NMR Facility - Chemistry Department

Training - Level1

Everyone seeking access to the facility’s NMR instruments starts with Level1 training, which enables access to our autosampler-equipped spectrometer. Leveraging our automation for data collection, we focus on magnet lab safety, proper sample preparation, detailed data processing techniques, and facility policies. Interaction with the spectrometer only takes about ten minutes, and emphasizes the different experiments and available and which are best for users’ purposes.

For the occasional NMR user, we’ve found this provides enough expertise and access to acquire meaningful data without delving into the details of spectrometer operation or NMR theory.

Topics Covered

PART 1A – Orientation, Safety, & Success
(~2 hrs)


  • Trainees and the facility manager introduce themselves to one another, discussing prior NMR experience
  • Identification of research aims


  • Identification of all spectrometers and their lab locations, discussion of each instrument’s functions and top-level capabilities
  • Mention of the EPR spectrometer, query for interest
  • Identification of the support lab in Searle, equipped with fume hood, bench, sink, and eyewash station, enabling titrations and reaction initiation for kinetics studies


  • Identification of exits, fire extinguishers, safety contact information posters, first aid kits, cleaning/spill supplies
  • Review of PPE requirements (labcoats prohibited, safety glasses optional, gloves optional but only fresh)
  • Lab-specific chemical safety
    • Response procedures for broken samples, including reporting procedure (send text or email to facility manager)
    • Requirement for use of secondary container for transporting samples
    • Introduction to the community craft table with safety flasks and decoration supplies
  • Magnet safety – Cryogens (frostbite and suffocation hazards, quench identification and response, hazards of weekly nitrogen fills)
  • Magnet safety – Magnetic field
    • Discussion of problematic/safe metals, risks of various medical devices/implants
    • Identification of 5-gauss radius safety limit and its significance
    • Caution about variability of 5-gauss radii in other facilities
  • Identification of major spectrometer components (magnet, autosampler and components, console, computer, probe)
    • Special attention and procedures for handling samples broke in the carousel or magnet
    • Advice on when to press the emergency stop button on the autosampler
    • Discussion of when and how to report instrument problems and work with the manager to resolve them
    • Clear distinction between the console and computer buttons in case power cycling of one is required; clear identification of the “Do not ever touch this” cover on the magnet

Success with Sample Preparation

  • Discussion of probe geometry and alignment of sample using sample depth gauge
    • Proper use of the depth gauge, proper handling of the spinner, and cleaning the sample
    • Importance of filling detection region with sample
    • Importance of excess material (0.6 mL); contrast with UV/Vis requirement; discussion of magnetic susceptibility
    • Hands-on practice inserting and aligning a normal sample
    • Hands-on practice realigning a sample with small volume
    • Tactile evaluation of spinner grip strength, then contrast with a too-loose spinner-sample interaction
      • Cautionary tale: the $10K+downtime consequences of a tube that is too loose
    • Discussion of suspended particles resulting in broad lines; relationship to magnetic susceptibility
  • NMR tube selection (economy vs precision glass, MHz ratings); economics factors deciding whether to reuse NMR tubes
  • Procedure for cleaning and drying NMR tubes, including Wilmad oven-drying guidelines
  • Discussion of when to discard NMR tubes (scratched, chipped, bent, too narrow, too contaminated)
  • Discussion of NMR tube CAP quality and economics
  • Best practices for sample labeling
    • At least include initials
    • Acceptable: Sharpie on cap, Sharpie on upper part of tube, Lab tape securely wrapped around upper part of tube, Paint pen on cap
    • Unacceptable: Paint pen on tube, Sharpie or paint pen where it would contact the spinner
  • NMR tube length requirements (< 8.75″ / 222 mm); cautionary discussion about bumping the red safety lever on the autosampler
    • J. Young tubes OK if they fit the length requirement
    • Regular tube sealed with electrical tape over cap often OK

Sample Management

  • Never take samples that do not belong to you out of the lab. You may take your own samples at any time
  • OK to move others’ samples from carousel to TOP SHELF racks next to magnet, ONLY IF the sample’s experiments are ALL marked “finished” in Icon software.
  • CYCLE: Every week, the racks on the top shelf get moved down to the bottom shelf. Then the samples from the bottom shelf (which had been there for a week and some days on the top shelf) are considered ABANDONED and moved to the “TAKE ME” beaker.
  • All samples in the TAKE ME beaker may be taken by any user who agrees to dispose of the contents properly.

Part 1B: Submitting samples
(~30 min)

  • Orientation to the ICON-NMR interface in Bruker’s Topspin software
    • Login panel, importance of logging in, use of the “Next User” button
    • Upper part: For sample submission, able to delete entries
    • Lower part: Completed Samples, like a log
  • Viewing completed spectra
    • Double-clicking on a completed spectrum in lower part of ICON – intended just for a quick check
    • DO NOT ANALYZE spectra on the spectrometer computer; permissions disabled for phasing, pick peaking, integrating
    • CAUTION: Do not close the Topspin window after viewing a spectrum! This shuts down all data acquisition.

Sample Submission columns, actions, best practices:

  • “Holder” column: designates sample position in carousel
  • “Status” column: Available, Finished, Queued, Running, Failed, AutoCalibrate
    • Use any “Available” position
    • Any sample with ALL experiments “Finished” may be converted to Available with the Delete button
    • Failed samples cannot be deleted, except by the admin. Their owners may return and fix whatever problem arose and try again.
    • Position #60, “AutoCalibrate”. is RESERVED for the admin and cannot be used by researchers. It is used for automatic weekly reshimming and performance checks.
    • Hands-on introduction to the AutoCalibrate sample, which MUST STAY IN THE CAROUSEL. It is in a unique orange spinner labeled “DO NOT MOVE”, is permanently sealed, bears a bar code, and the tube is glued to the orange spinner. Don’t touch it!
  • “Name” and “Number”: Sample name and Spectrum number are described
    • File location location is identified, and Data structure is discussed: Freely-named sample folder with each spectrum assigned a numerically-named subfolder.
    • Best practices on sample naming, advocating adoption of a convention that will preserve order in a single folder containing hundreds or thousands of sample names. Recommended:
      • <initials><lab notebook number>-<page number>-<A/B/C…, 1/2/3…, or other identifiers>
      • <initials>YYMMDD_<A/B/C…, 1/2/3…, or other identifiers>
    • Default experiments number = 10. Can be changed if desired. Incremented automatically when experiments added to the sample.
  • “Solvent” – Pull-down menu to select solvent
    • Default = CDCl3
    • Must choose a solvent in the list. If using a mixed solvent not in the list, choose the one with more deuterium
    • “No-D” deuterium-free samples are supported, but the user must choose either the protiated version of the solvent (e.g. C6H6 instead of C6D6) or “None”. Choosing “None” will provide a spectrum with unoptimized lineshape and requires manual referencing.
    • The facility manager welcomes new solvents to be added to the list, but the user must provide a sample of that solvent for calibration.
  • “Experiment” – Pull-down menu to select experiment(s)
    • Default = PROTON8
    • Most common experiments at top of list: PROTON8, PROTON1, PARAMAG-1H, CARBON, CARBON-3.5hr
    • Discussion of PROTON8 experiment. 8 scans, 30 deg excitation in vector model, 36 sec total acquisition. Decent S/N and integral accuracy for typical sample (~5 mg of 150 MW material).
    • Contrast with PROTON1: 1 scan w/ 17 second relaxation delay D1, 90 deg excitation. Great for accurate integrals, plenty of S/N in one scan.
    • Discussion of relationship between S/N improvement and NS, number of scans.
    • Identification of the “=” button, which enables changing of parameter values. Discussion of meaning of NS, D1, O1P, SW, and AQ
    • Identification of the “Title” button, which enables addition of a meaningful comment/title to each spectrum, e.g. “1H 1D”, “13C 1D”
    • Adding an experiment, noting change in experiment number
    • Distinction between CARBON and CARBON-3.5hr: only parameter difference is NS, but longer one is nighttime-only
    • Discussion of usage rules, Day Queue / Night Queue, per-sample time limits, per-day & per-night time limits. Spelled out here:
    • Identification of the Day/Night queue toggle button
    • Setup of common “SW-optimized” 2D experiments (COSY-opt, HSQC-opt, HMBC-opt, etc.). Requirement of PROTON8 experiment and pitfalls that cause failure. Identification of equivalent non-optimized “-manual” experiments in list.
    • Identification of heteronuclear experiments and nomenclature, e.g. difference between PHOSPHORUS and PHOSPHORUS_noHdec. See instrument pages for their available nuclei. https://chemnmrlab.uchicago.edu/instruments/
    • Identification of 1H-15N HSQC, HMBC
    • Identification of WATER experiment for automated solvent suppression of 90/10 H2O/D2O samples
    • Identification of PROTON_T1 experiment (and FLUORINE_T1 and PHOSPHORUS_T1 on 400-2). Discussion of the importance of measuring T1 for accurate quantitation. Tragedy avoidance: no one wants a situation where a manuscript reviewer asks whether quantitative conclusions are justified by 1H NMR integrations, and it turns out the D1+AQ settings were not long enough to provide high-quality integration.
  • Assessing total experiment time. Discussion of extra steps in data acquisition that take time (sample changing, tuning, locking, shimming without going into detail about what those processes are). Identification of the current “Day” and “Night” queue totals and the more informative “Busy Until” time. Also identification of “Current” sample identifier.
  • Clicking “Submit” to add experiments to the queue.
  • Clicking “Cancel” and “Edit” to remove experiments from the queue and resubmit.
  • Discussion of the “Copy” function.
  • Importance of logging out by clicking “Next User” button. Avoids next user submitting samples in your account.

Queue Management – For Problem Recovery Only

  • Build awareness of Start and Stop queue-control buttons.
  • Only use when troubleshooting a stopped queue and in conversation with the facility manager. Not for ordinary use.
  • Ignore the top row of software menu buttons. Those are admin-only.

PART 2: Data Processing and Assignment Strategy
(~90 min)

Orientation to LAN Environment

  • Local Area Network (LAN) architecture. Spectrometers on LAN only, cannot access directly from outside.
  • “Datastation” servers visible to campus network and VPN-activated computers outside
  • Flow of data from acquisition on spectrometer computer, automatic copying to datastations
  • Prohibition of USB drives for data transfer; all facility computers disabled
  • Data transfer via free online tools Cyberduck, FileZilla, and similar. See this for protocols:
  • Datastations also available for processing by users.

Datastation Working Environment, Spectrum File Structure

  • Logging in to datastation, orientation to workspace
  • Identification of Home folder, where to find data and data of labmates
  • Navigation to example dataset, discussion of samplename/samplenumber structure
  • Viewing contents of experiment folder, identifying fid file, other files, processed data folder
  • Web browser home page = UChicago Box login, opportunity to drag data folders into Box
  • Desktop icons for Topspin and MNova

Data Processing in Topspin 3: 1H 1D of Carvone

  • Opening Topspin, orientation to layout: top-row tabs, buttons within each tab, spectrum manipulation buttons, data browser
  • Data browser and association of experiment numbers with pulse sequence names and text comments/titles
  • Opening the 1D 1H spectrum, identification of the familiar spectrum as “frequency-domain” data. Walkthrough of different spectrum-specific tabs: procpars, acqupars, etc. Use of the command line to retrieve individual parameter values (e.g., D1).
  • Special attention to FID, identification as “time-domain” raw data. Discussion of detection mechanism in probe with vector model of NMR, oscillating sine waves undergoing exponential decay as magnetizations return to equilibrium along Z axis.
  • Simple fourier transform to yield frequency-domain data, examination of odd “out-of-phase” appearance.
  • Academic discussion of the nature of “phasing”. Signal detection, realization that FID signals are audio frequency with frequencies relative to center of spectrum; significance of O1P and SW. Necessity for acquiring two FIDs, real and imaginary, and balancing the two via “phasing” to yield normal frequency-domain spectrum.
  • Practical demonstration of manual phasing, discussion of its use in conjunction with automatic phasing, mention that MNova also handles auto and manual phasing.
  • Discussion of baseline correction, mention of MNova capability
  • Discussion of digital resolution and zero-filling. Practical demonstration of changing parameter SI
  • Discussion of window multiplication / line broadening / apodization to improve S/N without re-acquiring data. Practical demonstration of changing parameter LB.
  • Data evaluation – tutorial on measurement of line-width-at-half-height w/cursors and with “peakw” command. Aim for CDCl3, TMS, or sharpest singlet to be < 1.0 Hz. Effects of LB on measurement.
  • Chemical shift referencing, first to solvent, then to IUPAC-preferred TMS, noticing the difference.
  • ETHICS of chemical shift referencing, including responsibility of the chemist to accurately identify the peak being used for referencing, and to accurately specify the correct value for its chemical shift.
  • Brief demonstration of peak picking and integrating in Topspin. Advocating for MNova’s multiplet analysis for combination of peak-picking and integration that recognizes multiplets, which MNova can export as journal-ready assignments once assigned.
  • How to change Spectrum Display Preferences to remove peak labels and change spectrum colors. Mention that the software uses some color-coding, which may cause problems for people who are color-blind, and the display tools here can be adjusted to make the user perceive data more comfortable and effectively.

Survey of Common 1D and 2D Experiments for Assignment

  • All experiments performed on same sample, S-carvone 10.0 mg in 1.0 mL CDCl3 + 0.03% TMS
  • 1D 13C experiment
    • Impression of S/N for default-parameter 6-minute 400 MHz data
    • Note that all 13C signals are singlets; display pulse sequence and identify 13C channel, relaxation delay D1, excitation pulse, acquisition, then 1H channel with decoupling during acquisition. Discussion of 1H decoupling to achieve 13C singlets.
    • Note of 1H transmitter activity during D1 to build up 13C magnetization via NOE. Refer to 13C spectrum, noting different classes of signal intensity due to enhancement of 13C’s with 1H’s attached.
    • Discussion of how the NOE factor makes normal 13C 1D spectra unsuitable for quantitation by integration. Direct attention to spectrometer’s CARBON-quant experiment, which has not NOE buildup and uses a longer D1 period, making it suitable for reasonably accurate integration.
    • Examine solvent peak, characterized by 2H splitting. Discuss why 2H is not decoupled and why it is a 1:1:1 triplet.
  • 1D 13C DEPT135 experiment.
    • Introduction as a 2-minute automated 13C-experiment that only shows 13Cs with 1Hs attached.
    • Positive peaks = CH and CH3, negative = CH2
    • Showcase utility by examining peaks close in frequency but opposite sign
    • Mention that DEPT45 and DEPT90 exist, but 135 is preferred
    • RESPONSIBILITY of the chemist to determine whether phasing must be flipped 180°
  • 2D 1H-13C HSQC experiment, “S” = “single”
    • Inquire whether trainees have encountered 2D NMR
    • Orientation to 2D HSQC, analogy to togographic map with longitude and latitude coordinates <=> 1H and 13C frequencies. Rule = presence of a crosspeak indicates that the 1H at that X-axis frequency is covalently bound to the 13C at the Y-axis frequency.
    • (This part of the training involves a lot of physically touching the monitor screen)
    • Selection of blue (positive) crosspeak with most deshielding, assignment to corresponding 1H AND 13C atoms
    • Note existence of blue and red crosspeaks representing positive and negative intensity – same relationships with CH/CH3 and CH2 groups as DEPT135.
    • Examine close pair of red crosspeaks. Red = CH2, two peaks = separate chemical shifts, frequency indicates vinyl CH2 group with 1H in different chemical environments. Cannot yet assign E/Z 1H’s.
    • Examine other aliphatic crosspeaks. Identify CH and two methylene groups – each with two 1H frequencies associated with same 13C. Make assignment of CH.
    • Identify CH3 peaks. Consider possible relationships between each 1D methyl 1H peak and the two 1D methyl 13C peaks; would you be comfortable assigning which 1H was attached to which 13C with no other information? 2D HSQC tells you definitively which is attached to what.
  • 2D 1H-13C HMBC experiment, “MB” = “multiple bond”
    • Orientation to 2D HMBC, indicating crosspeaks appear between 1Hs and 13C two, three, or four bonds away (sometimes five in some aromatic systems).
    • Great utility: correlations of 13Cs with no 1Hs bound, but with 1Hs nearby (connected through bonds)
    • Examination of crosspeaks connecting the carbonyl 13C with various 1Hs. Confirm some assignments, make new assignments, including distinction of CH2 groups. KEY: Assign resonances for both CH3 groups based on which is close to the carbonyl 13C.
  • 2D 1H-1H COSY experiment, “COrrelation SpectroscopY”
    • Orientation to 2D COSY, noting that since both axes represent 1H resonances, there is a “diagonal” corresponding to the peaks in a 1D 1H spectrum. Off-diagonal crosspeaks appear when two 1H resonances are sscala-coupled with one another.
    • Point out superior, quicker method for connecting networks of coupled spins, relative to measuring precise coupling constants and constructing splitting trees.
    • Confirm final 1H assignments, mentioning that distinction of each 1H within the CH2 groups requires analysis of scalar coupling constants.

Certification Quiz

Quiz Review

  • After trainees are basically finished, we review the answers together to clear up any misunderstandings.
  • Quizzes are kept by the facility manager as a record of safety training, but are not graded.