Probe Type Target Temperature

Transcript

Tips & Tricks around temperature 40. NMR Benutzertagung 2016 Karlsruhe Frank Schumann Bruker BioSpin AG, Schweiz Innovation with Integrity Temperature in NMR • NMR experiments are sensitive to small changes (of less than 10mK) • even small deviations of the sample temperature have an impact on the quality of the NMR spectra (line broadening and shifting of the NMR lines) • • Precise, stable and reliable regulation required Stability of the experiment temperature subject of different influences Change of the ambient temperature Sample heating cause by RF pulses (heating of the coils) Convection flow inside the sample (10mm samples) Evaporation and condensation of the sample liquid November 9, 2016 Temperature Regulation • Temperatur sensor not inside the sample – offset of the sample temperature (CryoProbe around 2K for RT range) CryoProbes: • Direct flow • Higher VT gas flow required compared to RT • Excellent thermal insulation November 9, 2016 CryoProbes: Temperature range 2 different temperature ranges for CryoProbes: Probe Type Target Temperature Standard -40°C…+ 80°C Elevated Temperature (ET) 0°C…+ 135°C Probe Type Target Temperature Requested Flow [l/h] 1.7mm 0°C…+ 80°C 400 5mm -40°C…+ 80°C 0°C… + 135°C 670-800 10mm 0°C… + 135°C 670-800 November 9, 2016 Spinners • Standard (-40°C…+80°C) POM: 0- 80°C November 9, 2016 KEL-F: 0- 120°C Elevated (0°C… +135°C) Ceramic: down to -80°C up to 135°C SmartVT: Immunity to external VT gas and temperature variations Sensor/pneumatics and heater units • Gas flow, pressure, etc. measured and regulated… Probe interfaces • • • Excellent ‘digital’ temperature sensor electronics Plug & Play intelligent probe interfacing Dual sensor probes support (MAS) November 9, 2016 SmartVT: Intelligent VT gas flow management Ultra stable VT electronics Temperature sensor within 5  4 bar November 9, 2016 4  3 bar 3  2 bar 2  1 bar +/-15mK SmartVT: Bruker ‘self tune’ Features • Small overshoot (~1-2K, Bio samples) • Automatic heater power evaluation November 9, 2016 SmartCooler: 2nd Generation NMR cooler (BCU I/II) Cooler technology • • • Better probe interface Different cooling power modes TopSpin 3.0 integrated operation Benefits • • Best cooling with new Probes Lower NMR sample temperatures Good to know • • Compatible with all VTU and probes Maintenance free November 9, 2016 SmartCooler (BCU): New cooler technology Johnston type plug • • • No icing No leakage Adapters available for other probes Operational Modes • • • • “Remote” BCU controlled by the TopSpin 3.0 “Flush/0” Purge BCU and probe No cooling power “1” Min. cooling power (RT temperatures) “2” Max. cooling power (Low temperatures) November 9, 2016 SmartCooler and SmartVT: Improved sample temperature stability x64 SmartVT SmartCooler More than 2x better 100mM Sucrose / 5mm TCI700 spin-echo difference (HMQC without gradients, non-decoupled, refocused) November 9, 2016 Different operation modes Operation at room temperature (common operation mode in NMR) • BCU I (1, 2) Operation at temperatures below room temperature (max. -40°C) • BCU II mode (1, 2, 3) Operation at elevated temperatures • BCU I (« Flush ») Working at low and elevated temperatures: • Temperature offset higher compared to room temperature • Regulation Mode: slope limited; max. 5 K per min • Watch shim coil temperature during operation (less critical) November 9, 2016 Sample temperature stabilization November 9, 2016 Offset of the sample temperature: Temperature correction • • • • Compounds with exchangeable protons (methanol, glycol) Distance of two signals in 1Hspectrum is temperature dependent TOPSPIN: ‘calctemp D’: measure distance and calculate real temperature automatically CryoProbes: 99.8%D methanol CH3 Methanol OH D 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 Findeisen & Berger, Magn. Reson. Chem., 2007, 45, 175-178. November 9, 2016 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 ppm VTU display (edte) November 9, 2016 Save and enable temperature correction November 9, 2016 Temperature Artefakt in NMR • TOCSY (sucrose sample) Not enough dummy scans November 9, 2016 Problem • TOCSY and NOESY (sucrose sample) November 9, 2016 Solution – NMR Thermometer • TOCSY (sucrose sample) November 9, 2016 • TOCSY and NOESY NMR Thermometer Maintaining the sample temperature NMR Thermometer Standard time experiment start 4.22 4.20 4.18 4.16 4.14 4.12 4.10 4.22 4.08 4.20 4.06 ppm 4.16 4.18 4.14 4.12 Spectra of 10 mM raffinose in D2O with ~50 mM NaAc-d3 November 9, 2016 4.10 4.08 4.06 ppm H2O: ~ 0.01ppm / K @600MHz = 6Hz / K NMR Thermometer - Introduced in 2012 Hardware and software SmartVT DigiLockTM AVANCE III HD November 9, 2016 TopSpin3.2 NMR Thermometer Temperature measurement inside Probe sensor (outside) Inside the sample Add thermometer substance (temperature sensitive) Temperature sensor T November 9, 2016 T How does the NMR Thermometer work? November 9, 2016 How to choose the right substance? Compounds (typical concentrations 10 – 50 mM) C4D9OD 1.2 ppm edlock: T_* - solvents November 9, 2016 NMR ThermometerTM for bio-NMR deuterated micelles November 9, 2016 NMR ThermometerTM for bio-NMR deuterated micelles Sample heating due to CC-spinlock • monitored temperatures: target = green sample internal = yellow HCCH-TOCSY experiment 50ms spinlock duration recycle delay = 1 sec sample: protein in DHPC, acqueous solution November 9, 2016 Limits of the NMR thermometer - substance (concentration) requirements • • Shift difference D between field and frequency lock Signal ratio field and frequency lock signal Intensity ratio D November 9, 2016 Limits • S/N (2H): about 200:1 to obtain reliably lock-in performance compare S/N(2H) 1% D2O on TCI-CP600 > 1000:1 (0.2% considered to be the lower limit) • Depending on the shift difference and the signal intensity ratio of the lock signals Rule of thumb: Shift difference 3ppm -> ~1.5ppm -> approx. 2x concentration of the NMR thermometer substance necessary November 9, 2016 NMR Thermometer - features November 9, 2016 Application fields Main applications: • NMR experiments with heating (RF, rotation) • Keep temperature, small temperature range, absolute temperature not that important Other applications: • Same temperature between different systems • Bigger temperature range, absolute (exact relative) temperature • Measurements of temperature dependent parameters (kinetics, thermodynamics) • exact (absolute) temperature November 9, 2016 NOESY-HSQC, TOCSY-HSQC, CPMG-HSQC TCI CryoProbe 800MHz NMR thermometer OFF ON 0.5mM 13C/15N November 9, 2016 labeled ubiquitin/ 50mM Naac-d3 in H2O/D2O 95/5% HR-MAS on biological samples November 9, 2016 Same temperature at different systems Easy temperature referencing November 9, 2016 Temperature Correction Flow change 550 to 500l/h • time MeOD (99.8%), pseudo 2D NMR Thermometer off November 9, 2016 NMR Thermometer on Long time temperature stability NMR thermometer temperature [K] OFF 298.05 298.05 298.04 298.04 298.03 298.03 298.02 298.02 298.01 298.01 298 298 297.99 297.99 297.98 297.98 297.97 297.97 297.96 297.96 ON 297.95 297.95 0 2 4 6 8 10 0 2 1.1mK 0.9mK 50mM Naac in H2O/D2O 95/5% at 600MHz AVIIIHD TCI-CP November 9, 2016 6 8 10 time [h] time [h] T-stab.: 4 Probe Sensor NMR Therm. Automation November 9, 2016 Flowbar available in TS3.5 (since pl4) • November 9, 2016 The flowbar uses the AU- program nmr_therm Flowbar: Show regulation parameters November 9, 2016 Flowbar: Operation modes November 9, 2016 Flowbar: Tools Automatic temperature correction November 9, 2016 NMR Thermometer manual included November 9, 2016 New function – “Keep temperature” • Create T_ solvent (2 signals) • Lock on that solvent November 9, 2016 New function – “Keep temperature” • Execute AU-program keep_temp -> Creation of temperature-shift table (.xml) in background – lock - NMR-T. in regulation mode (automatically) November 9, 2016 Intermediate .xml in dataset November 9, 2016 New workflow – “Keep temperature” function • Start experiment November 9, 2016 Conclusion - Outlook • CryoProbes (5mm) come in two temperature ranges (-40°to 80°C and 0°to 135°C) • Well proven chillers (SmartCooler: BCU I/ II) and temperature regulation (SmartVT) available for highest temperature performance • Since TopSpin 3.5pl4 the NMR-T. flowbar is part of the TS release (including the nmr_therm AU program) • Easier access to the NMR-T. via the “Keep temperature” function (TS3.5pl6) for the majority of applications (heating experiments) • More to come November 9, 2016 Thanks to… • Wolfgang Bermel, Daniel Mathieu, Clemens Anklin, Helena Kovacs • Rolf Hensel • Marina Maurer, Werner Mausshardt, Eberhard Schweizer, Christof Johann, Peter-René Steiner November 9, 2016 Innovation with Integrity Copyright © 2011 Bruker Corporation. All rights reserved. www.bruker.com NMR thermometer using Naac (S/N-performance) Monitoring Experiment (SL, CPD) 600MHz TXI 600MHz QCI 850MHz TCI <1mK 1-3mK >200mM 50-100mM 50mM 1-2mK 3-6mK 200500mM <200mM 100mM 20mM <20mM 2-4mK 6-12mK 100200mM <100mM 50mM 10mM <10mM 4-8mK 12-30mK 50-100mM <50mM 20mM >8mK >30mK <50mM November 9, 2016 400MHz BBFO 600MHz BBFO