A Seminar on
Principle and Applications of Electron Spin Resonance (ESR) Spectroscopy Prepared by – Mr. Mohit G. Dewani (M.Pharm Q. A. – I Semester) Under the Guidance of Prof. (Mrs.) Mrinalini C. Damle At AISSMS College of Pharmacy, Kennedy Road, Near RTO, Pune. 1
CONTENTS Introduction to ESR Paramagnetism & Diamagnetism Principle of ESR The g – Value Maxwell – Boltzmann distribution Presentation of ESR spectrum Hyperfine splitting Multiple Resonance techniques ESR and NMR Applications of ESR
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Introduction
Electron Spin Resonance (ESR) also known as Electron Magnetic Resonance (EMR) or Electron Paramagnetic Resonance (EPR) is a branch of absorption spectroscopy in which radiations having frequency in the microwave region (0.04 – 25 cm) is absorbed by paramagnetic substances to induce transitions between magnetic energy levels of electrons with unpaired spins. ESR is shown by atoms having odd number of electrons. ESR is based on the fact that atoms, ions, molecules or molecular fragments which have an odd number of electrons exhibit characteristic magnetic properties. An electron has a spin and due to spin there is magnetic moment. 3
ESR
NMR 4
Substances with unpaired electrons
Stable Paramagnetic substances – E.g. NO, NO2
Unstable Paramagnetic substances E.g. Free radicals or radical ions which can be produced either as intermediates in a chemical reaction or by irradiation of a stable molecule with a beam of nuclear particles or with UV or X-ray radiation. 5
Paramagnetism & Diamagnetism
Paramagnetic substances exhibit an ESR spectrum because the molecules contain an odd number of electrons and hence the number in the two spins states is not identical. The spin of an unpaired electron induces a field that reinforces the applied field. e.g; Iron oxide, platinum, tungsten, sodium, lithium. Some molecules fail to exhibit an ESR spectrum because they contain an even number of electrons and the number in the two spin states is identical or spins are parallel. Hence magnetic effects of electron spin are cancelled. Such substanes are Diamagnetic. e.g.; water, mercury, lead, copper, silver, bismuth. 6
Principle of ESR
In ESR, energy levels are produced by the interaction of the magnetic moment of an unpaired electron in a molecule ion with an applied magnetic field. Every electron has a magnetic moment and spin quantum number (1/2), with magnetic components ms = +1/2 and ms = -1/2. In presence of an external magnetic field, electron's magnetic moment aligns itself either parallel (ms = -1/2) or antiparallel (ms = +1/2) to the field (Zeeman Levels), each alignment having a specific energy. The parallel alignment corresponds to lower energy state and the difference between this two energy levels (Zeeman Splitting) is given by –
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ΔE = hν = gβH
Existence of 2 Zeeman levels and the possibility of inducing transitions from lower energy level to higher energy level, is very basis of ESR spectroscopy. 8
The g - Value
g is proportionality factor / spectroscopic splitting factor / Lande’s spitting factor. It is a measure of ratio between frequency and magnetic field. The value of g for free electrons is 2.0023, which may vary by 0.0003. In ionic crystals, value of g vary from 0.2 – 0.8. The reason is that unpaired electrons are localized in a particular orbital of the atom and orbital angular momentum couples with spin angular momentum giving rise to a low value of g in ionic crystals. The g-factor in ESR is analogous to chemical shift in NMR. 9
Maxwell – Boltzmann Distribution
If a sample containing unpaired electrons is in thermodynamic equilibrium in a magnetic field, there will be a population difference between the two energy levels given by Boltzmann law, n1 / n2 = exp (–ΔE / kT) = exp (–gβH / kT) where, n1 and n2 are the populations in the upper and lower level respectively, leading to an excess population in lower level, k is the Boltzmann’s constant, T is temperature (in Kelvins). 10
Presentation of ESR Spectrum
For Absorption Intensity against strength of magnetic field First Derivative curve – First derivative (slope) of absorption curve Vs strength of magnetic field. 11
Hyperfine Splitting
The ESR spectrum exhibits hyperfine splitting which is caused by the interactions between the spinning electrons and adjacent spinning magnetic nuclei. When a single electron interacts with one nucleus, the number of adjacent splittings will be equal to (2I + 1), where I is the spin quantum number of the nucleus. For n equivalent nuclei, the electron signal will split up into (2nI + 1) multiplet.
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a) b)
Effect of External field on the energy states for an electron Effect of a nuclear spin ½ of proton on these energy states 13
Fig : The spectrum of a free electron showing the single peak corresponding to a transition between the energy levels (w.r.t. a)
Fig : The ESR spectrum showing two peaks corresponding to two transitions (w.r.t b) 14
For a radical having N1 equivalent nuclei, each with a spin of I1, and a group of N2 equivalent nuclei, each with spin of I2, the number of lines expected is (2N1I1 + 1) (2N2I2 + 1) As an example, CH3 – O - CH3 CH2 – O – CH3 + H The methoxymethyl radical, H2C(OCH3), has two equivalent 1H nuclei each with I = 1/2 and three equivalent 1H nuclei each with I = 1/2, and so the number of lines expected is; (2N1I1 + 1) (2N2I2 + 1) = [2(2)(1/2) + 1][2(3)(1/2) + 1] = [3][4] = 12 Hence, 12 peaks have been observed. 15
Figure: ESR Spectrum (First Derivative) for H2C(OCH3) radical
The two equivalent methyl hydrogens will give an overall 1:2:1 EPR pattern, each component of which is further split by the three methoxy hydrogens into a 1:3:3:1 pattern to give a total of 3 x 4 = 12 lines, a triplet of quartets. The smaller coupling constant is due to the three methoxy hydrogens, while the larger coupling constant is from the two hydrogens bonded directly to the carbon atom bearing the unpaired electron.
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Multiple Resonance Techniques
Multiple resonance techniques are used to improve the effective resolution of an ESR spectrum. In this techniques, the change in intensity of the ESR line upon irradiation with a second frequency are observed. It includes ENDOR, ELDOR & TRIPLE.
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ENDOR
Electron Nuclear Double Resonance In this technique, the sample is irradiated simultaneously with a microwave frequency and a radiofrequency. The radiofrequency is then swept while observing the ESR spectrum under microwave frequency conditions. Most suitable when there occurs broadening of normal ESR lines due to large variety of nuclear energy levels. Also suitable, when more precise values of hyperfine couplings are desired. 18
TRIPLE ENDOR experiment can be extended to the use of two simultaneous radiofrequency fields, called TRIPLE experiment. 19
ELDOR
Electron Double Resonance In this techniques, the sample is irradiated simultaneously with two microwave frequencies and the signal height thus obtained is the measure of the difference of the two microwave frequencies.
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Sr. No.
ESR
NMR
1.
Transition occurs at frequencies in the microwave region (0.04 – 25 cm).
Transition occurs in the radio frequency region (>25cm).
2.
Applicable to those molecules Applicable to those molecules which have their electrons having all their electrons unpaired. paired.
3.
The two different energy states are produced due to the alignment of the electron magnetic moments relative to the applied field and transition between these 2 energies takes place on the absorption of a quantum of radiation in the microwave region.
The two different energy states are produced due to alignment of the nuclear magnetic moments relative to the applied field & transition between these two energies takes place upon application of radio frequency field to the 21 appropriate frequency.
Applications
In Biological Systems Study of Free Radicals Study of Catalysts Spin Labels Study of Inorganic Compounds Reaction Velocities & Mechanisms Naturally Occurring Substances Conducting Electrons Analytical Applications 22
In Biological Systems
Presence of free radicals in healthy and diseased tissues has been studied by ESR. Transition metal ion if present, can also be studied. e.g; In case of Fe3+ ions of Hemoglobin, change in its valence state may be studied by ESR. Some typical systems which have been studied by ESR are hemoglobin, nucleic acids, enzymes, irradiated chloroplasts, riboflavin (before and after UV irradiation), and carcinogens. Role of free radical in photosynthesis, has been provided by the observation of a sharp ESR resonance line.
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Study of Free Radicals
A free radical is a compound which contains an unpaired spin such as methyl radical produced through the break up of methane.
CH4 CH3 + H
Methyl radical, has three 1H nuclei each with I = 1/2, and so the number of lines expected is, 2nI + 1 = 2(3)(1/2) + 1 = 4 4 peaks are observed in the proportion of 1:3:3:1. 24
Figure: ESR Spectrum (First Derivative) for methyl radical
In case of I = 1/2 nuclei (e.g., 1H, 19F, 31P), the line intensities produced by a population of radicals, each possessing N equivalent nuclei, will follow Pascal's triangle.
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Radical
Hyperfine lines
Relative Intensities
CH2OH
3
1:2:1
CH3CHOH
5
1:4:6:4:1
(CH3)2COH
7
1 : 6 : 15 : 20 : 15 : 6 : 1
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Study of Inorganic Compounds
E.g; [NO(SO3)2]2- yields a triplet in its ESR spectrum in chloroform. This arises from the interaction between the spin of the unpaired electron and the spin of a 14N nucleus (I=1), conforming that this electron is mainly localised on the nitrogen atom. (2I + 1) = 2 x 1 + 1 = 3
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Study of Catalysts
In a study of heterogeneous catalysis, the spin trapping technique has been used to prove the presence of radical species on a catalyst surface. e.g; In a study of palladium metal catalyst supported on alumina, it was shown that hydrogen is dissociatively chemisorbed by trapping hydrogen atoms with PBN (α-phenyl-N-t-butyl nitrone).
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Spin Labels
There are number of free radicals called Spin Labels which can attach themselves to particular sites in biological systems and produce spectra which provide information on changes in the chemical and physical characteristics in the neighborhood of the site. They can also be inserted into a cell membrane which provides information on the activities of the membrane at various depths below the surface. Label must have chemical activity & a group of atoms to form bridges between free radical and a particular group. Spin label must contain a nitroxyl group or a transition metal complex providing unpaired electrons. E.g; 2,2,6,6 tetramethyl-4-piperadone-1-oxyl (TEMPOL) has an unpaired electron on NO group. 29
The unpaired electron on oxygen of NO group of a nitroxyl compound strongly interacts with nuclear spin of nitrogen atom to produce a 3 line hyperfine spectrum. As for Nitrogen (I=1), it has one equivalent proton so, (2nI + 1) = 2 x 1 x 1 + 1 =3 Table: Selected spin labels and the groups on macromolecules to which spin label is attached
Group on the Macromolecule
Spin Labels
Heme
RCN (cyanide)
Myosin
ATP-R
Lysozyme
CH3CO-NHR
Methionine
BrCH2CO-NHR
Cystein Immidazole
I-CH2CO-NHR
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X + Transient Free radical
ST Spin trap
(paramagnetic)
(Diamagnetic)
=
XST long-lived spin adduct (paramagnetic)
Spin trap must react at relatively fast trapping rates. The radical adduct must have a reasonable half-life.
Commonly used spin traps : DMPO (Dimethyl pyridine N-oxide) PBN (α-phenyl-N-t-butyl nitrone). 31
In Toxicology –
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Conducting Electrons
ESR spectroscopy has been used to detect conduction electrons in solutions of alkali metals in liquid ammonia, alkaline earth metals, alloys (e.g; small amounts of paramagnetic metal alloyed with another metal).
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Reaction Velocities & Mechanisms
ESR is also found to be useful for determination of mechanisms and kinetics of reaction. The molecular interactions that exist e.g; between solvent and solute (environment) can also be studied by ESR spectroscopy. Special cells have been used in ESR spectroscopy, in which radicals are produced by irradiation with UV, gamma or X-rays or by electrolytic redox reactions.
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Fig; ESR Spectrum (First Derivative) for ethyl radical
E.g; Ethyl radicals are produced when ethyl alcohol is irradiated with X-ray radiation. Spectrum shows five lines which conforms the formation of ethyl radicals. The 5 lines thus obtained are in the proportion of 1:4:6:4:1 35
(…contd)
This technique can also be used to study very rapid electron exchange reactions. e.g; addition of naphthalene to a solution of naphthalene radical anion. This causes the broadening of the hyperfine component of ESR resonance line, which can thus be employed to calculate the rate constant for the exchange between naphthalene and naphthalene radical anion. 36
Analytical Applications Determination of Mn2+ – ESR spectrum of Mn2+ ions shows six lines. The multiplicity is given by 2I + 1, where I is 5/2. i.e; 2 x 5/2 + 1 = 6. This ions can be measured and detected even when present in trace quantities. Determination of Vanadium – Traces of vanadium in petroleum oils cause corrosion in combustion engines and furnaces and alter the catalytic cracking of petroleum during processing. ESR spectrum shows an 8 line spectra (I is 7/2); 2I + 1 = 2 x 7/2 + 1 = 8.
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(…contd)
Determination of Polynuclear hydrocarbons – ESR spectroscopy has been used to estimate polynuclear hydrocarbons which are first converted into radical cations and then absorbed in the surface of an activated silica-alumina catalyst. Free radicals so formed are then analysed. E.g; Naphthalene, anthracene, dimethylanthracene, perylene, etc. In case of Naphthalene negative ion, there are two sets (alpha & beta) of 4 equivalent protons each. (2nI+1)(2nI+1) = (4+1)(4+1) = 25 lines. 38
Naturally Occurring Substances
Minerals with transition elements [e.g; ruby (Cr/Al2O3)] Minerals with defects (e.g; quartz) Hemoglobin (Fe) Petroleum Coal Rubber
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Bibliography 1.
2.
3.
4.
5.
Cazes J.; Ewing’s Analytical Instrumentation Handbook (2005), 3rd edition, Marcel Dekker Publication, New York, pp. 349-390. Lindon J. C., Tranter G. E., Holmes J. L.; Encyclopedia of Spectroscopy and Spectrometry (2000), Vol. I, published by – Academic Press – London, U.K., pp.190-197,430-469. Conners K. A.; A Textbook of Pharmaceutical Analysis (1932), 3rd edition, John Wiley & Sons, A Wiley – Interscience Publication, New York, P. 299. Khopkar S. M., Basic Concepts of Analytical Chemistry (2008), 3rd edition, New Age International Publishers, New Delhi, p. 400 Anjaneyulu Y., Chandrashekhar K., Manickam V.; A Textbook of Analytical Chemistry (2006), published by – 40 Pharma Book Syndicate, Hyderabad, pp. 719-742.
Bibliography (…contd) 6.
7. 8. 9. 10 11
Banwell C. N.; Fundamentals of Molecular Spectroscopy (1983), 3rd edition, published by – Mc-Graw Hill Book Company, London, pp. 299-311. Chatwal G. R., Anand S. K.; Instrumental Methods of Chemical Analysis (2007), 5th edition, Himalaya Publishing House, Mumbai, pp. 2.245-2.271. Sharma B. K.; Instrumental Methods of Chemical Analysis (2006), 25th edition, Goel Publishing House, Meerut, pp.s-737 – s-773. www.uottawa.ca/publications/interscientia/inter.2/spin.html (accessed on - 12-09-2009). http://en.wikipedia.org/wiki/Electron_paramagnetic_resonanc e (accessed - 12-09-2009). http://www.ncbi.nlm.nih.gov/pubmed/3039940 (accessed on - 23-09-09) 41
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