0 2 1 0.5 0 0.0 0 5 10 15 20 25 Time [min] 0 5 10 15 20 25 Time [min] 474.1 Time=19.21min 465.1 Time=16.10min 309.2 100 200 300 400 500 600 700 800 900 m/z 100 200 300 400 500 600 700 800 900 m/z 303.0 LC-NMR/MS with D2O H-D exchange as an analytical tool M: 464 H-Dacidic: 8 D adduct: 2 474 Chromatography with water Chromatography with D2O [M+H]+ = 465 [M (D8)+D]+ = 474

  39. 0 9.50 9.75 10.00 10.25 10.50 10.75 11.00 11.25 11.50 Time [min] 0 6 8 10 12 14 16 18 Time [min] How to use MS in a LC-(SPE)-NMR setup UV 200-500nm A C B • Peak C • Trivial, change by RT 0.25min or 30% intensityhas no effect on trapping. • Peak B • Loose 10% intensity- peak is lost. • Shift A by 0.1min later– A instead of B is collected • Requires high setting of threshold,only part of the peak is collected.

  40. 0 9.50 9.75 10.00 10.25 10.50 10.75 11.00 11.25 11.50 Time [min] 0 6 8 10 12 14 16 18 Time [min] How to use MS in a LC-(SPE)-NMR setup m/z 215.5-216.6 UV 200-500nm A C B • Solution • Define MS chromatogramm for the peakof interest, here m/z 215.5-216.6 • Result is a simple chromatogramwith just one peak. • Define collection based on this mass chromatogramm. • RT-Time shift of 0.25min orintensity change of 50% willhave no effect. • Note: It is possible to usethe mass chromatogram for detection of Peak B andthe UV chromatogram forthe detection of Peak C.

  41. LC-SPE-NMR Multi-trappingRequirements and Results

  42. trapping (1B1) RT(3.59) trapping (1B2) RT(9.94) trapping (1B3) RT(11.76) trapping (1B4) RT(15.04) trapping (1B5) RT(15.84) trapping (1B6) RT(16.63) trapping (1B7) RT(17.06) trapping (1B8) RT(17.62) end trapping (1B1) RT(3.87) end trapping (1B2) RT(10.31) end trapping (1B3) RT(12.13) end trapping (1B4) RT(15.27) end trapping (1B5) RT(16.18) end trapping (1B6) RT(16.88) end trapping (1B7) RT(17.30) end trapping (1B8) RT(17.72) 0 2 4 6 8 10 12 14 16 Time [min] LC-SPE-NMR Multitrapping CH3CN 6 x 20ul injection of pepperextract. Peak at 3.59min elutes with34.8% CH3CN 6x trapping on samecartridgeGP Resin, 2x10mm. Eluted with CD3CN into60ul RT Probe at 500MHz

  43. LC-SPE-NMR Multitrapping 6x SPE, NS 256 Sino 210:1 Increase SINO by 6 1x SPENS 256Sino 36:1 6x SPE, NS 8 Sino 35:1 ReduceNMR time by factor 1/30

  44. LC-SPE-NMR Multitrapping 1x SPE NS 256 Sino 36:1 6x SPE, NS 8 Sino 35:1 Reduce chemical background from solvent.

  45. Applications

  46. UV @ 370 nm ? ? Auto MS/MS  same separation! O H H O O O H g l y c o s i d e O H O 5 10 15 20 25 Time [min] Q u e r c e t i n - 3 - s u g a r LC-NMR/MS Apple peel extract A. Lommen, M. Godejohann, D. P. Venema, P. C. H. Hollman, and M. Spraul. Application of Directly Coupled HPLC-NMR-MS to the Identification and Confirmation of Quercetin Glycosides and Phloretin Glycosides in Apple Peel. Anal. chem., 2000, 72, 1793-1797.

  47. Aromatic protons: Q-3-glu Q-3-gal Sugar protons: MeOH HDO Q-3-gal Q-3-glu LC-NMR/MS Quercetin-C6-glycosides Q-3--D-glucoside H O O O H H O O H O O O O H H O O H H O Q u e r c e t i n - 3 - ß - D - g l u c o p y r a n o s i d e M = 4 6 4 Q-3--D-galactoside H O O O H H O O H O O H O O O H O H H O Q u e r c e t i n - 3 - ß - D - g a l a c t o p y r a n o s i d e M = 4 6 4

  48. Chromatoraphy on 250 x 2mm column, flow rate 200ul/min, 20ul injected UV @ 280nm EIC m/z 463: Quercetin C6 glycosides EIC m/z 465: Phloretin C6 glycoside 0 10 20 30 40 Time [min] LC-NMR vs. LC-SPE-NMR

  49. Result from former LC-NMR run injection volume 100ul, 128 scans Result from LC-SPE-NMR; injection volume 20ul single loading, 128 scans OH protons H O O O H H O O H O O H O O O H O H H O Q u e r c e t i n - 3 - ß - D - g a l a c t o p y r a n o s i d e M 4 = 4 6 LC-NMR vs. LC-SPE-NMR

  50. LC-NMR vs. LC-SPE-NMR Injection volume 20ul single loading, 128 scans Injection volume 20ul 4x loading, 128 scans

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