EMX User`s Manual
Contents
1. An EPR Primer 2 This chapter is an introduction to the basic theory and practice of EPR spectroscopy It gives you sufficient background to under stand the following chapters In addition we strongly encourage the new user to explore some of the texts and articles at the end of this chapter You can then fully benefit from your particular EPR application or think of new ones Basic EPR Theory 2 1 Introduction to Spectroscopy 2 1 1 During the early part of this century when scientists began to apply the principles of quantum mechanics to describe atoms or molecules they found that a molecule or atom has discrete or separate states each with a corresponding energy Spectroscopy is the measurement and interpretation of the energy differences between the atomic or molecular states With knowledge of these energy differences you gain insight into the identity struc ture and dynamics of the sample under study We can measure these energy differences AE because of an important relationship between AE and the absorption of electro magnetic radiation According to Planck s law electromagnetic radiation will be absorbed if AE hv 2 1 where h is Planck s constant and v is the frequency of the radia tion EMX User s Manual Basic EPR Theory Figure 2 1 Transition associated with the absorption of electromagnetic energy The absorption of energy causes a transition from the lower energy state to the higher2. To acquire a spectrum we change the frequency of the electromagnetic radiation and measure the amount of radiation which passes through the sample with a detector to observe the spectroscopic absorptions Despite the apparent complexities of any spectrometer you may encounter it can always be simplified to the block diagram shown in Figure 2 8 Source Sample Detector Figure 2 8 The simplest spectrometer 2 10 i Basic EPR Practice Figure 2 9 shows the general layout of a Bruker EPR spectrom eter The electromagnetic radiation source and the detector are in a box called the microwave bridge The sample is in a micro wave cavity which is a metal box that helps to amplify weak signals from the sample As mentioned in Section 2 1 2 there is a magnet to tune the electronic energy levels In addition we have a console which contains signal processing and control electronics and a computer The computer is used for analyzing data as well as coordinating all the units for acquiring a spec trum In the following sections you will become acquainted with how these different parts of the spectrometer function and inter act Cavity and Console Sample Figure 2 9 The general outlay of an EPR spectrometer EMX User s Manual 2 11 Basic EPR Practice Signal Out aN amp Detect etector Diode wa jai a Reference E Arm ae Source N E Cavity F
3. How do all of these properties of a cavity give rise to an EPR signal When the sample absorbs the microwave energy the Q is lowered because of the increased losses and the coupling EMX User s Manual Basic EPR Practice changes because the absorbing sample changes the impedance of the cavity The cavity is therefore no longer critically coupled and microwave will be reflected back to the bridge resulting in an EPR signal The Signal Channel 2 2 4 EPR spectroscopists use a technique known as phase sensitive detection to enhance the sensitivity of the spectrometer The advantages include less noise from the detection diode and the elimination of baseline instabilities due to the drift in DC elec tronics A further advantage is that it encodes the EPR signals to make it distinguishable from sources of noise or interference which are almost always present in a laboratory The signal channel a unit which fits in the spectrometer console contains the required electronics for the phase sensitive detection The detection scheme works as follows The magnetic field strength which the sample sees is modulated varied sinusoi dally at the modulation frequency If there is an EPR signal the field modulation quickly sweeps through part of the signal and the microwaves reflected from the cavity are amplitude modu lated at the same frequency For an EPR signal which is approxi mately linear over an interval as wide as the modulation am
4. see only the microwave radiation coming back from the cavity The circulator at point C is a microwave device which allows us to do this Microwaves coming in port 1 of the circulator only go to the cavity through port 2 and not directly to the detector through port 3 Reflected microwaves are directed only to the detector and not back to the microwave source We use a Schottky barrier diode to detect the reflected micro waves point E in the figure It converts the microwave power to an electrical current At low power levels less than 1 micro watt the diode current is proportional to the microwave power and the detector is called a square law detector Remember that EMX User s Manual 2 13 Basic EPR Practice electrical power is proportional to the square of the voltage or current At higher power levels greater than 1 milliwatt the diode current is proportional to the square root of the microwave power and the detector is called a linear detector The transition between the two regions is very gradual For quantitative signal intensity measurements as well as opti mal sensitivity the diode should operate in the linear region The best results are attained with a detector current of approximately 200 microamperes To insure that the detector operates at that level there is a reference arm point F in the figure which sup plies the detector with some extra microwave power or bias Some of the source power is t
5. useful information however it does not tell us much about the molecular structure of our sample Fortunately the unpaired electron which gives us the EPR spectrum is very sensitive to its local surroundings The nuclei of the atoms in a molecule or complex often have a magnetic moment which produces a local magnetic field at the electron The interaction between the electron and the nuclei is called the hyperfine interaction It gives us a wealth of informa tion about our sample such as the identity and number of atoms which make up a molecule or complex as well as their distances from the unpaired electron B 0 Electron i Nucleus B 0 Electron I Nucleus Figure 2 5 Local magnetic field at the electron By due to a nearby nucleus EMX User s Manual 2 7 Basic EPR Theory Equation Figure 2 5 depicts the origin of the hyperfine interac tion The magnetic moment of the nucleus acts like a bar magnet albeit a weaker magnet than the electron and produces a mag netic field at the electron By This magnetic field opposes or adds to the magnetic field from the laboratory magnet depend ing on the alignment of the moment of the nucleus When By adds to the magnetic field we need less magnetic field from our laboratory magnet and therefore the field for resonance is low ered by By The opposite is true when B opposes the laboratory field For a spin 1 2 nucleus such as a hydrogen nucleus we observe that our single EP
6. we could keep the electromagnetic radiation frequency constant and scan the magnetic field See Figure 2 4 A peak in the absorp tion will occur when the magnetic field tunes the two spin states so that their energy difference matches the energy of the radiation This field is called the field for resonance Owing to the limitations of microwave electronics the latter method offers superior performance This technique is used in all Bruker EPR spectrometers AE S i Absorption J _ By Figure 2 4 Variation of the spin state energies as a func tion of the applied magnetic field EMX User s Manual 2 5 Basic EPR Theory The field for resonance is not a unique fingerprint for identifi cation of a compound because spectra can be acquired at several different frequencies The g factor hv g 2 4 HgBo being independent of the microwave frequency is much better for that purpose Notice that high values of g occur at low mag netic fields and vice versa A list of fields for resonance for a g 2 signal at microwave frequencies commonly available in EPR spectrometers is presented in Table 2 1 a is gaia Bres G L 1 1 392 S 3 0 1070 X 9 75 3480 Q 34 0 12000 W 94 0 34000 Table 2 1 Field for resonance Byes for a g 2 signal at selected microwave frequencies sD BROKER EPL Basic EPR Theory Hyperfine Interactions 2 1 3 Measurement of g factors can give us some
7. R absorption signal splits into two signals which are each By away from the original signal See Figure 2 6 a B gt a B gt Figure 2 6 Splitting in an EPR signal due to the local magnetic field of a nearby nucleus If there is a second nucleus each of the signals is further split into a pair resulting in four signals For N spin 1 2 nuclei we will generally observe 2N EPR signals As the number of nuclei gets larger the number of signals increases exponentially Some times there are so many signals that they overlap and we only observe one broad signal i Basic EPR Theory Signal Intensity 2 1 4 So far we have concerned ourselves with where the EPR signal is but the size of the EPR signal is also important if we want to measure the concentration of the EPR active species in our sam ple In the language of spectroscopy the size of a signal is defined as the integrated intensity i e the area beneath the absorption curve See Figure 2 7 The integrated intensity of an EPR signal is proportional to the concentration iii Figure 2 7 Integrated intensity of absorption signals Both signals have the same intensity Signal intensities do not depend solely on concentrations They also depend on the microwave power If you do not use too much microwave power the signal intensity grows as the square root of the power At higher power levels the signal diminishes as well as broadens with increa
8. a Hall probe placed in the gap of the magnet It produces a voltage which is dependent on the magnetic field perpendicular to the probe The relation ship is not linear and the voltage changes with temperature however this is easily compensated for by keeping the probe at a constant temperature slightly above room temperature and char acterizing the nonlinearities so that the microprocessor in the controller can make the appropriate corrections Regulation is accomplished by comparing the voltage from the Hall probe with the reference voltage given by the other part of the control ler When there is a difference between the two voltages a cor rection voltage is sent to the magnet power supply which changes the amount of current flowing through the magnet windings and hence the magnetic field Eventually the error 2 22 i Basic EPR Practice voltage drops to zero and the field is stable or locked This occurs at each discrete step of a magnetic field scan The Spectrum 2 2 6 We have seen how the individual components of the spectrome ter work Figure 2 19 shows how they work together to produce a spectrum Spectrum Y axis Intensity X axis Bg Signal Field J Channel Controller Magnet Cavity and Sample Figure 2 19 Block diagram of an EPR spectrometer EMX User s Manual 2 23 Suggested Reading Suggested Reading 2 3 Instrumentation Theory This chap
9. apped off into the reference arm where a second attenuator controls the power level and conse quently the diode current for optimal performance There is also a phase shifter to insure that the reference arm microwaves are in phase with the reflected signal microwaves when the two signals combine at the detector diode The detector diodes are very sensitive to damage from excessive microwave power and will slowly lose their sensitivity To pre vent this from happening there is protection circuitry in the bridge which monitors the current from the diode When the cur rent exceeds 400 microamperes the bridge automatically pro tects the diode by lowering the microwave power level This reduces the risk of damage due to accidents or improper operat ing procedures However it is good lab practice to follow cor rect procedures and not rely on the protection circuitry 2 14 Basic EPR Practice The EPR Cavity 2 2 3 In this section we shall discuss the properties of microwave EPR cavities and how changes in these properties due to absorption result in an EPR signal We use microwave cavities to amplify weak signals from the sample A microwave cavity is simply a metal box with a rectangular or cylindrical shape which resonates with microwaves much as an organ pipe resonates with sound waves Resonance means that the cavity stores the microwave energy therefore at the resonance frequency of the cavity no microwaves will be r
10. eflected back but will remain inside the cavity See Figure 2 11 f Reflected Microwave a lt Av Power Vres vyv Figure 2 11 Reflected microwave power from a resonant cavity Cavities are characterized by their Q or quality factor which indicates how efficiently the cavity stores microwave energy As Q increases the sensitivity of the spectrometer increases The Q factor is defined as 2m energy stored a ea ee S O 2 Q energy dissipated per cycle 2 5 where the energy dissipated per cycle is the amount of energy lost during one microwave period Energy can be lost to the side walls of the cavity because the microwaves generate electrical currents in the side walls of the cavity which in turn generates EMX User s Manual 2 15 Basic EPR Practice heat We can measure Q factors easily because there is another way of expressing Q Q 5 gt 2 6 where Ves is the resonant frequency of the cavity and Av is the width at half height of the resonance Sample Sample Stack Stack Microwave Magnetic Field Microwave Electric Field Figure 2 12 Magnetic and electric field patterns in a stan dard EPR cavity A consequence of resonance is that there will be a standing wave inside the cavity Standing electromagnetic waves have their electric and magnetic field components exactly out of phase i e where the magnetic field is maximum the electric field is mini mum and vice versa The spatial distr
11. energy state See Figure 2 1 In con ventional spectroscopy V is varied or swept and the frequencies at which absorption occurs correspond to the energy differences of the states We shall see later that EPR differs slightly This record is called a spectrum See Figure 2 2 Typically the fre quencies vary from the megahertz range for NMR Nuclear Magnetic Resonance AM FM and TV transmissions use elec tromagnetic radiation at these frequencies through visible light to ultraviolet light Radiation in the gigahertz range the same as in your microwave oven is used for EPR experiments t fiiv hv Absorption v Figure 2 2 A spectrum i Basic EPR Theory The Zeeman Effect 2 1 2 The energy differences we study in EPR spectroscopy are pre dominately due to the interaction of unpaired electrons in the sample with a magnetic field produced by a magnet in the labo ratory This effect is called the Zeeman effect Because the elec tron has a magnetic moment it acts like a compass or a bar magnet when you place it in a magnetic field Bg It will have a state of lowest energy when the moment of the electron u is aligned with the magnetic field and a state of highest energy when u is aligned against the magnetic field See Figure 2 3 The two states are labelled by the projection of the electron spin M on the direction of the magnetic field Because the electron is a spin 1 2 particle the parallel
12. ibution of the amplitudes of the electric and magnetic fields in the most commonly used EPR cavity is shown in Figure 2 12 We can use the spatial sep aration of the electric and magnetic fields in a cavity to great advantage Most samples have non resonant absorption of the microwaves via the electric field this is how a microwave oven works and the Q will be degraded by an increase in the dissi pated energy It is the magnetic field that drives the absorption in 2 16 i Basic EPR Practice EPR Therefore if we place our sample in the electric field min imum and the magnetic field maximum we obtain the biggest signals and the highest sensitivity The cavities are designed for optimal placement of the sample We couple the microwaves into the cavity via a hole called an iris The size of the iris controls the amount of microwaves which will be reflected back from the cavity and how much will enter the cavity The iris accomplishes this by carefully match ing or transforming the impedances the resistance to the waves of the cavity and the waveguide a rectangular pipe used to carry microwaves There is an iris screw in front of the iris which allows us to adjust the matching This adjustment can be visu alized by noting that as the screw moves up and down it effec tively changes the size of the iris See Figure 2 13 Wave guide Cavity Figure 2 13 The matching of a microwave cavity to waveguide
13. if the modulation amplitude is too large larger than the linewidths of the EPR sig nal the detected EPR signal broadens and becomes distorted See Figure 2 15 A good compromise between signal intensity and signal distortion occurs when the amplitude of the magnetic field modulation is equal to the width of the EPR signal Also if we use a modulation amplitude greater than the splitting between two EPR signals we can no longer resolve the two sig nals Time constants filter out noise by slowing down the response time of the spectrometer As the time constant is increased the noise levels will drop If we choose a time constant which is too long for the rate at which we scan the magnetic field we can dis tort or even filter out the very signal which we are trying to extract from the noise Also the apparent field for resonance will shift Figure 2 16 shows the distortion and disappearance of a signal as the time constant is increased If you need to use a long time constant to see a weak signal you must use a slower scan rate A safe rule of thumb is to make sure that the time needed to scan through a single EPR signal should be ten times greater than the length of the time constant an Time Constant NY B 0 Figure 2 16 Signal distortion and shift due to excessive time constants 2 20 Basic EPR Practice For samples with very narrow or closely spaced EPR signals 50 milligauss This u
14. igure 2 10 Block diagram of a microwave bridge Basic EPR Practice The Microwave Bridge 2 2 2 The microwave bridge houses the microwave source and the detector There are more parts in a bridge than shown in Figure 2 10 but most of them are control power supply and security electronics and are not necessary for understanding the basic operation of the bridge We shall now follow the path of the microwaves from the source to the detector We start our tour of the microwave bridge at point A the micro wave source The output power of the microwave source cannot be varied easily however in our discussion of signal intensity we stressed the importance of changing the power level There fore the next component at point B after the microwave source is a variable attenuator a device which blocks the flow of micro wave radiation With the attenuator we can precisely and accu rately control the microwave power which the sample sees Bruker EPR spectrometers operate slightly differently than the simple spectrometer shown in the block diagram Figure 2 8 The diagram depicts a transmission spectrometer It measures the amount of radiation transmitted through the sample and most EPR spectrometers are reflection spectrometers They measure the changes due to spectroscopic transitions in the amount of radiation reflected back from the microwave cavity containing the sample point D in the figure We therefore want our detector to
15. plitude the EPR signal is transformed into a sine wave with an amplitude proportional to the slope of the signal See Figure 2 14 p AA First Derivative Figure 2 14 Field modulation and phase sensitive detec tion 2 18 i Basic EPR Practice The signal channel more commonly known as a lock in ampli fier or phase sensitive detector produces a DC signal propor tional to the amplitude of the modulated EPR signal It compares the modulated signal with a reference signal having the same frequency as the field modulation and it is only sensitive to sig nals which have the same frequency and phase as the field mod ulation Any signals which do not fulfill these requirements i e noise and electrical interference are suppressed To further improve the sensitivity a time constant is used to filter out more of the noise Phase sensitive detection with magnetic field modulation can increase our sensitivity by several orders of magnitude how ever we must be careful in choosing the appropriate modulation amplitude frequency and time constant All three variables can distort our EPR signals and make interpretation of our results difficult Modulation Amplitude gt Figure 2 15 Signal distortions due to excessive field modulation EMX User s Manual 2 19 Basic EPR Practice As we apply more magnetic field modulation the intensity of the detected EPR signals increases however
16. sing microwave power levels This effect is called saturation If you want to measure accurate linewidths lineshapes and closely spaced hyperfine splittings you should avoid saturation by using low microwave power A quick means of checking for the absence of saturation is to decrease the microwave power and verify that the signal inten sity also decreases by the square root of the microwave power EMX User s Manual 2 9 Basic EPR Practice Basic EPR Practice 2 2 Introduction to Spectrometers 2 2 1 In the first half of this chapter we discussed the theory of EPR spectroscopy Now we need to consider the practical aspects of EPR spectroscopy Theory and practice have always been strongly interdependent in the development and growth of EPR A good example of this point is the first detection of an EPR sig nal by Zavoisky in 1945 The Zeeman effect had been known in optical spectroscopy for many years but the first direct detection of EPR had to wait until the development of radar during World War II Only then did scientists have the necessary components to build sufficiently sensitive spectrometers scientific instru ments designed to acquire spectra The same is true today with the development of advanced techniques in EPR such as Fourier Transform and high frequency EPR The simplest possible spectrometer has three essential compo nents a source of electromagnetic radiation a sample and a detector See Figure 2 8
17. state is designated as M 1 2 and the antiparallel state is M 1 2 By By Figure 2 3 Minimum and maximum energy orientations of u with respect to the magnetic field Bo EMX User s Manual 2 3 Basic EPR Theory From quantum mechanics we obtain the most basic equations of EPR 1 E 8 Ug BoM 35 8 Hp Bo 2 2 and AE hv g UpBo 2 3 g is the g factor which is a proportionality constant approxi mately equal to 2 for most samples but varies depending on the electronic configuration of the radical or ion Up is the Bohr magneton which is the natural unit of electronic magnetic moment Two facts are apparent from equations Equation 2 2 and Equa tion 2 3 and its graph in Equation Figure 2 4 e The two spin states have the same energy in the absence of a magnetic field e The energies of the spin states diverge linearly as the mag netic field increases These two facts have important consequences for spectroscopy e Without a magnetic field there is no energy difference to measure e The measured energy difference depends linearly on the magnetic field i Basic EPR Theory Because we can change the energy differences between the two spin states by varying the magnetic field strength we have an alternative means to obtain spectra We could apply a constant magnetic field and scan the frequency of the electromagnetic radiation as in conventional spectroscopy Alternatively
18. sually only happens for organic radicals in dilute solutions we can get a broadening of the signals if our modulation frequency is too high See Figure 2 17 The broad ening is a consequence of the Heisenberg uncertainty principle YA A v gee i D a SF lt J ms Sf for nae 100 kHz fj Pa ace B gt Figure 2 17 Loss of resolution due to high modulation frequency The Magnetic Field Controller 2 2 5 The magnetic field controller allows us to sweep the magnetic field in a controlled and precise manner for our EPR experiment It consists of two parts a part which sets the field values and the timing of the field sweep and a part which regulates the current in the windings of the magnet to attain the requested magnetic field value The magnetic field values and the timing of the magnetic field sweep are controlled by a microprocessor in the controller A field sweep is divided into a maximum of 4096 discrete steps called sweep addresses At each step a reference voltage corre sponding to the magnetic field value is sent to the part of the controller that regulates the magnetic field The sweep rate is EMX User s Manual 2 21 Basic EPR Practice controlled by varying the waiting time between the individual steps Microprocessor Reference Voltages 347G N Figure 2 18 A block diagram of the field controller and associated components The magnetic field regulation occurs via
19. ter is a brief overview of the basic theory and practice of EPR spectroscopy If you would like to learn more there are many good books and articles that have been written on these subjects We recommend the following Poole C Electron Spin Resonance a Comprehensive Treatise on Experimental Techniques Editions 1 2 Interscience Publishers New York 1967 1983 Feher G Sensitivity Considerations in Microwave Paramag netic Resonance Absorption Techniques Bell System Tech J 36 449 1957 Knowles P F D Marsh and H W E Rattle Magnetic Reso nance of Biomolecules J Wiley New York 1976 Weil John A J R Bolton and Wertz J E Electron Paramag netic Resonance Elementary Theory and Practical Applications Wiley Interscience New York 1994 A more extensive bibliography is found in last chapter of this manual 2 24 i
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