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Title: Nuclear Magnetic Resonance (NMR) Spectroscopy: A Comprehensive Review Title: Nuclear Magnetic Resonance (NMR) Spectroscopy: A Comprehensive Review Running title: NMR-A review Abstract Nuclear Magnetic Resonance (NMR) is one of the most significant diagnostic tools that have been developed in the past few decades. A broad range of biological and nonbiological samples ranging from an individual cell to organs and tissues can be investigated through NMR. Many aspects of this technique are still under research and many functions of the NMR are still pending better understanding and acknowledgement. Therefore, this review is aimed at providing a general overview of the main principles of NMR, types of this technique, and the advantages and disadvantages of NMR spectroscopy. This article also provides an insight into the ongoing developments in the field of NMR spectroscopy for future applications. Keywords: Nuclear magnetic resonance; computer assisted tomography; multiple sclerosis; electromagnetic radiation; metabolism; vibrational energy. 1. Introduction X-rays have been routinely used in the field of medicine and dentistry to detect abnormalities, fractures, and to observe healing tissues but this worthy detection tool comes with a risk of exposing the patients to excessive radiations. Although x-rays are swift and painless, still, long term exposure to their radiations could cause harmful effects including cellular damage. Many new powerful analytical tools have been developed in the recent years which are able to deliver precise results with minimal potential damage to the body tissues.1 Page 1 of 19 NMR was first discovered in 1940’s.2 During the next few decades, it underwent metamorphosis with new knowledge to create this device suited for various fields like chemistry, medicine, and dentistry and resulted in grabbing six Nobel prize in the field of spectroscopy 3 and the first application of NMR to study metabolism in a living tissue dates back to 1970’s. In the present era, with ongoing improvements in magnetic and equipment technologies, NMR spectroscopy and imaging can be utilized to explore an extensive range of biological activities. NMR spectroscopy makes full use of the magnetic properties of assured atomic nuclei and is widely being used in the field of physics and chemistry. Most researchers utilize NMR to determine the detailed molecular structures of solids, liquids, and gases as it gives thorough information about the organization, motion, response state, and chemical atmosphere of molecules. 4 In dentistry, this technique is predominantly beneficial to explore the structure of amorphous glasses and dental cements. 5 It is also commonly used to review the fluoridation of apatite surfaces in the tooth structure. 6 2. Basic mechanism of action of NMR The basic principal of NMR is that the structural and chemical composition of different substances can be determined by their nuclei, which have their distinctive magnetic field. Every nuclei of a material is electrically charged and this property allows the nuclei to spin due to its particular characteristic and quantum effect. The strength of the external magnetic field causes electrically charged nucleus to move from a lower energy level (E1) to a higher energy level (E2) and the difference between E2 and E1 is symbolized as ΔE which is dependent on the power of the magnetic field and size of the nuclear field moment. 7 Page 2 of 19 The NMR signal is attained by the electromagnetic radiation rhythm with a frequency denoted by ‘v’ causing the nuclei to move to a higher energy level (E1 to E2). When this electromagnetic radiation is stopped, it causes the nuclei to relax and accomplish thermal equilibrium. This release of energy from the nuclei is recorded in the form of spectra on the computer and this spectra is exclusive for every nuclei and is equivalent to the energy levels between the two states (E2 and E1) (Fig. I) (Keeler, 2007). 2.1. Nuclear spin The protons and neutrons of an atom exhibit spin. In some materials, the protons and neutrons exhibit paired spin like carbon 12C, oxygen 16O and sulphur 32S and in these cases, they cancel each other out thus, causing the nucleus not to spin. In some materials the number of protons and neutrons in an atom are unpaired like in proton 1H, phosphorus 31P and fluoride 19 F. If the total of the protons and neutrons in an atom sums up to be an odd number, than the nuclei will show half integer spin like 1/2, 3/2, 5/2. In cases where number of neutrons and protons are both odd, the spin would be an integer spin like 1, 2, 3.8 2.2. Applied magnetic field and nuclear moment The spin of the protons and neutrons creates a magnetic moment in the nucleus of an atom. This magnetic moment could be towards the externally applied magnetic field or away from it. This applied magnetic field is denoted by B0. The more desired direction of the nuclear moment is towards the magnetic field which is at lower energy level denoted as α compared to the path opposite to the magnetic field denoted as β5. The magnetic field is accountable for retaining the distinction in the energy levels. If it does not happen, all the orientations of the spin of 21+1 would be of equal energy.9 Page 3 of 19 2.3. Magnetic field strength NMR requires a magnetic field that is both strong and uniform. The magnetic field strength is measured in Tesla or MHz. NMR needs reference nuclei to represent the strength of the magnetic field, which is mostly of high resonance frequency. 2.4. Chemical shift Becker, 1999 proposed that whenever the outer magnetic field is applied, it has a dissimilar result on the electrons as compared to its nucleus. 10 The movement of the electrons creates a magnetic field in and around the nucleus. This magnetic field created is different in direction as compared to the outer magnetic field. Any change in the magnetic field causes a similar change in the spectrum of the NMR too. This sum of the shift is controlled by nature of the nucleus and nature of the motion of electrons in its surrounding atoms and molecules. This phenomenon is called “chemical shift” and a reference compound is needed to measure the chemical shift. 11 Chemical shift make it possible to determine and differentiate magnetically inequivalent nuclei present in a molecule. 2.5. Spin – spin coupling The nuclei close to each other induce an incident called “spin – spin coupling” which happens due to the difference in nuclei’s magnetic field direction. This direction could be either towards or opposing the magnetic field, causing the splitting of NMR signals. This magnetic field direction could either strengthen or fade the signals of NMR. The signals of the NMR can split into two or more components depending upon the specific nuclei having characteristic distance and relative potency. 12 2.6. Spin relaxation Page 4 of 19 Spin relaxation is the comeback of energy levels to stability. This occurs due to the loss of resonance signals with the passage of time after releasing the resonance frequency. There are two relaxation processes allowing nuclear spin to return to stability. These are spin – lattice relaxation and spin - spin relaxation. 13 Spin lattice relaxation, also called Longitudinal magnetic relaxation happens due to loss of energy of the excited nuclei to the surrounding molecules which cause a change of spin among their energy levels. The energy conveyed to the molecular frame is typically in the way of transitional or vibrational energy.T1 represents the time for a specific nucleus to come back to its thermal stability. A number of the elements such as structure, molecular mass, temperature, and solution could influence this procedure. 14 Spin – spin relaxation, also called transverse relaxation happens when energy lost from the nuclei cause loss of signals and the energy lost is conveyed to some other nearby spin active nuclei. Spin – spin relaxation time (T2) is the half-life of this procedure. 3. Types of NMR spectroscopy Solid state NMR is used for chemical analysis in order to recognize any changes in the structure during phase transitions and different transformations in a solid state. The main technique frequently used in a solid state NMR is magic angle spinning (MAS). Magic angle is described as the angle which the sample makes with the direction of magnetic field when it is lowered down into the vertical tube of the probe used in the NMR. When the sample moves down in the tube, the neighboring magnet tilts to an angle of 54.74, which is called the magic angle. This magic angle makes the resolution of the sample more precise by making the broader lines of the NMR narrower. 15 At this angle the nuclear bipolar interactions among the magnetic moments of nuclei averages to zero. This Page 5 of 19 results in the signals to become narrower giving isotropic values and spinning side bands which can be used to identify the chemical shift of the nuclei for structural determination of solid materials. 3.1. Phosphorus NMR In the solid state NMR, phosphorus is one of the isotopes used to study the molecules and structures of different samples. This valuable tool has a medium sensitive nucleus that gives sharp lines and has a wide chemical shift range.31P NMR has an influential approach which has been used in various studies including natural ecosystems, forests, agriculture, fertilizers and marine sediments. As 31P is the only naturally occurring phosphorus isotope, all phosphorus species present in a certain sample can be detected by this NMR. 16The area under each peak in NMR experiments should be proportional to the number of the particular type of phosphorus nucleus, allowing the NMR to identify the different phosphorus forms present in the sample. Compound classes of phosphorus which could be identified in this NMR include ortho-phosphate di esters, pyrophosphate, polyphosphate, phosphonates, ortho-phosphate monoesters, and ortho phosphates. 17 3.2. Proton NMR Proton is the initial and the most frequent atom to be used in NMR spectroscopy. It is also called 1H or hydrogen-NMR. With this technique, researchers are now capable to investigate the complete composition of an organic material. 1H NMR spectra gives information about the different varieties of hydrogen present in the molecule and also gives information about its adjacent surroundings.18 1H NMR spectrum of main materials shows small chemical shift range for usual compound being studied. This chemical shift Page 6 of 19 ranges from +14 to -14ppm. It also has a broad difference in the extent of coupling constant. 19 3.3. 29 Si MAS NMR Silicon is an essential element and its 29 Si isotope, which is used in 29 Si NMR has4.70% natural occurrence with ½ spin nucleus. It is another spectroscopic technique used to investigate the structures of organic compounds. Its value of magnetic moment is a little low causing a low resonance frequency. The predominance of 29 Si NMR shifts is present in a range from +50 to 200 ppm. 20 3.4. 19F MAS-NMR Isotopes of fluoride are naturally present in very less quantities except for the 19 F isotope. Fluorine-19 is the only constant isotope of fluorine found in large quantities. Due to its good nuclear qualities and a great quantity, it is used in 19 F MAS NMR technique.19F NMR technique is very rapid when compared to 1H NMR technique and without a doubt, 19 F nucleus is one of the most amenable NMR nucleus. Fluorine has a spin of ½ nucleus. Its nucleus in molecules is usually encircled by 9 electrons and its binding energy is 147801 keV. The sensitivity of 19 F NMR spectroscopy to its chemical shifts (in order to study the fine details of the local surrounding) are much high for fluorinethus, making it very reactive to NMR measurements with an extremely broad chemical shift range. 21 19 F NMR spectroscopy selectively identifies the surroundings of just the fluorine atoms in the study samples, allowing direct detection of the probable structural ranges in which fluorine may be present within the sample in all forms. Most frequently 19F NMR spectroscopy is Page 7 of 19 utilized to study the structure of fluorine containing materials. Interaction of fluoride with hydroxyapatite forming fluorapatite can be studied with this technique.Yesinowski and Mobley, 1983 verified the capacity of this NMR to differentiate between fluorapatite, fluorohydroxyapitite, and calcium fluoride in massive phase and also on hydroxyapatite surfaces. 0.1%. 22 19 23 19 F NMR can identify fluoride even in very small concentrations starting from F NMR technique has also been used to study the metabolism of drugs containing fluoride in them. 24 3.5. 13C NMR spectroscopy This technique is a significant tool to recognize carbon atoms in any organic material. It also gives detailed information regarding the chemical structure of the organic compound being studied. 13C is an isotope of carbon which has a spin quantum number of 1/2 and is only 1.1% naturally present and this isotope can be detected by 13C NMR.13C NMR is less responsive to carbon in view of the fact that the main isotope of carbon is 12C, which is not magnetically active therefore, it cannot be detected through this technique. The intensities of the signals in carbon NMR are not usually comparative to the number of corresponding 13Catoms. They are strongly reliant on the numerals of adjacent spins. 25 Magnets utilized in C NMR have a usual diameter of 10mm and its usual range of chemical shift is much larger compared to proton NMR.13 C NMR can be used to find out the composition of different molecules and is also used in the drug industry to verify drug purity. 3.6. 27 Al MAS NMR 27 Aluminum MAS-NMR has a natural abundance of 100% and it has a 5/2 nuclear spin. The nucleus of aluminum is very responsive giving wide lines over the broad range of chemical shift. The main application of this NMR is to identify the existence of aluminum Page 8 of 19 and to observe the probable structural changes of the different varieties of aluminum. Therefore, it is a very useful tool to identify the structure of amorphous dental materials. In a previous study performed on glass carbomerionomer cement, 27Al NMR has been used to observe alteration of Al (IV) into Al (VI) in the setting glass carbomer cement. 26 4. Advantages of NMR spectroscopy 4.1. Non-invasiveness The fundamental quality of nuclear magnetic resonance is its non-invasiveness. Due to NMR the studies of biological cells and tissues is now possible without damaging the sample. The fact that both spectrum and imaging can be obtained without destroying the sample, is noticeably the greatest advantage of NMR in vivo studies. 27 4.2. Lack of ionizing radiation Another major advantage of NMR is its lack of ionizing radiation. There are many techniques being used in vivo studies involving ionizing radiations. NMR has made it possible to avoid the exposure from radiation, which could be harmful for both the researcher and the subject. NMR has the capability to utilize isotopes which are stable such as carbon – 13 to measure metabolic fluxes instead of radioactive compounds. Moseley in 1982 observed that NMR not only minimizes the exposure to observer and subject from the harmful rays but also eliminates the need to dispose of the radioactive tissues and other materials that might be contaminated during the study. 28 Thus the application of NMR can ensure the safety of the employees as well as reduces experimental cost due to the removal of discarded radioactive substances. 4.3. Adjustability Page 9 of 19 Extensive variety of processes can be investigated through NMR due to the flexibility of the particular technique that can be applied. This technique not only gives information about the physiology of the tissues but it also gives great images of those tissues. With NMR, a single study can be performed with the same basic technique in both humans and animals, which is important to increase the translation of information. The computed tomography (CT) scan technique can only provide the imaging but not the metabolic or anatomic details. 29 Whereas, NMR spectroscopy has the capability of acquiring a wide variety of information. 4.4. Detailed structural analysis Over the period of time, NMR has played a major responsibility in determining the mechanisms and chemical connections at a molecular level. This technique has helped to obtain information regarding the minute details about the physical and chemical characteristics of structures. 30 NMR can also analyze the parameters of chemical shift and it can give details on the local bonding environment around a particular atom, which could be calculated for extended period of times with NMR 31 . It utilizes the pseudo wave function to get information about large compoundstructures. 32 NMR has the capability to assist studies of biochemical processes conducted in vivo, which is not efficiently achieved with other imaging techniques. Lee et al., 1995 proposed that NMR is a better-quality technique as compared to X-ray diffraction (XRD) in determining archaeological bone structure. 33 In food industry, NMR has been utilized to observe the features of various food products. 34 4.5. As a diagnostic tool CT scan images of cranium are restricted by artifacts but this limitation does not occur with NMR. In the field of medicine, NMR gives the benefit of identifying pediatric tumors, Page 10 of 19 hematomas, and other pathologies. 35 Since multiple sclerosis is a very tricky disease to identify, NMR has become the prime diagnostic device for multiple sclerosis. 36 NMR has a particular use for certain body areas like brain where it produces very detailed and definite images showing delineation between grey and white matter. 37 whereas, some tissues such as bone, having low water percentage cannot emit strong signals to create images for NMR. 38 Also, NMR is apparently victorious in identifying breast cancer at an early stage. According to a radiologist at Cleveland, a mammogram cannot differentiate between a small cancer and a spot, when there are multiple cysts in the breast but with NMR, this distinction is possible. 39 NMR technique also gives good images of fatty tissues and large quantity of fat creates very fine images. Therefore, researches on bone marrow shows much potential. Also, diagnosis of vascular diseases is promising with the NMR 40 as it enables the detailed structural analysis of the surfaces of blood vessels and their abnormalities 5. Disadvantages of NMR spectroscopy Although NMR technique has numerous advantages, few disadvantages are also present. 5.1. Presence of high magnetic field surroundings is essential An unavoidable outcome when performing the NMR technique is the requirement to perform in a surrounding which has a high magnetic field. The presence of the magnetic field can affect the proper functioning of monitors and computer controlled devices. If any sharp object like scalpel, scissor, or stapler is present in the NMR magnetic field area, it can get attracted to the magnet field which can cause severe injuries to the workers. Nowadays, monitoring devices being used in the magnetic field area are being designed to function properly under the magnetic field. 41 Also instruments being used in NMR studies Page 11 of 19 are made non-ferromagnetic to reduce the problems encountered with high magnetic field surroundings. 42 5.2. Cost of performing NMR NMR system cannot be purchased by a single investigator or for a single research because of its high cost. Expenses increase further with the size of the NMR magnet and maintenance costs of NMR are also high. 43 As a result, majority of the NMR systems are often purchased by a group of researchers through programs which are approved by research centers and also by funding grants. 44 6. Future directions Further improvements to make more efficient NMR magnets should continue as it is immensely upgrading the knowledge about the wide variety of different biophysical processes. Advancements in the NMR technology, particularly in the imaging technique and machinery design are needed to totally utilize its qualities and to make this research technique more effective and accurate. Additional work should be performed to gain information about the functional details of NMR as it would help to demonstrate more extensive details of structures, functions, and metabolism of any material being investigated. The utilization of this valuable technique is limited because of its cost and availability. In future, research centers and other medical programs should make its availability easy for the researchers so that they can gain full benefits of NMR in their studies. It is now essential for hospital management to do preparations for the modification of NMR services and employment. Growth in the clinical area must go together with the proper expansion in the management area, to make full use of this facility. Page 12 of 19 7. Conclusion Considering the potential advantages of the NMR technique, it can be concluded with authority that it has become a preferred choice of technique for many procedures. NMR is still a growing technology and it is being anticipated that few new discoveries are now just around the corner. References 1. Moseley I. Recent development in imaging techniques. Br Med J. 284 (1982), 1141-1144. 2. Grant MD, Harris KR. Encyclopedia of Magnetic Resonance, Volume 9, Advances in NMR. Wiley Publishers (2002). 3. Freeman R. Magnetic Resonance in Chemistry and Medicine. Oxford University Press, 1st ed. (2003). 4. Mark Wainwright Analytical Centre - University of Southern Wales, Sydney. Background and Theory Page of Nuclear Magnetic Resonance Facility (2011). 5. Zainuddin N, Karpukhina N, Law RV, Hill RG. Characterization of a remineralising Glass Carbomer® ionomer cement by MAS-NMR Spectroscopy. Dent Mater. 28 (2012), 1051-58. Page 13 of 19 6. 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Monitoring of task performance during functional magnetic resonance imaging of sensorimotor cortex at 1.5 T. Magn Reson Imaging 14 (1996), 51–58. Figure captions Fig. I: Showing a line in the spectrum is related with a transition between two energy levels (E2 and E1) (Keeler, 2007). Page 18 of 19 Page 19 of 19