University of Kurdistan Food Quality Evaluation Methods (FQEM) Lecturer: Kaveh Mollazade, Ph.D. Department of Biosystems Engineering, Faculty of Agriculture, University of Kurdistan, Sanandaj, IRAN. Lecture 4: Nuclear Magnetic Resonance Spectroscopy / Magnetic Resonance Imaging

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 1 Contents This lecture will cover: –An introduction to nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) –Principles of NMR –NMR spectrum interpretation –Apparatus and devices Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 2 Introduction  Magnetic resonance is a phenomenon that occurs between atomic particles and an external magnetic field.  The atomic particles responsible for this interaction are the electrons and the nucleus.  The interaction between the atomic particles and an external magnetic field is similar to what happens when iron filings are placed near a bar magnet. The filings become oriented and a magnetic field is induced in the metal. However, unlike the filings, the physical orientation of the atomic particles is not altered.  At most common magnetic field strengths only the magnetic moment of the atomic particles is induced.

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 3 Introduction  The phenomenon of resonance is observed in these systems because they absorb and emit energy at specific frequencies.  The specific frequency depends on the individual atomic particle and the strength of the applied magnetic fields.  Work discussed in this chapter will focus on using the nucleus as the atomic particle, and in this case the phenomenon is referred to as nuclear magnetic resonance (NMR).

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 4 Introduction  Common nuclei with magnetic moments include 1 H, 13 C, 31 P, 15 N, and 23 Na.  The most commonly studied nucleus in food systems is the 1 H.  In both medical and food applications of NMR, the technique is referred to simply as magnetic resonance (MR). The term nuclear is omitted so that patients/consumers will not confuse this technique with nuclear procedures that use radioactive materials. MR is a safe experimental procedure and does not harm or alter the sample, operator, or environment

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 5 Introduction  MR is a very useful spectroscopy because the signal emitted from a sample is sensitive to the number of nuclei, to the chemical and electronic surroundings of the nuclei at the molecular level, and to the diffusion or flow of the nuclei.  This sensitivity to a range of sample characteristics is responsible for the wide use of MR in chemistry, biochemistry, biotechnology, petrophysics, plastics, engineering, building materials, consumer products, medicine, and food technology.  When MR is used to make internal images of objects it is commonly called magnetic resonance imaging (MRI). MRI is a spectroscopic technique based on the magnetic properties of nuclei. MRI is the extension of MR spectroscopy to obtain the signal as a function of spatial coordinates within the sample.

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 6 Principles of NMR  Two common types of NMR spectroscopy are used to characterize organic structure: 1 H NMR is used to determine the type and number of H atoms in a molecule; 13 C NMR is used to determine the type of carbon atoms in the molecule.  The source of energy in NMR is radio waves which have long wavelengths, and thus low energy and frequency.  When low-energy radio waves interact with a molecule, they can change the nuclear spins of some elements, including 1 H and 13 C.

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 7 Principles of NMR  When a charged particle such as a proton spins on its axis, it creates a magnetic field. Thus, the nucleus can be considered to be a tiny bar magnet.  Normally, these tiny bar magnets are randomly oriented in space. However, in the presence of a magnetic field B 0, they are oriented with or against this applied field. More nuclei are oriented with the applied field because this arrangement is lower in energy.  The energy difference between these two states is very small (<0.1 cal).

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 8 Principles of NMR  In a magnetic field, there are now two energy states for a proton: a lower energy state with the nucleus aligned in the same direction as B 0, and a higher energy state in which the nucleus aligned against B 0.  When an external energy source (h ) that matches the energy difference (  E) between these two states is applied, energy is absorbed, causing the nucleus to “spin flip” from one orientation to another.  The energy difference between these two nuclear spin states corresponds to the low frequency RF region of the electromagnetic spectrum.

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 9 Principles of NMR  Thus, two variables characterize NMR: - An applied magnetic field B 0, the strength of which is measured in tesla (T) - The frequency of radiation used for resonance, measured in hertz (Hz), or megahertz (MHz).

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 10 Principles of NMR  The frequency needed for resonance and the applied magnetic field strength are proportionally related:  NMR spectrometers are referred to as 300 MHz instruments, 500 MHz instruments, and so forth, depending on the frequency of the RF radiation used for resonance.  These spectrometers use very powerful magnets to create a small but measurable energy difference between two possible spin states.

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 11 Principles of NMR  Schematic of an NMR spectrometer:

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 12 Principles of NMR  Protons in different environments absorb at slightly different frequencies, so they are distinguishable by NMR.  The frequency at which a particular proton absorbs is determined by its electronic environment.  The size of the magnetic field generated by the electrons around a proton determines where it absorbs.

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 13 Principles of NMR  Only nuclei that contain odd mass numbers (such as 1 H, 13 C, 19 F and 31 P) or odd atomic numbers (such as 1 H and 7 N) give rise to NMR signals. This means they have angular momentum.  Modern NMR spectrometers use a constant magnetic field strength B 0, and then a narrow range of frequencies is applied to achieve the resonance of all protons.

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan H NMR—The spectrum  An NMR spectrum is a plot of the intensity of a peak against its chemical shift, measured in parts per million (ppm).

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan H NMR—The spectrum  NMR absorptions generally appear as sharp peaks.  Increasing chemical shift is plotted from right to left.  Most protons absorb between 0-10 ppm.  The terms “upfield” and “downfield” describe the relative location of peaks. Upfield means to the right. Downfield means to the left.  NMR absorptions are measured relative to the position of a reference peak at 0 ppm on the  scale due to tetramethylsilane (TMS). TMS [Si(CH ₃ ) ₄ ] is a volatile inert compound that gives a single peak upfield from typical NMR absorptions.

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan H NMR—The spectrum  The chemical shift of the x axis gives the position of an NMR signal, measured in ppm, according to the following equation:  By reporting the NMR absorption as a fraction of the NMR operating frequency, we get units, ppm, that are independent of the spectrometer.

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan H NMR—The spectrum  Four different features of a 1 H NMR spectrum provide information about a compound’s structure: - Number of signals - Position of signals - Intensity of signals - Spin-spin splitting of signals

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan H NMR—Number of signals  The number of NMR signals equals the number of different types of protons in a compound.  Protons in different environments give different NMR signals. Equivalent protons give the same NMR signal.  To determine equivalent protons in cycloalkanes and alkenes, always draw all bonds to hydrogen.

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan H NMR—Number of signals  The number of 1 H NMR signals of some representative organic compounds:

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan H NMR—Position of signals  In the vicinity of the nucleus, the magnetic field generated by the circulating electron decreases the external magnetic field that the proton “feels”.  Since the proton experiences a lower magnetic field strength, it needs a lower frequency to achieve resonance. Lower frequency is to the right in an NMR spectrum, toward a lower chemical shift, so shielding shifts the absorption upfield.

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan H NMR—Position of signals  The less shielded the nucleus becomes, the more of the applied magnetic field (B 0 ) it feels.  This deshielded nucleus experiences a higher magnetic field strength, to it needs a higher frequency to achieve resonance.  Higher frequency is to the left in an NMR spectrum, toward higher chemical shift— so deshielding shifts an absorption downfield.  Protons near electronegative atoms are deshielded, so they absorb downfield.

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan H NMR—Position of signals  How chemical shift is affected by electron density around a nucleus:

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan H NMR—Position of signals  Shielding and deshielding effects:

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan H NMR—Position of signals

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan H NMR—Chemical shift values  Protons in a given environment absorb in a predictable region in an NMR spectrum.

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 26  The chemical shift of a C—H bond increases with increasing alkyl substitution. 1 H NMR—Chemical shift values

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 27  In a magnetic field, the loosely held  electrons of the double bond create a magnetic field that reinforces the applied field in the vicinity of the protons.  The protons now feel a stronger magnetic field, and require a higher frequency for resonance. Thus the protons are deshielded and the absorption is downfield. 1 H NMR—Chemical shift values

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 28  In a magnetic field, the  electrons of a carbon-carbon triple bond are induced to circulate, but in this case the induced magnetic field opposes the applied magnetic field (B 0 ).  Thus, the proton feels a weaker magnetic field, so a lower frequency is needed for resonance. The nucleus is shielded and the absorption is upfield. 1 H NMR—Chemical shift values

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 29  Regions in the1H NMR spectrum: 1 H NMR—Chemical shift values

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan H NMR—Intensity of signals  The area under an NMR signal is proportional to the number of absorbing protons.  An NMR spectrometer automatically integrates the area under the peaks, and prints out a stepped curve (integral) on the spectrum.  The height of each step is proportional to the area under the peak, which in turn is proportional to the number of absorbing protons.  Modern NMR spectrometers automatically calculate and plot the value of each integral in arbitrary units.

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan H NMR—Intensity of signals

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 32  Consider the spectrum below: 1 H NMR—Spin-spin splitting

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan H NMR—Spin-spin splitting  Spin-spin splitting occurs only between nonequivalent protons on the same carbon or adjacent carbons.  Let to consider how the doublet due to the CH2 group on BrCH2CHBr2 occurs: o When placed in an applied electric field, (B 0 ), the adjacent proton (CHBr2) can be aligned with (  ) or against (  ) B 0. o Thus, the absorbing CH2 protons feel two slightly different magnetic fields—one slightly larger than B 0, and one slightly smaller than B 0. o Since the absorbing protons feel two different magnetic fields, they absorb at two different frequencies in the NMR spectrum, thus splitting a single absorption into a doublet.

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 34  The frequency difference, measured in Hz between two peaks of the doublet is called the coupling constant, J. 1 H NMR—Spin-spin splitting

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan 35  Let now to consider how a triplet arises:  When placed in an applied magnetic field (B 0 ), the adjacent protons H a and H b can each be aligned with (  ) or against (  ) B 0.  Thus, the absorbing proton feels three slightly different magnetic fields—one slightly larger than B 0, one slightly smaller than B 0, and one the same strength as B 0. 1 H NMR—Spin-spin splitting

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan H NMR—Spin-spin splitting

Food Quality Evaluation Methods– Department of Biosystems Engineering – University of Kurdistan Kurdistan Nature Zrebar Lake, Marivan