FT-NMR

This is regarding the Fourier Transform NMR helpful for the analysis in the Pharmaceutical field and this is helpful to the Masters students as this topic is in the syllabus and the presentation gives the complete and detail idea of various aspects of FT-NMR.

1. Rutuja Dattatray Chougale, Research Scholar, M. Pharm Semester I, Tatyasaheb Kore College of Pharmacy, Warananagar. 1 Fourier Transform NMR (FT-NMR)

2. Introduction 2 Nuclear magnetic resonance (NMR) spectroscopy is a very important spectroscopic method that provides crucial information relating to chemical structure of molecule. NMR is a technique which gives information about the type and number of magnetically distinct protons in a molecule. Nuclear magnetic resonance spectroscopy is a spectroscopic method in which, under appropriate conditions, a sample placed in a magnetic field, absorbs electromagnetic radiation in the radiofrequency ( rf ) region. The frequency of absorption is governed by the characteristics of the sample and it is a function of certain nuclei constituting the sample. Example : Proton NMR ( 1 H-NMR) spectroscopy gives information about hydrogen nuclei, and one can determine number of distinct types of hydrogen nuclei as well as information regarding the nature of immediate chemical environment for each type of 1 H nucleus.

3. Why Fourier Transform NMR? 3 In a CW-NMR instrument operating on field sweep or frequency sweep modes, the nuclei undergo excitation one at a time. In the case of 1 H-NMR, chemically di stinct type of protons (phenyl, vinyl, methyl and so on) are excited individually and come to resonance in succession independently of other nuclei. The scanning continues till all varying types of protons have come to resonance one by one. The NMR spectrum obtained therefore, consists of a series of peaks plotted on the abscissa (x-axis) corresponding to variation in the field or frequency. As we look at first one type of hydrogen and then another scanning until all of the types have come into resonance. A disadvantage with this mode of operation is that the spectrum takes several minutes to be recorded.

4. Pulsed Fourier Transform NMR Spectrophotometer 4 An alternative approach followed very commonly in modern sophisticated NMR instrument is the pulsed Fourier Transform NMR. In this method, all transitions are stimulated simultaneously within a fraction of a second by irradiating the sample (at a fixed B ) with a powerful but short burst of rf energy (called a pulse) (approximately 10  s) that contains the complete range of frequencies required to cover the varying types of protons (e.g., spread around 90 MHz in an instrument with a 2.1 T magnet or around 100MHz for a 2.3 T instrument).

5. Fourier Transform NMR Spectroscopy 5 The Fourier Tran sformation is the basic mathematical calculation necessary to convert the data in time domain (interferogram) to frequency domain (NMR Spectrum). i.e., time domain  Intensity v/s Time. Frequency domain  Intensity v/s Frequency. It was developed by JEAN BAPTI SE JOSEPH FOURIER.

6. Pulsed Fourier Transform NMR Spectrophotometer 6

7. Theory of FT-NMR 7 When magnetic nuclei are placed in a magnetic field and irradiated with a pul se of radio frequency close to their resonant frequency, the nuclei absorb some of the energy and precess like little tops at their resonant frequencies.

8. Theory of FT-NMR 8 This precession of many nuclei at slightly different frequencies produces a complex signal that decays as the nuclei loses the energy they had gained from the pulse. This signal is called as free induction decay (FID) or transient ,it contains all the information needed to calculate a spectrum. The free induction decay can be recorded by a radio receiver and a computer in 1-2 seconds and many FIDs can be averaged in few minutes. A computer converts the average transients into a spectrum.

9. Pulsed Fourier Transform NMR Spectrophotometer 9 The powerful pul se excites all the magnetic nuclei in the molecule simultaneously as they absorb their own respective frequencies out of the pulse and all the signals are collected at the same time with a computer where these frequencies couple to give beats. On discontinuation of the pulse, the excited nuclei begin to relax to their original spin states by losing their excitation energy. In this process, the nuclei re-emit the absorption energies and coupling energies a electromagnetic radiation. As one molecule contains several different types of 1 H nuclei (protons), many different frequencies of electromagnetic radiation are simultaneously emitted. This emission is called a free-induction decay (FID) signal. The intensity of FID rapidly decays with the time as all the absorbed frequencies are eventually emitted by the excited nuclei.

10. Pulsed Fourier Transform NMR Spectrophotometer 10 The observed FID is an interference signal between the radiofrequency source and the frequency emitted by the excited protons. This output is digitized in a computer and individual frequencies due to different nuclei are extracted out from the interference pattern (interferogram) by on a computer by a mathematical method called a Fourier-transform analysis. The Fourier transform separates the FID into its sine or cosine wave components. The wavelength is given as:  = 1/ nucleus -  pulse

11. Schematic diagram of FT-NMR spectrometer with a super-conducting magnet 11

12. Instrumentation 12

13. Components of FT-NMR 13 A radio transmitter coil that produces a short powerful pulse of radio waves. A powerful magnet that produces strong magnetic fields. The sample is placed in a glass tube that spins so the test material is subject to uniform magnetic field. A radio receiver coil that detects radio frequencies emitted as nuclei relax to a lower energy level. A computer that analyses and record the data. In Fourier transform NMR instruments, the tube fits within the bore of the superconducting magnet or a solenoid and spins about the z-axis which is vertical. In other words, the probe is parallel to the z-axis of the magnet which is cooled with liquid helium surrounded by liquid nitrogen in a large Dewar flask.

14. Sen s itivity: Signal to noise ratio 14 Sensitivity of NMR technique is assessed in terms of the signal to noise ratio. Noise refers to the randomelectronic signals usually visible as baseline fluctuations.

15. Sen s itivity: Signal to noise ratio 15 The signal to noise ratio (S/N) indicates the sensitivity of an FT-NMR experiment S/N = N s T 2  exc (B   det ) 1/3 (n s ) 1/2 /T Here, Ns = Number of spins in the system or sample amount T2 = Transverse relaxation time(determine line width)  exc and  det = Magnetogyric ratio of excited and detected nuclei n s = Number of scans B  = Strength of external magnetic field T = Temperature of the sample

16. Modifications in FT-NMR 16 In most labs, the cheapest and easiest route is to increase the number of scans n s, but this increases the experiment time proportionately (Commonly used method in 13 C NMR). Routinely, sample tubes with 5mm (outer diameter) are available which use about 10 mg of sample dissolved in about 0.5 mL solvent. For greater sensitivity, microprobes are available with outer diameters 1.0mm, 2.5mm or 3.0mm. As low as 100 ng of an average molecular weight compound may be analyzed in a 1.0 mm tube (volume 5 L) in a 600 MHz in strument. The development of cryogenically cooled probes ( cryo -probes) has significantly decreased sample amount requirements. These have built in first-stage receivers and rf coils which are cryogenically cooled (approx. 20 K), and result in almost 4 times improvement s in S/N values.

17. Modifications in FT-NMR 17 Higher field instruments (higher B  ) conceptually provide higher sensitivity. For a fixed concentration (N s ), sample requirement becomes almost 2.8 times lesser on a 600 MHz instrument compared to 300 MHz instrument in order to obtain spectra with identical S/N : S/N = N s 600 /N s 300 = (600/300) 1/3 2.8 Thus, if smaller sample amount is available, one should go in for highest field instruments with the smallest possible diameter cryo-probe. A cryo-capillary flow microprobe can be used to dissolve few nanograms of the sample in approximately 1 L of the solvent for highest sensitivity.

18. Advantages of FT-NMR 18 FT-NMR is much faster than CW-NMR method. An entire spectrum is recorded, digitized and transformed within a few seconds. In comparison, a CW spectrum takes 5 to 10 minutes. The signal stand out clearly with low background noise. With a computer and fast measurement, FID signal can be repeated and averaged with a repetition every 2 seconds. As noise is random, its intensity does not increase when repeated measurements of the spectrum are added together. Hence, S/N ratio improves as number of scans ‘n’ is increased. S/N = f n

19. Advantages of FT-NMR 19 Pulsed NMR is more sensitive and weaker signals can be measured by this method. The possibility of repeated FID measurements is an enormous advantage in cases where signals are weak in intensity and which have a great amount of noise associated with them. Due to greater sensitivity, much lower concentrations of samples can be analyzed compared to CW-NMR instruments. This is particularly useful for biological samples where only microgram quantities may be available for analysis. Sparingly soluble compounds can also be analyzed due to high sensitivity of the techniques as even small number of nuclei in solution can give reasonably sharp peaks. This is useful for NMR studies on nuclei with low abundance and small magnetic moments (e.g., 13 C, 15 N, 17 O).

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