1. 05- NMR Instrumentation 1.Overview of the JEOL LA400 2.Superconducting magnet 3.The magnetic field and homogeneity 4.Field frequency lock 5.Pulse program 6. Decoupler 7. Radiofrequency generator 8. Probe 9. Amplifier, detector and receiver 10. Analog to digital converter 11. Sensitivity and Resolution 12. Peak shape

2. 05-NMR Instrumentation (Dayrit)2 Introduction Compared with other spectroscopic methods, NMR provides the richest amount of structural information. However, in order to properly obtain and process complex information, it is necessary to use sophisticated techniques. NMR is, in many ways, the most difficult spectroscopic technique to learn. To a great degree, the quality of the NMR result is dependent on the skill and know-how of the operator. If one is doing a routine analysis of known compounds, one can run the NMR as a “black box”. However, if one needs to determine a structure, or wishes to use NMR to its fullest advantage, then one should master its operation.

3. 05-NMR Instrumentation (Dayrit)3 1. Overview of the NMR instrument Main features of the JEOL LA400: 9.4 T SCM, long hold magnet; 54 mm bore 2-channels: high frequency and low frequency tunable frequencies temp control: -70 ~ +150 °C

4. 05 NMR Instrumentation (Dayrit)4 Tunable frequencies in the high and low frequency range.  13 C

5. 05-NMR Instrumentation (Dayrit)5 1. Overview of the NMR instrument 1. Pulse program 2. RF oscillator / carrier freq. ( r )‏ 3. Amplifier (A)‏ 4. RF pulse generator 5. NMR excitation 6. NMR signal ( 0 ) to pre-amp. (PA) 7. Amplified signal 8. Reference frequency 9. RF receiver 10. Digitization and processing

6. 05-NMR Instrumentation (Dayrit)6 2. The Superconducting Magnet At higher fields (>5 T), SCMs give superior stability and are actually less expensive to operate. The superconducting coils are immersed in liquid helium (boiling point: -269 o C; 4 K) in an inner dewar, which is surrounded by an outer dewar of liquid nitrogen (bp: -196 o C; 77 K). Both liquid cryogens require periodic refilling. Once the SCM is filled and started, it should run continuously for many years unless the superconductivity quenches. Magnex, 9.4 T SCM

7. 05-NMR Instrumentation (Dayrit)7 2. The Superconducting Magnet

8. 05-NMR Instrumentation (Dayrit)8 3. Magnetic Field Homogeneity The magnetic field must be very homogeneous over the observed volume of the sample in order that all of the spins within the observation volume have the same magnetic field. A lack of homogeneity means that the various spins experience different magnetic fields. For high resolution NMR, the tolerance of homogeneity is 1 part in 10 9, or 1 ppb. The SCM coil is made up of special alloys containing Cu, and other metals.

9. 05-NMR Instrumentation (Dayrit)9 3. Magnetic Field Homogeneity The SCM coils do not produce a sufficiently homogeneous magnetic field. In order to improve the homogeneity, a small secondary magnetic field is added using shim coils*. For liquid samples, the NMR tube is rotated rapidly in order to average out the inhomogeneities in the perpendicular direction. A "shim" is a thin piece or wedge of thin tapered pieces of material, usually wood or metal, to make fine adjustments on the fit of separate parts which need to be joined together. In the making of bells, shims are used for fine-tuning the resonance. Just like the shims used for bells, NMR *shim coils enable fine adjustments of the magnetic field gradient. Whenever a new sample is inserted into the probe, there are slight changes in the magnetic field homogeneity due to the NMR tube, the solvent, sample, etc.

10. 05-NMR Instrumentation (Dayrit)10 3. Magnetic Field Homogeneity SCMs can have from 13 to over 20 shim coils depending on the instrument design. The coils whose field is aligned along the vertical axis of the magnet are called the Z-gradient coils, designated, Z 1, Z 2, Z 3, etc. The Z 0 coil functions as a fine magnetic field adjustment. The field in the horizontal plane is adjusted via the higher order corrections designated as: X, Y (first order); XZ, YZ, XY, and X 2 -Y 2 (second order); XZ 2 and YZ 2 (third order) and others. Because the shim adjustments interact, shimming is an iterative process.

11. 05-NMR Instrumentation (Dayrit)11 4. Field/Frequency Lock The preparations for measuring an NMR spectrum are similar in some ways to the tuning of a radio to the desired radio station. First, one must tune into the desired radio station; analogously, the experimenter must locate the signal of the target nucleus, a process which is appropriately called tuning. Second, the radio signal should be locked in. Generally, the radio station operator does this for you by seeing to it that the frequency of transmission does not drift; some modern digital radios, nevertheless, have a radio signal locking feedback device. Analogously, the NMR spectrometer must be locked onto the desired nucleus. The NMR lock must be very accurate and precise if we are to have high resolution.

12. 05-NMR Instrumentation (Dayrit)12 4. Field/Frequency Lock There are two aspects to maintaining a good lock: a stable magnet and an efficient lock system. For SCMs at 400 MHz, the magnetic field stability (drift) is <8 Hz/hr. This stability is better than 1 part in over 40 x 10 6, but is insufficient for the requirements of a long NMR experiment. A field/frequency lock fixes the reference lock signal using a deuterated ( 2 H) solvent so that the lock signal is derived from the sample itself (internal lock). The alternative is to use an external lock which places a substance containing usually 2 H or 19 F at a close distance away from the sample.

13. 05-NMR Instrumentation (Dayrit)13 4. Field/Frequency Lock A feedback is used in the field/frequency lock. The receiver is equipped with a phase sensitive detector which is set on the dispersion mode signal of the lock signal. An output voltage of zero indicates the precise resonance condition of the reference. A positive or negative drift in the magnetic field is detected as a positive or negative voltage, respectively. This voltage is converted into a correcting signal on magnetic field via the Z 0 shim coil.

14. 05-NMR Instrumentation (Dayrit)14 5. Pulse Program The pulse program is the software interface which the operator uses to set instructions for the NMR. This is what the operator uses to perform the NMR experiment. The operator must some knowledge of NMR to be able to set the parameters appropriately. The most important parameters that have to be set are the following: type of NMR experiment (pulse sequence) pulse width (pulse angle) number of accumulations resolution (Hz/point) spectral width pulse delay

15. 05-NMR Instrumentation (Dayrit)15 5. Pulse Program The excitation of the nuclear spins is accomplished using both pulses and continuous irradiation of RF energy. The pulses programs are stored in the NMR software and these instructions are sent to the transmitter for implementation. In order to implement an NMR experiment, one must have the appropriate pulse program, NMR hardware, and processing software. Many pulse programs were designed to overcome imperfections of NMR hardware. Some pulse programs require certain types of NMR hardware for them to work.

16. 05-NMR Instrumentation (Dayrit)16 One controls the NMR experiment by selecting the appropriate pulse program and parameters.

17. 05-NMR Instrumentation (Dayrit)17 5. Pulse program  The pulse angle depends on the pulse width which is the duration of the pulse, designated t p. The pulse width is empirically determined and depends on the particular sample. The pulse width results in the tipping of the magnetization vector by a certain pulse angle. The most common pulse angles are: 45 o, 90 o, 180 o, and 270 o (- 90 o ).  M can be tipped down to the –z direction with a 180 o resulting in a spin population inversion. This corresponds to an excited energy condition where the equilibrium populations of the spin states N  and N  are inverted.

18. 05-NMR Instrumentation (Dayrit)18 5. Pulse Program

19. 05-NMR Instrumentation (Dayrit)19  The same pulse angle can be achieved by means of a hard pulse or a long, weak pulse (a soft pulse). A typical hard pulse lasts for several  s, while a soft pulse can last for several ms. Read pulses are usually hard pulses, while double resonance pulses are usually soft pulses.  The pulse phase is the axis from which the NMR pulse is implemented. Pulses can be applied along the  x’or  y’ axes.  The detector is placed along the y’-axis and the pulse sequence is designed so that the final position of the magnetization vector is along this axis. For this purpose, many pulse sequences must incorporate a read pulse which places the vector along the y’- axis immediately prior to the collection of the FID. This pulse which is usually applied along the x’-axis.

20. 05-NMR Instrumentation (Dayrit)20  In a multipulse experiment, time delays, t D, are inserted between pulses or other elements of the sequence to allow the spins to evolve. If the time delay is variable and incremented, this is referred to as t 1 in a 2-dimensional NMR experiment. If the time delay is fixed, this is usually a mixing time.  The acquisition time, AQT, refers to the time allocated for the collection of the FID. This normally lasts from about half a second to a few seconds.

21. 05-NMR Instrumentation (Dayrit)21  A pulse delay, PD, of about 3-5 times T 1 is added after the acquisition time before repeating the pulse sequence. This is necessary to allow the spin system to return to its equilibrium value so that M = M o (this also means that M x = M y = 0). This requirement gives rise to practical difficulties for nuclei with very long T 1 relaxation times, such as quaternary carbons which can have T 1 s of up to 100 seconds! One possible solution is to use smaller pulse angles so that relaxation time is shorter.  Note: T 1  T 2

22. 05-NMR Instrumentation (Dayrit)22 Illustration of two simple pulse programs. The basic components of a pulse program are: the pulse, delay time, acquisition time, relaxation delay, and decoupler.

23. 05-NMR Instrumentation (Dayrit)23 - zero filling - spectral width - no. of accum - T 1 and  -  - 0

24. 05-NMR Instrumentation (Dayrit)24 6. Decoupler All nuclei with I  0 have magnetic properties and therefore interact with other magnetically-active nuclei (“spin-spin coupling”). The NMR spectrum contains information regarding all of these interactions. Depending on what one needs, this can be good or bad. The technique of removing contributions from coupling is called decoupling. One of the earliest innovations in NMR was the introduction of multiple irradiation in CW 1 H NMR. This allowed the experimenter to specifically irradiate a specific proton frequency while taking the entire spectrum. If the coupling information being removed is from the same nucleus, the technique is called homonuclear decoupling.

25. 05-NMR Instrumentation (Dayrit)25 6. Decoupler If the nuclei being decoupled and observed are different, the technique is heteronuclear decoupling. There are several ways of carrying out heteronuclear decoupling: broadband decoupling (the standard 13 C NMR), selective heteronuclear decoupling, off-resonance decoupling, and gated decoupling. Broadband decoupling requires the application of decoupling energy over a wide band. For example, in a 1 H-decoupled 13 C spectrum in a 400 MHz machine, decoupling has to be applied over at least 4,000 Hz. The most common technique used is noise modulation wherein the decoupler energy is distributed uniformly over the 1 H range by random modulation. Today, there is a many sophisticated and clever ways of implementing decoupling and multiple irradiation.

26. 05-NMR Instrumentation (Dayrit)26 7. Radiofrequency Generator In order to carry out the NMR experiment, three independent RF fields are needed: for observation, for secondary irradiation (e.g., for decoupling), and for locking. These three frequencies may or may not be set for the same nucleus. The three frequencies are independently synthesized from the RF generator (master oscillator). The switch combines the pulse program with the RF frequencies.

27. 05-NMR Instrumentation (Dayrit)27 7. Radiofrequency Generator The power that is needed depends on several factors: 1. the bandwidth (in Hz) that needs to be excited; 2. the desired pulse angle; 3. the probe characteristics; and 4. sample. In pulse NMR, the RF field, B 1, should be uniform over the entire bandwidth of the nuclei under observation. Since the RF field tapers off at the ends, the RF field should be set for a wider range of frequencies. The strength of the RF field, B 1, generated is proportional to the square of the power used: B 1  w 2. If one wishes to double the field strength, the power must be increased by four times. This, unfortunately, generates more heat. A typical performance specification for a 1H 90° pulse at 400 MHz is 15  s at 50 W power.

28. 05-NMR Instrumentation (Dayrit)28 8. Probe The sample is placed in an NMR tube which is nested in the probe. The probe houses the coils and associated RF electronics. The probe is designed to carry out the following functions: 1. to hold the spinning NMR tube in a precise manner; 2. to efficiently transmit the RF energy, B 1, to the sample (pulse); 3. to pick up the resulting NMR signal for transmission to the receiver (observe); 4. to transmit energy for decoupling; and 5. to provide for an efficient third frequency for locking. In recent years, there has been much effort put into optimizing probe performance. There are specific probes for different NMR tubes, most commonly 5 mm and 10 mm. NMR probes are designed to operate at temperatures ranging from -100 to +150 o C for variable temperature experiments.

29. 05-NMR Instrumentation (Dayrit)29 8. Probe Since there are two channels (HF and LF), the probe should have two coils. The coils can be arranged in two ways: standard probe: LF coil inside and HF coil outside inverse probe: HF coil inside and LF coil outside Probe innovations: Pulsed field gradient (PFG) probe Microcoil probes.

30. 05-NMR Instrumentation (Dayrit)30 9. Amplifier, Detector and Receiver The receiver must amplify the signal linearly without distortion and added noise, from an initial magnitude of nanovolts to microvolts, to tens of volts required by the analogue-to-digital converter (ADC). Noise can arise from various sources such as the transmitter and receiver themselves, the NMR coils (which are actually radio antennas which can vibrate), and external noise (e.g., lightning, fluctuations or spikes in the line current, etc.). In radio technology, the function of the detector is to subtract the carrier frequency from the signal. In the case of a NMR, the detector has to subtract the carrier frequency, o (which is at the Larmor frequency of 400 MHz in the case of 1 H at 9.4 T), so that we will observe only  (which has values in Hz).

31. 05-NMR Instrumentation (Dayrit)31 9. Amplifier, Detector and Receiver When setting up an NMR experiment, the position of the reference frequency, o, has to be set. There are two choices: at the upper edge of the frequency range of interest or in the middle. The first option requires a wider bandwidth which collects more noise than necessary. All modern NMR spectrometers implement the second option of placing the reference frequency in the middle of the spectral range. This has two important advantages: first, there is less noise by virtue of the narrower range; and second, this requires a lower digitization speed. However, this has to address the problem of distinguishing the signals which occur on either side of the reference frequency (quadrature detection).

32. 05-NMR Instrumentation (Dayrit)32 10. Analog to Digital Converter (ADC)‏ The ADC enables the conversion of electrical signals (volts at small time intervals) into numerical form. There is an inherent difficulty in adequately representing a continuous analogue signal in terms of discrete numbers. The conversion of the analogue FID into digital form is implemented by converting the voltage into binary form (y-axis) as discrete points over time (x-axis). There are two important characteristics of the ADC: the number of bits used in the binary representation (the word length) and the digitization rate. The first word length determines the digitizer resolution and dynamic range, while the digitization rate determines the maximum spectral width and digital resolution.

33. 05-NMR Instrumentation (Dayrit)33 10. Analog to Digital Converter (ADC)‏ As already mentioned, we can make the analysis of the signal easier by subtracting the reference frequency from the total signal. So instead of processing frequencies of about 400 MHz, we need to only process signals generally in the fractions of Hz up to the 4-5 kHz range (a 10 ppm spectral range is equivalent to 4000 Hz). In order to cover a spectral width of 4000 Hz, the digitization rate of our ADC has to be at least: (2 x 4000 Hz) = 8 kHz, or a dwell time of 1.25 x 10 -4 sec. The factor of 2 arises from the requirement of the Nyquist theory. In order to properly record a waveform, the Nyquist theory tells us that at least two data points are needed for each cycle. Failure to do so results in the improper recording of frequencies leading to frequency dependent anomalies known as aliasing or folding.

34. 05-NMR Instrumentation (Dayrit)34 In this example, sampling is carried out at 100  s intervals (10 kHz). This gives a Nyquist frequency of 5 kHz. Since the two waves have frequencies of 4.5 kHz (solid line) and 1.2 kHz (dotted line), then this sampling rate is adequate and we can represent the two frequencies correctly. Sampling rate: 10 kHz Solid line: 4.5 kHz Dotted line: 1.2 KHz 10. Analog to Digital Converter (ADC)‏

35. 05-NMR Instrumentation (Dayrit)35 In this example, we have two signals: 7 kHz and 3 kHz. If we sample with a frequency of 10 kHz, the 3 kHz signal will be correctly detected, but the 7 kHz signal will be aliased or folded. To correctly record the FID, our ADC has to run at a minimum of twice the highest frequency signal present. Sampling rate: 10 kHz Solid line: 3 kHz Dotted line: 7 KHz 10. Analog to Digital Converter (ADC)‏

36. 05-NMR Instrumentation (Dayrit)36 The requirement for digitization rate for 1 H work is easy to meet since the spectral range of the 1 H nucleus is narrow and therefore requires a relatively slow ADC rate. In comparison, a 230 ppm spectral width for 13 C at 100 MHz requires a digitization rate of at least 2 x 23000 Hz = 46 kHz. Standard ADCs are usually rated for at least 100 kHz which means a maximum spectral width of 50 kHz. The second aspect important characteristic of the ADC is the number of bits it uses in the binary representation. The number of bits of data used limits both the sensitivity of the NMR experiment. The sensitivity is related to the dynamic range which is defined as the ratio of the biggest to the smallest signal. 10. Analog to Digital Converter (ADC)‏

37. 05-NMR Instrumentation (Dayrit)37 In practice, the maximum voltage range that is outputted in the FID is adjusted to the highest value of the digitizer; and the smallest signal that is recorded is that which gives a response of 1-bit. This is what is sometimes referred to as the digitizer resolution (not to be confused with the digital resolution). A sufficiently large digitizer is needed in order that all signals are measured. An inadequate digitizer can result in the nondetection of some peaks. In samples which contain both very large and very small signals (for example a dilute solution in an aqueous solvent), very small signals may be present but not be detected because of the limitation of the dynamic range. 10. Analog to Digital Converter (ADC)‏

38. 05-NMR Instrumentation (Dayrit)38 NMR instruments are available with an ADC resolution of 16 bits. (The ADC resolution, however, is adjustable for each NMR run.) A 16-bit digitizer, covers the range 0 to 1111111111111111 (=2 16 ) in binary. Since one digit is reserved for the sign of the signal (+/-), the actual range is from 0 to 2 16 - 1. With a 16-bit ADC, the largest signal will be 32767 and the smallest is 1. The digital resolution (given in Hz/point) refers to the accuracy with which the frequency of the signal can be presented in digitized form. According to theory, the resolution is also related to the length of time that we allow to record the FID; this is called the acquisition time. In general, the longer the acquisition time, the better our resolution: digital resolution, Hz = (1 / acquisition time, s)‏ 10. Analog to Digital Converter (ADC)‏

39. 05-NMR Instrumentation (Dayrit)39 11. Sensitivity and Resolution “Sensitivity” is one of the most important goals of a spectroscopic technique. Mathematically, sensitivity is measured using the dimensionless signal-to-noise ratio (S/N): (sensitivity, S/N) = 2.5 S ht / N pp where 2.5 is a factor obtained from statistics S ht is the signal height N pp is the peak-to-peak height at the noise level

40. 05-NMR Instrumentation (Dayrit)40 11. Sensitivity and Resolution Resolution = (frequency range / no. of points), ( Hz / point ) The key goal of all spectroscopic methods is to obtain the highest resolution and sensitivity. However, there are theoretical and practical aspects that need to be considered. The resolution set by the NMR parameters is given as:

41. 05-NMR Instrumentation (Dayrit)41 Some NMR parameters favor both resolution and sensitivity: magnetic field strength and homogeneity. Others parameters affect resolution and sensitivity in reciprocal ways: spectral width, acquisition time and ADC. In these cases, the operator needs to decide how much resolution and sensitivity are needed and then select the parameters accordingly. The trade-off between sensitivity and resolution is thus: Other phenomena, such as nuclear relaxation and kinetics, also affect sensitivity and resolution. (sensitivity, S/N)  1 / (resolution, Hz / point)‏ 11. Sensitivity and Resolution

42. 05-NMR Instrumentation (Dayrit)42 12. Peak Shape Comparison between normalized Lorentzian (solid) and Gaussian (dashed) distributions. Note that the area under both curves is 1. 1 J CH = 209 Hz

43. 05-NMR Instrumentation (Dayrit)43 NMR parameters that affect resolution and sensitivity.

44. Overview of JEOL LA 400 NMR operation Sequence Part of the NMR 1. Load sample, Tune probe  Probe 2. Lock  Field/frequency lock 3. Shim, spin  Magnetic field homogeneity 4. NMR experiment proper  Pulse program [RF generator, transmitter and amplifier decoupler, detector and receiver, ADC] 5. Data processing  Host computer Data Storage and Processing Network