CONTENTS Content Page no. UV-Vis Spectroscopy 2 IR Spectroscopy 3-14 NMR Spectroscopy 15.28 Mass Spectrometry 29-45 Affinity Chromatography 46-48 Electrophoresis 49-61 X-Ray Crystallography 62-67 Immunological Assay 68-70 Note: | have not been able to include some chapters/topics prescribed in the syllabus due to lack of time, Fortunately, the excluded topics are the ones whose reference materials are easily obtained from relevant sources.UV-VISIBLE SPECTROSCOPY CHOICE OF SOLVENTS: ‘The choice ofthe solvent to be used in ultraviolet spectroscopy is quite important. The first criterion for a good solvent is that it should not absorb ultraviolet radiation in the same region as the sub- stance whose spectrum is being determined, Usually solvents that do not contain conjugated sys- tems are most suitable for this purpose, although they vary as o the shortest wavelength at which they remain transparent to ultraviolet radiation. Table 7.1 lists some common ultraviolet spec~ troscopy solvents and their cutoff points, oF mininum regions of transparency. OF the solvents listed in Table 7.1, water, 95% ethanol, and hexane are most commonly used. Each is transparent in the regions of the ultraviolet spectrum where interesting absorption peaks from sample molecules are likely to occur. A second criterion for a good solvent is its effect am the fine structure of an absorption band. Figure 7.5 illustrates the effects of polar and nonpolar solvents on an absorption band. A nonpolar solvent does not hydrogen bond with the solute, and the spectrum of the solute closely approx {mates the spectrum that would be produced in the gaseous state, where fine structure is often ‘observed. In a polar solvent, the hydrogen bonding forms a solute-solvent complex, and the fine structure may disappear TABLE 7.1 SOLVENT CUTOFFS Acctonitle 190. nm ineHexane 201 am, Chloroform 240 Methanol 205 Cyclohenane 195 house 19s 1ADionane 215, Water 190 95% Etunol 205 ‘Trimethyl phosplisie 210 220 35 360 280 ‘Wavelength (am) FIGURE 7.5 Uheaviolet spectra of phenol in ethanol and in isooctane. (From Coggeshall, N.D., and EM. Lang. J. Am. Chem Soe., 70 (1948): 3288. Reprinted by permission.)IR SPECTROSCOPY TRIS ar Teas ROO R San ene cer ere eT ee ey eng Le ny eer eee eee Drom a as Peete perenne eet ee ee a molecule, and these are accempanind by changes the eat See 200 nm <9 400 nn <——— 600 new BLUE RED shor Wtaveiength (x) Fig: L.A portion of EMR spectrum showing relationship of vibratonal infrared to other types of radiationeee ers Se ee tet ere rey I el eee eee ee ed ee oe ee ee a ner nr) which can he decribed by Hooke’ tn of simple harmon Ihe spring has a force constant, K and masves m; and ma the eds, then the theoretical vibration frequency, vis given by re Pore ters Fee em feet teen TerRo nt Se eres nr Pierre crscar ey See et et Reena Cee a etme ene ote es een cee cet tee Rete nae eet Cee eee a eee ee eLearn ee ee ee ed ee ee en en een et ee al Se eee ne ee eee eum ener omer rey due to charge separation and will interact with the fleld. E.g HICLee ee a anne ee en ee non prep ereer aren n ee ete eee ne ee ee Steet eee need Se ee Set Se ete eee re See eee Be ye een OER er ny en erent erent Oper meee wenpoy eee ery Uren peor per Eee ener eer tne ee nt cee erent eet MODES OF VIBRATION: (sce Ravi Sankar’s book ) SAMPLE HANDLING: PREPARATION OF SAMPLES FOR INFRARED SPECTROSCOPY To determine the infrared spectrum of a compound, one must place the compound in a sample holder, or cell. In infrared spectroscopy this immediately poses a problem. Glass and plastics absorb strongly throughout the infrared region of the spectrum. Cells must be constructed of ionic ‘substances—typically sodium chloride or potassium bromide, Potassium bromide plates are more ‘expensive than sodium chloride plates and have the advantage of usefulness in the range of 4000 to 400 cm“. Sodium chloride plates are used widely because of their relatively low cost. The practical range for their use in spectroscopy extends from 4000 to 650 cm, Sodium chloride begins to ab- sorb at 650 em”, and any bands with frequencies less than this value will not be observed. Since few important bands appear below 650 cm, sodium chloride plates are in most common use for routine infrared spectroscopy. Liquids. A drop of a liquid organic compound is placed between a pair of polished sodium chloride of potassium bromide plates, referred to as salt plates. When the plates are squeezed gently, a thin liquid film forms between them. A spectrum determined by this method is referred to as neat spectrum since no solvent is used. Salt plates break easily and are water soluble. Organic com- pounds analyzed by this technique must he free of water. The pair of plates is inserted into a holder which fits into the spectrometer, Solids. There are at teast three common methods for preparing a solid sample for spectroscopy, ‘The first method involves mixing the finely ground solid sample with powdered potassium bro- ‘mide and pressing the mixture under high pressure. Under pressure, the potassium bromide melts and seals the compound into a matrix. The result is a KBr pellet which can be inserted into a holder in the spectrometer. The main disadvantage of this method is that potassium bromide ab- sorbs water, which may interfere with the spectrum that is obtained. If a good pellet is prepared, the spectrum obtained will have no interfering bands since Potassium bromide is transparent down to 400 cm=',‘The second method, a Nujol mull, involves grinding the compound with mineral oil (Nujol) to create a suspension of the finely ground sample dispersed in the mineral oil. The thick suspension is placed between salt plates. The main disadvantage of this method is that the mineral oil ob- scures bands that may be present in the analyzed compound, Nujol bands appear at 2924, 1462, and 1377 em” (p. 30). ‘The third common method used with solids is to dissolve the organic compound in a solvent, Most commonly carbon tetrachloride (CCl,), Again, as was the case with mineral oil, some regions of the spectrum are obscured by bands in the solvent. Although it is possible to cancel out the vent from the spectrum by computer of instrumental techniques, the region around 785 cm"? is often obscured by the strong CCl stretch that occurs there. THE INFRARED SPECTROMETER ‘The instrument that determines the absorption spectrum for a compound is called an infrared spectrometer or, more precisely, a spectrophotameter. Two types of infrared spectrometers are in common use in the organic laboratory: dispersive und Fourier transform (FT) instru- ments. Both of these types of instruments provide spectra of compounds in the common range of 4000 to 400 cm. Although the two provide nearly identical spectra for a given compound, FT infrared spectrometers provide the infrared spectrum much more rapidly than the dispersive instroments. ‘The following are the essential components of an infra-red spectrophotometer. 1. Light source. 2. Monochromator and optical materials 3. Sample holder 4. Detector and 5. Instrument for recording the response (Recorder) Infra Red Radiation Sources: The infra red radiation sources are the hot bodies, continuously emitting the radiations, which approximate a black body radiator in their emission properties. (a) Incandescent lamp : A closed wound nichrome coil ean be raised to incandescence by resistive heating, A black oxide film formed on the coil give acceptable emmisivity. In this the temperature can be reached up 1100”. The nichrome coil docs not require water cooling. It requires little or no maintenance and gives long service, This source is recommended where reliability is essential. Though this source is simple and rugged, it is less intense than some other infra-red radiation sourcos. A rhodium wiro heater scaled in a ceramic cylinder has also been used as a source of infra red radiatios (©) Nernst glower : In IR spectroscopy, Nernst glower is the most commonly used source of radiation. It is constructed by fusing mixture of oxides of metals like zirconium, yttrium and thorium. They are moulded in the form of hollow tubes or rods about 1-8 mm in diameter and 2-6 em in length. The ends of the rods are cemented to short ceramic tubes for mounting and short platinum leads are provided for power connections.Nernst glowers are fragile. They have negative coefficient of resistance and they are preheated as to be conductive. Thus they are provided with auxiliary heaters. In order to prevent overheating they are provided with ballast, but they should also be protected from draft and at the same time ventilation is needed to remove surplus heat. ‘Tho energy output of Nernst glower is predominantly concentrated between 1-10 j. with relatively low energy beyond 10 j.. Radiation intensity is approximately thrice that of nichrome and globar sources, except in the near infra red region. ‘The main advantages of Nernst glower is that it emits infra red radiations over a wide wavelength range and the intensity of radiation remains steady and constant over a long period of time. Secondly, it can be used in air as it is not oxidised (©) Globar Source : It is a rod of sintered silicon carbide 6-8 mm in diameter and 50 mm in length. It is self starting and is electrically heated. The operating temperature is about 1300". It has a positive coefficient of resistance and can conveniently be controlled with a variable transformer. It is often enclosed in a water cooled brass tube, with a slot provided for the emission of radiations. It emits maximum radiation at 5200 em”, In comparison with Nernst glower the Globar is a less intense source below 10 y. The two sources are comparable to about 16 y, and the Globar is superior beyond about 15 y. (d) Mercury Are : In the very far infra red region. i.e. beyond 50 j1 (200 em), black body type sources lose effectiveness as their radiations decreases with the fourth power of wavelength. Mercury are gives intense radiation in this region. It is enclosed in a quartz jacket to reduce loss. The output from mercury are is similar to that of black body sources, but additional radiation is emitted from a plasma which enhances the Jong wavelength output. (©) Tungsten Filament Lamp : This source is useful for near infra red region only. 2, Monochromators: ‘The radiation source emits radiations of various frequencies. As the sample in IR spectroscopy absorbs only at certain frequencies, it is therefore, necessary to select desired frequencies from the radiation source and reject the radiations of other frequencies. This selection is achieved by means of monochromators. The monochromators are of two types (i) Prism monochromators and (ii) Grating monochromators, (i) Prism Monochromators : These are favoured because of greater range and simplicity Neither glass nor quartz is sufficiently transparent to infra red radiations and therefore other materials like halogen salts are used in prism monochromators as they are transparent to infra red radiations Quartz prisms are used only in the near infra red region (0.8 - 3 y). It is absorbed strongly beyond 4p.‘The great bulk of analytical work in the infra-red region is done using crystalline sodium chloride as the prism material. It has high dispersion in the region between 5-15 4 and adequate upto 2.5 41 crystalline potassium bromide and cesium bromide are satisfactory for far infra red region (15 4 to 40 4). In the near infra red region (1-S j!), lithium fluoride is used as prism material. All the commonly used prism materials except quartz are water soluble and are easily scratched. These materials must be protected from moisture either by using dessicants or by placing in a sealed housing which is evacuated. In the infra red spectrometers the focusing of the radiations is achieved by using concave mirrors rather than prisms. These mirrors can be prepared from various materials like metals or glass coated aluminium. The main advantage of these materials is that it has no chromatic abbration and are also sturdy. Besides concave mirrors plane reflecting mirrors are also used. The prism monochromator may be a single pass monochromator or a double pass ‘monochromator as shown in figures 24.4 and 24.5 respectively, Exit st Fig. 244 Single pass monochromator Exit sit U Plane mitror Arenirance sit Fig. 24.5 : Double pass monochromator ‘Single pass monochromator: ‘The sample is kept at or near the focus of the beam, just before the entrance monochromator. The radiation from the source after passing through the sample cies an * strikes the off-axis parabolic Littrow mirror 3, This renders the radiation -parallel and transmitted to the prism 'C’. The dispersed radiation after reflecting from a plane mirror returns through the prism second time and focuses into the exit slit of the monochromator and then to the detector part of the instrument.Double pags monochromator: In the double pass monochromator, there occurs a total of four passes through prism as shown (1) (2) (3) and (4) in the figure 24.5. “Tha dsbls pus mensch omar eed ‘more resolution of radiation as compared to mono pass monochromator. — In both mono and double pass monochromators, sodium ehlorid i ‘employed for the entire region from 4000 650 em'(2.5 to 15.4). nt St Prisms are Prisms of lithium fluoride and calcium fluor the significant stretching vibrations are located. (i) Grating monochromators 1 The grating is essentially series of parall lines cut out into a plane surface. It is usually constructed from glass or pate whieh looted with aluminium. In order to minimise greater amounts of scattered radiations and the unwanted radiati ratios ine emg ther spectral orders, the gratings are blaze to concentrate the ide give more resolution in the region where A grating is generally used in combination with a small prism which acts as order sorter. Sometimes filters transparent over a limited wavelength range are incorporated with atings. Grating monochromators have certain advantages over prism monochromators as : ( The grating construction material is not attacked by moisture and is not subjected to etching where on the salt prisms are affected by moisture and can be subjected to etching. (ii) Secondly grating monochromators can be used over considerable wavelength range and (iii) Grating monochromators are sturdy and long lasting. 3. Sampling: ‘see previous pages) 4. Detectors: There are two types of detectors used in infra red spectrophotometry and they are (a) thermal detectors and (b) photodetectors. (a) Thermal detectors : When the infra radiations falls on these detectors, they cause heating which gives rise to a potential difference which is measured. This potential difference depends upon the amount of radiation. The thermal detectors commonly used are thermocouples, bolometers and thermisters and Golay cell or Golay detector. (i) Thermocouple + It is the most commonly used detector in infra red spectrophotometry. Thermocouples are basically the dissimilar strips of metals Joined together at one end. Thermocouples are constructed in various ways. In ‘one of the thermacouple detectors two fine wires of metals which have different thermoclectrical properties are welded with blackened gold foil, and which absorbs the radiations. One welded joint (cold junction) is kept at constant temperature and the other welded joint (hot junction) is exposed to radiations. ‘This exposure of hot junction causes a rise in its temperature. Thus, as the two junctions are at different temperatures, it causes a potential difference which is proportional to degree of heating of hot junction (or amount of radiations falling on the hot junction). (ii) Bolometers : They are constructed from metals or semiconductors. In this large change of electrical resistance depends on temperature. When the radiationsfall on bolometer, its temperature changes which cause change in the resistance of the conductor. This change in resistance depends upon the amount of radiations falling on the bolometer. Bolometer is made in one arm of the wheatstone bridge and a similar strip of metal is used as balancing arm of the bridge, which is not exposed to infra red radiations. When no infra red radiations fall on the bolometer, the bridge remains balanced. As the radiations fall on the bolometer, the bridge becomes unbalanced due to change in electrical resistance and thus the electrical current flows through galvanometer G. The amount of current flowing through galvanometer is « measure of the intensity of the radiations falling on the detector. The response time for bolometer is 4 m sec. The schematic representation is given below. Fig. 24.7 Both thermocouples or bolometers are fitted in ateel housing having potassium bromide or cesium iodide window and it is evacuated, which decreases the Gin to bolometers. They are tho resisters made by fusing several metallic oxides. These shows a negative thermal coefficient of electrical resistance. (iv) Golay cell or Golay detector: Golay cell is now-a-days used in seve commercial spectrophotomoters. It consists of a small metal cylinder, one end of which closed by blackened metal plate and the other with a metallised diaphragm. A light beam falls on the diaphragm which reflects to phototube. ‘The cylinder is filled with non-absorbing gas like xenon. When the radiations fall on blackened metal plate it is heated, which causes the expansion of gas which in turn affects the diaphragm (motion of the diaphragm). This causes the change in the output of cell received by the phototube, which can be modulated according to the power of the falling radiations on Golay cell. ‘Thermocouples and Golay detectors possesses similar sensitivity in the mid infra red region. () Photon detectors : Photon detectors are widely used in near infra red region. They consist of suitable semiconductors like lead sulphide, lead telluride or germanium which are non-conducting at lower energy state, When the radiations fall on thesethey are raised to higher level which ean conduct and produce a signal which is proportional to the amount of radiation. In those there is a drop of electrical resistance and if small voltage is applied there is a large increase in current which can be amplified and indicated on a meter or recorder. 5. Recorder: In infra red recording spectrophotometers as the sample absorbs some energy, the sample beam and reference beam differs in their radiant energics. Then detector system generates the signal which is normally amplified and goes to servometer. ‘The servometor which is connected to attenuator comb blocks the part of reference beam till energies of reference and sample beams are equal and thus beam balance is achieved (i.¢. optical null). The attenuator ‘comb is tied mechanically to the pen of the recorder and paper driver. They are synchronised with the automatic rotation of wavelength mirror. The transmittance of the sample is recorded as a function of wavelength. FIGURE 2.3 A schemutic diagram ofa dispersive infrared spectrometer,FT-IR: Instrumentation ‘Several Fourier transform spectrometers are available commercially. Their prices lie in the 535,000 to $120,000 range (including the dedicated computer for performing the Fou- rier transformation) Drie Mechaniam. A requirement for satis factory interferograms (and thus satisfactory spectra) is that the speod of the moving mirror be relatively constant and its position exactly known at any instant. The planarity ‘of the mirror must also remain constant during its entire sweep of 10 em or more. In the far-infrared region, where the wave- lengths are in the micrometer range, die- Placement of the mirror by a fraction of a wavelength, and socurate measurement of its Position, can be accomplished by means of & motor-driven micrometer screw. A more Precise and sophisticated mechanism is re- quired for the mid- and near-{alrared regions, however. Here, the mirror mount is generally floated of air cushions held within close- fitting stainless steel sleeves (sc Figure 8-22) ‘The mount is driven by an electromagnetic coil similar to the voice coil in a loudspeaker: = slowly increasing current in the coil drives mirtor at constant velocity. After reaching its terminus, the mirror is returned rapidly to the starting point for the next sweep by a rapid reversal of the current. The length of travel varies from 2 to about 18 em: the scan Fates range from 0.05 em/s to 4 em/s. Two additional features of the mirror system are necessary for successful operation in the infrared regions, The first of these is 2 means ‘of sampling the interferogram at precisely ‘spaced retardation intervals. The second is.a method for determining exactly the zero-fetardation point to permit signal averaging. If this point is not known pre- cisely, the signals. from repetitive sweeps ‘would not be fully in phase; averaging would tend to degrade rather than improve the ‘signal. ‘The problem of precise signal sampling ‘and signal averaging is accomplished in mod- em instruments by using three interferom- ters rather than one, with a single mirror mount holding the three movable mirrors.Beam Splitters. Beam splitters are con- structed of transparent materials with re- fractive indices such that approximately 50% of the radiation is reflected. A widely used material for the far-infrared region ia a thin film of Mylar sandwiched between two plates of a low refractive-index solid. Thin films of ‘germanium of silicon deposited on ocsium fodide or bromide, sodium chloride, oF potas- ‘sium bromide are satisfactory for the mid- infrared region. A film of iron(tIl) oxide is deposited on calcium Muoride for work in the ‘near-infrared. Soarces and Detectors. The sources for Fourier transform infrared instruments are similar to those discussed earlier in this chap- ter. Generally, pyroelectric detectors must be employed because their response times are bined and pass on to the detector. EE we Computer Interterogram: the signal theFACTORS: Factors Influencing Vibrational Frequency t From the discussion above we know that the probable frequency of absorption can be calculated by the Hook's law. However, it has been observed that the calculated value of frequency of absorption is not exactly equal to the experimental value, There are many factors which are responsible for shifts in vibrational frequencies. (a) The frequency shift may occur due to the effect of molecule in the immediate neighbourhood of bond, (b) Change in force constant of bond due to electronic structure and (©) Duo to different states of the same substance eg. solid, liquid or gas (vapour). The energy of vibration and thus the wavelength of its absorption peak is influenced by other vibrations in a molecule. The influence and extent of coupling of vibrations plays significant role. APPLICATIONS: (see book)CHAPTER-2 NMR SPECTROSCOPY NMR spectroscopy is the study of spin changes at the nuclear level when a radiofrequency energy is absorbed in the presence of magnetic field. Quantum numbers and their role in NMR: Many atomic nuclei have a property called spin: the nuclei behave as if they were spinning. In fact, ‘any atomic nucleus that possesses either odd mass, odd atomic number, or both has a quantized spin angular momentum and a magnetic moment. The more common nuclei that possess spin include |H, FH. '4C, 'IN. '20. and "SF. Notice that the nuclei of the ordinary (most abundant) isotopes of carbon and oxygen, 'ZC and '§O, are not included among those with the spin property. However, the nucleus of the ordinary hydrogen atom, the proton, does have spin. For cach mucleus with spin, the number of allowed spin states it may adopt is quantized and is determined by its nuclear spin quantum number, J. For each nucleus, the number /is a physical constant, and there are 2/-+ | allowed spin states with in- tegral differences ranging from +! to ~1. The individual spin states fit into the sequence HL Dye lt Det Equation 3.1 For instance, a proton (hydrogen nucleus) has the spin quantum number = | and has two allowed spin states (2() + 1 = 2) for its nucleus: ~! and +4. For the chlorine nucleus, J = {and there are four allowed spin states [2@)+ 1 = 4}: =f, +}, +4. Table 3.1 gives the spin quantum numbers of several nuclei. TABLE 3.1 SPIN QUANTUM NUMBERS OF SOME COMMON NUCLEI wm ie Ke 40 4N fo Sr owe ie Seca sae 1 ee Ss quantum number ms Os fie cE ye ee Number of spin states 92 Feb oe Pz sce on In the absence of an applied magnetic field, all the spin states of « given nucleus are of equivalent ‘energy (degenerate), and in a collection of atoms, all of the spin states should be almost equally Populated, with the same number of atoms having each of the allowed spins, ABSORPTION OF ENERGY ‘The nuclear magnetic resonance phenomenon occurs when nuclei aligned with an applied fleld are induced to ubsorty energy and change their spin orientation with respect to the applied field. Figure 3.5 illustrates this process for a hydrogen nucleus. “The energy absorption is a quantized process, and the energy absorbed must equal the energy difference between the two states involved. Emanes = (E- 4 sete — Fee sate) Equation 3.2 In practice, this enengy difference is a function of the strength of the applied magnetic Neld, Bo, as illustrassa jaa | f FIGURE 3.3 Theypinsatesofapmioninite Note ‘Applied told thcose sed in the presence ofan applied magne field, Energies Alignments _The stronger the applied magnetic field, the greater the energy difference between the possible spin states: AE=f(Bo) Equation 33 The magnitude of the energy-level separation also depends on the particular nucleus involved. Bach nucleus (hydrogen, chlorine, and so on) has a different ratio of magnetic moment to angular mo- ‘mentum, since each has different charge and mass. This ratio, called the magnetogyric ratlo, 7, is 0 constant for cach nucleus and determines the energy dependcnce on the magnetic elds BE=f (785) =hv Equation 34 Since the angular momentum ofthe nucleus is quantized in units of 12m, the fil equation takes form ry s (55) Bo ‘Solving for the frequency of the absorbed energy, Equation 3.5 v= (2) a equation 26 THE MECHANISM OF ABSORPTION (RESONANCE) “To understand the nature of a nuclear spin transition, the analogy of a child's spinning top is useful. Protons absorb encrgy because they begin to precess in an applied magnetic field. The phenomenon of precession is similar to that of a spinning top. Owing to the influence of the earth's gravitational field, the top begins to “wobble,” or precess, about its axis (Fig. 3.7a). A spinning nucleus behaves in a similar fashion under the influence of an applied magnetic field (Fig. 3.7b). ‘When the magnetic field is applied, the nucleus begins to precess about its own axis of spin with angular frequency «, which is sometimes called its Larmor frequency. The frequency atFIGURE 3.7 (A top precessig in the earth's gravitational Seld; (0) the precession ofa spinning nucleus rsuking Sean the influence of an applied mapretic field. which a proton precesses is directly proportional to the strength of the applied magnetic field; the stronger the applied field, the higher the rate (angular frequency, «) of precession. For a pro- ton, if the applied field is 1.41 Tesla (14,100 Gauss), the frequency of precession is approxi- mately 60 MHz, Since the nucleus has a charge, the precession generates an oscillating electric field of the same frequency. If radiofrequency waves of this frequency are supplied to the precessing proton, the en. ‘ergy can be absorbed. That is, when the frequency of the oscillating electric field component of the incoming radiation just matches the frequency of the electric field generated by the precessing nu- 60 Maz 7 1 1 = 6OMHE ! Absorption occurs "AS < > bye ne - { J 8, 14.100 gauss FIGURE 3.8 The nuclear magnetic resonance process; absorption occurs when VatINSTRUMENTATION: A line diagram of the instrument of NMR spectrophotometer along with its components is shown in Fig. 25.5. Pig. 25.5 Magnet : The strong magnet provides stable and homogencous field. The magnet size is 15 inches in diameter and is capable of producing strong fields (upto 23.500 gauss for 100 MHz work) If the magnetic field is not homogencous, the nuclei in the different parts of the recess with different frequencies, thereby producing broad signal imple Radio Frequency Oscillator (Transmitter) and Sweep Generator : The RF oscillator coil is installed perpendicular to the magnetic field and transmits radiowaves of some mixed frequency such as 60, 100, 220 of 300 MHz. Since the large magnet as well as the Radio Frequency oscillator both produce fixed fields, a sweep generator is installed to supply a variable de current to a sccondary smaller magnet ‘This allows us to vary (or sweep? the total applied magnetic field over a small range. RF receiver (detector) and Recorder The coil of the RF receiver or detector is installed perpendicular to both the magnetic field and the oscillator coil and is tuned to the same frequency as the transmitter. When the precession frequency is matched with the radio frequency the nuclei induces electromagnetic field (emf in the detector coil by virtue of the change in magnetic flux following: nuclear Mipover. This signal is amplified and sent to a recorder, The recorder gives a spectrum as a plot of the strength of the resonance signal on the ¥ axis Vs strength of the magnetic field on X axis. The strength of the resonance signal is directly Proportional to the number of nuclei resonating at that particular ficld strength ‘The area of the peak 1s therefore a direct measure of number of resonating nuclei and hence most of the instruments are equipped with automatic integrator which can record peak areas in the form of superimposed integration trace on the chart.‘The measurement of exact strength of continuously sweeping magnetic field is difficult task, hence it is difficult to assign a peak position on absolute scale. Thus, the method used is to record the peak position in relation to the position of an arbitrary standard lines (internal standard) ‘The tetramethyl silane (TMS ~ (CH,), Si) is used as internal standard for most of protons and is ndded to the sample before recording the spectrum. Sample und Sample Holder : A 1~30 mg sample is generally used in the form of dilute solution (2 - 10%). The solvent should not contain hydrogen of its own, Sample holder is a glass tube about 5 mm in diameter and is 15 ~ 20 cm in length.FACTORS AFFECTING CHEMICAL SHIFT 1, Inductive Effect : A proton is said to be deshielded if it is attached with an electro negative group. Greater the electronegativity of the atom, greater the deshielding effect and more will be the 8 value. CH, - CHy 098 CHy- Cl 3.055 | CH; -F 425 Thus electronegative groups deshield the proton. As the distance from the electronegative atom increases, the deshielding effect diminishes.2, Van der Waal's Deshielding + In overcrowded molecules it is possible that some proton may be occupying sterically hindered positions. Clearly the electronegative cloud of bulky group will tend to repell the electron cloud surrounding the proton, Thus, such a proton will be deshielded and will resonate at slightly higher values of 8 than expected. This is considered as Van der Wai deshielding. 3. Anisotropic effect (Space Eifect) : Magnetic field developed by x bond is stronger in one direction than other is oriented in stich a ‘The induced magnetic field around carbon is dimagnetic and paramagnetic in the direction of alkene proton. Thus proton will feel greater field strength and hence effect occurs at lower field, 4. Hydrogen Bonding: Hydrogen atom exhibiting the property of hydrogen bonding in a compound absorbs at a Jow field in comparison to the one which does not show hydrogen bonding. The hydrogen bonded proton being attached to highly electronegative atom will have smaller electron density around it (deshielded) hence resonate at downfield. 5, Concentration, Solvent and Temperature Effect : In CCl, and CdCl; chemical shift of proton attached to carbon is independent of concentration and temperature while protons of ~ OH, ~ NH;, ~ SH groups exhibit a substantial concentration and temperature effect due to hydrogen bonding. Intermolecular hydrogen bonding is less affected than intermolecular bonding by concentration change. Both types of hydrogen bonding are affected by temperature variations. ‘SPIN-SPIN COUPLING: ‘The spins of neighbouring groups of nuclei in molecule are said to be coupled if their spin states mutually interact. The interactions, which involve electrons in the intervening bonds, result in small variations in the effective magnetic fields experienced by one group of nuclei due to different orientations of the spin angular momenta and magnetic moments of those in the neighbouring group or groups, and vice versa. These lead to the splitting of the resonance signal into two or more components that are shifted slightly upfield and downfield respectively from the position in the absence of coupling, the probabilities of each being roughly the same because the permitted nuclear spin energy levels are almost equally populated. Thus, the resonance signals for two single adjacent nuclei with substantially different chemical shifts are each split into two component peaks of equal intensity. (Spln-Spin Splitting) On observing the NMR spectra of compounds it is seen that the signals are split into umber of lines. ©.g. in CHyCH, OH the signals given arc ‘Singlet for OH Quartet for CH, ‘Triplet for CHy We say that each signal will split into doublet, triplet, quartet depending on the number of Protons present on adjacent carbons. The multiplicity of lines is related to the number of Protons on neighbouring groups. A simple rule (a + 1 rule) is used to find the multiplicity. Count the number of neighbouring protons and add 1. ”Splitting of the spectral lines arise because of coupling interaction between neighbour protons and is related to the number of possible spin orientations that these neighbours can Consider Cinnamic acid representing 2 vicinal protons, H, and Hy. These protons, having different magnetic environments, come to resonance at different positions in unit spectrum. They do not give rise to single peaks (singlets) but doublets. The separation between the lines of each doublet is equal, this spacing is called coupling constant ‘J’. The resonance position for A depends on its total magnetic environment, part of its ‘magnetic environment is the nearby proton B, which is itself magnetic, and the proton B can either have its nuclear magnet aligned or opposed with proton A. The two spin orientations of B create 2 different magnetic fields around A. Therefore the proton A comes to resonance, not ‘once, but twice and proton A gives rise to a doublet. Similarly with proton B the mutual magnetic influence between protons A and B is not transmitted through space, but via the electrons in the intervening bonds. The nuclear spin of A couples with electron spin of C-H, bonding electrons these in turn couple with C-C bonding electrons and then with C-Hy bonding electrons. The coupling is eventually transmitted to the spin of Hy nucleus THE COUPLING CONSTANT Section 3.15 discussed the splitting pattem of the ethyl group and the intensity ratios of the multi plet components but did not address the quantitative amount by which the peaks were split. The dis tance between the peaks in a simple multiplet is called the coupling constant, J. The coupling constant is a measure of how strongly a nucleus is affected by the spin states of its neighbor. The spacing between the multiplet peaks is measured on the same scale as the chemical shift, and the coupling constant is always expressed in Hertz (Hz). In ethyl iodide, for instance, the coupling con- stant J is 7.5 Hz. To see how this value was determined, consult Figures 3.26 and 3. 4, . “The spectrum in Figure 3.26 was determined at 60 MHz; thus, each ppm of chemical shift (5 unit) represents 60 Hz. Inasmuch as there are 12 grid lines per ppm, each grid line represents (60 Hz)/12 = Singlet 1 Doublet rou Triplet 121 Quartet 13°31 Quintet 14 6 41 Sextet 1s 1010 5 1 FIGURE 3.33 Pascal's triangle. Septet 16 1S 20 15 6 1 »FIGURE 3.26 The 'MNMR spectrum of ethyl iodide (60 MHz), yuu Coupting constant aosee ‘measured in He (Chamical siti. a -———______- con ct gap —* (Chemical shi erenco FIGURE 3.34 The detnition ofthe coupling constants in the ethyl spliting pattern. $ Hz. Notice the top of the spectrum. It is calibrated in eycles per second (cps), which are the same as Hertz, and since there are 20 chart divisions pet 100 cps, one division equals (100 cpsy/20 = S cps = 3 Hz. Now examine the multiplets. The spacing between the component peaks is approximately 1.5 chart divisions, so iy eS He. JUS div SE 275 He n‘That is, the coupling constant between the methyl and methylene protons is 7.5 Hz. When the pro- tons interact, the magnitude (in ethyl iodide) is always of this same value, 7.5 Hz. The amount of coupling is constant, and hence J can be called a coupling constant, ‘The constant nature of the coupling constant can be observed when the NMR spectrum of ethyl iodide is determined a1 both 60 MHz and 100 MHz. A comparison of the two spectra indicates that the 100-MHz spectrum is greatly expanded over the 60-MHz spectrum. The chemical shift in Hertz for the CHy and CH; protons is much larger in the 100-MHz spectrum, although the chemical shifts in 8 units (ppm) for these protons remain identical to those in the 60-MHz spectrum. Despite the expansion of the spectrum determined at the higher spectrometer frequency, careful examination of the spectra indicates that the coupling constant between the CHy and CH; protons is 7.5 Hz in both spectra! The spacings of the lines of the triplet and the lines of the quartet do not expand when the spectrum of ethyl iodide is determined at 100 MHz. The extent of coupling between these 1wo sets of protons remains constant irrespective of the spectrometer frequency at which the spectrum was determined (Fig. 3.35), For the interaction of most aliphatic protons in acyelic systems, the magnitudes of coupling con- stants are always near 7.5 Hz. Compare, for example, 1,1,2-trichloroethane (Fig. 3.25), for which J = 6 Hz, and 2-nitropropane (Fig. 3.27), for which J = 7 Hz. These coupling constants are typical for the interaction of two hydrogens on adjacent sp°-hybridized carbon atoms. Different types of pro- tons have different magnitudes of J, For instance, the cis and trans protons substituted on a double bond commonly have values of approximately Jey = 10 Hz. and Jirans = 17 Hz. In ondinary come pounds, coupling constants may range anywhere from 0 to 18 Hz. ‘The magnitude of J often provides structural clues, For instance, one can usually distinguish be- tween a cis olefin and a trans olefin on the basis of the observed coupling constants for the vinyl Protons. Table 3.9 gives the approximate values of some representative coupling constants. A more ‘extensive list of coupling constants appears in Appendix 5. Before closing this section, we should take note of an axiom: the coupling constants of the ‘groups of protons that split one another must be identical. This axiom is extremely useful in inter- Dreting a spectrum that may have several multiplets, each with a differeat coupling constant. Ja 75 Ke io Me 7 J 75He 163 He FEGURE 3,35 Anuilustation ofthe relationship between the chemical shift and the coupling constant, ™NUCLEAR MAGNETIC DOUBLE RESONANCE / SPIN SPIN DECOUPLING: It is @ powerful tool for simplifying a spectra. In a complex molecule if several of the constants have nearly the same values; or if the long range coupling is present or if absorption gives multiplets then it becomes very difficult to determine structure. A proton spin couples with neighbouring proton because it has sufficient life time in a given spin state. If life time of a spin is reduced i.e. if the exchange between spin states of nuclei is speeded up then little information about the neighbouring nuclei will be obtained, HOH tJ 4m case of ruch compounds, where Hy and Hy are in different environments - CG - \ Hy Hy therefore 2 doublets at different field strengths are observed. If Hq is irradiated with strong correct radio frequency #o that the rate of its transition between the two energy states becomes Jarger then the life time of this nucleus in any one spin state will be too short to resolve coupling with Hy. In such a ease Hy, proton will have one time average view of Hy and hence Hy will come to resonance only once and Hp will appear as a singlet and not doublet. Time d; is needed to resolve the two lines of a doublet which is related to J. Thus, formation of a doublet is possible if each spin state of Hy has a life time greater than dy. Due to double irradiation life time becomes still less and thus coupling is not possible. So it results in a singlet by spin-spin decoupling, FT-NMR OR PULSED NMR: In NMR, the radiofrequency energy can be introduced either by continuous wave (CW) scanning of the frequency range or by pulsing the entire range of frequencies with a single burst of radiofrequency energy. The two methods result in two distinct classes of NMR spectrometers viz. CW NMR spectrometers and FT or pulsed NMR spectrometers, In Fourier transform (FT) or pulse NMR studies, an instrument with a 2.1-Testa magnetic field uses a short (1 to 10 41 sec) bursts of 90MHz energy to accomplish. The source is tured on and off very quickly, generating a pulse similar to that shown below. On—>P]<— on a Time FIGURE 3.14 A shor pulse, According to a variation of the Heisenberg Uneertainity Principle, even though the frequency of the oscillator generating this pulse is set to 90MHz, if the duration of the pulse is very short, the frequency content of the pulse is uncertain because the oscillator was not on long enough to establish a solid fundamental frequency. Therefore, the pulse actually contains a range of *frequencies centred around the fundamental frequency. This range of frequencies is great cnough to excite all of the distinct types of hydrogens in the molecule at once with this single burst of energy. When the discontinued, the excited nuclei begin to lose their excitation energy and return to their original spin state or relax. As each excited nucleus relaxes, it emits electromagnetic radiation. Since the molecule contains many different nuclei, many different frequencies of electromagnetic radiation are emitted simultaneously. This emission is called a free induction decay (FID) signal, The intensity of the FID decays with time as all of the nuclei eventually lose their excitation. The FID is a superimposed combination of all the frequencies emitted and can be quite complex. The individual frequencies due to different nuclei are extracted by using a computer and a mathematical method ealled Fourier transform (FT) analysis. ‘Therefore, the FID is the superimposition of many different frequencies, each of which could have 1 different decay rate. The FT analysis will separate each of the individual components of this signal and convert them to frequencies, The FT analysis breaks the FID into its separate sine or cosine wave components. This procedure is too complex to be carried out by eye or by hand and it requires a computer, Pulsed FT NMR spectrometers have computers built into them that not only ‘can work up the data by this method but can control all of the settings of the instrument, Fig. The appearance of the FID when the decay is removed. ‘The pulsed FT method described here has several advantages over the CW method. It is more sensitive, and it can measure weaker signals. Five to 10 minutes are required to scan and record a CW spectrum; a pulsed experiment is much faster, and a measurement of an FID can be performed in a few seconds. With a computer and fast measurement, itis possible to repeat and average the ‘measurement of the FID signal. This isa real advantage when the sample is small, in which case the FID is weak in intensity and has a great amount of noise associated with it. Noise is random elec tonic signals that are usually visible as fluctuations of the baseline in the signal (Fig. 3.17). Since noise is random, it normally eancels out of the spectrum after many iterations of the spectrum are are added together.