Innovation with Integrity

UK NMR Users’ Meeting 2016

Matteo Pennestri

Tips & Tricks around temperature

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

December 5, 2016

Temperature Regulation

• Temperatur sensor not inside the sample – offset of the sample temperature (CryoProbe

around 2K for RT range) CryoProbes: • Direct flow

• Excellent thermal insulation

• Higher VT gas flow required compared to RT

December 5, 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

December 5, 2016

Spinners

• Standard (-40°C…+80°C) Elevated (0°C… +135°C)

POM: 0- 80°C KEL-F: 0- 120°C Ceramic: down to -80°C up to 135°C

December 5, 2016

SmartVT: Intelligent VT gas flow management

December 5, 2016

Ultra stable VT electronics

Temperature sensor within +/-15mK

5 4 bar 4 3 bar 3 2 bar 2 1 bar

SmartVT: Bruker ‘self tune’

Features • Small overshoot (~1-2K, Bio samples) • Automatic heater power evaluation

December 5, 2016

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

December 5, 2016

SmartCooler: 2nd Generation NMR cooler (BCU I/II)

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)

December 5, 2016

SmartCooler and SmartVT: Improved sample temperature stability

December 5, 2016

100mM Sucrose / 5mm TCI700 spin-echo difference (HMQC without gradients, non-decoupled, refocused)

x64

SmartVT SmartCooler

More than 2x better

Temperature artifact in 2D experiments

• TOCSY (sucrose sample)

Not enough dummy scans

December 5, 2016

Problem

• TOCSY and NOESY (sucrose sample)

December 5, 2016

Solution – NMR Thermometer

• TOCSY (sucrose sample) • TOCSY and NOESY

December 5, 2016

4.084.104.124.144.164.184.204.084.104.124.144.164.184.20

Standard NMR Thermometer

NMR Thermometer Maintaining the sample temperature

Spectra of 10 mM raffinose in D2O with ~50 mM NaAc-d3

experiment start

time

H2O: ~ 0.01ppm / K @600MHz = 6Hz / K

December 5, 2016

DigiLockTM SmartVT TopSpin3.2

NMR Thermometer - Introduced in 2012 Hardware and software

AVANCE III HD

December 5, 2016

NMR Thermometer Temperature measurement inside

Probe sensor (outside)

T

Temperature sensor

Inside the sample

T

Add thermometer substance (temperature sensitive)

December 5, 2016

How does the NMR Thermometer work?

December 5, 2016

Compounds (typical concentrations 10 – 50 mM)

How to choose the right substance?

C4D9OD 1.2 ppm

December 5, 2016

edlock: T_* - solvents

NMR ThermometerTM for bio-NMR deuterated micelles

December 5, 2016

• monitored temperatures: target = green sample internal = yellow

HCCH-TOCSY experiment 50ms spinlock duration recycle delay = 1 sec sample: protein in DHPC, acqueous solution

NMR ThermometerTM for bio-NMR deuterated micelles Sample heating due to CC-spinlock

December 5, 2016

Limits of the NMR thermometer - substance (concentration) requirements

∆

Intensity ratio

• Shift difference ∆ between field and frequency lock • Signal ratio field and frequency lock signal

December 5, 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

December 5, 2016

NMR Thermometer - features

December 5, 2016

Application fields

Main applications:

• NMR experiments with heating (RF, rotation) • Keep temperature, small temperature range, not important absolute

temperature

Other applications: • Same temperature between different systems

• Bigger temperature range, absolute (exact relative) temperature

• Measurements of temperature dependent parameters (kinetics, thermodynamics) • exact (absolute) temperature

December 5, 2016

NOESY-HSQC, TOCSY-HSQC, CPMG-HSQC TCI CryoProbe 800MHz

NMR thermometer OFF ON

0.5mM 13C/15N labeled ubiquitin/ 50mM Naac-d3 in H2O/D2O 95/5% December 5, 2016

HR-MAS on biological samples

December 5, 2016

Same temperature at different systems Easy temperature referencing

December 5, 2016

Temperature Correction Flow change 550 to 500l/h

December 5, 2016

NMR Thermometer off NMR Thermometer on time

• MeOD (99.8%), pseudo 2D

Long time temperature stability

0.9mK

tem

pera

ture

[K]

time [h] time [h]

NMR thermometer OFF ON

NMR Therm. Probe Sensor 1.1mK T-stab.:

50mM Naac in H2O/D2O 95/5% at 600MHz AVIIIHD TCI-CP

297.95

297.96

297.97

297.98

297.99

298

298.01

298.02

298.03

298.04

298.05

0 2 4 6 8 10297.95

297.96

297.97

297.98

297.99

298

298.01

298.02

298.03

298.04

298.05

0 2 4 6 8 10

December 5, 2016

Automation

December 5, 2016

Flowbar available in TS3.5 (since pl4)

• The flowbar uses the AU- program nmr_therm

December 5, 2016

SampleJet

• Holds 5 racks with each 96 tubes • Standard 96 well format

compatible with common automation robots

• Tube diameter automatically recognized

• Outer ring up to 99 tubes (3 spinner positions)

• No spinner needed

5. Dezember 2016

Can we store our samples refrigerated ?

SampleJet with cooling option

5. Dezember 2016

NEW

• Storage of cold and warm samples at

the same time. Racks can be temperature regulated individually.

• Outer ring for 99 tubes (5mm) at room temperature

• Extended firmware for faster automation.

Speed up automation

• Samples at 6ºC - Measurements are at 37ºC

• Preheating station (at 37ºC)

• Extended firmware temperature regulation «on hold» during sample change

5. Dezember 2016

T

min

~1- 1.5min

~1K

SampleCase

5. Dezember 2016

• Very popular with open access routine labs

• Cost-effective and robust sample changer

• 24 sample positions

• Convenient and safe sample access at user height

• Random access for sequential and batch mode automation

• Almost all samples in tubes (1mm – 10mm ∅, up to 9’’ length)

• IconNMR & TopSpin control

• Compatible with CyroFit and MAS

Temperature relevant samples SampleCase Cooled

• Keeps NMR samples refrigerated

(6ºC +/- 2ºC) before and after experiments

• temperature range 4 – 40ºC • Requires 2nd BCU-I or equivalent

cooling unit

• TopSpin implementation

5. Dezember 2016

IconNMR temperature handling

5. Dezember 2016

Temperature relevant samples Polymer applications

• Mix and heat up to 120-130ºC in heat block for several hours (overnight) to dissolve the polymer

• Homogenize sample (stick, heat gun)

• NMR experiments at 120-130ºC

5. Dezember 2016

New: SampleCase Heated

• Up to 24 individual heated sample

positions (number of positions selectable)

• Temperature setting controlled via TopSpin software (range: max. 125ºC +/- 5ºC)

• Proven heating control technology

• Supports all common sample-tubes in spinners suitable for high temperature operation

5. Dezember 2016

Implementation in TopSpin3.5

5. Dezember 2016

Implementation in TopSpin3.5

5. Dezember 2016

IconNMR: Configuration Temperature handling

5. Dezember 2016

Comparison of heated and not heated

5. Dezember 2016

~2.5-3min

~ 1.5min

Holder not heated Holder heated

5min MeOD sample at 313K pseudo 2D

Innovation with Integrity

Copyright © 2011 Bruker Corporation. All rights reserved. www.bruker.com

NMR thermometer using Naac (S/N-performance)

Monitoring Experiment (SL, CPD)

400MHz BBFO

600MHz BBFO

600MHz TXI

600MHz QCI

850MHz TCI

<1mK

1-3mK >200mM 50-100mM 50mM

1-2mK

3-6mK

200-500mM

<200mM 100mM 20mM

<20mM

2-4mK

6-12mK

100-200mM

<100mM 50mM 10mM <10mM

4-8mK 12-30mK 50-100mM <50mM 20mM

>8mK

>30mK <50mM

December 5, 2016