Analytical Techniques Past Paper PDF 2021

Analytical Techniques (11%) =========================== (Mass Spectrometry, Infrared (IR) Spectroscopy, Nuclear Magnetic Resonance (NMR) Spectroscopy, Thin layer Chromatography and High Performance Liquid chromatography (HPLC) ------------------------------------------------------------------------...

Analytical Techniques (11%) =========================== (Mass Spectrometry, Infrared (IR) Spectroscopy, Nuclear Magnetic Resonance (NMR) Spectroscopy, Thin layer Chromatography and High Performance Liquid chromatography (HPLC) -------------------------------------------------------------------------------------------------------------------------------------------------------------------------- - **Thin Layer Chromatography** (TLC) (Scope: principle, instrumentation and applications) - **High Performance Liquid Chromatography** (HPLC) (Scope: principle, instrumentation- components of basic HPLC system, application of high performance liquid chromatography) - **Spectroscopy** (Scope: mass spectrometry: Principle, instrumentation, application, Infrared (IR) Spectroscopy: principle, instrumentation, application, Nuclear magnetic resonance (NMR) spectroscopy: principle, equivalent and non-equivalent proton, chemical shift, spin-spin coupling, (n+1) rule, instrumentation, interpret NMR spectra of simple aliphatic hydrocarbons, application Thin Layer Chromatography (TLC) =============================== Thin layer chromatography is a technique used to isolate non-volatile mixtures. The experiment is conducted on a sheet of aluminium foil, plastic or glass which is coated with a thin layer of adsorbent material (stationary phase). The material usually used is aluminium oxide (Al2O3/alumina), cellulose or silica gel. The most commonly used binders are gypsum and plaster of Paris. \[Binders make the adsorbent stick to the glass plate.\]. On completion of the separation, each component appears as spots separated vertically Each spot has a retention factor (R~f~) expressed as: The factor affecting retardation or retention factor are the solvent system, amount of material spotted, adsorbent and temperature, TLC is one of the fastest, least expensive, simplest and easiest chromatography technique. Thin layer chromatography (TLC) is similar to both paper and column chromatography, having features of both. The stationary phase consists of a thin film of adsorbent that is spread as a coating over a support such as glass plate in a thin even layer. However, TLC differ from the column chromatography in the following respects: i. It does not need any glass column. ii. The mobile phase ascends through the thin layer of adsorbent by capillary action and does not descend through the column under gravity as in case of column chromatography. It is because of this reason that TLC is also known as **reverse column chromatography**. Principle of Thin Layer Chromatography -------------------------------------- Like other chromatographic techniques, TLC depends on the separation principle. The separation relies on the relative affinity of compounds towards the mobile and stationary phases. The movement occurs in a such a way that the compounds which have a higher affinity to the stationary phase move slowly while the other compounds travel fast. Therefore, the separation of the mixture is attained. On completion of separation process, the individual components from the mixture appear as spots at respective levels on the plates. Their character and nature are identified by suitable detection techniques. TLC based on the principle of separation through adsorption type. The separation relies on the relative empathy of compounds towards the mobile phase and a stationary phase. ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 1. **Thin Layer Chromatographic Plates (stationary phase)**: Ready-made plates are used which are chemically inert and stable. The stationary phase is applied on its surface in the form of a thin layer. The stationary phase on the plate has a fine particle size and also has a uniform thickness. 2. **Thin layer Chromatography Chamber:** Chamber used to develop plates. It is responsible to keep a steady environment inside which will help in developing spots. Also, it prevents the solvent evaporation and keeps the entire process dust-free. 3. **Thin Layer Chromatography Mobile phase:** Mobile phase is the one that moves and consists of a solvent mixture or a solvent. This phase should be particulate-free. The higher the quality of purity, the development of spots is better. \[The choice of mobile phase or solvent for separation depends upon the adsorbent and the components to be separated. The solvent selected should be of greater polarity than the sample. Some commonly used solvent for TLC are petroleum ether, acetone, diethyl ether, benzene, ethyl acetate, amyl alcohol etc.\]. 4. **Thin Layer Chromatography Filter Paper:** It has to be placed inside the chamber. It is moistened in the mobile phase. Separation Technique of TLC --------------------------- - The stationary phase that is applied to the plate is made to dry and stabilise. - To apply sample spots, thin marks are made at the bottom of the plate with the help of a pencil. - Apply sample solutions to the marked spots. - Pour the mobile phase into the TLC chamber and to maintain equal humidity, place a moistened filter paper in the mobile phase. - Place the plate in the TLC chamber and close it with a lid. It is kept in such a way that the sample faces the mobile phase. - Immerse the plate for development. - Remember to keep the sample spot well above the level of the mobile phase. Do not immerse it in the solvent. - Wait till the development of spots. Once spots are developed, take out the plate and dry them. - The sample spots can be observed under UV light chamber. Advantages of TLC ----------------- i. It is very quick (separation time 15-50 minutes) and easy to perform. ii. It gives highly reliable results. iii. It requires very small quantity of the substance (of the order of a fraction of a milligram). iv. It can also use for quantitative analysis on a small scale. v. It can also use to follow the progress of a reaction. vi. TLC plates can be heated to higher temperature without causing any damages to it. Disadvantages ------------- i. It cannot be used for volatile compounds having boiling points less than about 373 K. ii. It cannot be used for the purification of compounds on a large scale. iii. The thin layer chromatography plates do not have long stationary phase. iv. When compared to other chromatographic techniques, the length of separation is limited. v. The results generated from TLC are difficult to reproduce. vi. Since TLC operates as an open system, some factors such humidity and temperature can be consequences to the final outcome of the chromatogram. vii. It is only a qualitative analysis technique and not quantitative. viii. The detection limit is high and therefore cannot be used TLC for lower detection limit. Application of TLC ------------------ 1. The qualitative testing of various medicines such as sedatives, local anaesthetics, anticonvulsant tranquilisers, analgesics, antihistamines, steroids, hypnotics is done by TLC. 2. TLC is extremely useful in biochemical analysis such as separation or isolation of biochemical metabolites from its blood plasma, urine, body fluid, serum etc. 3. TLC can be used to identify natural products like essential oils or volatile oil, fixed oil, glycosides, waxes, alkaloids etc. 4. It is widely used in separating multicomponent pharmaceutical formulations. 5. It is used to purify of any sample and direct comparison is done between the sample and the authentic sample. 6. It is used in the food industry to separate and identify colours, sweetening agents, and preservatives. 7. It is used in the cosmetic industry. 8. It is used to study if a reaction is complete. High Performance Liquid Chromatography (HPLC) ============================================= High-performance liquid chromatography is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. Principle of HPLC ----------------- HPLC is an improvised form of column liquid chromatography. Instead of a solvent being allowed to drop through a column under gravity, it is forced through under high pressure upto 400 atm. which makes the process faster. The basic principle of HPLC includes the interaction of the compound with other chemical species in a characteristic manner and separation of a sample into its constituent parts due to the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used. Instrumentation of HPLC: Components of Basic HPLC system -------------------------------------------------------- 1. **Solvent (Mobile phase)** The solvent (mobile phase) used in HPLC is a mixture of polar and non-polar liquid components whose concentration varies and depends upon the concentration of the composition of the sample used. Precaution must be taken to keep solvent free of dissolved gas which can bubble out of solution during mid separation.  Solvent reservoir ----------------- **Function:** Reservoir holds the solvent which acts as a mobile phase. It is filled with a gas diffuser through which helium can be bubbled. High pressure pump ------------------ **Function: -**Pump is used to force the solvent (mobile phase) and sample through the column at a specific flow rate which is expressed in ml/min. Multiple piston pumps is used over a syringe pump (though it has a greater control of flow rate) to generate high pressure and maintain constant pulse free flow rate because syringe pump is unable to produce as much pressure as piston pump can produce. Sample Injector system ---------------------- **Function:** The injector introduces the liquid sample into flow stream of the mobile phase. An auto sampler is used when many samples have to be analysed or when manual injection is not practical. Column (stationary phase) ------------------------- **Function:** Column is considered as the heart of chromatograph because column's stationary phase separates the sample components using various physical and chemical properties. Column contain specific packing material (stationary phase). The columns which are packed with solids like SiO2 or Al2O3 are called **homogenous columns** while those which contain a liquid are called **bonded columns.** In bonded columns the liquid is bonded to a solid support. Detectors --------- **Function:** The detector is located at the end of column and can detect the individual molecule that comes out (elutes) form the column. A detector serves to measure the amount of those molecules so that chemist can quantitatively analyse that sample components. The detector provides an output to a recorder or computer that result in the liquid chromatogram (i.e., the graph of the detector responses). Detector used in HPLC is a UV absorption detector because most medium to large molecules absorb uv radiations. Data collection devices ----------------------- Computer is used as data collector device. The computer not only controls all the molecules of the HPLC instrument but it takes the signal form the detector and uses it to determine the time of elution (retention time) of the sample components (qualitative analysis) and the amount of sample (quantitative analysis). Separation Technique of HPLC ---------------------------- i. Small volume of the liquid sample is injected into a tube which is packed with tiny particles (stationary phase / column). In the tube the individual components of sample are move down the packed tube (column) with a liquid (mobile phase) forced through the column pressure delivered by the pressure pump. ii. The components are separated from one another by the column packing that involves various chemical and / or physical interactions between their molecules and the packing particles. iii. The separated components are detected at the exit of the tube (column) by a flow - through device (detector) that measures their amount. An output from the detector is called a "liquid chromatogram (LC) Applications of HPLC -------------------- i. **For the separation of chemical and biological compounds that are non- volatile and thermally unstable.** The typical non-volatile compounds are: a. Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol) b. Salts like NaCl and K3PO4 c. Proteins like egg white or blood protein d. Organic chemicals like polymers (e.g. polystyrene, polyethylene) e. Heavy hydrocarbons like asphalt or motor oil. f. Many natural products such as ginseng, herbal medicines, plant extracts. g. Thermally unstable compounds like trinitrotoluene (TNT), enzymes. ii. **For the qualitative analysis / identification of chemical compound in the sample: -** The most parameter for compound identification is its retention time (time taken for a particular compound to elute from the column after injection) which depends on the chemical structure, molecular weight and some other molecular parameter. The retention time depends on the detector used. iii. **For the quantitative analysis / measurement of the amount of compound in a sample: -** In order to make a quantitative assessment of the compound, a sample with a known amount of the compound is injected and its peak height or peak area is measured. In many cases, there is a linear relation between the height or peak area and the amount of sample. iv. **For the preparation of pure compounds: -** It can be done by collecting the chromatographic peak at the exit of the detector and concentrating the compound by removing or evaporating the solvent. v. **For the determination of trace compounds.** vi. **The other applications of HPLC include:** - **Pharmaceutical Applications** 1. To control drug stability. 2. Tablet dissolution study of pharmaceutical dosages form. 3. Pharmaceutical quality control. - Environmental Applications -------------------------- 4. Detection of phenolic compounds in drinking water. 5. Bio-monitoring of pollutants. - Applications in Forensics ------------------------- 6. Quantification of drugs in biological samples. 7. Identification of steroids in blood, urine etc. 8. Forensic analysis of textile dyes. 9. Determination of cocaine and other drugs of abuse in blood, urine etc. - Food and Flavour ---------------- 10. Measurement of Quality of soft drinks and water. 11. Sugar analysis in fruit juices. 12. Analysis of polycyclic compounds in vegetables. 13. Preservative analysis. Applications in Clinical Tests ------------------------------ 1. Urine analysis, antibiotics analysis in blood. 2. Analysis of bilirubin, biliverdin in hepatic disorders. 3. Detection of endogenous neuropeptides in extracellular fluid of brain etc. Mass Spectrometry ================= Mass spectrometry is an analytical technique in which the vapours of chemical species are bombarded with energetic electrons that ionises chemical species into molecular ion and fragmented ions and sorts the ions based on their mass to charged ratio (m/e). **Mass spectrum** of a substance is a plot between m/e of the ions versus relative abundance. In which m/e ratio are taken along the "abscissa" (X-axis) and relative abundance are taken along the "ordinate (Y-axis) (m/e). m/e is a dimensionless ratio of the mass number of the given particle to the charge by the particle. Calculation of m/e ratio of the following molecular ion. -------------------------------------------------------- The molecular ions mostly have charge equal to +1, thus m/e ratio is equal to mass of the ion. −2e −H+ E.g. NH3 + e →−−→ \[NH3\] →−−→ NH2+→−−−→ NH^+^→−−→N^+^ E.g. C H ^+^ = 72/1 E.g. C H ^+^ = 44/1 Principle of mass spectrometry ------------------------------ The gaseous molecules of a sample substance are bombarded with high energy electrons (70ev) to produce molecular ions. The molecular ion being energetic is fragmented further in steps to produce smaller positive ions called **daughter ions** which possess certain m/e ratio. The molecular and daughter ions having different m/e values are passed through electric and magnetic field and are separated from one another. The separated molecular and daughter ions are detected in proportion to their relative abundance and thus the mass spectrum is obtained. The highest intensity peak found in mass spectrum is called **base peak** and is always given the numerical value 100. The intensities of all other peaks are expressed relative to the highest of the base peak. Mass Spectrum of NH3 -------------------- Molecular ion -------------------- ---- -- -- -- m/e value 14 Relative intensity A parent ion is formed when one electron is removed from the parent molecule of the substance. The m/e value of the parent ion is equal to the molecular mass of the compound. The mass spectrograph is designed to perform three basic functions. These are 1. To vaporize the compounds of varying volatility. 2. To produce ions from the neutral compounds in vapour state. 3. To separate ions according to their m/e ratio to record them Instrumentation of Mass Spectrometry ------------------------------------ a. **Ion Source** The vapour of the substance produced in the source at the pressure of 10^-6^ mm Hg is passed in the ion chamber through slit A and are bombarded by a stream of electrons having energy about 70 eV produced form a tungsten filament. Due to bombardment, molecule lose electron to form parent ion radical. When the energy used for bombarding is around 70eV, the fragmentation in the neutral parent molecule takes place and the fragment ions are produced. The minimum energy required to ionise an atom or a molecule is called ionisation potential. Mass analyser ------------- The positively charged ions are accelerated by applying an accelerating potential of the order of 8 KV and passed into the mass analyser. The positive ions travel with high velocities through mass analyser and are differentiated on the basis of m/e ratio. Ion detector ------------ The ions which are separated by the analyser are detected and measured electrically or photographically. The ions fall on detector through collecting slit and the spectrum is recorded by using a fast scanning oscillography  Mass Spectrum of Neopentane --------------------------- Structure of neopentane: CH3 Different fragment ions obtained from molecular ion (C5H12+) and the relative intensity of mass spectrum of neopentane are: +-------------+-------------+-------------+-------------+-------------+ | Fragment | C5H9 | | \+ | | | ions | | | | | +=============+=============+=============+=============+=============+ | m/e | 57 | | | | +-------------+-------------+-------------+-------------+-------------+ | Relative | 100 | | | | | intensity | | | | | +-------------+-------------+-------------+-------------+-------------+ Application of mass spectrum ---------------------------- 1. **Determination of molecular** The molecular mass of the compound is obtained by interpreting the M^+^ peak in the mass spectrum. The molecular mass obtained is usually the mass corresponding to common isotopes of the elements. Interpreting mass spectra and fragmentation pattern of molecular ions. ---------------------------------------------------------------------- In case an organic compound gives fragment ion as well as parent peaks in pairs which are two units apart; a. If the pair of peaks is in the intensity ratio 1:3, the compound contains chlorine. b. If the pair of peaks appears in the intensity ratio 1:1, the compound contains bromine. The intensity of M^+1^ peak is valuable to know the number of carbon as well as nitrogen atom. Incase nitrogen is absent the number of carbon atom can be calculated by dividing relative intensity of M^+1^ peak by 1:1. If M+ is scaled to 100%, then the M+1 intensity/1.1% gives an estimate for the number of carbons in the compound ---------------------------------------------------------------------------------------------------------------- If M^+2^ peak of the parent ion looks larger than M^+1^, the compound may contain sulphur, chlorine or bromine Infra-red spectroscopy (IR Spectrometry) ======================================== Principle of infrared spectrometry. ----------------------------------- The atoms in a molecule are not held rigidly and are in a state of vibrational and rotational motion. The vibration of the atoms may be of two types namely stretching and bending. Stretching Vibration -------------------- The vibration in which distance between the two atoms increases or decreases but the atoms remain in the same bond axis. Types of stretching vibration Symmetrical stretching ---------------------- The movement of the atoms with respect to a particular atom in a molecule is in the same direction. Unsymmetrical/Asymmetrical stretching ------------------------------------- The vibration in which one atom approaches the central atom while the other departs from it. Bending Vibration ----------------- The vibration in which position of the atoms changes with respect to original bond axis. Types of bending vibration. --------------------------- c. **Scissoring** The bending vibration in which two atoms approach each other Rocking ------- The bending vibration in which the movement of atoms takes place in the same direction Wagging ------- The bending vibration in which the two atoms move up and down the plane with respect to the central atom (out of plane). Twisting -------- In bending vibration, one atom moves up the plane while the other atom moves down the plane with respect to the central atom. Bending vibration requires lesser energy and occurs at higher wavelength than stretching vibration. When IR light is passed through the sample, the molecule absorbs certain frequency of IR radiation and get excited from lower vibrational and rotational levels to higher levels, Hence the amplitude of vibration of the bond increases. Every bond and every functional group have a specific absorption frequency which is calculated to excite the molecule to higher vibrational or rotational level. Lighter atoms will allow the oscillation to be faster -- *higher energy.* This is especially true of bonds to hydrogen -- C -- H, N -- H and O -- H. Stronger bonds will have higher energy oscillations Triple bonds \> double bonds \> single bonds in energy A graph between amounts of absorption (emission) of IR light against frequency (wavelength) of a light is called IR spectrum. Instrumentation of IR Spectrometry ---------------------------------- a. **Production of IR radiation** A Nernst glower (a rod made up of sintered mixture of oxides of Zr, Y and Er) or a rod of silicon carbide (globar) is electrically heated to 1500^o^C to produce infrared. Prism (made up of NaCl or alkali metal halides) or grafting is used to obtain monochromatic light. Glasses or quartz cannot be used for prism material --------------------------------------------------- Because glass or quartz absorb strongly most of the infra-red region, thus NaCl is used which is transparent to infrared radiation. Moreover, NaCl is hygroscopic therefore is protected from the condensation of moisture. Sample preparation ------------------ The sample is taken in the form of KBr disc (a mixture of the sample and KBr) or The sample is grind with nujol (highly purified form of petroleum) to obtain nujol mull or the sample is dissolved in pure chloroform or carbon tetrachloride (CCl4) (solvent). Samples are not taken in solid form because it scatters the radiation too much. ------------------------------------------------------------------------------------------------------ c. **Recording of Spectra** The sample to be investigated is placed in the path of IR radiation. The intensity of transmitted light is measured automatically and a graph between the intensity of transmitted light and wavelength is obtained. Two regions of IR Spectrum -------------------------- a. **Finger Print Region** The region below 1500cm^-1^ rich in many absorption shoulders which are caused by bending vibration. In this spectrum the bending vibration is usually more than the number of stretching vibration. Some substances containing the same functional group show similar absorption above 1500 cm^-1^ but their absorption position differ in the finger print region; therefore, such compounds can be easily distinguished by comparing their finger print region. It helps to identify the structure of the compound. Functional group region: ------------------------ The region of 4000 -- 1430 cm^-1^ related to stretching vibrational changes in the functional groups in a molecule is called functional region. It helps to identify the functional groups present in the compound.  Wave number ----------- Percentage of transmission -------------------------- The amount of a particular frequency of IR radiation transmitted through the sample after being absorbed or without being absorbed. E.g. A transmission of 20 % means, 80 % is absorbed by the sample while 20 % is transmitted. Negative evidence is very reliable in interpreting the IR spectrum ------------------------------------------------------------------ Because if an absorption frequency corresponding to a particular group is missing from the spectrum, it is almost sure that the group is absent. E.g. If there is no absorption in the region 1900 -- 1600 cm^-1^, the carbonyl group must be absent in the compound. **Hydrogen bonding changes the position of absorption in the infrared spectroscopy** Hydrogen bonding brings about a remarkable download shift in the wave number of absorptions, for example alcohol in the vapour state (isolated molecule) shows O-H stretching at about 3600 cm^-1^ whereas that in the associated form gives a broad absorption band at 3200cm^-1^. Stronger the hydrogen bonding, more broadening of the band occurs at much lower wave number. Distinguish the type of hydrogen bonding by infra --red spectroscopy -------------------------------------------------------------------- Yes, it can be done by taking the infrared spectra of the given compound at two different concentrations. If there is a shift in the absorption position of a particular peak or band, then the type of the hydrogen bonding in the substance is intermolecular otherwise is in intra molecular. Intra molecular H bonding in a substance does not depend upon the concentration of the solution. Applications of infra-red spectroscopy -------------------------------------- i. **Identification of an organic compound: -** The identity of organic compound is confirmed from its finger print region (1400 -- 900 cm^─1^) exactly matches with the known spectrum of that compound. Organic compounds containing have the same functional groups may have similar absorption above the 1500 cm^─1^ but differ considerably in the finger print region. ii. **Identification of structure of unknown compounds:** - All major function groups absorb at their characteristic wave numbers. From the absorption frequencies, the probable structure can be predicted. iii. **Qualitative analysis of functional group: -**The presence or the absence of absorption bands help in predicting the presence of certain functional groups in the compound. iv. **Distinction between intramolecular and intermolecular hydrogen bonding: -** Organic compounds in solid and liquid states contain active hydrogen like R-OH, R-COOH etc. undergoes intermolecular hydrogen bonding and exists as polymeric aggregates. The absorption of IR radiation in aggregate form occurs at lower frequencies and bands formed are relatively broad. When the polymeric aggregates dissolved in non-polar solvent, it breaks in dimmers and monomers. Due to this, the O−H str. absorption shifts to higher frequencies and peaks become sharp. Intramolecular hydrogen bonded compound does not show any shift in absorption on dilution. Hence IR spectroscopy helps to distinguish between intramolecular and intermolecular hydrogen bonding. v. **Quantitative estimation of organic mixtures:** - It can be done by: a. measuring the intensities of absorption bands characteristic of each component. b. knowing the optical density of the absorption bands for a pure component. vi. **Study of chemical reaction: -** This technique is quite useful for studying chemical reaction. For example: Reduction of ketone (2- butanone) to secondary alcohol (2- butanol). Ketone forms a strong band about 1710 cm^−1^. When it is subjected to reduction, it forms 2- butanol which absorb at 3300 cm^−1^ due to O ─ H str. The progress of reaction can be studied from time to time and the reduction will complete when a strong band due to C ═ O str. will be missing and only a band due to O − H str. is present. vii. Study of keto-enol tautomerism ------------------------------ viii. **To establish the structure of complex molecules: -** ix. **Conformational analysis: - (a)** IR spectroscopy is quite useful in determining the relative stability of various confirmations of cyclic compounds. **For example:** Cyclohexane exists in both chair and boat form. Chair form is more stable than boat form. **(b)** IR spectroscopy helps to distinguish the axial and equatorial substituent of cyclohexane. Equatorial substituent usually absorbs at a higher frequency than the axial substituent. The higher absorption frequency of C − X bond in the equatorial position is due to less steric interaction of C − X bond with adjacent hydrogen atom. x. **Identification of geometrical (cis and trans) isomerism: -** A vibration is infra-red active only if it causes a change in dipole moment of the molecule and the intensity of absorption depends upon the change in the dipole moment. For most of the vibrations, the change in dipole moment occurs in the cis isomer but in trans isomer, the dipole moment zero due to its non-polar nature. xi. **Identification of Rotational isomerism: -** IR spectroscopy help in the detection of Gauche (skew) \[CH2- rocking vibration bands at 1235 cm^−1^\] and staggered (trans)confirmation \[CH2- rocking vibration bands at 1291 cm^−1^\] For example: 1, 2-dichloroethane. --------------------------------- In Gauche (skew) form, CH2- rocking vibration bands appear at 1235 cm^−1^ and dominate at higher temperature. In staggered (trans) confirmation, CH2- rocking vibration bands appear at 1291 cm^−1^ and predominate at lower temperature. The relative intensities of the absorption bands depend upon temperature. The rocking vibrations at 1291 cm^−1^ become less intense as the temperature is raised. From the relative intensities of the bands, their relative abundance at a particular temperature can be estimated. xii. **Detection of impurities in a compound: -** Detection of impurities in a compound can be done by comparing its spectrum with the spectrum of authenticated sample of compound. Pure sample always consists of sharp peaks and bands while the impure sample will consist of poor band and some additional bands. Characteristic absorption frequencies of various functional groups. ------------------------------------------------------------------- +-----------------+-----------------+-----------------+-----------------+ | **Types of | **Class of | **Frequency | | | vibration** | Compound** | (cm--1)** | | +=================+=================+=================+=================+ | C―H Str. C―H | Alkanes Alkenes | 2960 -- 2850 | | | Str. C―H Str. | Alkynes | | | | C―H Str. C―H | Aromatics | 3100 -- 3010 | | | Str. | Aldehydes | | | | | | -- 3300 | | | | | | | | | | 3150 -- 3020 | | | | | | | | | | \~ 2820 | | | | | | | | | | 2775 -- 2720 | | +-----------------+-----------------+-----------------+-----------------+ | C═C Str. C═C | Alkenes | 1675 -- 1600 | | | Str. C═O Str. | Aromatics | | | | C═O Str. C═O | Alkynes | 1600 -- 1450 | | | Str. C═O Str. | Aldehydes | | | | C═C Str. C═C | Ketones | 2260 -- 2100 | | | Str. C═O Str. | Carboxylic | | | | | acids Easters | 1740 -- 1720 | | | C═O Str. | | | | | | Amide | 1725 -- 1700 | | | | Anhydrides Acid | | | | | chlorides | 1750 -- 1730 | | | | | | | | | | 1680 -- 1630 | | | | | | | | | | 1850 -- 1800 | | | | | | | | | | 1790 -- 1740 | | | | | | | | | | \~ 1790 | | +-----------------+-----------------+-----------------+-----------------+ +-----------------+-----------------+-----------------+-----------------+ | O―H Str. | Alcohols and | 3650 -- 3580 | | | | phenols (dilute | | | | O―H Str. O―H | solution) | 3550 ‒ 3200 | | | Str. | | | | | | Alcohols and | 2700 ‒ 2500 | | | | phenols | | | | | (hydrogen | | | | | bonded) | | | | | | | | | | Carboxylic acid | | | +=================+=================+=================+=================+ | N―H Str. N―H | 1^0^ amines, | -- 3500 | | | Str. N―H Str. | amides (free) | | | | | (two bands) | -- 3400 | | | N―H Str. | | | | | | 1^0^ amines, | 3500 ‒3300 | | | | amides | | | | | (Hydrogen | 3310 ‒ 3140 | | | | bonded) | | | | | | | | | | 2^0^ amines, | | | | | amides Free | | | | | (one band) | | | | | | | | | | 2^0^ amines, | | | | | amides | | | | | (hydrogen | | | | | bonded) | | | +-----------------+-----------------+-----------------+-----------------+ | C≡N | Nitriles | 2260 ‒ 2220 | | +-----------------+-----------------+-----------------+-----------------+ | | Nitro compounds | 1620 ‒ 1535 | | | | Asymmetric | | | | | Symmetric | 1375 ‒ 1275 | | +-----------------+-----------------+-----------------+-----------------+ Str. = stretching s = strong w = weak m = medium v = variable and b = broad. Nuclear Magnetic Resonance (NMR) Spectroscopy ============================================= Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy or magnetic resonance spectroscopy, is a spectroscopic technique to observe local magnetic fields around atomic nuclei. Principle of NMR spectrometry ----------------------------- The nuclei of certain atoms behave as if they are spinning charges and create a magnetic field and behave as it were a tiny bar magnet. The proton of hydrogen nucleus behaves as a tiny bar magnet. When a proton in an organic molecule is placed in a strong magnetic field it can align with the field (alpha spin state) or against it (beta spin state). If the proton is aligned with the magnetic field, it is said to be in more stable low energy state or alpha spin state. If the proton is aligned against the magnetic field it is said to be in less stable high energy state or beta spin state. If the energy in the form of radio waves of exactly the right frequency is supplied to the proton of more stable low energy state, radiation will be absorbed and the nucleus will flip (shift) and align against the applied magnetic field in the less stable high energy state. The absorption is recorded in the form of NMR spectrum. NMR spectrum ------------ A graph of absorption of energy versus magnetic field strength is called NMR spectrum. +-----------------------------------+-----------------------------------+ | **Equivalent proton** | **Non-equivalent proton** | +===================================+===================================+ | 1. 2. 3. 4. | 1. 2. 3. | +-----------------------------------+-----------------------------------+ +-----------------------------------+-----------------------------------+ | | **Deshielding** | +===================================+===================================+ | 1. 2. | 1. 2. | | | | | 𝛿.(upfield) | of 𝛿.(downfield) | +-----------------------------------+-----------------------------------+ More is the Deshielding of proton; more is increase in the value of 𝛿. The Deshielding of a proton is caused when the proton is surrounded by the electronegative atoms or groups. The shift in the position of signal is caused due to shielding and deshielding of protons. Chemical shift -------------- The shift in position of NMR signal, compared to standard substance (TMS) as result of shielding and deshielding by electrons are called chemical shift. Chemical shift is expressed by two scales i.e. delta and tau scale. Both of them are in parts per million. And tau (𝜏) = 10 − 𝛿 δ = In delta scale, the position of TMS (Tetra Methyl Silane) is taken as zero and most chemical shift have the values between 0-10. Smaller the value of delta indicates a small downfield shift whereas a large delta value indicates a large downfield shift. If the observed shift from TMS is 250Hz and the operating frequency of the instrument is 100MHz.Calcualte the value of delta. ----------------------------------------------------------------------------------------------------------------------------- ∆𝑣 = 250 Hz delta =^250^ ^𝑥^ ^10^6 = 2.5 ppm 1MHz = 10^6^ Hz 1Hz = 1cps A signal is obtained at 120 cps downfield with reference to TMS using 60 MCPS (60 × ----------------------------------------------------------------------------------- Delta = ^120^ ^𝑥^ ^10^6 = 2 ppm peaks is called spin-spin coupling. The splitting takes place because of magnetic interaction between neighbouring protons. Splitting of signals takes place when there are non-equivalent protons on the adjacent carbon atoms. No splitting is observed if all the protons are equivalent CH3CH2 CH2Cl CH2Cl -- CH2Cl CH3 -- CH3 Splitting pattern: (n+1) rule ----------------------------- (n+1) rule states, the number of smaller peaks which are obtained due to the splitting of a peak by neighbouring protons which is one more than the total number of non-equivalent protons present on directly adjacent carbon atoms. The peaks obtained due to splitting are not of equal heights. Instrumentation: Components of NMR Spectrometer =============================================== NMR spectrometer consists of a magnet, radio frequency source, a detector and an amplifier. The function of detector is to note the energy being transferred from the radio frequency source to the nucleus. The sample under examination is taken in a glass tube and placed between the pole faces of magnet. Radio frequency radiations obtained from the RF source are allowed to fall on the sample and the energy is transferred from the RF source to the RF detector. In practice RF is held constant but the strength of magnet filed is varied as the magnetic field strength increases, the signal or the detector is amplified by amplifier and produces a peak (signal on the chart paper). The number of peak (signal) obtained on the chart paper is called **NMR spectrum.** The number of peaks on NMR spectrum indicates the number of different types of protons present in the sample molecule. Peak Area of Signals: Area under Signals or Peaks. -------------------------------------------------- Example: NMR spectrum of benzyl alcohol, C6H5 -- CH2−OH The spectrum of benzyl alcohol has four signals (peaks) iii. The signal at 𝛿 = 0 (far right) is the standard reference (TMS- tetramethyl silane \[(CH3)4Si\]) iv. The signal at 𝛿 = 7.3 is a single sharp peak which is due to the absorption of **five** chemically equivalent protons of phenyl group (− C6H5 −). Hence total area = 5 v. The signal at 𝛿 = 4.6 is the single sharp peak which is due to the absorption of **two** chemically equivalent protons of methylene (− CH2 −) group**.** Hence total area = 2 (doublet) (iv)The signal at 𝛿 = 2.4 is the single sharp peak which is due to the absorption of **one** proton of hydroxyl (− OH −) group. Hence total area = 1 (singlet) The number of areas in the NMR spectrum indicates the number of equivalent protons which produce the signals (peaks). The peak areas of different signals are proportional to the heights of the peaks. The peak areas are measured by an automatic electronic integrator. i. **Quantitative analysis** ii. **Identification of structural isomers** iii. **Distinction between cis- trans isomers and conformation: -** The *cis* and *trans* isomer of a compound can be easily distinguish as the concerned protons have different have different value of chemical shift. iv. **To distinguish between intramolecular and intermolecular hydrogen bonding: - Intermolecular** hydrogen bonding depends on the nature of solvent, concentration of solution and temperature but intramolecular hydrogen bonding does not depend on concentration of solution. Thus, on the basis of the difference of their dependence on the concentration of the solution, the two types of bonding can be distinguished. Both types hydrogen bonding shifts the absorption for a concerned proton down field. v. **Detection of electronegative atom or groups: -**The presence of electronegative atom or groups in the neighbourhood of a proton cause deshielding and the signal is shifted downfield. Greater the electronegativity of the adjacent atom, smaller is the tau value of absorption for the concerned proton. vi. **Identification of functional group: -** Every functional groups gives a characteristic signal NMR spectrum. By studying the chemical shift of compound, it becomes possible to establish what kind of functional group present in the compound. vii. **Comparison of two compounds: -** Two compounds showing same NMR spectrum must be structurally identical. viii. **Determination of number of protons in a molecule: -**Peak area under the peaks gives number of protons. Interpretation of NMR spectrum on some aliphatic hydrocarbon ------------------------------------------------------------ 1. **Ethyl bromide.** (CH3−CH2−Br)  The following peaks can be identified in the spectrum i. Triplets, 𝛿 = 1.7, 3H ii. Quartet, 𝛿 = 3.4, 2H The triplet at 𝛿 1.7 is give by three methyl protons which are magnetically equivalent and are coupled with the methylene protons to give an upfield triplet. The quartet at 𝛿 3.4 is from the two equivalent methylene protons which are coupled with the three methyl protons to produce a downfield quartet as a result of deshielding influence of bromine. The relative areas under the respective signals are in the ratio of the number of protons involved i.e., 3 : 2. Acetaldehyde ------------ The spectrum of acetaldehyde contains the following peaks: iii. Double,𝛿, 2.2, 3H iv. Quartet,𝛿, 9.8, 1H The doublet at 𝛿 2.2 is due to three equivalent protons of methyl group coupled with a single proton of aldehyde (− CHO) group. The aldehyde group proton absorbs far downfield at 𝛿 9.8. The signal appears in the form of a quartet due to coupling by the three protons of the neighbouring methyl groups. The peaks area in 3:1 ratio. 3. **Isopropyl bromide** (CH3−CHBr−CH3) The spectrum of isopropyl bromide contains the following peaks: v. Doublet, 𝛿, 1.75.6H vi. Multiplet, 𝛿, 4.3, 1H All **six** protons on the methyl groups are equivalent and different from the proton of -- CHBr − group. The **six** protons give rise to an upfield signal at 𝛿 = 1.75 which is split into a doublet due to coupling with the lone proton of -- CHBr − group. The proton of -- CHBr − group gives a downfield signal at 𝛿 = 4.3 which is split into a multiplet by the six protons on adjacent carbons (two CH3 groups). The two peak area is in the ratio 6:1. Medical and Industrial Application of NMR spectroscopy ------------------------------------------------------ Magnetic Resonance Imaging (MRI) is a medical diagnostic technique which combines strong magnetic field, radiowaves and computer technology. MRI is used to create maps of biological compounds which provide basic biomedical and anatomical information such as image of any part (e.g., heart, arteries, veins etc.) of the internal body. This information is used for early diagonosis of many diseases especially brain and CNS. Principle of MRI is based on the random distribution of protons which have magnetic properties. In medical practice, MRI is preferred for diagonosing most diseases of brain and central nervous system.