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EMX USER'S MANUAL - University of Warwick

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1. position to maximize Q your sample coupled Optimize Optimize Optimize field Optimize receiver time constant modulation microwave gain for selected power scan time Instrument related factors Optimize AFC Operate the EMX Minimize _ Minimize modulation in a stable microphonics electromagnetic depth environment interference Allow the Minimize Flow nitrogen Shield Isolate spectrometer temperature gas through the and turn to warm up fluctuations in cavity system off the noise the lab source Do not place vibrating objects on bridge Avoid air drafts Tighten sample collets and waveguide stabilizer Make sure iris screw is not loose Factors to consider when optimizing your EMX for sensitivity EMX User s Manual 6 4 Cos BRUKER Optimizing Sensitivity Optimizing Sensitivity 6 2 Instrumental Factors 6 2 1 For better spectrometer stability keep the spec trometer away from windows and ventila tion ducts Minimize microphonics Microphonics are unwanted mechanical vibrations in the spectrometer Depending on the nature and frequency of the microphonics these vibrations may generate noise in your EPR spectrum The most com mon microphonic sources include the cavity the sample and the bridge Prevent microphonic noise by securing the waveguide with the wave
2. Figure 2 15 Signal distortions due to excessive field modulation EMX User s Manual 2 19 Cos BRUKER Basic EPR Practice As we apply more magnetic field modulation the intensity of the detected EPR signals increases however 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
3. Get ready to acquire a noise spectrum Click the RUN button in the tool bar to acquire the noise spectrum Frequently the baseline will drift since 200 mW micro wave power is going to heat up the cavity and the sample Wait a few minutes to achieve thermal equilibrium Check the tuning and coupling of the system Retune the system if necessary You may also have a rather large offset due to the excessive power and high gain Use the interactive box to make the offset adjustment so that the indicator of the receiver level is in the middle Click the left mouse button on Set Parameters to Spectrum move the pointer to the noise measurement window and click again If you experience overshoots or undershoots you EMX User s Manual 9 11 Cos BRUKER Signal to Noise Ratio Test can set a 2 5 second delay time in the Experimental Options box as in Step 4 Acquire a noise spectrum Click the RUN button in the tool bar and acquire the noise spectrum again Two horizontal lines will automatically emerge indicating the noise level If the baseline still drifts you can click the lin ear baseline correction button to compensate for linear drifts MWINEPR ACQUISITION Signal Noise Measurement M E Eile Parameter Acquisition Processing View Options Window Info OSie e4 6 CERI Beis Ishi el z Biel Biz AFC Receiver Level MORERA Frequency 9 81 T aa Pa LEVELLED Attenuation 0 dB Lo ne a Power 200
4. EMX User s Manual Cos BRUKER Standard Samples Standard Samples 8 1 Standard samples are useful for system performance tests spec trometer calibration and quantitative concentration measure ments Ideally the standard sample should contain stable long lived paramagnetic species be easily prepared under consistent and controlled methods and should be fully characterized with respect to all spectroscopic parameters such as relaxation times and hyperfine and fine structure splittings In addition the reso nance line should be narrow and preferably homogeneous Unfortunately the universal standard sample has not been found Many standards have been suggested and each has its own par ticular merit The standard samples supplied with every Bruker spectrometer are discussed below DPPH a a diphenyl B8 picryl hydrazyl 8 1 1 DPPH serves as a reference both in the solid state and in the liq uid state when dissolved in benzene or toluene mineral oil The line width measured from the solid is subject to exchange nar rowing and thus varies from under 1 gauss to over 4 gauss depending on the solvent that was used for recrystallization It has a g factor of 2 0036 0 0003 When dissolved in solution a quintet with unresolved hyperfine couplings is observed as the spin exchange narrowing is reduced as the sample is diluted A small single crystal of DPPH is an ideal sample for calibrating the phase and the field mod
5. YA Signal Field a Channel Controller JU ci Cavity eel Figure 2 19 Block diagram of an EPR spectrometer EMX User s Manual 2 23 Cos BRUKER Suggested Reading Suggested Reading 2 3 Instrumentation Theory This chapter 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 EMX User s Manual 2 24 Getting Started 3 This chapter contains basic operating instructions for first time users of a Bruker EMX spectrometer It describes basic opera tion of an EMX spectrometer with an X band bridge 9 1 9 9 GHz This chapter will guide you from a completely shut down spectrometer to a hardcopy of your spectrum
6. Low W Paramagnetic Resonance Vol 2 Proceedings of the First International Conference held in Jerusalem Aca demic Press New York London 1963 Mabbs F E D Collison Electron Paramagnetic Resonance of d transition Metal Compounds in Studies in Inorganic Chemistry Vol 16 Elsevier Science Amsterdam 1992 McConnell H M B G McFarland Quart Rev Biophys Vol 3 91 136 1970 McDowell C A Editor Magnetic Resonance MTP Interna tional Review of Science Vol 4 Butterworths London University Park Press Baltimore Physical Chemistry Series 1972 McGlynn S P Ti Azumi M Kinoshita Molecular Spectros copy of the Triplet State Prentice Hall New York 1969 EMX User s Manual 10 11 BRUSER Bibliography McLachlan A D Electron Spin Resonance Harper and Row New York 1969 McLauchlan K A Magnetic Resonance Oxford University Press Don Mills Ontario Oxford Chemistry Series 1974 McMillan J Electron Paramagnetism Reinhold New York 1960 McWeeny R Spins in Chemistry Academic Press New York 1970 Manenkov A A R Orbach Spin Lattice Relaxation in Ions Sol ids Harper and Row New York Evanstown London 1966 Memory J D Quantum Theory of Magnetic Resonance Parame ters McGraw Hill New York 1968 Microwave and Radio Frequency Spectroscopy General Dis cussion of the Faraday Society Aberdeen University Press Aberdeen 1950 Mims W B The Linear
7. p Time Constant B gt Figure 2 16 Signal distortion and shift due to excessive time constants EMX User s Manual 2 20 BRUSER Basic EPR Practice For samples with very narrow or closely spaced EPR signals 50 milligauss This usually 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 12 5 kHz a AN yo a 100 kHz 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 BRUSER Basic EPR Practice controlled by varying the waiting
8. DataType gt O reset 500 1000 1500 2000 2500 3000 3500 4000 4500 Figure 9 17 Measure the signal height of the cavity background signal 18 Measure the weak pitch signal Click the weak pitch spectrum and repeat the same procedure to get the signal height of the weak pitch signal EMX User s Manual 9 24 BRUSER Cavity Background Signal Test 19 Calculate the result The ratio of the cavity back ground signal over the weak pitch signal is the test result cavity background signal high val low val s 1 weak pitch signal high val low val 4 The ratio must be less than 1 4 to meet the specifications If the ratio is greater than 1 4 contact your local Bruker EPR service representative EMX User s Manual 9 25 BRUSER Cavity Background Signal Test EMX User s Manual 9 26 Bibliography 10 Abragam A The Principles of Nuclear Magnetism Clarendon Press Oxford International Monographs of Physics 1961 Abragam A B Bleaney Electron Paramagnetic Resonance of Transition Ions Clarendon Press Oxford 1970 Abragam A M Goldman Nuclear Magnetism Order and Dis order Clarendon Press Oxford 1982 Aleksandrov I G The Theory of Nuclear Magnetic Resonance Academic Press New York 1966 Alekseev B F Yu V Bogachev V Z Drapkin A S Serdjuk N B Strakhov S G Fedin Radiospectroscopy of Natural Substances by EPR and NMR Norell Mays Landing
9. File name Folders st_pitch par c winepr data john_spc Save file as type Drives Parameter par fa J c micron z Figure 4 35 The File Save As dialog box If the chosen filename were already used by another file a warning box gives you the opportunity to decide whether or not to replace the existing file with the present spec trum See Figure 4 36 Pressing No cancels the save process and allows you to select another name or folder File Save As C WINEPR DATA JOHN_SPC st_pitch par This file already exists Do you wantto replace it Yes Figure 4 36 Warning dialog box for overwriting files Disk Housekeeping 4 7 2 The hard disk is the major storage device for storing acquired spectra It can rapidly become cluttered and unwieldy These type of problems are easily overcome if you implement an effi cient folder system and if you back up your hard disc periodi cally EMX User s Manual 4 32 BRUSER Spectrum Files Subdivide your folders when you are saving many spectra Make sure that the folders have descriptive names so that it will be eas ier to find data in the future For labs where many people use the instrument it helps keep your data separate from those of others Folders facilitate easy data transfer to floppy or to other comput ers via ethernet or Kermit Creating folders is easy with either My Computer or Windows Explorer Consult your Microsoft Windows docume
10. ih ih M Console Power Switch ead Figure 3 4 The location of the console power switch AN 3 Turn on the power for the computer The details of turning on the computer depend on the type of the PC you have Consult your PC user s manual Make sure you do not have a floppy disk in the floppy disk drive when you turn the PC on EMX User s Manual 3 8 ph ER Turning the Spectrometer On 4 Turn on the tap water for cooling There are usually two valves one for the supply and one for the return or drain Consult the local instrument or facilities manager if you are not sure where the valves are 5 Identify your power supply There are two types of power supplies and you must identify which type you have See Figure 3 5 The small ER 080 081 power supplies are located directly below the console and are accessed by opening the glass door If you have this type of power supply go to Step 6 The Bruker ER 082 083 085 086 magnet power supplies are larger They A B amp _ Electricity Power On On Power On Off Switch S E The small ER 080 081 The large ER 082 083 085 086 power supply Go to step 6 power supply Go to step 7 Figure 3 5 Two types of power supplies EMX User s Manual 3 9 BRUSER Turning the Spectrometer On are not under the console
11. 10 3 _ s 20 10 10 20 G 3477 4 3477 6 3477 8 3478 0 3478 2 3478 4 3478 6 J SPECTR7 PAR Pm E SPECTR6 PAR FA ES f 10 3 10 _ PEN Bais x10 3 3477 4 3477 6 3477 8 3478 0 3478 2 3478 4 3478 6 6 Figure 4 18 Perylene radical cation spectrum acquired with 1024 or 4096 points EMX User s Manual 4 17 Cos BRUKER Time Scans Time Scans 4 4 Not all radicals are very stable in fact most are very reactive species and will disappear via chemical reactions Many people are interested in the kinetics of these reactions The EMX soft ware has a Time Scan option to study the time behavior of such changing systems The magnetic field is kept fixed at a specified value and the EPR signal intensity is monitored as a function of time The first task is to acquire a field swept spectrum before acquir ing the time scan This spectrum is usually acquired under steady state conditions or before the chemical reactions have started BRACE EAF Va Duplicate Button Static Field Button Figure 4 19 Interactive Change of Static Field Parameter and Duplicate but tons in the tool bar In order to determine where to set our static field we must first duplicate the field swept spectrum by clicking the Duplicate button in the tool bar Activate the new spectrum window and click the Interactive Change of Static Field Parameter but ton in the tool bar See Figur
12. 7 Cavity Figure 2 10 Block diagram of a microwave bridge EMX User s Manual 2 12 Cos BRUKER 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
13. Experiment x ee Y no Y Sweep gt Hall Center Field 3420 00 G Receiver Gain KH 2 00 10 a Sweep Width 200 0 G Modulation Frequency 100 00 kHz Static Field 3480 000 H G Modulation Amplitude 1 00 E G Signal Channel Microwave Bridge Modulation Phase 0 00 deg Frequency 9 751000 GHz Offset 0 00 Power 2 00 mW Time Constant 0 64 msec Step 1 H db Conversion Time 5 12 E msec Temperature unit Sweep Time 5 24 sec Temperature H K Harmonic 1 Step 1 00 alik ar Resolution in X 1024 kK t a pune aaa Number of X Scans 1 a Angis ceg Resolution in Y 1 Step 1 000 deg a Repetitive Mode E Figure 3 14 The Experiment Parameter dialog box Changing a parameter followed by clicking trum in the active win dow The software is one to one correspon dence between a spec trum and parameters OK wipes out the spec 7 designed to maintain a 8 Check the field sweep parameters The parameters for the field sweep are listed in the Hall for Hall effect field controller section Edit the values of the parameters so they correspond to the ones in Figure 3 14 The Static Field parameter is not used in this experiment therefore you do not have to edit its value Check the Signal Channel Parameters Edit the values of the parameters so they correspond to the ones in Figure 3 14 Set the Microwave Power Enter a value of 2 0 in
14. Microwave reference phase If the microwave reference phase is not set properly you will not be able to critically couple the cavity Carefully follow the instructions in Section 3 4 when tuning the spectrometer Iris motor limits improperly set If the iris motor limits were improperly set the iris can not be screwed in suffi ciently Follow the procedure in Section 5 2 to properly adjust the iris motor limits Iris tip size When working with lossy samples it is advis able to use a larger iris tip to increase the coupling range of the cavity This is particularly important when working with flat cells or capillaries Contact your Bruker service represen tative for advice EMX User s Manual 7 5 Cos BRUKER Magnet Power Supply Shuts Down Magnet Power Supply Shuts Down 7 6 Insufficient cooling capacity Make sure that the heat exchanger is on and that there is sufficient cold water flowing through it Either the Ext or Temp warning LED s on the magnet power supply will light up with this fault Hall probe inserted with the wrong polarity The mag netic field will go to the maximum field Hall probe fallen out of the magnetic air gap If the Hall probe has fallen from the pole piece of the magnet the power supply may go to the maximum current value EMX User s Manual 7 6 Cos BRUKER Baseline Distortion Baseline Distortion 7 7 Linear baseline drifts The use of very large modulation f
15. New Jersey 1991 Alger R S Electron Paramagnetic Resonance Techniques and Applications Interscience Wiley New York 1968 Allen P S E R Andrew C A Bates Editors Magnetic Reso nance and Related Phenomena Proceedings of 18th Ampere Congress North Holland and American Elsevier Publishing Co New York 1975 Allendoerfer R D Magnetic Resonance M T P Inter Rev Sci Phys Chem Sec 2 Vol 4 McDowell C A Editor But terworth Publications London 1972 pp 29 53 Al tshuler S A B M Kozyrev Electron Paramagnetic Reso nance New York 1964 C P Poole Editor Al tshuler S A B M Kozyrev Electron Paramagnetic Reso nance in Compounds of Transition Elements 2nd Ed Halsted New York 1974 Andrew E R Nuclear Magnetic Resonance Cambridge Univer sity Press 1954 EMX User s Manual BRUSER Bibliography Assenheim H M Introduction to Electron Spin Resonance Hilger and Watts London Hilger Monographs on ESR 1960 Atherton N M Electron Spin Resonance Theory and Applica tion Halsted New York 1973 Atherton N M Principles of Electron Spin Resonance Ellis Horwood Ltd Chichester England 1993 Atkins P W M C R Symons The Structure of Inorganic Radi cals An Application of Electron Spin Resonance to the Study of Molecular Structure Elsevier Amsterdam New York 1967 Averbuch P Editor Magnetic Resonance and Radio frequency Spectroscopy Proceedings of th
16. So MANUAL EMX USER EMX USER S MANUAL Dr Ralph T Weber Dr JinJie Jiang Dr David P Barr EPR Division Bruker Instruments Inc Billerica MA USA Manual Version 2 0 Software Version 2 3 Part Number 6130858 March 1998 EMX User s Manual Manual Version 2 0 Software Version 2 3 Copyright 1998 Bruker Instruments Inc The text figures and programs have been worked out with the utmost care However we cannot accept either legal responsibility or any liability for any incorrect statements which may remain and their consequences The following publication is protected by copyright All rights reserved No part of this publication may be reproduced in any form by photocopy microfilm or other proce dures or transmitted in a usable language for machines in particular data processing systems with out our written authorization The rights of reproduction through lectures radio and television are also reserved The software and hardware descriptions referred in this manual are in many cases registered trademarks and as such are subject to legal requirements This manual is part of the original documentation for the Bruker EMX spectrometer Preface 0 Bruker strives to supply you with instructional and accurate doc umentation We encourage you to tell us how we are doing Please send us your suggesti
17. 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 EMX User s Manual 2 10 BRUSER 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 Console Wa 7 Za Figure 2 9 The general outlay of an EPR spectrometer EMX User s Manual BRUSER Basic EPR Practice Signal Out N N NX Reference Arm Detector Diode Circulator
18. EMX User s Manual 5 26 BRgER Performing 2D Experiments units of 5 dB between each scan You will notice the scan number updating in the box in the upper right corner of the spectrum window See Figure 5 22 Spectr BEES 10 3 z1 Current Scan Display 3460 3480 3500 3520 G Figure 5 22 Current scan display 8 Transfer your 2D data set to WIN EPR By clicking the WIN EPR button you will launch the WIN EPR pro gram and automatically load your dataset See Figure 5 23 LC Se a at EAE WIN EPR Button Figure 5 23 Launching the WIN EPR program from Win Acquisition 9 Display your 2D data set Select 2D Processing from the WIN EPR System menu See Figure 5 24 Your data should automatically appear as seen in Figure 5 25 If your data does not appear as in EMX User s Manual 5 27 BROBER Performing 2D Experiments Figure 5 25 make sure the display mode is set to Stack Plot See Figure 5 26 Figure 5 24 Opening a 2D dataset EMX User s Manual 5 28 Cos BROBER Performing 2D Experiments WINEPR System 2D Processing Eile 2D Processing Parameters Display Options pease ores MU Coa PTD eeSNS UAL Ome Help 10 3 3450 0 3462 5 3475 0 3487 5 3600 0 3512 5 3525 0 F2 G 3437 5 Figure 5 25 Stack plot display of 2D dataset EMX User s Manual 5 29 BpGgeR Performing 2D Experiments WINEPR System 2D Processing Eile
19. EMX User s Manual Index 2nd 8 17 theory 8 4 to 8 7 cavity background signal test 9 14 to 9 24 dip 3 18 5 6 absence of 7 2 disconnecting 5 15 matching 2 17 5 9 to 5 10 errors 7 2 7 5 reconnecting 5 15 to 5 17 theory 2 15 to 2 18 comment 3 25 cooling water turning off 3 35 turning on 3 9 coupling See cavity matching cryostat drop in resonant frequency 5 6 6 1 improving S N 6 7 removal before calibration 8 8 D disk housekeeping 4 32 DPPH 8 2 to 8 3 drop down list 3 6 E EPR basic practice 2 10 to 2 23 basic theory 2 1 to 2 9 excessive time constant 6 9 exiting WIN EPR Acquisition 3 32 F field sweeps 4 10 to 4 17 finding EPR signals 6 1 to 6 3 fine tune 4 27 H heat exchanger turning off 3 34 to 3 35 turning on 3 10 to 3 11 verifying water flow 3 11 warning noises 7 17 helpful hints 6 1 to 6 14 how to use manual 1 6 to 1 7 hyperfine interaction 2 7 to 2 8 icons 4 4 info line 3 4 interactive spectrometer control 4 22 to 4 26 introduction 1 1 to 1 7 EMX User s Manual Index iris screw disconnecting 5 14 proper setting of limit switches 5 16 reconnecting 5 17 K keeping things neat 4 2 to 4 4 L lab conditions and stability 6 5 magnet power supply shut down due to fault 7 6 turning off 3 32 turning on 3 9 to 3 10 magnetic field controller 2 21 to 2 23 microwave bridge 2 12 to 2 14 microwave bridge control 4 27 to 4 30 microwave
20. Make sure that the pedestal is not in the cavity as it can give an EPR signal Tighten the top collet nut to firmly hold the sample tube in place and the bottom collet to firmly hold the pedestal Sample Tube a Collet and Collet Nut So Zz Pedestal ZL Figure 5 4 Cutaway view of a Bruker ER 4102ST cav ity EMX User s Manual 5 9 BRUSER Manually Tuning a Microwave Bridge A Steps 10 and 11 assume you have bias in the ref erence arm You usu ally do If the Bias slider bar See Figure 5 1 is all the way to the left side move it towards the mid dle to ensure sufficient bias Otherwise leave it be 10 11 Retune the microwave source Repeat the procedure of Step 6 You may notice a shift in the frequency width and depth of the cavity dip when you insert the sample This is an indication that the microwave field patterns in the cavity are perturbed by the sample and tube Lossy and conductive samples will appreciably perturb the field patterns resulting in large shifts in the resonant frequency Highly conductive samples tend to increase the resonant frequency by decreasing the effective cavity volume Lossy samples will decrease the resonant frequency because of their large dielectric constants Tune the signal reference phase Gunn Diode Microwave Sources While the dip is in the center of t
21. N Microwave Interactive Bridge Spectrometer Control Control Button Button Figure 5 5 The Interactive Spectrometer Control button Hall Signal Channel Center Field 3480 00 G Calibrated Sweep Width po H G Receiver Gain H e32 10 e E Sweep Address pos o Modulation Freg fioo o0 HHkrz 4 ail gt Modulation Amplitude 0 96 Eye Field 3480 000 G Modulation Phase 24 0 00 Haeg Setup Scan Offset 0 00 Hx Er ej Time Constant p12 HHmsec Sweep Width fioo oo H ConversionTime fiz Fe4 msec Harmonic ris MW Attenuator Eje fo dB Temperature j i K Set parameters to spectrum SCT Options Figure 5 6 The Interactive Spectrometer Control dia log box EMX User s Manual 5 13 BRUSER Changing EPR Cavities A 3 Setting the magnetic field to the minimum 4 value avoids the risk of i magnetizing your watch when changing cavities Set the modulation amplitude to zero Enter a value of 0 00 in the Modulation Amplitude box Set the magnetic field to the minimal value Enter in a value of 0 00 in the Sweep Width box and a value of 0 00 in the Center Field box Close the Interactive Spectrometer Control dia log box Click the Interactive Spectrometer Control the button labeled I in the tool bar The button toggles the dialog box on and off The Interactive Spectrometer Control dialog box will then disappear S
22. Signal Channel Calibrated Efo 10 a kHz Receiver Gain Modulation Freq Modulation Amplitude Modulation Phase Set Offset Time Constant Parameters to Spectrum Button Setup Scan I Enable 100 00 H SNAS B Sweep Width ConversionTime Harmonic he Set parameters fo spectrum SCT Options MW Attenuator Temperature Use of the Interactive Receiver Level Display to optimize spectrom eter parameters EMX User s Manual 4 26 Cos BRUKER Controlling the Microwave Bridge Controlling the Microwave Bridge 4 6 Auto Tune vs Fine Tune 4 6 1 The time saved by using Fine Tune instead of the full Auto Tune procedure can be particularly important when you are working with unstable and decaying samples LC St a at EALE Lei 2 In Chapter 3 we used the Auto Tune commands to tune the microwave bridge and cavity This routine tunes everything including the Bias Signal Phase Frequency and the cavity matching Quite often you do not need to adjust all these param eters For example unless you have a large change in microwave frequency the Bias and Signal Phase do not need to be adjusted The parameters that change more frequently are the Frequency and the matching of the cavity The Fine Tune rou tine optimizes only the Frequency and the matching iris posi tion and therefore is considerably faster than the
23. When you turn on your spec trometer it should be in Stand By mode which is indi cated by Stand By appearing in the Microwave Bridge EMX User s Manual BRUSER Manually Tuning a Microwave Bridge Control menu See Figure 5 1 If you have been acquir EMX User s Manual 5 2 BRgER Manually Tuning a Microwave Bridge AFC amp Diode Meter Microwave Frequency Bridge Slider Attenuation Control Display Button Bias Slider Signal Phase Slider Figure 5 1 The Microwave Bridge Control dialog box ing spectra already your bridge will probably be in Oper ate mode Click the Tune button in the dialog box to ly gt change to the Tune mode 3 Set the microwave attenuator to 25 dB The micro You may notice Wal wave attenuation is set by clicking the arrows on either LEVELED and side of the attenuation display See Figure 5 1 The UNCALIBRATED arrows on the left side change the attenuation in 10 dB appear in the bridge sta steps the arrows on the right side change the attenuation tus indicator Do not be in 1 dB steps alarmed by the UNCALIBRATED indi cator this is normal during Tune EMX User s Manual 5 3 Cos BRUKER Manually Tuning a Microwave Bridge There are two types of microwave sources The letter G in the micro wave bridge designation i e ER 041 XG on the front panel identifies a Gunn source T
24. field modulation amplitudes and variable temperature work allow the system to equilibrate under the same conditions as the experiment will be performed Carefully follow the procedure for positioning the sample inside the cavity This is particularly important for samples exhibiting a large dielectric loss Improper sam ple positioning can perturb the microwave field mode pat terns in the cavity resulting in less than optimum sensitivity Periodically check the iris coupling screw for tight ness of fit A worn iris screw thread will make the iris sus ceptible to microphonics which can modulate the cavity coupling Critically couple the cavity Best cavity performance is obtained with a critically coupled cavity Maximum transfer of power between the cavity and the waveguide occurs under this condition Optimize the AFC Adjust the AFC modulation depth to minimize the noise level observed in the absorption EPR spectrum at full incident microwave power Adjustments of the AFC MOD LEVEL potentiometer located on the rear of the microwave bridge Figure 6 4 should be made while in the Operate mode with the sample inserted and the spec trometer tuned as described in Section 3 4 You should make this adjustment for all experiments limited by signal to noise considerations The optimum AFC modulation depth is a function of the loaded cavity Q Consequently slight varia tions in the optimum setting may be anticipated If you are using
25. purging gas as the operating temperature departs further from room temperature Wait for the cavity to stabilize at each new operating temperature before recording the spectrum Retune the cavity to compensate for any frequency shift and re establish critical coupling at each temperature Background signal Your cavity cryostat sample tube or sample may be contaminated Call your local Bruker EPR Service representative for advice Never take the cavity apart to clean it EMX User s Manual 7 8 Cos BRUKER Excessive Noise Output Excessive Noise Output 7 8 Electromagnetic interference Verify that laboratory equipment is not a source of electromagnetic interference EMI If possible turn off all other equipment in the labora tory and observe spectrometer noise output Determine if radio microwave or TV broadcasting stations are operating in proximity to the spectrometer Record the noise level while operating at various times of the day and night EMI related noise will often be reduced at night Power line noise Check the noise content of the AC power lines feeding the spectrometer Line transients or momentary blackouts will drastically degrade the perfor mance of high gain detection systems such as EPR spectrom eters Ground loops Ground loops are very common and often difficult to avoid Disconnect accessory equipment espe cially if it is plugged into remote AC outlets and observe the noise level Turn
26. s Manual vii shen Table of Contents 2 22 Fhe Microwave Bridge srp men pronor pne roaie iaat AE Ee aee EO Ra a 2 12 22 3 The BPR Cavity nezem 3 cil Sis desea ier a BO eS 2 15 2 2 4 The Sinal Channeli Ee sede cicdeecte acne Genscesste odedeeltes cdaggdbiecsceots odeo Soleeeeee 2 18 2 2 5 The Magnetic Field Controller eee eeesccesccesneeeceeeeeeseeeeeceseeeseeeaeeenaes 2 21 2 2 6 The Spectrum soinnin niena ENEE A TERE EE aT 2 23 29 UCSC RACING tds sted e e E E RE E aa 2 24 3 Getting Started elect eet eae areata 3 1 3 1 Bret Tips on Windows 95 Gasicaucn anaes sale a a ai in 3 2 3 1 1 Dialog BOXES n 5 aise isl et si nti A de ie A eee 3 5 3 2 Turning the Spectrometer On 5 3 66 Oe ee ees 3 8 3 3 Removing and Inserting Samples eeeeceeseeeceeeeceseeeeceeeeeeeeteeeees 3 13 3 4 Tuning the Microwave Cavity and Bridge eeececeeeeeeeeeeeeeeteeees 3 17 3 5 Acquiring Spectra sang sce cncdasavtccvedonsasiseis stale naueukesnaveds vabuneetadaadesedlanecsaannans 3 20 3 6 Turning the Spectrometer Off scovexsnsstave nine ies senedoranedinntendeead anise aes 3 31 4 A Brief EMX Tutorial ssid cscctateeuaaetctan Mar ckeeetearenaieud yi 4 1 4 l Spectrum WANK WS aos reari e cece a EA cana on ae eee 4 2 4 1 1 Keeping Things Neatei deenen neee AE EEEE EE EECA 4 2 4 1 2 Creating a New Spectrum Window 20 0 cee eeeeeeceeeneeeeeseeeeeneeeseeeseeeaaecnaees 4 5 4 1 3 Transferring Parameters ienie EL E a E AEE 4 5 4 1 4 Resizing Spectrum Windows
27. tration 1 Install an ER 4102ST standard cavity See Section 5 2 for instructions The specification for the signal to noise ratio is based on an ER 4102ST standard cavity and using the weak pitch sample We strongly sug gest using the standard cavity for this standard test and keep a record and verify the specification periodically If you use other types of cavities to do the signal to noise ratio test the results and the settings will be different due to different Q values and the microwave field distribu tions of the cavities Insert the weak pitch sample Copy down the cali bration factor posted on the label of the weak pitch before you insert it The weak pitch sample should be inserted in the cavity until the bottom of the label and tape on the sample tube is flush with the collet You also should use the pedestal to hold the weak pitch rigidly Turn on the instrument and tune Turn on the instru ment if it is not on yet Tune the microwave bridge and the cavity It is best to wait several hours because the spec trometer is most sensitive and stable after it has achieved thermal equilibrium Set the AFC depth You can find the AFC depth adjust ment knob on the back of the microwave bridge See Figure 9 1 Full scale is ten Set the AFC depth or amplitude at around 2 Check the signal channel options settings Open the Signal Channel Options dialog box Make sure you have the right calibration file for the s
28. 14 9 2 1 Preparing for the Background Signal Test eee eeeeseeseeeneeeeseeeeeeneee 9 14 9 2 2 Performing the Background Signal Test cee ees ceseceseeceeceseceseenseeees 9 15 EMX User s Manual X shen Table of Contents TO BIDNO lge Te1Ah A EE 10 1 EMX User s Manual xi Introduction 1 This introduction describes the operation of a Bruker EMX EPR Electron Paramagnetic Resonance spectrometer No assump tions have been made about the background of the reader except that he or she has a general scientific or technical background Many of the elementary principles necessary for following the chapters are presented in a concise form This chapter starts with a list of EPR applications A brief description of the spectrometer and its capabilities follows The chapter concludes with an explanation of how to use this man ual EPR Applications 1 1 EPR has matured into a powerful versatile nondestructive and nonintrusive analytical method Unlike many other techniques EPR yields meaningful structural and dynamical information even from ongoing chemical or physical processes without influ encing the process itself Therefore it is an ideal complementary technique for other methods in a wide range of studies and appli cation areas Here is a list of some of the EPR applications which are commonly used EMX User s Manual Cos BRUKER EPR Applications Chemistry Physics 1 1 1 Kinetics of rad
29. 152 G Modulation Freq Sweep Width X Enable Na DPX MW Attenuator ZAA Time Constant ConversionTime Harmonic Setup Scan _ Offset i Hx msec z Eae Temperature 5 parameters to spectrum SCT Options Button Figure 4 26 The Setup Scan The rapid sweep for the Setup Scan may be a little too fast for some signals or parameters A time constant that does not distort EMX User s Manual 4 23 Cos BRUKER Interactive Spectrometer Control an EPR signal during a normal field sweep is usually too long for the rapid field sweep of the Setup Scan and will distort the signal See Figure 4 27 One means of dealing with this is to shorten the time constant Another means is to narrow the width of the Setup Scan so that you are looking at a narrower portion of the signal The time required to sweep through the spectrum is then longer The number in the Sweep Width box indicated in Figure 4 27 can be edited or varied with the arrows next to it The values are in percentage of the 50 G setup scan sweep W interactive Spectrometer Control Hall Center Field 3480 00 G Sweep Width poo ye Sweep Address 2174 g fi v Field 3480 152 G Setup Scan IX Enable Sweep Width 100 00 P Signal Channel Mi Ei Receiver Gain Modulation Freq Modulation Amplitud Modulation Phase Offset Time Const
30. 60 mW Time Constant 1310 72 H msec Step db Conversion Time 63 64 msec Temperature unit Sweep Time 167 77 sec Temperature f300 00 Sik Harmonic 1 Step 1 00 K E rs Resolution in X Goniometer Number of X Scans Angle deg Resolution in Y Step fi coo HH deg EH Wil Repetitive Mode Figure 9 14 Parameters for cavity background signal measurement 12 Set a time delay Set a 2 5 seconds time delay in the Experiment Options box as in Section 9 2 2 Step 3 13 Acquire a cavity background spectrum Click the RUN button in the tool bar to acquire the cavity back ground signal See Figure 9 15 EMX User s Manual 9 21 BpGgeR Cavity Background Signal Test 14 Save the spectrum Save the spectrum on the hard disk for future reference HB WINEPR ACQUISITION BACK_CVT PAR Joi Eile Parameter Acquisition Processing View Options Window Info Hesa kkhk aeae BIL lel o el AFC Receiver Level E or oe OOO e a O LEVELED Attenuation 3 dB Lt zm N O 100 8 mW Diode Hall Field MECAUBRATEDM ovaie 2200 e m 1000006 BACK_WKP PAR iol BACK_CVT PAR BEE 10 3 10 3 25 20 15 3470 3480 3490 3500 1000 2000 3000 4000 G Figure 9 15 Acquire the cavity background signal 15 Transfer the spectra to WinEPR Transfer the two spectra you acquired to WinEPR You can either open the WinEPR progr
31. Bard Electron Spin Resonance Spectros copy in Treatise on Analytical Chemistry 10 225 P J Elving Editor 2nd Ed John Wiley amp Sons New York 1983 Gordy W Techniques of Chemistry Vol 15 Theory and Appli cations of Electron Spin Resonance John Wiley and Sons New York 1979 Gorter C J Paramagnetic Relaxation Elsevier Publishing Co New York Amsterdam London and Brussels 1947 Gorter C J Editor Progress in Low Temperature Physics Annual Series Interscience New York began in 1957 Griffith O H A S Waggoner Nitroxide Free Radicals Spin Labels for Probing Biomolecular Structure Accounts Chem Res 2 17 24 1969 EMX User s Manual 10 7 BpGsen Bibliography Haar ter D Fluctuation Relaxation and Resonance in Magnetic Systems Oliver and Boyd London Edinburgh 1961 Harriman J E Theoretical Foundations of Electron Spin Reso nance Academic Press New York 1978 Hecht H G Magnetic Resonance Spectroscopy Wiley New York 1967 Hellwege K A Hellwege Editors Magnetic Properties of Free Radicals Springer Berlin 1967 Herak J N K J Adamic Editors Magnetic Resonance in Chemistry and Biology Lectures at the Ampere Int Summer School Basko Polje Yugoslavia June 1971 Marcel Dekker New York 1975 Hershenson H M Nuclear Magnetic Resonance and Electron Spin Resonance Spectra Index 1958 63 Academic Press New York 1965 Hill H A O P Day Ed
32. Bolton and Wertz J E Electron Paramag netic Resonance Elementary Theory and Practical Appli cations Wiley Interscience New York 1994 Weissbluth M The Triplet State in Molecular Biophysics B Pullman and M Weissbluth Editors Academic Press New York 1965 p 205 Weissbluth M Phonon Atom Interactions Academic Press New York 1989 Chapter 3 Weltner W W Jr Magnetic Atoms and Molecules Scientific and Academic Editions Van Nostrand Reinhold New York 1983 EMX User s Manual 10 18 BRUSER Bibliography Wertheim G K A Hausmann W Sander The Electronic Struc ture of Point Defect as Determined by Moessbauer Spec troscopy and by Spin Spin Resonance American Elsevier New York 1971 Wertz J E Nuclear and Electronic Spin Magnetic Resonance Chemical Reviews Vol 55 No 5 October 1955 Wertz J E J R Bolton Electron Spin Resonance Elementary Theory and Practical Applications McGraw Hill New York McGraw Hill Series in Advanced Chemistry 1972 Whiffen D H Quarterly Reviews London Vol 12 250 1958 Wilmhurst T H Electron Spin Resonance Spectrometers Adam Hilger Ltd London momograph 1967 Winter J Magnetic Resonance in Metals Oxford University Press 1971 Wyard S J Solid State Biophysics McGraw Hill New York 1969 Yariv A Quantum Electronics Wiley New York 1967 Chap ter 8 Yen T F Editor Electron Spin Resonance of Metal Complexes
33. D Processing Parameters Help aera ia pena Reset Scan 10 3 Contour 3 Li Density Plot Layout gt Stacked Plot DataType gt Scan Plot Settings 3437 5 3450 0 3462 5 3475 0 3487 5 3600 0 3512 5 3625 0 F2 G Figure 5 26 Setting the display mode to Stack Plot EMX User s Manual 5 30 Helpful Hints 6 Hints for Finding EPR Signals Cryostats shifts the res onant frequency of the cavity and hence the frequency of the spec trometer to a lower value The field for res onance of your EPR sig nals will therefore be lower than you would expect for a cavity with out a cryostat This chapter contains useful and helpful hints to get the most out of your EMX spectrometer and its hardware The first half of this chapter covers advice on what to do if you do not observe an EPR signal from your sample The second half of the chapter concerns itself with optimizing the performance of the EPR spectrometer for your particular sample and operating condi tions It is assumed that you are familiar with the material pre sented in Chapter 2 and Chapter 3 6 1 Make sure that the spectrometer is functioning properly If you followed the directions of Chapter 3 this should not be a problem There are many common mistakes Is the modulation cable connected properly to the cavity and console Is the waveguide gasket installed properly Is everything turned on Advice on troubleshoo
34. Delay Time 2 00 sec Condition Don t wait PLease Goniometer Sweep jaseline Correction Angle Flyback Off x Delay caling Value Save as Default Figure 9 11 Set Experimental Options 4 1 7 uy See Section 4 5 for help with interactive spectrometer control Q lt 6 Acquire a weak pitch spectrum Click the RUN but ton in the tool bar to acquire a weak pitch spectrum See Figure 9 12 Adjust Receiver Gain if needed If the weak pitch signal clipped return back to Step 2 and reacquire the spectrum Adjust the offset if needed If there is a large offset you can open the Interactive Spectrometer Control dia log box to adjust the offset to the proper position where the indicator of the Receiver Level is in the middle Do not forget to click the Set Parameters to the Spectrum button and move the pointer to the signal measurement window and click the left mouse button again Reacquire the spectrum EMX User s Manual 9 18 Cos BRUKER Cavity Background Signal Test 7 for future reference MWINEPR ACQUISITION BACK_WKP PAR Eile Parameter Acquisition Processing View Options Window Info Deis 4s Bes kL me ia Save the spectrum Save the spectrum on the hard disk BEE AFC Receiver Level OPERATE Frequency 982 GHz Power 100 6 mW Diode Hall Field MNCAUBRATEDM Qvae 0 CE o 3480 000G BACK_WKP PAR 10 3 3500 Figu
35. Electric Field Effect in Paramagnetic Resonance Oxford Press Oxford 1976 Minkoff G J Frozen Free Radicals Electron Spin Relaxation in Liquids Plenum Press New York 1972 Molin Yu N K M Salikhov and K I Zamaraev Spin Exchange Principles and Applications in Chemistry and Biology Springer Verlag Berlin New York 1980 Muus L T P W Atkins Electron Spin Relaxation in Liquids Plenum Press New York London 1972 Myers R J Molecular Magnetism and Magnetic Resonance Spectroscopy Prentice Hall Englewood Cliffs New Jer sey 1973 EMX User s Manual 10 12 BRUSER Bibliography NMR and EPR Selected Reprints American Institute of Phys ics New York Nelson S F in Free Radicals J K Kochi Editor Wiley New York 1973 Vol II Chapter 21 pp 527 594 Norman R O C Editor Specialist Periodical Reports Electron Spin Resonance The Chemical Society Burlington House London WIV OBN 1973 and following years Ohnishi S The Spin Label Technique Seibutsu Butsuri Vol 8 118 129 1968 Orton J W Electron Paramagnetic Resonance An Introduction to Transition Group Ions in Crystals Iliffe London 1968 Orton J W Reports Progr Phys 22 204 1959 O Reilly D E J H Anderson Magnetic Properties Reprinted from Physics and Chemistry of the Organic Solid State Vol II Edited by D Fox M Labes and A Weisberger Owens F J C P Poole Jr and H A Farach Editors M
36. New York 1972 Rado G T H Suhl Magnetism Vol II Part A Academic Press New York London 1965 Ramsey N F Nuclear Moments John Wiley and Sons New York Chapman and Hall Ltd London 1953 Ranby B J F Rabek ESR Spectroscopy in Polymer Research Springer New York 1977 Royal Society of Chemistry Electron Spin Resonance Special ist Periodical Reports Vol 1 Roy Soc Chem London 1971 Rozantsev E G Free Nitroxyl Radicals Plenum Press New York 1970 EMX User s Manual 10 14 BRUSER Bibliography Rozantsev E G V D Scholle Synthesis and Reactions of Stable Nitroxyl Radicals Synthesis 1971 pp 190 202 Salikhov K M A G Semenov D Yu Tsvetkov Electron Spin Echo and its Applications Novosibirsk Nauka 1976 Salikhov K A Yu N Molin R Z Sagdeev and A L Buch achenko Spin Polarization and Magnetic Effects in Radi cal Reactions Elsevier Amsterdam 1984 Schoffa G Electronensprinresonanz in der Biologie G Braun Karlsruhe 1964 Schumacher R T Introduction to Magnetic Resonance Ben jamin New York 1970 Schweiger A Pulsed Electron Spin Resonance Spectroscopy Basic Priciples Techniques and Examples of Applica tions Angew Chem Int Ed Engl 30 265 1991 Seitz F D Turnbull Editors Solid State Physics Advances in Research and Applications Vol 5 Academic Press New York 1957 Servant R A Charru Editors Electronic Magnet
37. Saving Files 4 7 1 The WIN EPR Acquisition program stores all the spectra you see on the screen in memory If you exit the application or turn your computer off they are gone forever You need to save your spectra to a disk for more permanent storage Saved spectra consist of two files The first file is the spectrum file spe file extension that is a binary file containing all the intensities y values of the spectrum The second file par file extension is a parameter file that is an ASCII file containing the parameter values used to acquire the spectrum Whenever you save or open spectra both the spectrum and parameter files are automatically saved or loaded After you have acquired a spectrum you may save it in any folder Click on the File menu bar and then click Save As An alternative would be to click on the Save button in the tool bar A dialog box appears that lets you choose a filename a destina tion folder and a destination disk drive See Figure 4 35 The spectrum to be saved is the spectrum that is presently active To select the appropriate disk drive click on the arrow on the Drives selector To select the appropriate folder click on the appropriate paths in the Folders selector The spectrum file name is selected by typing the filename in the File Name selec tor Clicking OK saves the spectrum on the hard disk or diskette EMX User s Manual 4 31 BRUSER Spectrum Files File Save As ME
38. Tile Horizontal command arranges all the open windows top to bottom such that they are all visible Figure 4 2 Q The Tile Vertical com mand arranges all the open windows side by side such that they are all visible Figure 4 3 E WINEPR ACQUISITION OHCTRL2 PAR Mim E Eile Parameter Acquisition Processing View Options Window Info uesia KES Se MoI eNA Se Ea OERE Frequency 9 52 GHz ails nee LEVELLED Attenuation 25 dB LEI m i Powers 0635 mW Diode Hall Field MECAUBBATEDNS ovale 7700 LI L OHCTRL2 PAR LEd ET ox 10 al 10 3 3440 3460 3480 3500 3440 3460 3480 3500 10 3 eres 3440 3460 3480 3500 Ajer 3 0 10 3440 3460 3480 3500 DMPO_OH PAR ME 10 3 100 0 100 3440 3460 3480 3500 10 3 3440 3460 3480 3500 Horizontal tiling E WINEPR ACQUISITION OHCTRL2 PAR Bee Eile Parameter EET Processing View Options Window Info Bsa SEE ENAME ee T Frequency 9 52 GHz ae Receiver Love ee eeo 25 dB LEI E Power 0 635 mw Diode Hall Field MECAUBPATEONE ovaie 7700 LI 3480000G OHCTRL2 PAR MIES DMRO OH PAR o Bema iol 10 a Se 3 10 3 L 3450 3500 G 3450 3500 G 3450 3500 OHCTRLIPAR BRIE 10 3 l P103 3500 G 3450 3500 G Vertical tiling EMX User s Manual 4 3 Cos BRUKER Sp
39. be reversed by clicking the right mouse button This action resets the display so that the spectrum is entirely in the window Pressing Ctrl R has the same result A second manner to zoom spectra is to use the rectangular scal ing option Clicking on the Zooming button in the tool bar acti vates or deactivates this option Click the left mouse button and drag the rectangle until it encompasses the region of interest Click the right mouse button and the region of interest will then expand to fill the spectrum window See Figure 4 7 When this option is active clicking the left mouse button toggles the cursor between the lower right and upper left corner of the zoom rect angle As you move the cursor the position of the rectangle cor ner moves with the cursor Clicking with the right mouse button expands the region encompassed by the rectangle to fill the whole window All the preceding zooming can be undone by clicking on the Reset command or clicking the right mouse button EMX User s Manual 4 7 BpGgeR Spectrum Windows SPECTR3 PAR ol x 10 3 400 300 200 100 0 100 200 400 3440 3460 3500 G 7 SPECTR3 PAR Mi E 10 3 150 100 100 150 200 3486 3488 3490 3492 3494 3496 3498 3500 G Figure 4 7 Rectangular zooming and its result EMX User s Manual 4 8 BpGsen Starting and Stopping Acquisitions Starting and Stopping
40. between the reference signal and the modulated EPR signal Because the calibration and adjustment of the modulation amplitude can affect the phase difference the first procedure is performed first You calibrate the modulation amplitude by overmodulating a narrow EPR signal A crystal of DPPH with a line width of approximately 1 G is a very good sample to use When the mod ulation amplitude is large compared to the line width the mag netic field modulation brings the sample into resonance before and after the magnet has reached the field for resonance This results in a broadening and distortion of the EPR signal See Figure 8 1 In the limit of an infinitesimally narrow EPR sig nal the peak to peak width of the first derivative EPR signal will be approximately equal to the peak to peak modulation ampli tude EMX User s Manual ph ER Calibration of the Signal Channel 6G v _ ie 8G e S Ss w 5 m S 4G l Sn ig eee e e ss J V v 1G Figure 8 1 The signal shape of the DPPH EPR signal as a function of the field modulation amplitude The first step of calibrating the modulation amplitude involves choosing the correct tuning capacitors The modulation ampli fier needs a bit of help to obtain large modulation amplitudes at modulation frequencies greater than 50 kHz This is a conse quence of the decreasing skin depth with increasing frequency The mod
41. boxes on either side of the attenuation display in the dialog box See Figure 3 8 Remove the sample If there already is a sample in the cavity remove it Loosen the top collet nut You do not need to remove it and carefully remove the sample from the cavity Pulling the sample tube out as straight as possi ble prevents you from destroying your valuable samples See Figure 3 9 Figure 3 10 and Figure 3 11 for details Sample Tube Pedestal Yi Figure 3 9 Cutaway view of a Bruker ER 4102ST cav ity EMX User s Manual 3 14 BRgER Removing and Inserting Samples lt ial O Figure 3 10 Loosening of the collet nut and removal of the collet N Collet Nut lt Figure 3 11 Right and wrong technique for removing a sample EMX User s Manual 3 15 Cos BRUKER Removing and Inserting Samples If this is your first time operating an EMX spectrometer we rec ommend that you use the strong pitch sample supplied with your instrument Our instructions in this chapter are based on using this sample Make sure that the ped estal is not in the cav ity as it can give an EPR signal Clean the sample tube to be inserted
42. complete Auto Tune procedure A good approach to take is to initially use the Auto Tune routine to make sure that the Bias and Signal Phase are set properly Then as you change samples providing they have similar prop erties or rotate your sample etc you can use the Fine Tune routine to tune the spectrometer You can either press the Fine Tune button in the tool bar Figure 4 30 or open the Micro wave Bridge Control dialog box and press the Fine Tune but ton Figure 4 31 Fine Tune Button Figure 4 30 The Fine Tune button in the tool bar EMX User s Manual 4 27 Cos BRUKER Controlling the Microwave Bridge W Microwave Bridge Control Loix Frequency 76 6 Attenuation E j ae Fey eo HH O Tune Bias 43 0 iia Operate e a CI 4 Fine Tune Signal Phase 46 8 Button ea of 3 Auto Tune a Fine Tune Stop Tuning F Dual Trace Figure 4 31 The Microwave Bridge Control dialog box You can set up an automatic fine tune before each sweep Open the Experiment Options dialog box by pressing the button in the tool bar Click the check box for MW Fine Tune Before Each Sweep See Figure 4 32 Click OK and the fine tune routine will be executed automatically before each scan Experiment Options SPECTRUM Lx Field Controller Microwave Settings Sweep Direction Up M Power Set x Flyback On Power Flyback Off x Settling Condition Wait LED off xl Fiel
43. cursor is in the window Spectrum windows may also be minimized to an icon at the bottom of the applica tion window The bar at the top of a window is the title bar Both the WIN EPR application window and the spectrum windows have title bars It shows the name of the application or of the spectrum window The color of the title bar indicates whether a window is active or not See above By clicking and dragging the title bar the window may be moved Other elements of the title bar are as follows A click on the maximize button of the application window expands it to fill the entire screen Clicking on the maximize but ton of the spectrum window expands it to fill the entire area of the application window You may restore the window to its orig inal size by clicking the restore button Clicking on the minimize button of the application window shrinks the window and places it on the task bar A click on the minimize button of the spectrum window shrinks it to an icon at the bottom of the application window EMX User s Manual 3 3 BRUSER Brief Tips on Windows 95 Restore Button Exit Button Control pa Menu icon Menu Bar Tool Bar Info Line Window Border A click on the restore button returns the window to its previous size and locations This button reverses the effect of using the maximize button A click on the exit button closes the window Double clicking this icon closes the windo
44. fi Step fi 00 SK Resolution in X fl 024 Sommerer Number of X Scans fi cE deg Resolution in Y fi Ste 1 000 ie p H Repetitive Mode M Figure 9 5 Parameters for signal measurement 4 Set a time delay Since a very long time constant is used set a delay time of 2 5 seconds to avoid overshoots or undershoots in the first few data points when you acquire the spectrum Open the Experimental Options dialog box found in the Parameter drop down menu and set the Delay before each sweep option and a delay of two to five seconds See Figure 9 6 We also advise you to select MW Fine Tune before each sweep option to ensure the acquisition is made under proper coupling conditions EMX User s Manual 9 8 Cos BRUKER Signal to Noise Ratio Test See Section 4 3 2 for help in setting center fields and Section 4 5 for help with interac tive spectrometer con trol Experiment Options Signal Noise M Field Controller Sweep Direction Flyback Settling Condition Field Offset Correction 0 00 G Fine m Tune a Option Wait LED off j Power Microwave Settings Power Flyback Temperature Unit Tolerance Temperature Flyback Condition 1 00 H K Off Fd Delay Time Delay before each sweep z 2 00 sec Don t wait h Lor Sweep Goniometer Angle Flyback off x Delay Save as Default Value jasel
45. in the dialog box See Figure 3 12 Clean the sample tube to be inserted into the cavity Wiping the outside of the sample tube with tissue paper is usually adequate Insert the sample tube carefully into the cavity If you have inserted the sample already proceed to Step 6 otherwise see Section 3 3 for details on how to do this Tune the bridge and cavity Pressing either of the arrow buttons on both sides of Auto Tune starts the auto matic tuning procedure See Figure 3 12 The up arrow starts by scanning the microwave frequency up in search of the cavity dip or frequency where the cavity reso nates The down arrow starts by scanning the microwave frequency down in search of the cavity dip If you are not sure if the search should start up or down do not worry The frequency will be scanned until its limit is reached and then scan in the other direction until the cavity dip is found The Auto Tune routine adjusts the frequency phase and bias of the bridge and the coupling matching of the cavity If there is an error message there may be something wrong with the instrument Notify the facility manager or see Chapter 7 for trouble shooting EMX User s Manual 3 18 BRUSER Tuning the Microwave Cavity and Bridge Close the Microwave Bridge Control dialog box Click the bridge controller button the button labeled MW in the tool bar The MW button toggles the dialog box open and closed The Microwave Bridge Co
46. left mouse button The command such as OK or Cancel is displayed in the center of the button EMX User s Manual 3 6 BRUSER Brief Tips on Windows 95 Arrow Buttons Slider Bar Scroll Bar OK Button Cancel Button The arrow buttons are used to change a variable in a discrete step wise fashion If the box has a white background the values may be edited as in an editable box Clicking the up or down arrow button increases or decreases the parameter with a fixed step size For example the step size for modulation amplitude is 0 1 Gauss Keeping the mouse button pressed repeats the action automatically If the background of the box were gray the up and down arrows next to the box move you through the allowed values for the variable sequentially You are then not able to edit the values The slider bar is used to vary a parameter continuously between its allowed limits For example it is used to vary the microwave source frequency from 9 1 to 9 9 GHz Clicking to the left or right of the square acts as a coarse adjustment while clicking the left or right arrows allows fine adjustments Keeping the mouse button pressed repeats the action automatically The value of the parameter is indicated graphically by the rectangle to supply you with visual feedback The parameter may be varied as well by clicking and dragging the square The scroll bar looks like a slider bar but functions differently It is used to view e
47. power optimization 6 13 7 11 microwave settings 4 29 read only 4 29 set 4 29 modulation amplitude optimization 6 11 connecting cable 5 16 disconnecting cable 5 14 N nitrogen purge 5 14 7 12 no signal 7 17 noise excessive 7 9 to 7 10 ground loops 7 9 interference 7 9 microphonics 7 10 worn iris screw 7 10 power line 7 9 variable temperature operation 7 10 not ready warning 7 2 O operating instructions 3 1 to 3 35 optimizing sensitivity 6 5 to 6 14 instrumental factors 6 5 to 6 7 parameter selection 6 8 to 6 13 P pitch samples 8 3 poor resolution 7 13 to 7 14 excessive microwave power 7 13 excessive modulation amplitude 7 13 excessive modulation frequency 7 13 EMX User s Manual Index excessive time constant 7 13 field inhomogeneity 7 14 poor sensitivity 7 11 to 7 12 AFC adjustment 6 6 calibration file 7 12 cavity choice 7 11 conversion time 6 9 electrical interference 6 5 excessive microwave power 7 11 insufficient receiver gain 6 8 matching 6 6 7 11 microphonics 6 5 loose iris screw 6 6 microwave power 6 13 modulation amplitude 6 11 sample position 6 6 7 12 time constant 6 9 water condensation 7 12 power computer turning on 3 8 console turning off 3 35 turning on 3 8 system turning off 3 35 turning on 3 8 printing output formatting 3 28 parameters 3 28 spectra 3 28 to 3 30 Q Q factor 2 15 to 2 16 7 11 R receiver gain optimization 6 8 7 12 resi
48. sample point D in the figure We therefore want our detector to 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 Cos BRUKER 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 det
49. 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 Bo Electron i Nucleus Bo Electron Nucleus Figure 2 5 Local magnetic field at the electron By due to a nearby nucleus EMX User s Manual 2 7 BRUSER 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 By opposes the laboratory field For a spin 1 2 nucleus such as a hydrogen nucleus we observe that our single EPR absorption signal splits into two signals which a
50. through one of your EPR lines is at least ten times the length of the time constant See Figure 6 6 EMX User s Manual 7 13 Notes Magnetic field inhomogeneities or gradients Extremely narrow lines less than 20 milliGauss may be lim ited by magnetic field irregularities Vary the position of the cavity in the magnet air gap If the linewidth changes check for magnetic objects in or around the magnet If possible suspend these objects by a string and watch for a deflection in the same field strength as used in the experiment Do not attempt this with the cavity in the magnet The force of a fer romagnetic object being pulled into the magnet air gap can cause serious damage to accessories in the air gap Spectrometer not thermally stabilized Be sure that the spectrometer has been turned on for several hours Verify that the laboratory conditions are within specified limitations i e temperature fluctuations etc 7 14 or BROKER Cos BRUKER Lineshape Distortion Lineshape Distortion 7 11 Microwave power too high The effect of saturating microwave fields is to broaden the resonance This is easily apparent for single structureless lines however small split tings may become unresolvable if strongly saturating levels of microwave power is used Lower the microwave power until you obtain a power independent lineshape Modulation amplitude too high Large field modulation will broaden the resonanc
51. time between the individual steps Regulator Microprocessor Reference Voltages Figure 2 18 A block diagram of the field controller and associated components The magnetic field regulation occurs via 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 EMX User s Manual 2 22 BROKER 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 Bo
52. time constant EMX User s Manual 6 14 Troubleshooting 7 This chapter lists some common problems you may encounter with your Bruker EMX EPR spectrometer Major hardware mal functions are not covered We concentrate on problems due to operator errors set up errors or protective circuitry The mate rial presented in Chapters 2 3 and 4 is useful in understanding much of what is discussed in this chapter Many problems are easily solved by the user The flow diagram on this page will help you diagnose the majority of problems that occur during the tuning phase of operation If you fail to find a solution to your problem after reading this chapter call your local Bruker EPR service representative No Tuning No Unable to picture dip couple cavity Power too Sample low not aligned Adjust Attenuation to 25 dB Adjust Bias Reference phase not set correctly Bridge switches Wron to standby k ong Iris motor automatically requency limits not set correctly N Waveguide Over voltage Insufficient gasket occured cooling misaligned Figure 7 1 Flow Chart for diagnosing problems EMX User s Manual Cos BRUKER No Cavity Dip not ready No Cavity Dip TA If a warning dialog box appears when y
53. to enter required input for the acquisition What follows is a description of the basic elements of a dialog box and how to use them Experiment Hall Standard Parameter Spectr X Field eee Time Scan Center Field Editable Box Step Frequency a 750000 GHz Power 00 mW Offset Time Constant Conversion Time Temperature unit Temperature Step Sweep Time Harmonic Goniometer Resolution in X Angle Step Number of X Scans Resolution in Y mek Repetitive Mode Figure 3 3 W Interactive Spectrometer Control Signal Channel Push oe pao E 6 X Calibrated Button po iverGain faz u e EH 0 00 G Receiver Gain 32 10 e Addi jos o Modulation Freq 100 00 HHH Modulation Amplitude 0 96 EHe E 3480 000 E Modulation Phase H jo 00 tae Setup Scan Offset 0 00 Time Constant 5 12 msec Sweep Width ConversionTime 5 12 HH msec Harmonic 1 MW Attenuator g Check Temperature Box Set parameters to spectrum SCT Options The parts of a dialog box EMX User s Manual 3 5 BRUSER Brief Tips on Windows 95 Editable Box Drop down List Check Box Push Button The editable box is a plain box with a white background As the name suggests you may edi
54. z Y no Y Sweep z Hall Signal Channel Center Field 3477 71 ia G Receiver Gain E 2 00 10 fe E Sweep Width 119 55 G Modulation Frequency 100 00 E kHz Static Field 3480 000 E G Modulation Amplitude 1 00 H G Microwave Bridge Modulation Phase 0 00 KY deg Frequency GHz Offset 0 00 Power mW Time Constant 20 48 msec Step i E db Conversion Time 40 96 Number Temperature unit Sweepilime aug of Temperature i K Harmonic 1 Scans Step po S K Resolution in X 1 024 5 S goniometer Number of X Scans mM amp augfia deg Resolution in Y 1 Step a gg Repetitive Mode r Save as Default Comment Figure 4 15 The Number of Scans parameter EMX User s Manual 4 14 BpGgeR Field Sweeps x10 3 20 10 0 10 20 3420 3440 3460 3480 3500 3520 G One Scan 10 3 400 300 200 100 0 100 400 2008440846048 3500 3520 S 3440 3460 3480 3500 3520 Sixteen Scans Figure 4 16 Improvement in signal to noise ratio through signal averaging EMX User s Manual 4 15 Cos BRUKER Field Sweeps Resolution 4 3 4 EPR spectra acquired with a computer consist of a list of mag netic field values and corresponding intensities If you have very narrow lines care must be taken that there are enough data points to fully characterize the lineshapes If there is not suffi cient resolution expanded sections of the spectrum will only be crude app
55. 1 Step 1 00 Ek Resolution in X 1024 kK SC Number of X Scans 1 H engl l deg Resolution in Y 1 1 000 ace deg Repetitive Mode r Figure 4 21 Selection of the Time Scan option There remains one more task before we can acquire the time scan we must disable the Automatic Baseline Correction The default option is to subtract the average value of the spectrum at the end of a scan This feature is convenient for field sweep spectra because the average value should be zero if you sweep through the complete EPR spectrum Subtraction of the average value therefore makes double integrations easier A zero average value is not necessarily true for a time scan You may also want to know the ratio of the initial and final intensity which would be impossible if the average value were subtracted To disable the Automatic Baseline Correction option open the Experi mental Options dialog box and click the box next to Automatic Baseline Correction so that the x disappears See Figure 4 22 EMX User s Manual 4 20 ph ER Time Scans Field Controller Microwave Settings Tp Sweep Direction Up l Power Set Zl Flyback On z Power Flyback Off z Settling Condition Wait LED off zl Field Offset Correction 0 00 G Temperature Unit Automatic Baseline E Tolerance K Temperature Flyback Off gt Delay Correction sec Condition Don t wait E S Dispy Settings i enoma Ba
56. 7 mW Diode Hall Field MERE vane 2500 CEI A Signal Noise Measurement olx 10 3 10 0 10 3490 3470 3480 signal trace Peak to peak height 34722 00 2 00 10 12 66 163 84 Receiver gain Microwave power mW Conversion Time ms noise trace noise 725 33 5 02 10 200 68 163 84 Receiver gain Microwave power mW 3500 G Conversion Time ms 103 0 2 50 75 Acquisition done Figure 9 9 Weak Pitch factor Signal noise ratio 100 125 sec Noise measurement and the final result EMX User s Manual 9 12 BRUSER Signal to Noise Ratio Test 10 Check the S N ratio On the right panel the results of the signal intensity and noise level measurements will automatically appear At the bottom of the panel the auto matically calculated signal to noise ratio will be displayed in the box See Figure 9 9 The signal to noise ratio should be higher than 330 to meet the specifications of the Bruker EPR instrument If the result is lower than this value consult Chapter 6 and Chapter 7 Sometimes a large cavity background signal can significantly decrease the test result Refer to the next section Section 9 2 and run a cavity background signal test to verify this If those hints do not help contact your local Bruker service repre sentatives EMX User s Manual 9 13 Cos BRUKER Cavity Background Signal Test Cavit
57. AFC CE Figure 5 19 A significant AFC needle deflection EMX User s Manual 5 23 BRUSER Fine AFC Tuning for Gunn Diode Bridges 5 Adjust the FINE AFC Potentiometer Turn the knob until the AFC needle is once again centered in the AFC meter Figure 5 20 AFC EM Adjust the 4 needle to the AFC center m m Figure 5 20 Centering the AFC meter Verify that the AFC needle remains centered Vary the microwave attenuation between 0 and 60 dB Note that there may be a drift at 0 dB caused by sample heating if you have a lossy sample in the cavity Also the needle may rush off to the left or right at low powers because the AFC loses lock In most cases the AFC will lock again at higher microwave power levels If not switching between Operate and Tune modes and back again at 30 dB atten uation will lock the AFC once more Then increase the attenuation more slowly than the previous time Repeat Step 2 through Step 6 until the needle remains centered Record the microwave frequency and FINE AFC potentiometer setting The setting is microwave fre quency dependent and reproducible If you record the set ting at that microwave frequency you need not perform this whole procedure every time you use low microwave power levels Because only the insertion of a cryostat sub stantially shifts the microwave frequency you will typi cally only need a setting for a cavity with and without a cryostat EMX Use
58. Acquisitions 4 2 There are three handy buttons in the toolbar for starting and stopping acquisitions The RUN button in the tool bar starts acquisitions You can also start the acquisition by clicking Start Acquisition Run in the pull down menu of the Acquisition menu Acquisitions may be stopped in two ways The red STOP button stops the acquisition immediately The green STOP but ton is used when signal averaging It stops the acquisition only after the end of a field sweep or time sweep has been reached See Figure 4 8 Stop Acquisition Button Deeja eea Ble GIS e e Run Acquisition Button Stop at End of Scan Button Figure 4 8 Start and stop commands in the tool bar EMX User s Manual 4 9 Cos BRUKER Field Sweeps Field Sweeps 4 3 Setting Parameters via Zooming 4 3 1 LC St a at Early lof J If you are searching for EPR signals from an unknown species the most prudent approach to find signals is to make a very broad scan with the center field set to a value where you expect to see a signal See Section 6 1 Hints for Finding EPR Sig nals This approach maximizes the probability of finding a sig nal in your field sweep If you are lucky the EPR signals will already be nicely centered in the field sweep most of the field sweep will contain EPR signals and not empty baseline and the receiver gain will be set perfectly Such luck rarely occurs The Interactive Change of C
59. Amp G at 100 kHz with the values listed for your cavity in Table 8 1 If the value obtained by the calibration routine exceeds the values listed in Table 8 1 first record the values for Mod Amp G and Mod Amp max because you will need them for the next step Then stop the calibration routine by clicking the STOP button in the Tool Bar twice If the value is less than or equal to the value listed in Table 8 1 allow the calibration routine to continue its task and proceed to Step 9 EMX User s Manual 8 18 BROER Calibration of the Signal Channel Maximum Mod Gauss Cavity at 100 kHz ER 4102ST 32 ER 4105DR 32 ER 41040R 32 ER 4116DM 10 ER 4103TM 16 ER 4108TMH 16 ER 4106ZRC 10 ER 4106ZRAC 10 ER 4107WZC 10 ER 4107WZAC 10 ER 41150DC 10 ER 41150DAC 10 ER 4122SHQ 15 ER 4114HT 10 ER 4117D MVT 10 ER 4117D R 10 ER 4109EF 10 Table 8 1 Maximum modulation amplitude for EPR cavities EMX User s Manual 8 19 BRUSER Calibration of the Signal Channel Mod Amplitude Limit Mod Amp max Set the Mod Amplitude Limit The Mod Amplitude Limit parameter in the Calibrate Signal Channel dialog box allows you to limit the size of the signal sent to the modulation amplifier to prevent any danger of burning out the coils To calculate a safer value for Mod Amplitude Limit use the following formula Max Mod Actual Mod where Mod Amp max is the value determined by the calibration routine Actual Mod is t
60. Application of Elec tron Spin Resonance Butterworths London 1958 Ingram D J E Spectroscopy at Radio and Microwave Frequen cies Butterworths London 1967 Ingram D J E Radio and Microwave Spectroscopy Butter worths 1975 Jeffries C D Dynamic Nuclear Orientation No 23 Inter science Publishers John Wiley and Sons New York 1963 Jones R A Y et al Techniques of NMR and ESR United Travel Press Ltd London 1965 Jost P C O H Griffith in Methods in Pharmacology Vol II Physical Methods F Chignell Editor Appleton Cen tury Crafts New York 1972 pp 223 276 EMX User s Manual 10 9 BRUSER Bibliography Jost P C A S Waggoner O H Griffith Spin Labeling and Membrane Structure in Structure and Function of Biolog ical Membranes Rothfeld Editor Academic Press New York 1971 Kaiser E T L Kevan Editors Radical Ions Interscience New York 1968 Kalmanson A E G L Grigoryan Spin Labels in EPR Investi gation of Biological Systems in Experimental Methods in Biophysical Chemistry Nicolau E Editor Wiley New York 1973 pp 589 612 Keijzers C P E J Reijerse J Schmidt Pulsed EPR A New Field of Applications Koninklijke Nederlandse Akademie van Wetenschappen 1989 Kevan L R N Schwartz Editors Time Domain Electron Spin Resonance Wiley Interscience New York 1979 Kevan L M K Bowman Modern Pulsed and Continuous wave Electron Spin Resonan
61. H YOU MUST CONTINUALLY MAINTAIN A HIGH STANDARD OF VIGILANCE Do not assume a cavalier attitude the substances with which you work present very real and very serious threats to your health and safety Adhere to all currently recommended guidelines for standard laboratory safety as promulgated by governmental codes and contemporary laboratory practice Inform yourself about the specific risks that are present when you handle actual or poten tial carcinogens cancer causing agents explosive materials strong acids or any liquids that are sealed in glass containers EMX User s Manual oo BREBER Chemical Safety Specifically Be extremely careful when you handle sealed glass samples that are rapidly heated or cooled The rapid cooling of some samples may result in the formation of a solid bolus in the sample tube that may make the tube prone to explosive rup ture Educate yourself about the temperature at which chemicals evaporate When a sample gets close to the temperature at which it evaporates it may quickly become volatile In general the safety threat posed by flying glass and vio lently escaping gases and liquids should not be underesti mated Wear safety glasses face masks and other protective clothing whenever there is any risk of spillage breakage or explosion Protective shields should also be employed when there is any risk of explosion Be sure that both storage and working areas are properly v
62. Hz A sufficiently large range of frequencies is then covered for most EPR experiments Choose the harmonics The signal channel can pro duce either a first harmonic first derivative or a second harmonic second derivative spectrum If you have no need for second harmonic spectra and wish to save a bit of time in the calibration routine you may deselect the option to calibrate the phase for the second harmonic by clicking the 2nd Harmonic box A cross in the box indi cates that the option is selected However the time sav ings are minimal and you never know when you may need a second harmonic display it is probably best to always calibrate the second harmonic phase EMX User s Manual 8 16 Cos BRUKER Calibration of the Signal Channel The ER 4105DR dual cavity is different from the ER 4116DM dual mode cavity The ER 4116DM has only one set of modulation coils Select the resonator In almost all cases the 1st Res onator should be selected The ER 4105DR dual cavity has two sets of modulation coils By selecting 1st Reso nator or 2nd Resonator you are selecting the set of modulation coils that are to be calibrated Start the calibration routine Click the Start button A new dialog box will appear See Figure 8 14 The spectrometer will then automatically calibrate the signal channel at each of the specified modulation frequencies Calibration File c winepr tpu st95 1 5f cal F
63. Norman Ed Vol 1 3 The Chemical Society London P B Ayscough Ed Vol 4 Speight J G The Application of Spectroscopic Techniques to the Structural Analysis of Coal and Petroleum Applied Spec troscopy Reviews 5 211 1971 Squires T L An Introduction to Electron Spin Resonance Aca demic Press New York 1964 Squires T L Introduction to Microwave Spectroscopy G Newnev London 1963 Standley K J R A Vaughan Electron Spin Relaxation Phe nomena in Solids Adam Hilger Ltd London 1969 EMX User s Manual 10 16 BRUSER Bibliography Stepin L D Quantum Radio Frequency Physics Translated by Scripta Technica Inc Edited by H H Stroke MIT Press Cambridge 1965 Stoneham A M Theory of Defects in Solids Calrendon Press Oxford 1975 Chapter 13 Strandberg M W P Microwave Spectroscopy Wiley New York 1954 Sugano S Y Tanabe H Kamimura Multiplets of Transition metal Ions in Crystals Academic Press New York 1970 Swartz H M J R Bolton and D C Borg Editors Biological Applications of Electron Spin Resonance Wiley Inter science New York 1972 Symons M C R Chemical and Biochemical Aspects of Electron Spin Resonance Spectroscopy J Wiley New York 1978 Symons M C R Electron Spin Resonance Specialist Periodical Reports Volumes 10 A B Royal Society of Chemistry London 1987 Talpe J Theory of Experiments in Paramagnetic Resonance Pergamon Press Oxf
64. Parameter box simply reflects the current microwave power This mode is useful when you have interactively optimized the microwave power for an EPR experi ment The opposite is true for the Set mode In this mode the micro wave power is set to whatever value is displayed in or typed into the microwave power display See Figure 4 34 The software determines the appropriate microwave attenuator value to obtain the specified microwave power This mode is useful when you want to set the microwave power to a specific value EMX User s Manual 4 29 ph ER Controlling the Microwave Bridge Standard Parameter Spectri x Experiment x EEC y nov sweep g Hall Signal Channel Center Field 3420 00 iuar Gain Efo 10 a Sweep Width kaian Microwave Frequency 100 00 H kHz Static Field A Amplitude 1 00 G Power Microwave Bride Phase poo deg L GHz Offset 0 00 Power mW Time Constant 0 64 E msec 3 751000 2 00 Step 1 db Conversion Time 5 12 HA msec Frequency Sweep Time 5 24 sec Temperature K Harmonic 1 F Step 1 00 K E Resolution in X 1024 E Goniometer Number of X Scans 1 Angle EJ ieg Resolution in Y l Step ee poo E deg Repetitive Mode a Temperature unit Figure 4 34 The Experiment Parameter dialog box EMX User s Manual 4 30 Cos BRUKER Spectrum Files Spectrum Files 4 7
65. Plenum Press New York 1969 Yordanov N D Electron Magnetic Resonance of Disordered Systems World Scientific Singapore 1989 Yordanov N D Electron Magnetic Resonance of Disordered Systems World Scientific Singapore 1991 Zahlan A B Editor Excitons Magnons and Phonons in Molec ular Crystals Beirut Symposium 1968 Zahlan A B and others Editors The Triplet State International Symposium on the Triplet State Cambridge University Press Cambridge 1967 EMX User s Manual 10 19 Index A acquiring spectra 1D spectra 3 20 to 3 30 2D spectra 5 23 to 5 28 additional techniques 5 1 to 5 28 AFC fine tuning Gunn bridges 5 19 to 5 22 trap filter 8 11 applications 1 1 to 1 4 biology and medicine 1 4 chemistry 1 2 ionizing radiation 1 3 materials research 1 3 physics 1 2 auto tune 4 27 bar menu 3 4 scroll 3 7 slider 3 7 title 3 3 tool 3 4 bias level adjustment 5 9 bibliography 10 1 to 10 19 box check 3 6 control menu 3 4 dialog 3 5 to 3 7 editable 3 6 button arrow 3 7 cancel 3 7 maximize 3 3 minimize 3 3 OK 3 7 push 3 6 restore 3 4 C calibration of signal channel and cavity calibration file names 8 15 reading in 5 17 DPPH sample positioning 8 10 dual cavity 8 17 harmonics 8 16 modulation amplitude 8 4 to 8 6 modulation amplitude limits 8 18 to 8 20 modulation frequency limits 8 16 modulation phase 8 6 practice 8 8 to 8 20 resonator Ist 8 17
66. QUISITION Spectr2 W Eile Parameter Acquisition Processing View Options Window Info OSES CREE ESR EES eee ae Inactive Spectrum Window Spectr 1 10 3 Active Spectrum Window Window Border Figure 3 2 O BERETS alolx Frequency Attenuation Power 0 00 GHz AFC Receiver Level 60 a CA o 0 000 nW Diode Hall Field iolxi 3500 G _ CAP 3493 12 G 2464 74 The parts of an applications window Window Border 3 1 EMX User s Manual 3 2 Cos BRUKER Brief Tips on Windows 95 Application Window Spectrum Window Title Bar oj Maximize Button E Minimize Button All Windows programs operate in an application window See Figure 3 2 WIN EPR Acquisition displays all its com mands and spectra in the application window Acquired spectra are displayed in a spectrum window You may have multiple spectrum windows open at the same time how ever only one is active at a time The active spectrum window is the one upon which operations will be performed Each spec trum window has its own set of instrument parameters The parameters viewed or edited in the menus correspond to those of the active spectrum window With default Microsoft Win dows colors the active windows have a blue title bar and the inactive windows have grey title bars You activate a window by clicking it while the
67. a finger dewar with a boiling refrigerant such as liquid EMX User s Manual 6 6 Cos BRUKER Optimizing Sensitivity nitrogen you should turn the AFC modulation level to maxi mum 50 OUTPUTS AFC MOD LEVEL 4 2 sou a as Ales S ae S Ey 0 RS MOTOR accessory 83 5 D y H Figure 6 4 Location of the AFC MOD LEVEL potentiometer Cryostats can protect your cavity from con tamination due to sam ple tube breakage Insert a cryostat in the cavity Quartz has a dielectric constant of 3 8 but a low dielectric loss Inserting high purity quartz sleeves such as the variable temperature dewar actu ally concentrates the microwave magnetic field intensity at the sample The increased field intensity produces an EPR signal that has a larger signal to noise ratio than is achieved in the absence of the dewar insert If your experiments approach the sensitivity limit and your samples are nonlossy you may benefit from the use of the variable temperature quartz insert dewar even if the experiment is run at room temperature EMX User s Manual 6 7 BpGsen Optimizing Sensitivity Parameter Selection 6 2 2 e Optimize the receiver gain You need to have sufficient receiver gain in order to see all the details in your spectrum Figure 6 5 shows the results of insufficient as well as exces s
68. abilizers EMX User s Manual 5 17 Cos BRUKER Changing EPR Cavities Steps 14 and 15 are used to set the limit switches in the iris motor The limit switches prevent you from screwing the iris in too far and thereby breaking the iris screw Make sure you connect the modulation cable to the MOD modulation connector and not the R S Rapid Scan con nector 13 14 15 16 17 Figure 5 12 Attach the appropriate size collet and pedestal on the cavity Screw in the iris Manually turn the iris screw until it is almost all the way in The iris screw will stop rotating It may be a good idea to back the screw out 1 2 turn after it hits the bottom This will further decrease your chances of accidentally breaking the iris screw dur ing the tune procedure Click and hold the down Iris Button Activate this button See Figure 5 1 until the iris motor stops this is the lower limit of the motor With the iris motor in its lower limit reattach the iris motor drive to the iris screw Connect the modulation cable to the cavity Reconnect the waveguide sections and tighten the stabilizers Do not forget to install the waveguide flange gasket between the two flanges make sure it is ori ented correctly See Figure 5 12 Position the cavity in the center of the magnet air gap by moving the bridge on the table Carefully tighten the stabilizers Be careful not to stress the waveguide
69. ace Frequency Attenuation gt Bias 43 0 al Attenuation Display C Operate Signal Phas Auto Tune a Figure 3 8 The Microwave Bridge Control dialog box 2 Switch the microwave bridge to Tune There are three states or modes for the microwave bridge Stand By Tune and Operate When you turn on your EMX User s Manual 3 13 Cos BRUKER Removing and Inserting Samples If a klystron bridge does not switch to Tune and the Stand By indicator is red wait a minute There is a time delay of approximately three minutes between the time the console is turned on and the time the klystron can be turned on This allows the klystron to warm up sufficiently Take care if you are wearing an analog mechanical watch The magnetic field in the air gap of the mag net is sufficiently strong to magnetize your watch Therefore to avoid damage to your watch remove your watch before put ting your hands in the magnet air gap spectrometer it should be in Stand By This is indicated by Stand By in green color appearing in the microwave bridge controller menu See Figure 3 8 If you have been acquiring spectra already your bridge will probably be in Operate Click the Tune button in the dialog box to switch the bridge to Tune Set the microwave attenuator to 25 dB The micro wave attenuation is set by clicking the arrow
70. agnetic Resonance of Phase Transitions Academic Press New York 1979 Pake G E Paramagnetic Resonance W A Benjamin Inc New York 1962 Pake G E T L Estle The Physical Principles of Electron Para magnetic Resonance Addison Wesley Reading Mass 1974 Peisach J W E Blumberg Electron Spin Resonance of Metal Complexes Plenum Press New York 1969 Petrakis L J P Fraissaird Editors Magnetic Resonance Intro duction Advanced Topics and Applications to Fossil Energy NATO ASI Sries C124 Reidel Dordrecht 1984 EMX User s Manual 10 13 BRUSER Bibliography Pilbrow J R Transition Ion Electron Paramagnetic Resonance Clarendon Press Oxford 1990 Poole C P Electron Spin Resonance A Comprehensive Treatise on Experimental Techniques First Ed Interscience Pub lishers New York 1967 Poole C P Electron Spin Resonance A Comprehensive Treatise on Experimental Techniques Second Ed J Wiley New York 1983 Poole C P H A Farach Editors Handbook of Electron Spin Resonance Data Sources Computer Technology Relax ation and ENDOR AIP Press New York 1994 Poole C P Editor Magnetic Resonance Reviews Vol I Gordon and Breach New York 1971 Poole C P and H A Farach Relaxation in Magnetic Reso nance Dielectric and Mossabauer Applications Aca demic Press New York 1971 Poole C P and H A Farach The Theory of Magnetic Reso nance Wiley Interscience
71. am and then load the data files you just saved or you can click the Transfer to WinEPR button in the tool bar which will automatically launch the WinEPR program and transfer the spectrum of the active window To transfer the other spectrum you need to activate that spectrum window in Acquisition program by clicking the EMX User s Manual 9 22 BRgER Cavity Background Signal Test spectrum window and then click the Transfer to WinEPR button in the tool bar again The WinEPR appli cation will appear See Figure 9 16 Data Tansfer Options Help ele Pe isa Algebra Show Document Figure 9 16 Transfer to the WinEPR for data processing 16 Click 1D processing under WINEPR System Select the cavity background spectrum 17 Measure the cavity background signal Click Expand under Display A box contains Expand Display Values will appear On the right side of the box there are low val and high val of the Y Scale The difference EMX User s Manual 9 23 BpGgeR Cavity Background Signal Test between these two values is the signal height of the cavity background signal See Figure 9 17 WINEPR System 1D Processing Eile 1D Processing Parameters Help Rear EET pales Keg Expand Display Yalues Split Screen ni Compare Scaling Start 100 00 Re 5100 00 View sath f Sealiag Aj low val 1663 5 high val fies O 5 Distance siz Display
72. andard Parameter Spectr1 Lx Experiment X Field Sweep a Y MW Power Sweep z no Y Sweep incremental Sweep Hall i Signal Channel Field Sweep Center Field Starting iver Gain Sweep Width Microwave ulation Frequency Power ulation Amplitude M Step Modulation Phase Microwave Value 780 ci Offset Power Sweep Power N 0 20 mW Time Constant 5 FS msec Step aa db Conversion Time 5 12 msec Temperature unit i SHEETS UGH 5 24 oars UT ets E Set the Number E Siep Mises ES of Scans to 5 foe g Goniometer Number of ENN 1 Angle GEG Resolution in Y T S Lom deg Repetitive Mode E Save as Default Comment Figure 5 21 Sample parameter settings for acquiring a 2D data set Set the starting microwave power to 0 2 milli watts Change the power in the power setting box to 0 2 milliwatts This will be the power that is used to acquire your first spectra See Figure 5 21 Use a step value of 5 dB By using a negative step value the power will increase in units of 5 dB between each scan See Figure 5 21 Set the number of spectra to be acquired to 5 In the Resolution in Y box change the setting to 5 This will program the spectrometer to acquire 5 scans See Figure 5 21 Click OK to close the window Click on Run to acquire your 2D data set This will initiate the first of five scans with the power increasing in
73. ant ConversionTime Harmonic KX Calibrated Efsa 10 B EA Time f Constant oo z Yo 10 24 2 HH msec HH msec wT a Set parameters to spectrum SCT Options Figure 4 27 A distorted Setup Scan EPR spectrum If the EPR signal is very weak or very broad the Setup Scan may not be the best way to optimize the signal For such cases EMX User s Manual 4 24 BRUSER Interactive Spectrometer Control Dlhe aS EAE ompa A you can use the Interactive Receiver Level Display option in conjunction with the Interactive Spectrometer Control dialog box to optimize your EPR signals First make sure you have an acquired spectrum and that its window is active Clicking the Interactive Receiver Level Display button in the tool bar Figure 4 28 creates a marker vertical line with a short hori zontal bar in the spectrum window The position of this marker in the spectrum determines the magnetic field i e changing its position changes the actual magnetic field The short horizontal bar indicates the receiver level at the magnetic field at which the marker is placed The marker is moved by placing the cursor at the desired position in your EPR spectrum and clicking the left mouse button Clicking the right mouse button makes the marker disappear Fix the cursor at the desired position in the spectrum and open the Interactive Spectrometer Control dia log box Now as you vary parameters such as r
74. ation file into the data system Also make sure that the Calibrated check button in the Interactive Spectrometer Control dia log box is not un checked Receiver gain or modulation not optimized See Section 6 2 2 Sample not positioned properly Center your sample in the cavity EMX User s Manual 7 12 Cos BRUKER Poor Resolution Poor Resolution 7 10 Microwave power set too high Saturating microwave power levels will broaden your resonance line Verify that the linewidth is independent of the microwave power level by recording the spectrum at various power levels Modulation amplitude set too high Large field modu lation amplitudes will broaden your resonance line particu larly as the modulation amplitude approaches the linewidth Reduce the modulation amplitude to ensure that the spectrum is independent of the modulation amplitude See Figure 6 8 Modulation frequency set too high The spectral reso lution is limited by the field equivalence of the modulation frequency used Reduce the modulation frequency to verify that the linewidth is independent of the frequency See Figure 2 17 Time constant too long for sweep time A larger time constant will begin to filter out the high frequency compo nents of your signal Consequently if the sweep rate is too fast relative to the time constant the spectrum will appear distorted and broadened To avoid this problem make sure that the time required to sweep
75. but free standing Skip to step 7 for instructions on turning your power supply on 6 Turn on the heat exchanger and magnet power supply Instructions for Small Power Supplies If you have a heat exchanger Not all systems require a heat exchanger you must first turn it on by activating the power switch See Figure 3 6 To turn the power supply on push the POWER ON OFF button See Figure 3 5 A Step 8 Power Switch A 1 ae hg Li Ea T La Figure 3 6 The location of the heat exchanger power switch EMX User s Manual 3 10 BRUSER Turning the Spectrometer On Turn on the heat exchanger and magnet power supply Instructions for Large Power Supplies On systems with large power supplies See Figure 3 5 B you need to first press the ELECTR ON button and then the POWER ON button Pressing the POWER ON button also starts the heat exchanger If not make sure that the power switch on the heat exchanger is activated See Figure 3 6 Verify the water flow Make sure that the valves con trolling the flow of water to the microwave bridge are open The valve handles should be parallel to the hose You can find them on either the left side or right side of the base of the magnet Follow the black water lines com ing from the back of the bridge Make sure that the water lines are properly installed Water coming out of the back of the bridge indicates a loose coolant tub
76. cases particularly if the micro wave attenuation is less than 40 dB the AFC works very well without any need for you to fine tune it If you are performing experiments in which low microwave powers are required fol lowing the instructions in this section will ensure that you will obtain optimal AFC performance Please note that this proce dure is not required for klystron bridges You can determine the type of bridge you have by looking at the model designation on the front plate of the bridge A model designation containing a G for example ER 041 XG indicates a microwave bridge with a Gunn diode microwave source In contrast a bridge with a model designation with a K such as ER 041 XK has a klystron microwave source The Fine tuning Procedure 5 3 1 1 Set the FINE AFC potentiometer to zero The potentiometer for the AFC can appear in two different locations on the bridge depending on when your bridge was manufactured See Figure 5 15 2 Tune the microwave bridge Follow the procedures in Section 3 4 for automatic tuning or Section 5 1 for man ual tuning The frequency bias phase and iris screw should be adjusted so that the needles of the AFC and Diode meters remain centered as you change the micro wave attenuation from 0 to 40 dB See Figure 5 16 and Figure 5 17 Note that there may be a drift at 0 dB EMX User s Manual 5 21 Cos BRUKER Fine AFC Tuning for Gunn Diode Bridges caused by sample heating i
77. ce John Wiley and Sons New York 1990 Kevan L L D Kispert Electron Spin Double Resonance Spec troscopy John Wiley and Sons New York 1979 Kliava J EPR Spectroscopy of Disordered Solids Zinatne Publ Moscow 1988 in Russia Knowles P F D Marsh H W E Rattle Magnetic Resonance of Biomolecules Wiley Interscience New York 1976 Kundla E E Lipmaa T Saluvere Editors Magnetic Reso nance and Related Phenomena Springer Verlag Berlin 1979 Kwiram A L Electron Nuclear Double Resonance in Annual Review of Physical Chemistry Annual Review Inc Palo Alto CA 1971 Vol 22 pp 133 170 EMX User s Manual 10 10 BRUSER Bibliography Kwiram A L in Magnetic Resonance M T P Int Rev Sci Phys Chem Ser 2 C A McDowell Editor Butterworths Publi cation London 1972 Vol 4 pp 271 316 Kurreck Harry Burkhard Kirste Wolfgang Lubitz Electron Nuclear Double Resonance Spectroscopy of Radicals in Solution VCH Publishers Inc 1988 Lancaster G Electron Spin Resonance in Semiconductors Hilger and Watts London 1966 Lebedev Ya S Atlas of Electron Spin Resonance Spectra Con sultant Bureau New York 1963 1964 Lichtenstein G I Spin Labeling Methods in Molecular Biology Wiley Interscience New York 1976 Low W Solid State Physics Vol 2 Supplement Academic Press New York London 1960 Low W Paramagnetic Resonance in Solids Academic Press New York 1960
78. ces the center field value with the magnetic field position of the marker For further details on this operation consult Section 4 3 2 of this manual MB WINEPR ACQUISITION Spectr1 Eile Parameter Acquisition Processing View Options Window Info esua eds Bel oe ei sie Ea 5 AFC Receiver Level Temperature MMOPERATENEE Frequency 9 77 GHz P LEVELLED Attenuation 25 dB Toa m T 300 00K PO 0 636 mW Diode Hall Field E ovane 2200 C 3406 480 Spectr Intensity 600 400 200 0 3410 3420 3430 3440 3450 3460 3470 3480 3490 3500 G NUM 3483 21 G Figure 8 11 The center field marker Acquire the spectrum once more The DPPH signal should now be nicely centered in the spectrum EMX User s Manual 8 14 epGsen Calibration of the Signal Channel Calibrating the Signal Channel 8 2 3 1 Open the Calibrate Signal Channel dialog box Click the Calibrate Signal Channel command in the Acquisition drop down menu A new dialog box will appear Calibrate Signal Channel OOOO OE c winepr tpu st9515al cal Frequency List Calibrate from 100 0 kHz to 6 kHz Increments 10 kHz Mod Amplitude Limit 100 0 Senay 1st Resonator IX 2nd Harmonic 2nd Resonator Start Cancel J Figure 8 12 The Calibrate Signal Channel dialog box 2 Enter the filename for the calibration file The cali bration file name usually consists of two or th
79. changing cavities fine tuning the AFC and per forming automated 2D experiments Manually Tuning a Microwave Bridge 5 1 A klystron bridge requires approximately three minutes to warm up after the console is turned on When the Stand By indicator is green the software allows you to switch to Tune mode The Auto Tune routine of the EMX software is effective at tun ing the cavity and bridge under most circumstances However there are some circumstances where automatic tuning may have difficulties Lossy samples such as water can be problematic particularly when you work at high microwave power levels Following these instructions will help you to tune the spectrom eter under these adverse conditions 1 Open the Microwave Bridge Control dialog box If this window is not already open click its button the button labeled MW in the tool bar The button toggles the dialog box open and closed The microwave bridge con trol dialog box will then appear See Figure 5 1 2 Switch the microwave bridge to Tune mode The bridge status indicator shows the three states or modes for the microwave bridge Stand By Tune and Operate See Figure 5 1 In Stand By the power to the micro wave source is shut off When you switch to Tune the source turns on and you produce a frequency sweep that allows you to see the dip of your cavity Switching to Operate causes power only at the resonant frequency to be transmitted to the cavity
80. d Offset Correction 0 00 G MW Fin Temperature Unit Tolerance MW Fine No Time Delay z Temperature Flyback tune before Delay Time 0 00 sec Condition each sweep P z Display Settings Goniometer automatic Baseline Correction Angle Flyback Off gt X automatic Scaling Figure 4 32 The MW Fine Tune Before Each Sweep check box EMX User s Manual 4 28 Cos BRUKER Controlling the Microwave Bridge Setting the Microwave Power 4 6 2 There are two options for setting the microwave power Set and Read Only Either option can be selected with the Microwave Settings drop down list in the Experiment Options dialog box See Figure 4 33 Experiment Options Spectr1 Lx Field Controller Microwave Settings Sweep Direction Up x Flyback Power et x Power Flyback Settling Condition Microwave Field Offset Correction Settings Temperature Unit Tolerance yk No Time Delay x Temperature Flyback Off x Delay Time 0 00 sec Condition Don t wait E Display Settings Goniometer automatic Baseline Correction Angle Flyback Off x X automatic Scaling Save as Default Figure 4 33 The Experiment Options dialog box In the Read Only mode the microwave power is set by adjust ing the attenuator in the Microwave Bridge Control or Interac tive Spectrometer Control dialog box The microwave power displayed in the Experiment
81. d scan range See Section 4 3 You also need to optimize your EPR signal using the method described later in this chapter If you still cannot find the signal you may have to adjust parameters such as the microwave power modula tion amplitude scan time etc Standard Parameter Spectr ix Experiment X Field Sweep x Y no Y Sweep Hall Signal Channel Center Field pasoo E G Receiver Gain Hafo 10 p Sweep Width poo 9 G Modulation Frequency jooo FH kHz Static Field 3480 0 He Modulation Amplitude 400 He Microwave Bridge Modulation Phase o 00 deg Frequency 7660000 GHz Offset o oo Power 10 0 mW Time Constant feres H msec Step 1 db Conversion Time 827 68 msec Temperature unit Sweep Time 33554 Hele Temperature Coo K Harmonic iz Step o Ek Resolution in X fi 024 Gonometer Number of X Scans fo ok Anale Z a geg Resolution in Y O E See ict Sj deg Repetitive Mode r g B 5 3 g 3 Figure 6 1 Parameters for finding an EPR signal from an organic radical EMX User s Manual Cos BRUKER Hints for Finding EPR Signals Standard Parameter Spectr1 Lx Experiment X Field Sweep z Y no Y Sweep z Hall Signal Channel Center Field 3100 0 He Receiver Gain fJfioo 10 5 EA Sweep Width 6000 00 G Modulation Frequency 100 00 Fl kHz Static Field 3480 0 E G Mod
82. determine which one is best for your sam ples Low cavity Q The cavity Q can be degraded because of improper sample positioning Having your sample positioned in the microwave electric field will reduce the sensitivity by degrading the cavity Q especially for samples with high dielectric loss This can happen if you are using flat cells or capillaries Observe the Q value read out in the microwave bridge dialog box when you are adjusting the sample posi tion Cavity not critically coupled Maximum power is trans ferred between the cavity and waveguide when the cavity properly matches the impedance of the waveguide i e is critically coupled A drastically undercoupled iris will not transmit power to the cavity and so will not excite EPR tran sitions A drastically overcoupled cavity will have a lower Q resulting in lower sensitivity These effects can occur when using lossy samples such as aqueous solutions or conducting samples EMX User s Manual BRUSER Poor Sensitivity Water condensation During low temperature operation water can condense inside the cavity Water being a high dielectric loss material will absorb the microwave power in the cavity and destroy the cavity Q Avoid condensation by using a purging nitrogen gas flow through the cavity Signal channel not calibrated The modulation ampli tude and phase of the signal channel may not be properly cal ibrated Make sure that you load the proper calibr
83. dth 0 00 Field ceiver Gain H 2 52 10 BE Sweep Address 1828 7 Modulation Freq 100 00 HHkrz af Ee gt Modulation Amplitude fico Hye Field 3474 000 G Modulation Phase 4 fo 00 HH deg Setup Scan Enable 0 00 Hx PE _ tant psa HH msec A Button _ Sweep Width 100 00 ial Time 5 12 Fe4 msec teve E E 4 Harmonic L MW Attenuator He fo dB Temperature 300 00 Hk Set parameters to spectrum SCT Options Figure 8 5 The Interactive Spectrometer Control dia log box 6 Optimize the DPPH sample position Move the sample tube up and down until the maximum signal inten sity is attained See Figure 8 4 Avoid moving the sam ple from side to side Perhaps the best technique is to loosen the collet nuts for the pedestal and sample tube and move the sample too low Then use the pedestal to slowly push the sample up Sometimes the process of moving the sample tube in the cavity can cause the AFC to lose lock Retune the frequency if this happens If the signal is clipped decrease the Receiver Gain When you have centered the DPPH sample secure the sample tube by tightening the collet nuts EMX User s Manual 8 10 Calibration of the Signal Channel If there is no spectrum available click on the New Experiment but ton in the tool bar to create a new spectrum See Figure 8 6 Transfer the parameters To set the parameter values to a spectrum click on the Set paramet
84. e 15th Colloque Ampere North Holland Amsterdam 1969 Axel F S Biophys Struct Mechanism 2 181 218 1976 Ayscough P B Electron Spin Resonance in Chemistry Meth uen London Barnes and Nobel New York 1967 Ayscough P B Electron Spin Resonance Volumes 1 5 Ameri can Chemical Society Bagguley D M S Editor Pulsed Magnetic Resonance NMR ESR and Optics Clarendon Press Oxford 1992 Bass A M H P Broida Editors Formation and Trapping of Free Radicals Academic Press New York 1960 Bencini A D Gatteschi Electron Paramagnetic Resonance of Exchange coupled Systems Springer Verlag Berlin 1990 Benedek G B Magnetic Resonance at High Pressure Inter science New York 1963 EMX User s Manual 10 2 BRUSER Bibliography Berliner L J Editor Spin Labeling Theory and Applications Academic Press New York 1976 Berliner L J Editor Spin Labeling II Theory and Applications Academic Press New York 1979 Berliner L J J Reuben Biological Magnetic Resonance Vol umes l Plenum New York 1978 Bernheim R An Introduction to Optical Pumping W A Ben jamin New York 1965 Bersohn M J C Baird An Introduction to Electron Paramag netic Resonance W A Benjamin New York Frontiers in Chemistry 1966 Bertini I R Drago ESR amp NMR of Paramagnetic Species in Biological and Relates Systems NATO Advanced Studies Institute Kluwer Boston 1980 Biels
85. e 4 19 Clicking this button cre ates a marker vertical line in the spectrum window that moves with the cursor See Figure 4 20 Place the cursor where you would like the static field to be and click with the right mouse button This action replaces the default static field value with the magnetic field position of the marker EMX User s Manual 4 18 Cos BRUKER Time Scans SPECTRI12 PAR L i 10 3 Figure 4 20 Interactive selection of the static field for a time scan Once the static field has been selected we must switch from a Field Sweep to a Time Scan Open the Experiment Parame ter dialog box and select Time Scan See Figure 4 21 Change the other parameters such as the Conversion Time and Time Constant so that they are appropriate for the time scales to be encountered with the chemical reaction EMX User s Manual 4 19 os i BROKER Time Scans Experiment X Y no Y Sweep z Field Sweep Time Scan Hall Signal Channel 4 Center Field 3477 71 KE G Receiver Gain 2 00 S e H Sweep Width 50 00 E G Modulation Frequency 100 00 kHz Static Field Baso 000 HHG Modulation Amplitude 1 00 Hse Microwave Bridge Modulation Phase 0 00 H deg Frequency 9 795000 GHz Offset 0 00 H Power 20 17 mW Time Constant 1 28 H msec Step 1 H db Conversion Time 5 12 H msec Temperature unit Sweep Time 5 24 ale Temperature i K Harmonic
86. e cavity This is the twinax cable labeled with a white connector and attached to the front of the cavity See Figure 5 8 Disconnect the nitrogen purge line from the port on the waveguide The port is half way down the waveguide attached to the cavity See Figure 5 8 Disconnect the iris motor shaft from the iris screw First unscrew the lock nut from the iris screw Lift the shaft upwards to disconnect Move the iris motor to the side where it is out of the way See Figure 5 9 Tris Screw Figure 5 9 Disconnecting the iris motor shaft from the iris screw EMX User s Manual 5 16 BRgER Changing EPR Cavities 11 Disconnect the cavity See Figure 5 8 While grasp ing the waveguide attached to the cavity with one hand unscrew the four waveguide screws joining the two sec tions of waveguide Loosen the waveguide stabilizers rotating the screws and carefully remove the cavity from the air gap of the magnet See Figure 5 10 Take care not to lose the gasket which was between the two wave guide flanges Seal the cavity with the solid collets and put the cavity in a safe clean place Figure 5 10 Loosening the waveguide stabilizers 12 Install the waveguide stabilizers on the new cav ity See Figure 5 11 Visually position them just above the magnet pole caps e Figure 5 11 Installing the waveguide st
87. e line Lower the modulation amplitude to a region where the lineshape is independent of the modulation amplitude See Figure 6 8 Time constant too long for sweep time used A safe rule of thumb is that the time required to sweep through an EPR line should be ten times the length of the time constant See Figure 6 6 Modulation frequency too high The modulation fre quency can determine the resolution of the experiment The spectral profile may also change due to the effect of molecu lar dynamics if saturating microwave fields are applied These effects are especially pronounced if the motional fre quency for the spin dynamics is similar to the applied modu lation frequency The technique of saturation transfer is based on this mechanism The spectral profile may change mark edly if the modulation frequency is varied while applying strong microwave fields See Figure 2 17 Magnetic field gradients These may produce highly asymmetric lineshapes Reposition the cavity within the mag net air gap to check the magnet for homogeneity Check for magnetic objects in or around air gap Magnetic field inho mogeneity could also broaden the response to obscure split tings by overlapping spectral components EMX User s Manual 7 15 BRUSER Lineshape Distortion Anisotropic g matrix A highly anisotropic g matrix natu rally produces asymmetric lines Background signal A strong background signal from contamination of t
88. e 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 BRGRER 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 higher 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 s
89. eceiver gain phase or microwave power you can monitor the signal intensity at that magnetic field value See Figure 4 29 Interactive Receiver Level Display Button Js Interactive Spectrometer Control Button Figure 4 28 The Interactive Spectrometer Control and Interactive Receiver Level Display button in the tool bar EMX User s Manual 4 25 Cos BRUKER Interactive Spectrometer Control The parameter values that you have carefully optimized have no effect on the spectrum that you have already acquired To use these new parameters for a new acquisition you need to set these values to a spectrum To set these values to a spectrum click on the Set parameters to spectrum button The cursor will turn into the letter P for Parameter Place the cursor on the target spectrum window and click the left mouse button to copy the parameters to that spectrum Reacquire a spectrum in that window the spectrometer will use the newly optimized parame ter set M2 WINEPR ACQUISITION Interactive Spectrometer Control Eile View Options Window Info Of esler eal kke Pele See OPERAE Frequency oe Power AERATED o vajue ap AFC aa LEII Diode Receiver Level Hall Field 34829233G Spectr2 10 3 Figure 4 29 W interactive Spectrometer Control Hall Center Field G Sweep Width 200 00 F G Sweep Address fsa oo 4 J 4 Field 3482 923 G Lol
90. ector with some extra microwave power or bias Some of the source power is tapped 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 EMX User s Manual 2 14 Cos BRUKER 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 t
91. ectrum Windows Icons allow quick access to many spectra in your application window A second approach to neaten the display is to iconize the spec trum windows so they require considerably less space on the screen See Figure 4 4 You lose the ability to see the contents of a window immediately however you gain almost instanta neous access to many more spectra than with the previous approaches The Arrange Icons command neatly organizes the spectrum windows at the bottom of the application window BB WINEPR ACQUISITION OHCTRL2 PAR File Parameter Acquisition Processing View Options Window Info BEE SiS eS 1x14 Bs EI ely Ma la OPERATE Frequency 9 54 GHz AFC Receiver Level LEVELLED Attenuation 25 dB Lo m a Rower 0 636 mW Diode Hall Field EE vane 7700 LI 3480000G OHCTRL2 PAR 10 3 2 Iconized spectra 3460 3480 3500 _DMeo c FaIsIeal OHCTRL AE DMPO c A DMPO 0 EE mo m Figure 4 4 Iconized spectra EMX User s Manual 4 4 Cos BRUKER Spectrum Windows Creating a New Spectrum Window Create New Spectrum Button 4 1 2 You may need to create a new window in which you can acquire a spectrum Simply click the Create New Spectrum button in the tool bar See Figure 4 5 A new spectrum window will appear having the default parameter set le EE EAE Duplicate Button Figure 4 5 Transferring Para
92. ee Figure 5 5 and Figure 5 6 Open the Microwave Bridge Control dialog box If this window is not already open click its button See Figure 5 5 in the tool bar The button toggles the dialog box open and closed The Microwave Bridge Control dialog box will then appear See Figure 5 7 Attenuation Display 8 Microwave Bridge Control Jo Frequency 76 6 ee ce a Bias 43 0 Iris E eC Stand By nal Phase 46 8 Ll Loh Button y 4 Auto Tris o Fine Tune Buttons Stop Tuning F Dual Trace Figure 5 7 The Microwave Bridge Control dialog box EMX User s Manual 5 14 BRUSER Changing EPR Cavities 6 Switch the microwave bridge to Stand By mode Click the Stand By button in the dialog box to change to the Stand By mode See Figure 5 7 Iris Motor NN F Modulation Cable Waveguide Screws f j Fa esa Waveguide Gasket Nitrogen Purge Port Iris Screw Figure 5 8 Connections on the ER 4102ST cavity EMX User s Manual 5 15 BRUSER Changing EPR Cavities Store the lock nut in a place where it will not be lost 10 Disconnect accessories If a variable temperature dewar assembly is installed disconnect the coolant trans fer line and the thermocouple connections from the cavity Disconnect the modulation cable from th
93. em contact your local Bruker EPR service representa tive EMX User s Manual Cos BRUKER Signal to Noise Ratio Test Signal to Noise Ratio Test 9 1 The signal to noise ratio test is an important part in maintaining your spectrometer It is also helpful in diagnosing possible prob lems you may encounter especially when you deal with very weak signals or quantify your EPR signals A standard signal to noise ratio test uses the ER 4102ST standard cavity and the weak pitch sample that was shipped with your spectrometer The test measures the EPR signal intensity peak to peak height of the weak pitch sample at low microwave power 12 db and then measures the noise level under the same conditions except higher microwave power 0 db and higher receiver gain to char acterize the noise better The formula for calculation of signal to noise ratio is G P S EN ye A 9 1 N Ay Gg Ps NTXC where Ag and Ay are the peak to peak height of the weak pitch and amplitude of the noise respectively G and Gy are the receiver gains used in signal and noise measurements respec tively We use their ratio to correct for the gain difference Ps and Py are the powers used in two measurements and we use the square root of the ratio of powers to correct for the power differ ence The factor of 2 5 translates the peak to peak noise level to a RMS Root Mean Square noise level T is the time constant in seconds and we use the square root of t
94. en tilated They should be equipped with powerful blowers and fume heads Store chemicals safely Avoid integrating containers of chem icals that may result in dangerous combinations Practice good housekeeping in work and storage areas Clean up spills and refuse promptly Do not leave volatile combus tible or acidic liquids exposed on counters benches or other work areas Make certain all chemical containers are properly labeled and classified and that especially hazardous materials are appro priately designated with clearly understood decals or warn ings EMX User s Manual oo BREBER Microwave Safety e Never taste or inhale unmarked chemicals e All laboratories should be equipped with fire doors fire extinguishers fire smothering materials and sprinkler sys tems or showers as well as a detailed fire safety plan Microwave Safety 0 3 As long as the microwaves are contained in metal structures microwaves can be very safe Here are some precautions which if followed will eliminate the possibility of injury due to the microwaves e Do not have an open waveguide when the microwave power is on e Switch the bridge to standby when you remove or change EPR cavities e Never look down an open waveguide when there is micro wave power The eyes are very susceptible to damage from microwaves EMX User s Manual vi BRGReR Table of Contents Table of Contents 0 4 0 Prefaci enove ern
95. en a a Aa sede EE E TEE lil Od Electrical Safety ised cont seSGpce saa ied canta a Aeterna en ata ec don ee beens iv OF Chemical Safety cely irrene s eneren e T S EErEE E TE NESES iv 0 3 Microwave Safety heen a aE enero E Aa bau E N TEE vi OFF Table of CONTENTS isei e ot a aa e aE E E ae vii 1 ArodUCHOM oaeee a EEE 1 1 KUEPRAppicati ons 6 ics te EEE TEE E A 1 1 LPT CHEMISE n stecte A NE ial asin tea esha E eet 1 2 TeV sPWYSICS sit Gatets saceethcesce O EA EEE 1 2 1s1 3 Materials Research is cy ceded etnies ee ee ae 1 3 1 1 4 Tomizing Radiation nseni en iier i 1 3 1 1 5 Biology and Medicine aine erneieren e e e e aiiai arla 1 4 1 2 The Spectrometer sorceres i n a o aa a REER 1 5 1 3 Usine this Mania rene mnan ea e a e a a e ee aka Weld s 1 6 1 3 1 How to Find Things sos elore linc e E EE E T 1 6 1 3 2 Typographical Conventions eeseseeeeeseeereesseseesresrssesteeresresrerrresresreesesressese 1 7 2 AMEPR PAINEL a e aa a e e 2 1 Zl BaSIGEPR PING OLY isch vue See e E E O E E E ns 2 1 2 1 1 Introduction to Spectroscopy eesesseesresseseeresrersisstestrstessestessesrtssresreerenreses 2 1 212 The Zeeman Efiect en tenn ee E a Eea aeaee anna 2 3 21 3 Hyperfine Interactions vena e a e E a a a a r 2 7 21 4 Signal Intensity asenn es ae ee ee ee el 2 9 22 Basie PPR Pracee Zags cays etag Gal oc cacao sie s aces ee 2 10 2 2 1 Introduction to Spectrometers ee ceeceecesecesecesseceseceeeeeseeeseeesaeeeaeessaes 2 10 EMX User
96. enter Field and Sweep Parameter button Figure 4 9 helps you to achieve the desired results on your second attempt The following procedure allows you to use a broad scan to optimize the center field sweep width and receiver gain so that you can acquire an aesthetically pleasing as well as meaningful spectrum Interactive Change Button Figure 4 9 The Interactive Change of Center Field and Sweep Parameter button in the tool bar EMX User s Manual 4 10 Cos BRUKER Field Sweeps In Figure 4 10 we have used a broad field scan to find our EPR signal Clicking the Interactive Change of Center Field and Sweep Parameter button in the tool bar creates a zoom rectan gle in the spectrum window This rectangle functions in the same manner as the rectangular scaling option described in Section 4 1 5 Surround the area that you would like to have in your spectrum and click the right mouse button The first thing you will notice is that the selected region will be zoomed or expanded to fill the entire spectrum window If you open the Experiment Parameter dialog box you will also notice that the receiver gain center field and sweep width have been adjusted such that the spectrum will fit in the spectrum window with optimal receiver gain SPECTRI PAR Mil Ei 10 3 20 Figure 4 10 The zoom rectangle for interactive adjust ment of parameters We are not done yet the expanded spectrum does not have the sa
97. ers to spectrum button The cursor will turn into the letter P for Parame ter Place the cursor on a spectrum window and click the left mouse button to copy the parameters to that spectrum Click the Interactive Spectrometer Control button in the tool bar to close the dialog box Deas CMA ZEHA EA New Experiment Button Figure 8 6 The New Experiment button Check the AFC Trap and High Pass Filters Click on the Signal Channel Options command in the Parameter drop down menu The Signal Channel Options dialog box will appear Signal Channel Options Bel ES Resonator Tuning Caps 32 KE c winepr tpu st9515al cal St t k H Resonator 1 Change File High Pass Filter Resonator 2 IX AFC Trap Filter Modulation Signal Input Lock in F external Acquisition Trigger Internal Internal Internal F Lock4n Integrator External External External Figure 8 7 The Signal Channel Options dialog box The AFC trap filter blocks any frequency signal compo nents at the AFC modulation frequency that may contrib ute to noise in the EPR signal The high pass filter suppresses low frequency signal components that may EMX User s Manual Cos BRUKER Calibration of the Signal Channel You do not need to edit the Static Field param eter for a signal channel calibration also contribute to added noise in the EPR signal These two filters infl
98. escribed in Section 5 4 a an et ad at GAGE Figure 6 9 Effect of using progressively higher power a c on an EPR spectrum EMX User s Manual 6 13 Cos BRUKER Optimizing Sensitivity Signal averaging With a perfectly stable laboratory envi ronment and spectrometer signal averaging and acquiring a spectrum with a long scan time and a long time constant are equivalent Unfortunately perfect stability is usually impos sible to attain and slow variations can result in considerable baseline drifts A common cause of such variations are room temperature changes or air drafts around the cavity For a slow scan the variations cause broad features to appear in the spectrum as shown in spectrum b of Figure 6 10 You can achieve the same sensitivity without baseline distortion by using the signal averaging routine with a small time constant and shorter scan time For example if you were to signal average the EPR spectrum using a scan time that was signifi cantly shorter than the variation time these baseline features could be averaged out In this case the baseline drift will cause only a DC offset in each of the scanned spectra Spec trum a shows the improvement in baseline stability through the use of short time scans with signal averaging when the laboratory environment is not stable Figure 6 10 a Signal acquired with short time sweeps and signal averaging b Signal acquired with long time sweep and long
99. f resonance b Slightly off reso nance c On resonance phase 180 off d On resonance phase 90 off e On resonance correct phase undercoupled f On resonance correct phase overcoupled On resonance correct phase critically coupled wm 8 EMX User s Manual 5 5 BRUSER Manually Tuning a Microwave Bridge EMX User s Manual 5 6 BRUSER Manually Tuning a Microwave Bridge Figure 5 3 Mode tuning pat terns for a klystron microwave source a Off resonance b Slightly off reso nance c On resonance phase 180 off d On resonance phase 90 off e On resonance correct phase undercoupled f On resonance correct phase overcoupled g On resonance correct phase critically coupled EMX User s Manual 5 7 Cos BRUKER Manually Tuning a Microwave Bridge The resonant frequency of a Bruker ER 4102ST cavity is usually approximately 9 8 GHz A cryostat will drop the frequency to approxi mately 9 4 GHz Tune the microwave source Adjust the Frequency slider bar to locate and center the mode pattern dip on the display monitor Clicking the left or right arrows will step the parameter value downwards or upwards Clicking to the left or right of the square steps the parameter value downward or upward faster than when using the arrows Keeping the mouse button pressed repeats the action auto ma
100. f you have a lossy sample in the cavity m m l J Fine sta Model ode Pot Designation PA a a SS BRUKER ER 041 XG MICROWAVE BRIDGE X BAND OS FINE AFC Fine AFC Pot ER 047 PH 7 BROAD BAND TONE e k 20Hzm6 5MHz x08 bax 3 a a SS owave ped lm f Wrs SS 3 O GA S FN FINE AFC vorr evam r F ar Ng i o 7 AOESSORY Jg FREQUENCY a COUNTER R mn Q amp Rs232 8 me A A WS WS J Figure 5 15 Two possible locations for the fine AFC potentiometer Lia Figure 5 16 Properly centered AFC meter EMX User s Manual 5 22 BRgER Fine AFC Tuning for Gunn Diode Bridges HB WINEPR ACQUISITION ST_PITCH PAR Eile Parameter Acquisition Processing Yiew Options Window Info DSSS XA Be Bo ls oe la OPERATE Frequency 9 76 GHz AFC Receiver Level LEVELLED Attenuation 20 dB ys mamm Power 2 012 mw Diode Hall Field MAANED ovo oi apc I m Meter Diode Meter Mm E Figure 5 17 Location of the AFC and diode meters 3 Switch the microwave attenuation from 40 dB to 50 dB The AFC meter may drift to the right See Figure 5 18 AFC E Figure 5 18 The AFC needle drifting towards the right 4 Increase the microwave attenuation slowly Increase the attenuation in 1 dB increments between 50 and 60 dB until you observe a significant deflection of the needle See Figure 5 19
101. ge Control dialog box If this window is not already open click its button the button labeled MW in the tool bar The button toggles the dialog box open and closed The Microwave Bridge Control dialog box will then appear See Figure 3 30 BS WINEPR ACQUISITION Microwave Bridge Control File View Options Window Info Cal Gs Ee olx esaa kkeken mE Frequency 0 00 GHz Attenuation Power Q Value AFC Lit Diode Microwave Bridge Control bquency Button Receiver Level Interactive Spectrometer Control Button 4 Button 3 Auto Tune a o Fine Tune Stop Tuning F Dual Trace bnalPhase Attenuation Display Figure 3 30 The Microwave Bridge Control dialog box Switch the microwave bridge to Stand By See Figure 3 30 Click the Stand By button in the dialog box to change to Stand By The microwave attenuator will be automatically set to 60 dB EMX User s Manual 3 31 BRUSER Turning the Spectrometer Off Exit the software first before turning the PC off A Close the Microwave Bridge Control dialog box Click the bridge controller button the button labeled MW in the tool bar The button toggles the dialog box open and closed The Microwave Bridge Control dialog box will then disappear Remove the sample from the cavity See Section 3 3 for details on how t
102. guide stabilizers Rigidly secure the sample in the cavity by tightening the collets on the cavity sample stack Do not place objects on the microwave bridge that may vibrate or are free to move Avoid placing a fre quency counter with a fan on top of the bridge Maintain a controlled environment for the best spectrometer performance Air drafts past the spectrom eter especially the cavity may induce temperature fluctua tions or microphonics from sample vibration Large fluctuations in the ambient temperature may degrade perfor mance by reducing the frequency stability of the cavity Very humid environments may cause water condensation You can reduce condensation inside the cavity by maintaining a con stant purging stream of dry nitrogen gas Note that excessive gas flow rates can generate microphonic noise through sam ple vibration Minimize electrical interference Noise pick up from electromagnetic interference EMI noise may be encoun tered in some environments You may be able to minimize EMI noise by shielding or perhaps by turning the noise source off if generated by equipment near the spectrometer There is often less EMI at night EMX User s Manual 6 5 BRUSER Optimizing Sensitivity Allow the spectrometer to warm up One hour is usu ally adequate to achieve a stable operating temperature For maximum stability under extreme operating conditions such as any combination of high microwave power high magnetic
103. h time scans Interactive adjustment of spectrometer parameters is described in the fifth topic The sixth topic contains advice on options for controlling the microwave bridge The chapter ends with advice on saving files and how to export spectra to WIN EPR for more sophisticated post process ing EMX User s Manual ph ER Spectrum Windows Spectrum Windows 4 1 Keeping Things Neat 4 1 1 The Cascade com mand stacks all the open windows such that their title bars are all visible With an EMX spectrometer you can generate a very large num ber of spectra very quickly The spectrum windows can com pletely clutter your screen just as quickly There are several ways to keep things neat The first approach is to cascade or tile the windows to organize them better The commands for per forming these operations are found in the Window menu Per haps the best way to explain what each of these commands does is to look at the following examples See Figure 4 1 Figure 4 2 Figure 4 3 EE WINEPR ACQUISITION OHCTRL2 PAR M E3 Eile Parameter Acquisition Processing View Options Window Info J EAA EEE Hel GSI Ela CARATE oyaeoe 8400 Attenuation 13 dB Power 10 08 mW AFC CEI Diode Receiver Level Hall Field DMPO_CH3 PAR ETEA Pie E E BGI Figure 4 1 Cascaded spectra EMX User s Manual 4 2 Spectrum Windows The
104. hall 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 Thy hv Absorption a Je v D Vi V2 Figure 2 2 A spectrum EMX User s Manual 2 2 Cos BRUKER 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 B 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 state is designated as M 1 2 and the antiparallel state is M 1 2 B 0 Figure 2 3 Minimum and maximum energy orientations of u
105. hat the cavity stores the microwave energy therefore at the resonance frequency of the cavity no microwaves will be reflected back but will remain inside the cavity See Figure 2 11 Reflected Microwave gt lt Av Power res VP 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 a 2m energy stored 2 5 energy dissipated per cycle 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 BRUSER Basic EPR Practice heat We can measure Q factors easily because there is another way of expressing Q res i 3 2 6 where Ves is the resonant frequency of the cavity and Av is the width at half height of the resonance Stack YA N a am gt Ne Re ae 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 exactl
106. he EPR cavity or the sample can distort your EPR spectrum High conductivity High conductivity exhibited by sam ples with mobile electrons will result in asymmetric lines known as Dysonian lineshapes This results from a mixing of absorption and dispersion components induced in the sample itself Lossy samples If you put large lossy samples in a cavity you can also obtain Dysonian lineshapes Use progressively smaller capillaries until you obtain a symmetric lineshape Microwave reference phase The dispersion signal from easily saturated samples can be very large compared to the absorption signal To minimize the contribution of the disper sion signal carefully adjust the microwave reference phase In addition make sure that the AFC offset is close to zero Magnetic field drifts Magnetic field drift may produce an asymmetric or distorted line for samples exhibiting very nar row resonance linewidths This problem may arise for line widths less than 20 mG Use a field frequency lock system to eliminate field drift problems EMX User s Manual 7 16 BpGsen Warning Noises No Signal When Everything Works 7 12 e Check cables Make sure that all the cables are connected Check the modulation cable and the preamplifier cable e Sample position If you have a small sample make sure that the sample is centered in the cavity e Magnetic field values Are you using the correct field val ues to see your EPR signal If y
107. he cavity This number is located on either the front or back of the cavity Clicking Cancel returns you to the Experiment Options dialog box EMX User s Manual 5 19 Cos BRUKER Changing EPR Cavities Signal Channel Options olki Resonator Tuning Caps 32 4 c winepr tpu st9227 cal I SCT Selftest H Resonator 1 5 High Pass Filter IX AFC Trap Filter F external Acquisition Trigger F Lock in Integrator Open Calibration File File name st9227 call dm9304 cal dpphtest cal hall0121 cal mvt9406 cal st9227 cal st9515 cal st9515a cal st9515b cal List files of type Jscr Calibration Files ce Resonator 2 Modulation Signal Input Lock in Internal Internal Internal Change External External O External File 21x Eolders c winepr tpu acy Awinepr amp tpu Network Drives J c micron id Figure 5 14 The Experiment Options and Open Cali bration File dialog boxes Service engineers often save the calibration files in the c acquisit tpu directory during the installation of the spectrometer EMX User s Manual 5 20 Cos BRUKER Fine AFC Tuning for Gunn Diode Bridges Fine AFC Tuning for Gunn Diode Bridges 5 3 The AFC Automatic Frequency Control is the circuitry used to lock the microwave source frequency to the resonant fre quency of the cavity In most
108. he display adjust the Signal Phase slider bar See Figure 5 1 until the depth of the dip is maximized and the dip looks somewhat symmetric See Figure 5 2 We shall fine tune this phase later but this procedure gets us close to the correct phase Tune the signal reference phase Klystron Microwave Sources While the dip is in the center of the display adjust the Signal Phase slider bar See Figure 5 1 until the shoulders on each side of the dip appear to be approximately the same height and the dip looks somewhat symmetric See Figure 5 3 We shall fine tune this phase later but this procedure gets us close to the correct phase EMX User s Manual 5 10 BRUSER Manually Tuning a Microwave Bridge 12 13 14 Fine tune the microwave source frequency Click the Operate button in the dialog box to change to the Operate mode Adjust the Frequency slider bar until the needle of the AFC meter is centered You can locate the AFC meter by referring to Figure 5 1 Sometimes the needle may rush off to the right or left edges of the meter This happens when the AFC Automatic Frequency Con trol is no longer locked If this happens click the Tune button to return to the Tune mode Repeat Step 9 and then try again Adjust the bias level Change the microwave attenua tion to 50 dB Adjust the Bias slider bar See Figure 5 1 until the Diode meter needle is centered You can locate the D
109. he letter K designates a klystron source Perhaps the sur est method to identify the type of source is by comparing the mode pattern with either Figure 5 2 or Figure 5 3 Observe the mode pattern on the display moni tor Gunn Diode Microwave Sources This mode pattern is a display of the microwave power reflected from the microwave cavity and the reference arm power as a function of the microwave frequency The mode pat tern should resemble one of the mode tuning patterns in Figure 5 2 If the mode pattern amplitude is too small increase the microwave power in dB steps by decreasing the attenuation If the mode pattern amplitude is too large decrease the microwave power in 1 dB steps by increasing the attenuation Observe the mode pattern on the display moni tor Klystron Microwave Sources This mode pat tern is a display of the microwave power reflected from the microwave cavity and the reference arm power as a function of the microwave frequency The mode pattern should resemble one of the mode tuning patterns in Figure 5 3 If the mode pattern amplitude is too small increase the microwave power in dB steps by decreasing the attenuation If the mode pattern amplitude is too large decrease the microwave power in 1 dB steps by increasing the attenuation EMX User s Manual 5 4 BRUSER Manually Tuning a Microwave Bridge Figure 5 2 Mode tuning pat terns for a Gunn diode microwave source a Of
110. he time constant to normalize the S N to a one second time constant C is the weak pitch correction factor that is printed on the label of the weak pitch sample The standard instrument settings for signal and noise measurements are listed in Table 9 1 There is a built in subroutine to measure the signal to noise ratio which has the default values of standard settings If you want to measure the amplitudes of the signal and noise on a print out by hand make sure that you use the same scale for both signal and noise spec EMX User s Manual 9 2 BROER Signal to Noise Ratio Test tra Otherwise you need to multiply the result by the ratio of the scales Signal Noise Parameter Measurement Measurement Modulation 8 0G 8 0G Amplitude Modulation 100 kHz 100 kHz Frequency Receiver Gain 2 0 x 10 5 0 x 10 Phase 0 0 Time Constant 1310 72 ms 1310 72 ms Conversion Time 163 84 ms 163 84 ms Center Field 3480 G 3300 G X Axis Setting Field Sweep Time Scan Sweep Width 50 G Resolution 1024 points 1024 points of X Axis Microwave 12 dB 0 dB Attenuation Table 9 1 Parameters for Signal Noise Measurements EMX User s Manual 9 3 BpGsen Signal to Noise Ratio Test Preparing for the S N Test 9 1 1 The calibration factor is found on the weak pitch sample s label It is listed as C Co X factor calibration It is usually approxi mately equal to one and corrects for variations in the sample concen
111. he value for Mod Amp G determined by the calibration routine and Max Mod is the maximum modulation amplitude listed for your cavity in Table 8 1 Return to Step 1 e g start the calibration routine again and enter this new value for Mod Amplitude Limit Continue from Step 2 through Step 7 as before Finish the Calibration When the routine is finished the message Acquisition Done will appear in the info line Double click the control menu box in the upper left hand corner of the window to close the window The sig nal channel is now calibrated for your cavity and the data saved in the calibration file The next time that you start the WIN EPR Acquisition software this calibration file will be the default calibration file EMX User s Manual 8 20 System Performance Tests 9 This chapter describes procedures for testing the performance of your Bruker EPR spectrometer The first test measures the spec trometer s sensitivity The procedure is especially designed to test as many of the components of the spectrometer as possible with one simple test It therefore gives you a good indication of the overall health of your spectrometer It is also an excellent cri terion for comparing the sensitivity of different spectrometers The second test measures the background signal of the cavity Should your spectrometer or cavity not meet specifications first consult Chapter 7 on troubleshooting If none of the hints solve the probl
112. hniques c ceeeeeneeeeeeeeeeeeeeeeeeeeeeeeeeeees 5 1 5 1 Manually Tuning a Microwave Bridge cesceeseeeeeseeeeeeeeeeeeeeneeeeneees 5 1 5 2 Chansing EPR Cavities eee a eens a oats eases ees 5 11 5 3 Fine AFC Tuning for Gunn Diode Bridges ceeeceeeeeeceeteeeeeteeeees 5 19 5 3 1 The Fine tuning Procedure percioc i a 5 19 5 4 Performing 2D EX PeMmMentss sass te2 sence pad cosaesasemniadsaie aGuewt eae ese 5 23 6 Helpful FATS drian eset dar uuda nada O anar rE 6 1 6 1 Hints for Finding EPR Signals seesseeeeeeseeesessereesseesrsssessresrersesrresseseeess 6 1 6 2 Optimizing Sensitivity senscie isanos i ena n a i ao ia S aa 6 5 6 2 1 Instrumental Factors ue moeorensererceoin as Eees ECER E E EAE 6 5 6 2 2 Parameter Select mire sic cicezeesecss ri E E ES a E EEEE 6 8 7 Tro bleshooting reesen e tant Aus 7 1 Tek ponot ready e en bald E A AEA ARRS 7 2 1 2 INO Cavity DIP ecciesie e a E E OTER 7 2 EMX User s Manual ix BRGBeR Table of Contents T3 Tuning Error neen e ed apni eta aides ays aa eae 7 3 TA ING s Tun Pict re cerita a a e Ga tage acum cea sue cera cone eee 7 4 7 5 Unable to Critically Couple G avitycc scsgcueecegesdetev ow ener reers enero 7 5 7 6 Magnet Power Supply Shuts Down 0 ceescceesseccesneceeeeecceeeeecesteeeeeaas 7 6 Tel Baseline Distortion ssis iio Seo esas lade io i e EEEE ESSES 7 7 17 89 Excessive Noise COUUDIIL airesin a TEETE s 7 9 TF POOL SENSIAVILY vcsscdadecscassacceve
113. ic Resonance and Solid Dielectrics Proceeding of the 12th Colloque Ampere North Holland Publishing Co Amsterdam 1964 Sixl H Festkoerperspektroskopie II Resonanzspektroskopie Hochschulverlag Stuttgart 1979 Sigel H A Sigel Metal Ions In Biological Systems ENDOR EPR and Electron Spin Echo for Probing Coordination Spheres Vol 22 Marcel Dekker Inc New York and Basel 1987 Skobel tsyn D V Editor Quantum Electronics and Paramag netic Resonance Plenum Press New York EMX User s Manual 10 15 BRUSER Bibliography Slichter C P Principles of Magnetic Resonance Harper and Row New York Evanstown London 1963 2nd Edition Springer Verlag Berlin and New York 1978 3rd Edition 1989 Smidt J Editor Magnetic and Electric Resonance and Relax ation Proceedings of the 11th Colloque Ampere Amster dam North Holland Publishing Co 1963 Snipes W Editor Conference on Electron Spin Resonance and the Effects of Radiation on Biological Systems Gatlin burg Tennessee 1965 National Academy of Science National Research council 1965 Sorin L M V Vlasova Electron Spin Resonance of Paramag netic Crystals Plenum Press New York Spaeth J M J R Niklas and R H Bartram Structural Analysis of Point Defects in Solids An Introduction to Multiple Magnetic Resonance Spectroscopy Springer Verlag Ber lin 1982 Specialist Periodical Reports Electron Spin Resonance R O C
114. ical reactions Polymerization reactions Spin trapping Organo metallic compounds Catalysis Petroleum research Oxidation and reduction processes Biradicals and triplet states of molecules 1 1 2 Measurement of magnetic susceptibility Transition metal lanthanide and actinide ions Conduction electrons in conductors and semiconductors Defects in crystals e g color centers in alkali halides Optical detection of magnetic resonance excited states of molecules Crystal fields in single crystals Recombination at low temperatures EMX User s Manual 1 2 Cos BRUKER EPR Applications Materials Research lonizing Radiation 1 1 3 Degradation of paints and polymers by light Polymer properties Defects in diamond Defects in optical fibers Laser materials Organic conductors Influence of impurities and defects in semiconductors Properties of novel magnetic materials High T superconductors Coo compounds Behavior of free radicals in corrosion 1 1 4 Alanine radiation dosimetry Control of irradiated foods Archaeological dating Short time behavior of organic free radicals produced by radiation Radiation effects and damage Radiation effects on biological compounds EMX User s Manual 1 3 Cos BRUKER EPR Applications Biology and Medicine 1 1 5 Spin label and spin probe techniques Spin trapping Dynamics of biomolecules using saturation transfer tech niques Free radicals in living tis
115. ields can produce large eddy currents in the cavity side walls These currents can interact with the magnetic field to produce a torque on the cavity and create a resonant fre quency shift A linear field dependent or modulation ampli tude dependent baseline is indicative of such an effect This phenomenon should not be observed if the cavity end plates are properly fitted and torqued Do not attempt to adjust the torque on the plates Contact your local Bruker EPR service representative Slowly and randomly varying baseline The use of high microwave power or large modulation fields can heat the cavity and the sample The ensuing thermal drifts in the coupling of the cavity as well as the frequency of the cavity can result in a fluctuating offset in the signal Allow the tuned cavity and sample to come to thermal equilibrium before per forming the final tuning of the cavity Once the cavity is equilibrated and properly tuned under the equilibrated condi tion you can start acquiring a spectrum Avoid air drafts around the cavity as they can randomly change the tempera ture of the cavity and sample and hence the baseline of the spectrum EMX User s Manual 7 7 Cos BRUKER Baseline Distortion Variable temperature operation Cavity frequency and coupling instability may be induced during variable tempera ture operation especially at very low or very high tempera tures Increase the flow rate of the cavity and waveguide
116. in the tool bar EMX User s Manual 8 12 BRUSER Calibration of the Signal Channel 11 Adjust the Receiver Gain Monitor the Receiver Level while the scan is running See Figure 8 9 If the needle deflects more than 1 4 of the display lower the Receiver Gain Reacquire the spectrum and lower the Receiver Gain until the needle does not deflect more than 1 4 of the display You may have to repeat this last step a few times HS WINEPR ACQUISITION ST_PITCH PAR File Parameter Acquisition Processing View Options Window Info olx CEEA CREO EDT GLE EAER OPERATE Frequency 9 76 GHz R Receiver Level Cee Attenuation 20 dB CI l PO 2 012 mW Diode Hall Field CALIBRATED 33200006 Q Value 2700 Receiver Level Figure 8 9 The Receiver Level display 12 Set the center field To interactively set the center field click the Interactive Change of Center Field Parameter button in the tool bar See Figure 8 10 Ohsas ka Ee TE e Interactive Change Button Figure 8 10 The Interactive Change of Center Field Parameter button in the Tool Bar Clicking this button creates a marker vertical line in the spectrum window that moves with the cursor Place the cursor where you would like the center field to be and click with the right mouse button See Figure 4 13 This EMX User s Manual 8 13 ph ER Calibration of the Signal Channel action repla
117. ine Correction Figure 9 6 Set Experimental Options Acquire a signal spectrum Click the RUN button in the tool bar to acquire a weak pitch spectrum See Figure 9 7 If the spectrum is off center you can use the center field tool to set the correct field center If there is a large offset you can open the Interactive Spectrometer Control dialog box to adjust the offset to the proper posi tion where the indicator of the Receiver Level is in the middle Do not forget to click the Set Parameters to the EMX User s Manual 9 9 ph ER Signal to Noise Ratio Test Spectrum button and move the pointer to the signal mea surement window and click the left mouse button again E WINEPR ACQUISITION Signal Noise Measurement Pale Es Eile Parameter Acquisition Processing View Options Window Info Ds eS DIEZIZIo Sleals ELI lel 2 Sle EI n AFC Receiver Level i E a LEVELLED Attenuation 12 dB Lit m ee Poer 12 66 mw Diode Hall Field ARAE ovaue 2500 D m 3485 440G Signal Noise Measurement of x 10 3 signal trace Peak to peak height 34722 00 10 Receiver gain 200 10 Microwave power 12 66 mW 0 Conversion Time 163 84 ms noise trace 10 noise Receiver gain 5 02 10 Microwave power 200 68 mW 3470 3480 3490 3500 G Conversion Time 163 84 ms SS SSSeoro 1_0 aaoouaas 103 linear Baseline Correction 50 Weak P
118. ing connector If this happens turn off the heat exchanger and power sup ply and tighten the leaky connector Condensation on the magnet coils may indicate an improperly adjusted thermo stat in the heat exchanger If the magnet power supply shuts itself off the thermostat may not be set properly or you do not have sufficient cold water flowing through the heat exchanger Start the WIN EPR Acquisition Application You should have Microsoft Windows already running Consult your Windows documentation for details Locate the task bar on the desk top See Figure 3 7 The position of the bar can be on the top bottom left or right edge depending on how Windows is set up Point to or click on Start and a drop down list will appear Point to or click on Programs a cascading menu will appear Point to or click on WIN EPR and then click on Acquisit to start the program The program will then initialize all the mod ules of the EMX spectrometer You can also double click the Acquisit shortcut icon to start the program If the EMX User s Manual Cos BROBER Turning the Spectrometer On Bae My Computer GET EPR jo fg Documents ia G Settings a Dp x m S o Asiant F Capture 0 1 A Acquisit shortcut has not been set up consult your Win dows documentation to learn how to set up a shortcut Sin t Accessories Adobe Acrobat T Cec488 Corel5 7 InControl Tools 95 75 InCont
119. inter and Daela eksiz EAL omea H Print Button Figure 3 27 Print button Windows documentation for further details regarding the options Clicking OK starts the document printing and closes the dialog box Clicking Cancel closes the dialog box without printing anything The output from the printer will look similar to Figure 3 29 Figure 3 28 The Print dialog box Printer Default Printer HP LaserJet 4P on LPT1 Print range Cancel Selection Pages Erom po To Zz Help Print quality josi Copies o T Collate copies EMX User s Manual 3 29 BpGgeR Acquiring Spectra FR Acomisit pate 1995 05 11 Time 14 38 Filename EPR_SPEC JOHN_sPc st_PITcH PAR Comment Strong Pitch Spectrum for EMX Manual ton Intensity Parameter List operator 4 9 spectroscopist Resonator 1 4 5t9239 cal Genter Field 3480 000 sweep Width 200 000 Resolution 512 points Micro Frequency 756 SHz Power 997 ma Beceiver_ Receiver Gain 2 00e 004 Phase 0 00 deg Harmonic 1 Mod Frequency 100 00 kHz maA anplitude 1 00 e z z Time constant Sweep Time 600 T T T T T T T T T 1 3380 3400 3420 3440 3460 3480 3500 3520 3540 3560 3580 s Figure 3 29 Typical output from the printer EMX User s Manual 3 30 Cos BRUKER Turning the Spectrometer Off Turning the Spectrometer Off 1 3 6 Open the Microwave Brid
120. into the Cavity It is vital to avoid contaminating the microwave cavity as a paramagnetic contaminant may produce spuri ous EPR signals or distorted base lines Wiping the out side of the sample tube with tissue paper is usually adequate Insert the sample tube carefully into the cavity See Figure 3 9 and Figure 3 10 Make sure you have the appropriate collet size for your sample tube size The tube should be slightly loose before you tighten the collet nut The bottom of your sample should rest in the indenta tion on the pedestal This ensures that your sample is cen tered horizontally The height can be adjusted by loosening the bottom collet nut and moving the pedestal up and down Tighten the top collet nut to firmly hold the sample tube in place and the bottom collet to firmly hold the pedestal Tune the cavity Details on this procedure are given in the next section EMX User s Manual 3 16 BpGsen Tuning the Microwave Cavity and Bridge Tuning the Microwave Cavity and Bridge 3 4 i Open the Microwave Bridge Control dialog box If this window is not already open click the button labeled MW in the tool bar The MW button toggles the dialog box open and closed See Figure 3 12 W WINEPR ACQUISITION Microwave Bridge Control Microwave File View Options Window Info Bridge Sie a2 DIE Bal eee ei Bile Ee STANDBY Frequency C Receiver Level Control Hi H Dialog mao Ne MG es 2 POWST d Microwa
121. iode bridges Reference microwave power too low klystron or Gunn diode bridge Carefully adjust the Bias slider bar of the Microwave Bridge Control dialog box until you observe a tuning mode pattern on the display Microwave bridge controller automatically switches from Tune to Stand By klystron or Gunn diode bridge There is insufficient cooling for the microwave source The protection circuitry will shut the microwave source off if the temperature rises too high Make sure that the valves for the coolant lines leading to the bridge are open See Section 3 2 Make sure that the heat exchanger is on and has sufficient water flow Microwave bridge controller automatically switches from Tune to Stand By klystron bridge only There is protection circuitry which protects the microwave source from voltage spikes To reset the protection circuitry turn the console power off for approximately three seconds and turn it on again The voltages used in the Gunn diode bridge are not sufficiently high to require this type of protection circuitry EMX User s Manual Cos BRUKER Unable to Critically Couple Cavity Unable to Critically Couple Cavity 7 5 Sample position If too much of a lossy sample is in the microwave electric field in the cavity you will not be able to critically couple the cavity Move the sample until the cou pling becomes better The sample position is particularly crit ical for flat cells and capillaries
122. iode meter by referring to Figure 5 1 The center corresponds to 200 microamperes of diode current Sometimes particularly when the cavity has a low Q the AFC meter may rush off either to the right or left and lose lock at 50 dB In most cases the AFC will lock again at higher microwave power levels If not switching between Operate and Tune modes and back again at 30 dB attenuation will lock the AFC once more Match the cavity For maximum sensitivity we need to critically couple or match the cavity to the waveguide Critical coupling results in a maximum power transfer between the waveguide and the cavity It also means that no incident microwaves are reflected back from the cav ity If the cavity and waveguide are truly matched the reflected microwave power seen by the detector should remain constant i e 0 when we vary the attenuation This is the criterion we use for critical coupling You control the coupling or matching of the cavity by adjust ing the iris screw First increase the microwave power by 10 dB i e attenuator setting 40 dB Click the Tor J iris buttons for the iris screw motor until the diode current again returns to 200 microamperes i e The needle is EMX User s Manual 5 11 Cos BRUKER Manually Tuning a Microwave Bridge centered Repeat the procedure 10 dB steps in the atten uator setting and adjust the current to 200 microamperes with the iris screw until you have reached an attenuat
123. ion The weak pitch samples from Bruker Instruments have a nominal concen tration of 101 spins per centimeter The samples are calibrated and the correction factor is printed on the side of the tube This sample is prepared for the purpose of measuring instrument per formance owing to its high stability however it is not meant as a quantitative spin counting standard EMX User s Manual 8 3 Cos BRUKER Calibration of the Signal Channel Calibration of the Signal Channel 8 2 Basic Theory You need to carefully calibrate your spectrometer s signal chan nel reference phase and modulation amplitude in order to obtain maximum sensitivity minimum distortion and quantitatively reproducible measurements The EMX027 in conjunction with the WIN EPR Acquisition software make this calibration easy to perform The results of the calibration are saved on disk for future use We recommend recalibration at least once a year to ensure quantitative and reproducible results Each cavity or reso nator has its own individual calibration file therefore this pro cedure must be followed for each cavity 8 2 1 Calibration of the signal channel involves two separate yet inter dependent procedures The first procedure is to calibrate the peak to peak modulation amplitude For the sake of brevity modulation amplitude will be used in place of peak to peak mod ulation amplitude The second procedure is to calibrate the phase difference
124. itch factor 1 02 0 2 X r Signal noise ratio 50 0 25 50 75 100 125 sec SPECTR Iie E3 Figure 9 7 Signal measurement 6 Activate the noise measurement Click the lower window to activate the noise measurement window 7 Check the parameters Open the parameter dialog box Make sure the X axis is set to Time Scan the power is 200 mW gain is 5 x 10 and the field center at 3300 G EMX User s Manual 9 10 Cos BRUKER Signal to Noise Ratio Test The other parameters should be similar to that in signal measurement See Figure 9 8 Standard Paramoter Signel Nolse Meesuroment Experiment X Time Scan M Y no Y Sweep Z Hall Signal Channel S P N 5 E Center Field 3300 00 G Receiver Gain Ke 5 02 10 Sweep Width p o0 G Modulation Frequency 100 00 kHz Static Field 3300 000 E G Modulation Amplitude e 00 G Microwave Bridge Modulation Phase o oo deg Frequency e 795000 GHz Offset fao o0 H Power 200 68 mW Time Constant fisto zz E9 msec Step 1 db Conversion Time 63 64 msec Temperature unit Sweep Time 167 77 sec Temperature 300 00 ik Harmonic fi Step 1 00 aik Resolution in X fl 024 woniometer Number of X Scans fi angie deg Resolution in Y fi Step 1 000 2 deg a Repetitive Mode y Figure 9 8 Parameters for noise measurement See Section 4 5 for help with interactive spectrometer control
125. itors Physical Methods in Advanced Inorganic Chemistry ESR NMR Moessbauer Wiley New York 1968 Hoff A J Advanced EPR Applications in Biology and Bio chemistry Elsevier Science Publishers B V 1989 Holtzman J L Spin Labeling in Pharmacology New York 1984 Hovi V Editor Magnetic Resonance and Related Phenomena Proceedings of the XVII the Congress Ampere North Holland Publishing Company Amsterdam 1973 Hudson R P Principles and Applications of Magnetic Cooling North Holland Publishing Company Amsterdam and American Elsevier Publishing Company New York 1972 EMX User s Manual 10 8 BRUSER Bibliography Hutchison Jr C A Determination of Organic Structures by Physical Methods Chapter 7 E A Braude and F C Nachod Editors Academic Press New York 1955 Hyde J S Paramagnetic Relaxation in Annual Review of Physi cal Chemistry Eyring H C J Christensen H S Johnston Editors Annual Reviews Inc Palo Alto CA 1974 Hyde J S Saturation Transfer Spectroscopy in Methods in Enzymology Enzyme Structure Part F C H W Hiss S N Timasheff Editors Academic Press New York 1978 Vol 49G No 19 pp 480 511 Ikeya M New Applications of Electron Spin Resonance Dating Dosimetry and Microscopy World Scientific Singapore 1993 Ingram D J E Free Radicals as Studied by Electron Spin Reso nance Butterworths London 1958 Ingram D J E Biological and Biochemical
126. ity Greno ble 1961 Gordon and Breach New York 1962 Dikanov S A Y D Tsvetkov Electron Spin Echo Envelope Modulation ESSEM Spectroscopy CRC Press Boca Raton 1992 Dixon W T Theory and Interpretation of Magnetic Resonance Spectra Plenum Press New York Dorio M M J H Freed Editors Multiple Electron Resonance Spectroscopy Plenum New York 1979 Drago R S Physical Methods in Chemistry W B Saunders Co Philadelphia 1977 Eaton G R S S Eaton Electron Paramagnetic Resonance in Ewing G W Editor Analytical Instrumentation Hand book Marcel Dekker New York 1990 EMX User s Manual 10 5 BRUSER Bibliography Eaton G R S S Eaton K Ohno EPR Imaging and In Vivo EPR CRC Press Boca Raton 1991 Ehrenberg A B G Malmstroem T Vaenngard Editors Inter national Conference on Magnetic Resonance in Biologi cal Systems Stockholm 1966 Pergamon Press London and New York 1967 Erbeia A Editor Resonance Magnetique Centre d actualisa tion scientifique et technique Monographies no 4 Recueil de travaux des sessions de perfectionement Insti tut National des sciences appliquees Lyon September 1967 Masson Paris 1969 Faraday Society Microwave and Radio Frequency Spectroscopy General Discussions of the Faraday Society Aberdeen University Press Aberdeen 1955 Fischer H H Heimgartner Editors Organic Free Radicals Proceedings of the Fifth International Sympo
127. ive receiver gain If the receiver gain is too low you will see the effect of digitization in the spectrum spectrum b whereas at high gain the signals will clip due to an overload in the signal channel spectrum c A good way to automati cally optimize your receiver gain is to use the set field center and field range button in the tool bar as described in Section 4 3 1 When you draw a rectangle around the entire spectrum the receiver gain is automatically set such that the newly acquired spectrum will fill the display completely oe fl ai aika rete ja perme Figure 6 5 Effect of using gain settings that are either a optimal b too low or c too high on an EPR spectrum EMX User s Manual 6 8 Cos BRUKER Optimizing Sensitivity Optimize the conversion time The conversion time you select will affect the dynamic range of your experiments The conversion time is actually the amount of time the ana log to digital converter spends integrating at one field posi tion before moving to the next field value in the sweep If you need to resolve lines that are very intense as well as lines that are very weak i e carbon 13 satellites within the same spec trum you will need to use a sufficiently long conversion time If the conversion time is too short the smaller signals will be lost in the steps of the digitizer The conversion time you select will also determine the sweep ti
128. joj x GHz AFC Receiver Level Si nal s CEI I 8 mW Diode Hall Field Measurement Cit Revco Parameters signal trace y j Peak to peak height Receiver gain 2 00 10 a Noise Microwave power 12 00 mW Measurement Conversion Time 163 84 ms Parameters noise trace noise Receiver gain 5 02 10 f G Microwave power 200 00 mW Weak Pitch Conversion Time 163 84 ms Calibration linear Baseline Correction Factor Weak Pitch factor Signal noise ratio 1 453e 006 Figure 9 4 Signal Noise Measurement Window 3 Activate the signal measurement Click the signal window the upper one A blue bar will appear on the right upper corner Check the parameter settings by open EMX User s Manual 9 7 ph ER Signal to Noise Ratio Test ing the Standard Parameter dialog box The parameters should look like those in Figure 9 5 Standard Parameter Signal Noise E Experiment X Field Sweep M Y no Y Sweep z Hall Signal Channel z 5 7 5 Center Field pasooo Ea Receiver Gain feo 10 Sweep Width 0 00 G Modulation Frequency 100 00 kHz Static Field 3483 116 G Modulation Amplitude e 00 G Microwave Bridge Modulation Phase foc deg Frequency s 766000 GHz Offset fo 00 Power 12 00 mW Time Constant 1310 72 H msec Step 1 db Conversion Time 63 64 msec Temperature unit Sweep Time 167 77 sec Temperature fF IK Harmonic
129. ki B H J M Gebicki Atlas of Electron Resonance Spec tra Academic Press New York 1967 Bleaney B K W H Stevens Reports Prog Phys 16 108 1953 Blinc R Magnetic Resonance and Relaxation North Holland Publishing Co Amsterdam 1967 Bloch F Editor Spectroscopic and Group Theoretical Methods in Physics North Holland Publishing Co Amsterdam and Interscience Wiley New York 1968 Bloembergen N Nuclear Magnetic Relaxation Drukkery fa Schotanus and Jens Utrecht 1948 Blois M S et al Free Radicals in Biological Systems Aca demic Press New York 1961 EMX User s Manual 10 3 BRUSER Bibliography Blumenfel d L A V V Voevodskii A G Semenov Applica tions of ESR in Chemistry Academic Nauk SSSR Sibirsk Old 1962 Blumenfel d L A W W Wojewolski A G Semenov Die Anwendug der Paramagnetischen Elektronen Resonanz in der Chemie Akademische Verlagsgesellschaft Leipzig 1966 Bowers K D J Owen Reports Progr Physics 18 304 1950 Boyer R F S E Keinath Editors Molecular Motion in Poly mers by ESR Harwood Academic Publishers New York 1980 Box H C Radiation Effects ESR and ENDOR Analysis Aca demic Press New York 1977 Buchachenko A L ESR of Stable Radicals Consultants Bureau New York 1965 Carrington A H C Longuet Higgins Quarterly Reviews Lon don 14 427 1960 Carrington A A D McLachlan Introduction to Magnetic Res ona
130. llow the other chapters of the manual A short list of references is given at the end of the chapter for more information This chapter is a simple how to section describing how to acquire the spectrum of sample It covers turning the spectrome ter on tuning the microwave cavity and bridge and acquiring spectra The step by step instructions lead you through the acqui sition of a strong pitch a standard sample EPR signal This tutorial introduces you to many of the convenient and com monly used features of the spectrometer The most basic operations of the Bruker EPR spectrometer are covered in Chapter 3 Chapter 5 explains further procedures such as how to change cavities how to tune a bridge manually and how to perform 2 D experiments General helpful hints for acquiring EPR spectra are presented in this chapter Before consulting this chapter you should be famil iar with the material in Chapters 2 and 3 It gives tips on where to find EPR signals as well as how to optimize the sensitivity of the spectrometer for your particular sample EMX User s Manual 1 6 BRUSER Using this Manual Chapter 7 Chapter 8 Chapter 9 Chapter 10 Sometimes things go wrong Chapter 7 gives some possible solutions to problems you may be having Many times problems appear to be the fault of the instrument however with the proper choice of operating conditions these problems often disappear This chapter describes
131. me That is the sweep time will be equal to the conversion time multiplied by the number of data points in the spectrum See selecting the number of data points below Optimize the time constant for the selected conver sion time The time constant filters out noise however if you choose a time constant that is excessively high relative to your sweep time you may actually filter out your signal You should adjust your time constant to fit the conversion time you have selected These two parameters are actually very related because the conversion time will determine the total sweep time You need to use a time constant that will be suf ficiently long to filter out undesirable noise yet short enough that you do not distort your signal Therefore if you want to use a longer time constant you will need to increase the scan time as well Figure 6 6 shows the effect of progressively increasing the time constant while maintaining the same sweep time All the spectra are at the same scale A safe rule of thumb is to make sure that the time needed to scan through an EPR signal i e one EPR line is ten times greater than the length of the time constant A time constant that is 1 4 that of the conversion time will guarantee that your spectrum is not distorted However for samples limited by a low signal to noise ratio you may want to make the time constant equal to the conversion time or greater EMX User s Manual 6 9 BRUSER O
132. me number of points as the original spectrum You also need to acquire the spectrum with the newly optimized parameters EMX User s Manual BROKER Field Sweeps Click on the RUN button in the tool bar The spectrum will then be nicely centered as in Figure 4 11 after being acquired with the optimized center field receiver gain and sweep width SPECTR3 PAR Lot 10 3 400 300 200 100 0 100 200 300 400 3420 3440 3460 3480 3500 G Figure 4 11 An optimized spectrum Setting Center Fields 4 3 2 Sometimes you may not have to change all the parameters such as receiver gain and sweep width setting the center field may be sufficient To interactively set the center field click the Interac tive Change of Center Field Parameter button in the tool bar See Figure 4 12 CS Jc tl 3 Ele Ge Interactive Change Button Figure 4 12 The Interactive Change of Center Field Parameter button in the tool bar EMX User s Manual 4 12 Bpigen Field Sweeps Clicking this button creates a marker vertical line in the spec trum window that moves with the cursor See Figure 4 13 Place the cursor where you would like the center field to be and click with the right mouse button This action replaces the center field value with the magnetic field position of the marker 10 3 20 10 10 3460 3480 3500 3520 3540 G Figure 4 13 The center field marker To acquire the s
133. meters In order to perform drag and drop func tions you have to turn off certain functions such as Zooming Change Center Field and Sweep Width Change Center Field Change Static Field and Interactive Receiver Level The Duplicate and Create New Spectrum buttons in the tool bar 4 1 3 You may often need the same set of parameters to acquire a whole series of spectra The parameters of one spectrum window can be easily transferred to another spectrum window This is easy accomplished by using the Duplicate button in the tool bar See Figure 4 5 Clicking this button creates a new spectrum window with parameters that are identical to the original active spectrum You can also drag and drop an EPR spectrum and its parameters to another spectrum window Click the spectrum window with the left mouse button and the pointer will change into a hand sign Drag to the spectrum window where you want to copy and the pointer will change into a spectrum sign Releas ing the mouse button will copy the spectrum and the parameters to the window EMX User s Manual 4 5 BpGsen Spectrum Windows Resizing Spectrum Windows 4 1 4 You can fit more spectra in the application window if you resize the spectrum windows to a smaller size Make sure they are large enough to see all their important details Click and drag the window borders to change the size of the spectrum You can still make them larger by clicking the Maximize butt
134. nce with Applications to Chemistry and Chemical Physics Harper and Row Chemistry Series 1967 Caspers W J Theory of Spin Relaxation Interscience New York 1964 Catoire B Editor Electron Spin Resonance ESR Applications in Organic and Bioorganic Materials 1990 Conference Proceedings Springer Verlag Berlin 1992 Clarke R H Editor Triplet State ODMR Spectroscopy Wiley New York 1982 Cohen G B Giovannini EPR of Magnetic Ions in Metals Conf Proc Haute Nendez Switz 3 5 Sept 1973 Uni versite de Geneve Geneve 1974 EMX User s Manual 10 4 BRUSER Bibliography Coogan C K Editor International Symposium on Electron and Nuclear Magnetic Resonance Melbourne 1963 Coogan C K N S Ham S N Stuart J R Pilbrow G V H Wil son Editors International Symposium on Electron and Nuclear Magnetic Resonance Melbourne 1969 Plenum Press New York 1970 Cross R C Editor Molecular Relaxation Processes Academic Press New York 1966 Czoch R A Francik Instrumental Effects in Homodyne EPR Spectrometers Horwood Chichester UK 1989 Dalal D P S S Eaton G R Eaton The Effects of Lossy Sol vents on Quantitative EPR Studies J Magn Res 44 415 1981 Dalton L R Editor EPR and Advanced EPR Studies of Biolog ical Systems CRC Press Boca Raton 1985 DeWitt C B Dreyfus P G de Gennes Editors Low Tempera ture Physics Les Houches Lecturers Univers
135. ntation for further details EMX User s Manual 4 33 Cos BRUKER Sending Spectra for Processing Sending Spectra for Processing 4 8 Sending Spectra to WIN EPR and SimFonia 4 8 1 Sv IES EAE EAE pl DIO Ls You can directly send a spectrum to WIN EPR for immediate processing Click the spectrum you want to process to activate the spectrum window and then click the Send Spectrum to WIN EPR button in the tool bar See Figure 4 37 The WIN EPR data processing program will open with the spectrum automatically loaded Consult the WIN EPR documentation about details of data processing Send Spectrum to WIN EPR Send Spectrum to SimFonia Figure 4 37 The Send Spectrum to WIN EPR button and Send Spectrum to SimFonia button in the tool bar You can also send a spectrum to the SimFonia program for sim ulation without quitting the Acquisit program Click the spec trum you want to simulate to activate that spectrum window and then click the Send Spectrum to SimFonia button in the tool bar See Figure 4 37 The SimFonia program will open with the spectrum automatically loaded Consult the SimFonia docu mentation for details about simulations EMX User s Manual 4 34 Additional Techniques 5 This chapter provides instructions for procedures that are routine for some users but may be infrequently encountered by others Specifically the chapter will describe manually tuning the EMX spectrometer
136. ntries in a list For example it is used in the Save As dialog box to choose subdirectories Clicking the up or down arrows scrolls the list up and down Keeping the mouse button pressed repeats the action automatically The position of the viewed entries in the list is indicated graphically by the square The list may be scrolled as well by clicking and dragging the square This button returns you to the original window or dialog box when clicked All the changes made in the dialog box are set This button returns you to the original window or dialog box when clicked All changes made in the dialog box are canceled EMX User s Manual 3 7 BpGsen Turning the Spectrometer On Turning the Spectrometer On 3 2 al 7 1 Turn on the power for the system How you do this O depends on how the electric power was hooked up when the spectrometer was installed Most likely you will acti vate the switch on the breaker box for the spectrometer If you are not sure how th l Breaker boxes are usually mounted on the wall Consult e electric power is ae connected consult the local instrument or facilities manager if you are not your local instrument sure where the breaker box is or facilities manager 2 Turn on the power for the console The power switch for the console is located in the lower right corner of the back of the console See Figure 3 4 The LED s on the various units in the console will light up
137. ntrol dialog box will then disappear See Figure 3 12 Proceed to the next section to learn how to acquire spectra EMX User s Manual 3 19 Cos BRUKER Acquiring Spectra Acquiring Spectra 3 5 CSc ad 3 dP WS SY e E Create New Spectrum Button 1 Follow the instructions of Section 3 2 through Section 3 4 You should have the spectrometer turned on a Bruker ER 4102ST standard cavity installed with a strong pitch sample in it and the microwave bridge and cavity tuned Experiment Parameter Button Figure 3 13 The Experiment Parameter and Create New Spectrum buttons Create a new spectrum window if needed If you have just started the Acquisition program it automatically presents you with an empty spectrum window containing default parameters If there is no empty spectrum window create one by clicking on the Create New Spectrum button in the toolbar See Figure 3 13 Open the Experiment Parameter dialog box in order to check the parameters If this window is not already open click its button in the tool bar See Figure 3 13 The button toggles the dialog box open and closed Check the experiment type Check to see if Experi ment X is set to Field Sweep See Figure 3 14 If it is set to Time Sweep change it to Field Sweep Experi ment Y must be set to No Y Sweep EMX User s Manual 3 20 Cos BRUKER Acquiring Spectra Standard Parameter Spectr Lx
138. o G Microwave Bridge Modulation Phase o 00 deg Frequency 8 814000 GHz Offset 2 00 H Power 100 80 mW Time Constant 1310 72 H msec Step 1 db Conversion Time 63 64 msec Temperature unit Sweep Time 167 77 sec Temperature 300 00 al K Harmonic fi Ea Step fi 00 aik Resolution in X fi 024 Soniometer Number of X Scans fl TIE gag Resolution in Y fi Step 1 000 I deg oH Repetitive Mode p Figure 9 10 Set the parameters for weak pitch sample 2 Set receiver gain properly Since the microwave power 3 db is higher than in the signal noise ratio test you may need to adjust the receiver gain accordingly The suggested receiver gain is 1 x 10 3 Set a time delay Since a very long time constant is used set a delay time of 2 5 seconds to avoid overshoots or undershoots in the first few data points when you acquire the spectrum Open the Experimental Options dialog box found in the Parameter drop down menu and set the Delay before each sweep option and a delay of two to five seconds See Figure 9 11 EMX User s Manual 9 17 BRUSER Cavity Background Signal Test Field Controller Microwave Settings lt lt Sweep Direction Up zi Power Flyback On zi Power Flyback Settling Condition Wait LED off z Field Offset Correction 0 00 G IX MW Fine tune before Temperature Unit Tolerance fico fey K Delay before each sweep Bd Temperature Flyback Off Fd
139. o do this Cover the upper collet or insert a blank collet plug Exit the WIN EPR Acquisition program Click on the File menu bar and then click Exit If there are any unsaved spectra you will be asked if you wish to save them It is important to exit the software in an orderly manner i e don t just turn the computer off before exiting the software because many instrument parameters are set to specific values for a safe shut down of the spectrome ter Identify your power supply There are two types of power supplies and you must identify which type you have See Figure 3 31 The small ER 080 081 power supplies are located directly below the console and are accessed by opening the glass door See Figure 3 31 A If you have this type of power supply go to Step 8 The Bruker ER 082 083 085 086 magnet power supplies are larger They are not under the console but free standing See Figure 3 31 B If you have this type of power sup ply skip to Step 9 for instructions on turning your power supply off EMX User s Manual 3 32 BRgER Turning the Spectrometer Off Electricity Power On Off A RB Power On Off Switch The small ER 080 081 The large ER 082 083 085 086 power supply Go to step 8 power supply Go to step 9 Figure 3 31 Two types of power supplies EMX User s Manual 3 33 BRUSER Turning the S
140. odulation Freg floo co Feqxrz Kil E gt Modulation Amplitude fo 96 Hye Field 3480 152 G Modulation Phase H jpo HH ieg Setup Scan Offset 0 00 Hx l Enable Time Constant pea HH msec Sweep Width fioo o0 a ConversionTime paz HJ nmsec Harmonic oOo MW Attenuator Ede p dB Temperature i K Set parameters to spectrum SCT Options Figure 4 25 The Interactive Spectrometer Control dia log box EMX User s Manual 4 22 Cos BRUKER Interactive Spectrometer Control It is best to move the Interactive Spec trometer Control dia log box up as high as possible before enabling the Setup Scan The Setup Scan display will then be as large as possible To supply immediate feedback the dialog box also has a Setup Scan option in which the magnetic field is rapidly swept up to 50 Gauss in order to display the EPR spectrum on the screen This is achieved by setting the main magnetic field with the field controller thereby setting the center field of the Setup Scan and sending current through the modulation coils of the cavity to produce the rapid sweep Click on the Enable button to activate the option Use the field slider bar to center your spectrum in the setup scan See Figure 4 26 Hall Signal Channel Center Field Sweep Width Calibrated ype P EA 100 00 HHkrz Modulation Amplitude So Modulation Phase by l HH deg Receiver Gain Sweep Address 7 4 aan Field 3480
141. 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 amplitude the EPR signal is transformed into a sine wave with an amplitude proportional to the slope of the signal See Figure 2 14 gt al i First Derivative Figure 2 14 Field modulation and phase sensitive detec tion EMX User s Manual 2 18 Cos BRUKER 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 vs
142. off the magnet power supply and observe the noise level If the noise level changes during either of these tests consult your local Bruker EPR service representa tive for alternate installation planning EMX User s Manual 7 9 Cos BRUKER Excessive Noise Output Microphonic generated noise Secure the waveguide and cavity assembly by using the plastic waveguide stabiliz ers Secure the sample firmly in the collet If you use a cry ostat make sure that the cryostat sits firmly in the cavity Make sure that an excessive nitrogen gas flow rate through the cryostat does not vibrate the sample Worn iris screw Check for a worn iris coupling screw An iris screw that does not fit snugly in the waveguide may gen erate noise by modulating the cavity coupling Replace the worn iris screw with a new one Boiling liquids If you are using a dewar with a boiling refrigerant such as liquid nitrogen you will need to increase the AFC modulation level EMX User s Manual 7 10 Cos BRUKER Poor Sensitivity Poor Sensitivity 7 9 Excessive microwave power The microwave power may be set too high which will cause your sample to satu rate Optimize the power for your sample by recording spec tra at a variety of power levels Wrong cavity type for sample The type of cavity you use for a particular sample can make a large difference in sensitivity Consult the Bruker literature on the full line of EPR cavities to
143. on Details are given in Section 3 1 Zooming Spectra 4 1 5 You can zoom in on specific areas of a spectrum in two ways The first way is to use the tools in the tool bar See Figure 4 6 Spectra can be increased or decreased in size by factors of two by clicking the expand and contract buttons Clicking on the off set arrows shifts the spectrum up and down Note these actions only affect the display not the actual data set Zooming Divide Button Times 2 by 2 A Button Button Wa TREES Shas Be Soe DE Offset Offset Plus Minus Button Button Figure 4 6 Some Display commands in the tool bar EMX User s Manual 4 6 BRUSER Spectrum Windows Times 2 Divide by 2 Offset Plus Offset Minus Reset Clicking the Times 2 command decreases the vertical display range by a factor of two This corresponds to enlarging the verti cal size of the spectrum by a factor of two Pressing function key F5 from the keyboard has the same result Clicking the Divide by 2 command increases the vertical dis play range by a factor of two This corresponds to reducing the vertical size of the spectrum by a factor of two Pressing func tion key F6 has the same result Clicking the Offset plus command shifts the spectrum upwards Pressing function key F7 has the same result Clicking the Offset minus command shifts the spectrum down wards Pressing function key F8 has the same result All of the above operations may
144. on a printer You will learn to acquire an EPR spectrum of a standard sample with the WIN EPR Acquisition Software There will also be recom mended precautions to prevent damage to the instrument All the components possess self protecting features however it is good lab practice to follow correct operating procedures and not rely on the protection circuitry No in depth knowledge of EPR is required however we recommend that you familiarize yourself with some of the material in Chapter 2 To help you in the fol lowing sections Figure 3 1 will assist you in identifying the various units which comprise the EPR spectrometer Console af 7 um A a BRUKER I Figure 3 1 Modules and components of the EMX spectrometer EMX User s Manual Cos BRUKER Brief Tips on Windows 95 Brief Tips on Windows 95 Not everyone may be familiar with Microsoft Windows The following section explains some basic aspects of Windows It is not meant to be an in depth treatise the Microsoft documen tation should be consulted for more details If you are already familiar with Windows you can easily skip this section If you have not used Windows before we highly recommend Microsoft s on line Windows tutorial The tutorial can be found under Help Title i Bar Menu ool Bar B ar ipa K W WINEPR AC
145. ons for improvements corrections or bug reports If there is anything you particularly liked tell us as well With your input and assistance Bruker can continually improve its products and documentation You can send your messages and correspondence via e mail FAX telephone or mail It is important to include the document name product name version number and page number in your response Here are the addresses and numbers to which you can send your messages e mail epr_applications bruker com FAX 978 670 8851 Tel 978 667 9580 mailing EPR Division address Bruker Instruments Inc 19 Fortune Drive Manning Park Billerica MA 01821 USA Thank you for your help EMX User s Manual oo BREBER Chemical Safety Electrical Safety 0 1 Do not remove any of the protective covers or panels of the instrument They are fitted to protect you and should be opened by qualified service personnel only Power off the instrument and disconnect the line cord before starting any cleaning work in the spectrometer Never operate the instrument with the grounding cord disconnected or by passed Facility wiring must include a properly grounded power receptacle Chemical Safety 0 2 Individuals working with hazardous chemicals toxic substances or enclosed liquid samples must take every precaution possible to avoid exposure to these agents As a general rule THINK OF THE CHEMICAL LABORATORY AS A HAZARDOUS ENVIRONMENT IN WHIC
146. or setting of 10 dB You will notice that as you increase the microwave power the diode current becomes more sensi tive to the position of the iris screw Another thing you may notice is that the AFC meter also changes with the iris screw position Simply adjust the frequency slider bar until the needle is centered again When you have reached 10 dB microwave attenuation adjust the Signal Phase slider bar until you achieve a local maximum in the diode current You should not have to adjust it very much Ver ify that you have achieved critical coupling by changing the microwave attenuation from 10 dB to 50 dB with vir tually no change in the diode current Repeat the matching and bias level adjustment procedures if necessary If you need to operate at power levels greater than 20 mW 10 dB set the attenuator to 0 dB and once again adjust the diode current to 200 microamperes with the iris screw The current can sometimes drift because the high micro wave power Starts to heat the sample If this happens wait a minute or two and readjust the coupling EMX User s Manual 5 12 BpGsen Changing EPR Cavities Changing EPR Cavities 5 2 i Open the Interactive Spectrometer Control dia log box If this window is not already open click its but ton See Figure 5 5 in the tool bar The button toggles the dialog box open and closed The Interactive Spec trometer Control dialog box will then appear See Figure 5 6 EACE EAE Baa
147. ord and New York International Series of Monographs in Natural Philosophy Vol 33 1971 Teh F Y Editor Symposium on Electron Spin Resonance of Metal Chelates Cleveland 1968 Electron Spin Reso nance of Metal Complexes Plenum Press New York 1958 Townes C H and A L Schawlow Microwave Spectroscopy McGraw Hill New York 1955 Ursu I La Resonance Electronique Dunod Paris 1968 Ursu I Resonata Electronic de Spin Bucuresti Editura Acad emiee Republicii Socialiste Romania 1965 EMX User s Manual 10 17 BRUSER Bibliography Ursu I Editor Magnetic Resonance and Related Phenomena Proceedings of the 16th Colloque Ampere Acad Socialist Repub Romania Bucharest 1971 Van Gerven L Editor Koninlijke Vlaamse Academie voor Wetenschappen Proceedings of the 13th Colloque Ampere North Holland Amsterdam 1965 Van Reijen L L Electron Spin Resonance Studies of Pentava lent and Trivalent Chromium Amsterdam 1964 Varian Associates Workshop on Nuclear Magnetic Resonance and Electron Paramagnetic Resonance Pergamon Press New York 1959 Vonsovskii S V Editor Ferromagnetic Resonance U S Dept of Commerce Washington 1965 Waugh J S Editor Advances in Magnetic Resonance Vol 1 Academic Press New York 1965 Weil John A Editor Electronic Magnetic Resonance of the Solid State The Canadian Society for Chemistry Ottawa Ontario Canada 1987 Weil John A J R
148. ou are using a cryostat remember that the microwave frequency drops and hence the field for resonance will also be lower Is the Hall probe posi tioned properly in the magnet Warning Noises 7 13 e High pitched noise from the heat exchanger The heat exchanger will emit a high pitched noise when it requires more distilled and deionized water Funny noises from the iris motor Stop turning the iris motor immediately You may be breaking the iris screw EMX User s Manual 7 17 EPR Spectrometer Calibration 8 For many experiments it is vital that your spectrometer is care fully calibrated For example it is essential to know the precise values of the magnetic field modulation amplitude in order to obtain quantitative EPR spectra The calibration procedures in this chapter enable you to measure the experimental conditions produced by the spectrometer with considerable accuracy This chapter is not meant to be a general overview of spectrome ter calibration and quantitative EPR Therefore we highly rec ommend the following references which discuss the topic in much greater detail e Poole C P Electron Spin Resonance a Comprehensive Trea tise on Experimental Techniques First Ed Interscience New York 1967 e Poole C P Electron Spin Resonance a Comprehensive Trea tise on Experimental Techniques Second Ed Wiley New York 1983 e Alger R S Electron Paramagnetic Resonance Interscience New York 1968
149. ou first start the Acquisition program with a message such as Field Control ler not ready or Signal Channel not ready you have probably forgotten to turn the console power supply on 7 2 Waveguide gasket installed improperly See Figure 5 12 for the proper orientation of the gasket Cavity undercoupled or overcoupled First look at the microwave frequency where you normally expect the cavity to resonate and then adjust the iris screw for better coupling This can occur when working with lossy samples such as aqueous solutions in flat cells or capillaries You need more microwave power If you are using insufficient microwave power it can be difficult to see the cavity dip We recommend setting the microwave attenuator at 25 dB for the best visibility You are not at the correct frequency By putting the sample in you will cause the cavity to resonate at a lower frequency Thus you will usually need to lower the frequency after you have placed the sample in the cavity in order to see the dip EMX User s Manual 7 2 Cos BRUKER Tuning Error Tuning Error 7 3 Both the auto tune and fine tune procedures of the microwave bridge controller will terminate with an appropriate error mes sage if a particular parameter cannot be set or optimized Here are the possible error messages Tuning Frequency Both the upper and lower limits of the frequency range i e 8 9 9 9 GHz have been reached and no defined dip ha
150. pectrometer Off 8 Turn off the heat exchanger and magnet power supply Instructions for Small Power Supplies Turn the power supply off by pushing the POWER ON OFF button See Figure 3 31A If you have a heat exchanger Not all systems require a heat exchanger you must turn it off by pressing the power switch See Figure 3 32 Skip to step 10 for instructions on turning the tap water off Power Switch _ _ eee Figure 3 32 The location of the heat exchanger power switch EMX User s Manual 3 34 BRUSER Turning the Spectrometer Off If you have many power outages or elec trical storms it is a very good idea to shut off power to the spec trometer 10 11 Turn off the heat exchanger and magnet power supply Instructions for Large Power Supplies On systems with large power supplies the power supply is not under the console but free standing See Figure 3 31B You need to first press the POWER OFF button and then the ELECTR ON button Pressing the POWER OFF button also turns the heat exchanger off Turn off the tap water for cooling There are usually two valves one for the supply and one for the return or drain Consult the local instrument or facilities manager if you are not sure where the valves are Turn off the power for the console The power switch for the console is located in the lower right corner of the back of the console See Fig
151. pectrum with the new center field click on the RUN button in the tool bar The newly acquired spectrum will then be nicely centered as in Figure 4 14 SPECTRS5 PAR joi 10 3 20 10 0 10 3440 3460 3480 3500 Figure 4 14 A centered spectrum EMX User s Manual 4 13 Cos BRUKER Field Sweeps Signal Averaging Keep in mind when measuring peak heights or double integration that you need to nor malize the results by the number of scans 4 3 3 If you are looking for very weak signals you can increase your signal to noise ratio by signal averaging This process involves repeatedly acquiring the spectrum and adding each spectrum together Actually this is not an average in the strict mathemati cal sense It is not normalized by the number of scans but is the sum of the individual spectra As a result the signal increases proportionally with N the number of scans Owing to the random nature of noise its increase will only be proportional to VN The resultant enhancement of signal to noise is then pro portional to VN In order to signal average we must open the Experiment Parameter dialog box Enter the number of scans you wish to average See Figure 4 15 In this example 16 scans results in a four fold improvement See Figure 4 16 Acquire the spec trum by clicking the RUN button in the tool bar Standard Parameter Spectr1 Lx Experiment X Field Sweep
152. 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 Cos BRUKER 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
153. ptimizing Sensitivity adea er oe Figure 6 6 Effect of using a progressively longer time constant a d on an EPR spectrum Selecting the number of data points The number of data points is the other parameter that will determine the appropriate sweep time A general rule is to make sure that you have at least 10 data points within the narrowest line that you are trying to resolve This means that for EPR signals with very narrow lines you will need to increase the number of data points that are collected for a given field sweep How ever if the lines of your EPR signal are sufficiently wide increasing the number of data points will not yield any addi tional information but will only result in longer sweep times With the EMX you can select 512 1024 2048 4096 or 8192 data points Remember you will probably want to increase EMX User s Manual 6 10 BRgER Optimizing Sensitivity the time constant by a factor of two as you double the number of data points Figure 6 7 shows the enhancement in resolu tion achieved by increasing the number of data points Figure 6 7 Expanded view of narrow lines in an EPR spectrum using 1024 points a or 8192 points b e Optimize the field modulation amplitude Excessive field modulation broadens the EPR lines and does not con tribute to a more intense signal Figure 6 8 shows the results of excessive field modulation You can see how some of the smaller line
154. r s Manual 5 24 Cos BRUKER Performing 2D Experiments Performing 2D Experiments 5 4 Using the WIN Acquisition software you can perform experi ments in which a second parameter i e in addition to the mag netic field can be varied For example you can perform a set of experiments in which the power is increased incrementally over several successive field scans Alternatively you might perform several consecutive experiments in which the temperature is ramped either up or down between each field scan You can then display the 2D dataset using WIN EPR This section will describe how to utilize the Acquisition software to create a 2D data set and how to display it in WIN EPR The procedure is more easily described by performing an example experiment that investigates the response of the strong pitch spectrum to microwave power 1 Insert the strong pitch sample Place the strong pitch sample into the cavity and tune the spectrometer as described in either Section 3 4 or Section 5 1 2 Open the Experimental parameter dialog box If this window is not already open click its button See Figure 5 21 in the tool bar The experimental parameter dialog box will then appear 3 Change the Y experiment setting The Y Experi ment setting will probably be set to No Y Experiment Change this by selecting MW Power Sweep See Figure 5 21 EMX User s Manual 5 25 Cos BRUKER Performing 2D Experiments St
155. re 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 i AE gt i Absorption ee B gt Figure 2 4 Variation of the spin state energies as a func tion of the applied magnetic field EMX User s Manual 2 5 BRUSER 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 HpBo 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 i Sa 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 B e for a g 2 signal at selected microwave frequencies EMX User s Manual 2 6 Cos BRUKER Basic EPR Theory Hyperfine Interactions 2 1 3 Measurement of g factors can give us some useful information however it does not tell us much about
156. re 9 12 Acquire a weak pitch signal 8 Open Microwave Control dialog box and set to Stand by bridge and cavity Remove the weak pitch sample and retune the EMX User s Manual 9 19 BRUSER Cavity Background Signal Test 10 Duplicate the weak pitch spectrum window Click the Duplicate button in the tool bar Eile Parameter Acquisition Processing View Options Window Info ihel k khera Bele 2 e 2 exile ela MNOPERATENEE Frequency 9 02 GHz AFC Receiver Level LEVELLED 3 a et Attenuation Power 100 6 i mW MNCALIBRATEDI Value 1 Diode Hall Field 0 CI o 3480 000G 10 3 1 x10 3 3470 3480 3490 3500 3510 Figure 9 13 Duplicate weak pitch spectrum 11 Change the Center Field and Sweep Width Open the Experiment parameter dialog box Change the Cen ter Field to 2600 G and the Sweep Width to 5000 G EMX User s Manual 9 20 BRgER Cavity Background Signal Test Other parameters should be the same as that for the weak pitch measurement See Figure 9 14 and Table 9 2 Experiment X Field Sweep M Y no Y Sweep z Hall Signal Channel Center Field 2600 00 Fc RecenenGein wo eam Sweep Width 5000 00 G Modulation Frequency f o0 00 fal kHz Static Field 2480 000 G Modulation Amplitude e 00 G Microwave Bridge Modulation Phase o oo deg Frequency 9 819000 GHz Offset 2 00 a1 Power 100
157. re each By away from the original signal See Figure 2 6 a B gt lt 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 EMX User s Manual 2 8 Cos BRUKER 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 ii 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 increasing microwave
158. ree letters that identify the type of cavity ST for ER 4102ST or TM for ER 4103TM followed by the serial number of the cavity This number is found on the back or front of the cavity Click on the Change File button to open the Open Calibration File dialog box Signal channel cali bration files are normally stored in the tpu subdirectory along with field controller and other calibration files Enter a filename and click OK EMX User s Manual 8 15 Cos BRUKER Calibration of the Signal Channel Better safe than sorry It is a good idea to cali brate the phase of the second harmonic when running the calibration routine Open Calibration File zix File name Folders p51 5al cal c winepr tpu Cancel st9515al cal 5 st9515b cal st9515c cal Help st9515d cal sto9122 cal test cal Newark tm4103 cal tm4103a cal List files of type Drives Jscr Calibration Files ce J c micron fd Figure 8 13 The Calibrate Signal Channel dialog box Set the frequency limits A calibration is required for each modulation frequency that you intend to use The standard signal channel the EMX 027 SCT H has a range of 100 kHz to 6 kHz in 0 1 kHz steps Most people will normally run all their spectra using 100 kHz modula tion but under some special circumstances other frequen cies may be desirable A good approach to take is to calibrate the signal channel every 10 kHz from 100 kHz to 10 k
159. req kHz Res TuningC nF Mod Amp max Mod Amp G Phase 1 Phase 2 50 0 1 25 00 103 Figure 8 14 The Calibration routine The calibration file consists of a table of parameter values and settings for each modulation frequency The first parameter is the modulation frequency The second col umn indicates the resonator that was selected in Step 5 For modulation frequencies greater than or equal to 50 kHz the optimal tuning capacitor value is listed in the third column The fourth column contains the value of Mod Amp max Mod Amp G in the fifth column is EMX User s Manual 8 17 BRUSER Calibration of the Signal Channel the measured maximum modulation amplitude when the corresponding Mod Amp max is used Phase 1 and Phase 2 in columns six and seven respectively are the phases at which the first and second harmonic signals are nulled Check Mod Amp G at 100 kHz The calibration routine performs its task sequentially starting with the highest modulation frequency and continuing for each selected modulation frequency As each parameter is determined it is displayed in the table See Figure 8 14 Wait until the 100 kHz calibration is completed and note the value in the fifth column of the table Mod Amp G This value can allow excessive current to flow through the modulation coils of the cavity at the maximum modula tion amplitude resulting in damaged modulation coils Compare the Mod
160. responds approximately to the vertical center of the cavity Open the Interactive Spectrometer Control dia log box Click the Interactive Spectrometer Control button in the tool bar and the dialog box will appear See Figure 8 5 We can now optimize some of the parame ters and adjustments for the calibration routine Set some parameters Set the Microwave Attenuator to approximately 25 dB The Time Constant needs to be set to a low value less than about 0 16 ms A Modula tion Amplitude of 1 Gauss is usually sufficient Set the Sweep Width to 100 Gauss A Receiver Gain of approximately 1 x 10 works well Click the Enable button for the Set Up Scan When this option is enabled the magnetic field is swept rapidly up to 50 Gauss to provide a real time display of the EPR spectrum on the screen Center your DPPH spectrum in the display Adjust the Field slider bar until the signal appears centered in the Setup Scan window For a microwave frequency of about 9 78 GHz DPPH resonates at 3480 Gauss Adjust the Receiver Gain so that the signal fills approximately half of the vertical display range Make sure that the sig nal channel is set to 100 kHz modulation and first har monic detection EMX User s Manual 8 9 BREBER Calibration of the Signal Channel W interactive Spectrometer Control BEE Hall i nal Channel Center Field 3474 00 Magnetic Calibrated Sweep Wi
161. riment Options dialog box Click on the OK button and the dialog box will then disappear Cancel exits the dialog box without saving the changes CO Sf Ol Le fru Run Button Figure 3 17 The Location of the RUN Button 12 Acquire a spectrum Click the RUN button to start an acquisition See Figure 3 17 If you have a similar spec trum like the one in Figure 3 18 congratulations You have successfully acquired an EPR spectrum You may notice that the EPR line is not nicely centered The next step will help you center your spectrum Spectr Bel 10 3 2 1 3325 3350 3375 3400 3425 3450 3475 3500 G Figure 3 18 An EPR spectrum of strong pitch EMX User s Manual 3 23 BRgER Acquiring Spectra is EINE EAN KETE Interactive Change Button Figure 3 19 The Interactive Change of Center Field Parameter button in the Tool Bar 13 Center a spectrum To interactively set the center field click the Interactive Change of Center Field Parameter button in the Tool Bar See Figure 3 19 Clicking this button creates a marker vertical line in the spectrum window that moves with the cursor See Figure 3 20 Place the cursor where you would like the Spectri MEE 10 3 2 1 3325 3350 3375 3400 3425 3450 3475 3500 G Figure 3 20 The center field marker center field to be and click with the right mouse button This action replaces the center field value with the mag ne
162. rning dialog box for overwriting files 16 Prepare to print the spectrum Turn the printer on Make sure a sheet of paper is loaded Refer to your printer is yout printer online documentation for details EMX User s Manual 3 27 Cos BRUKER Acquiring Spectra 17 Select the Output Formatting Click on the File menu bar and then click Output Formatting A cascading menu will appear as shown in Figure 3 26 Select Spec trum Parameters by clicking on it A checkmark next to it indicates the option is active Both the spectrum as well as the instrumental parameters will be displayed when you print the spectrum MB WINEPR ACQUISITION ST_PITCH PAR E Perameter Acquisition Processing View Options Window Info New Ctrl N Open Ctrl O Load Ctrl L Duplicate Ctrl D Close Save Ctrl S Save As SendTo Copy Ctrl C Print Ctrl P Print Preview Ou ting Spectrum 1ST_PITCH PAR 2C TEMP SPEC1 PAR 3 CAWINEPR DATA UNKNOWN PAR Exit 3325 3350 3375 3400 3425 3450 3475 3500 G Select the active spectrum and the acquisition parameter Figure 3 26 Selection of the Output Formatting 18 Print the spectrum Click on the Print button in the tool bar See Figure 3 27 A dialog box will then appear in which you enter the desired options and print your EMX User s Manual 3 28 ph ER Acquiring Spectra spectrum See Figure 3 28 Consult your pr
163. rol Tools Shortcuts 7 Mediamatics Software MPEG Al Norton AntiVirus QuickTime for Windows StartUp 9 Microsoft Exchange MS DOS Prompt QQ Windows Explorer r ES Acquisition Shortcut WIN EPR Acquisition Icon C8BoI 09 01 Figure 3 7 Starting the Win EPR Acquisition Application 10 Install an EPR cavity if there is not one presently installed The instructions in this chapter assume you are using a properly installed Bruker ER 4102ST cavity If there is no cavity installed seek the assistance of a knowl edgeable EPR colleague or refer to Chapter 5 to learn how to install a cavity 11 Proceed to Section 3 3 EMX User s Manual 3 12 Cos BRUKER Removing and Inserting Samples Removing and Inserting Samples 3 3 1 W WINEPR ACQUISITION Microwave Bridge Control Open the Microwave Bridge Control dialog box If this window is not already open click the button labeled MW in the tool bar The MW button toggles the dialog box open and closed The microwave bridge con trol dialog box will then appear See Figure 3 8 File View Options Window Info Brid Ces kke Hae JAAR ge STANDBY Frequency Receiver Level Control O UNLEVELED Attenuation 60 dB 7 Dialo ee Power 0 000 nw q Microwave 8 Bridge m Auto Tune Buttons Tune Button Control Button o Fine Tune Stop Tuning F Dual Tr
164. roximations of the actual signal The number of points in a spectrum can be chosen in the Experiment Parameter dia log box See Figure 4 17 The parameter to adjust is the Res olution in X Alas this is a misnomer what is actually listed is the reciprocal of the resolution Perhaps the best way of demon strating this effect is to look at the following two examples The first spectrum was acquired with only 1024 points See Figure 4 18 The second spectrum was acquired with 4096 points and reproduces the lineshapes much better than the first example See Figure 4 18 Standard Parameter Spectr Lx Experiment x EEE EG z Y no Y Sweep xl Hall Signal Channel 4 Center Field sazo00 Ha Receiver Gain foo 10 TS Sweep Width 200 0 E G Modulation Frequency 100 00 Fl kHz Static Field 3480 000 HH G Modulation Amplitude 1 00 G Microwave Bridge Modulation Phase 0 00 kK deg Frequency 9 751000 GHz Offset 0 00 Hx Power po mW Time Constant psa E msec Step 1 H db Conversion Time 5 12 H msec Temperature unit Sweep Time 5 24 sac Temperature K Harmonic 1 Step po E K Resolution in X 1024 Kk Goniometar Number of X Scans 1 E Angle Eo 8 deg 1 Step oo HH aeg Resolution in X Save as Default Comment cm Figure 4 17 The Resolution in X parameter EMX User s Manual 4 16 BpGgeR Field Sweeps SPECTR PAR B E f 10 3 30
165. s been detected Check manually if a dip can be found A very slight dip e g very lossy sample may not be detected by the auto tune routine Adjusting Ref Arm Phase The full 360 range of the signal phase has not resulted in an optimal phase setting Adjusting Ref Arm Bias The system is unable to set the diode current to 200 microamperes at 50 dB attenuation Adjusting AFC Lock Offset The system is unable to set the AFC lock offset to zero Check the back of the bridge to make sure the AFC is on If this error occurs during fine tune try auto tune Critically coupling cavity The iris motor has reached both of its limit switches and has been unable to obtain a diode current of 200 microamperes Check if the iris motor is still connected to the screw and that the limit switches have been set properly See Section 5 2 If you are using a flat cell when this happens it is likely that you need to adjust the position of the flat cell It is easier to optimize the cavity dip if you adjust the flat cell while you are looking at the tuning picture If this error occurs during fine tune try auto tune EMX User s Manual 7 3 Cos BRUKER No Tuning Picture No Tuning Picture 7 4 Tune mode delay period not expired klystron bridge only After you turn on the spectrometer a delay of approximately three minutes is required before a klystron will activate as you switch from Stand By to Tune This does not apply to Gunn d
166. s in spectrum a were lost in spectrum b even after increasing the modulation only slightly A good rule of thumb is to use a field modulation that is approximately the width of the narrowest EPR line you are trying to resolve Keep in mind that there is always a compromise that must be made between resolving narrow lines and increasing your EMX User s Manual 6 11 BRgER Optimizing Sensitivity signal to noise ratio If you have a very weak signal you may need to sacrifice resolution i e by using a higher field mod ulation in order to even detect the signal However if you have a high signal to noise ratio you may choose to use a much lower field modulation in order to maximize resolu tion TF Figure 6 8 Effect of using progressively higher field modulation a d on an EPR spectrum EMX User s Manual 6 12 BRUSER Optimizing Sensitivity e Optimize the microwave power level The intensity of an EPR signal increases with the square root of the micro wave power in the absence of saturation effects When satu ration sets in the signals broaden and become weaker EPR signals with very narrow lines are particularly susceptible to distortion by excessive power Figure 6 9 shows the result of excessive microwave power You should try several micro wave power levels to find the optimal microwave power for your sample A convenient way to find the optimum power is to use the 2D experiment routine d
167. sdiceaitersaateceatsidveaeedevska tasters a 7 11 TLO Poor Resolution jsscticcceiats ie oak eae etisalat ielcdeemaupieds 7 13 TAN Laine shape Distro asninn eda ceived cae Sides Gade sees Seta zodehes 7 15 7 12 No Signal When Everything WorkS cceeeesseceestecceeeeeceeeeeeeeteeeees 7 17 Dy Lo Warming NOISES esesten ee can E ataean R 7 17 8 EPR Spectrometer Calibration ssssseesseesneeneeennr rnr rnn erenn 8 1 8 1 Standard Samples mssicsiississsssiisarsiensiisisn eissii 8 2 8 1 1 DPPH Q a diphenyl B picryl hydrazyl ee eeceesceeseeeteeeneeeeeeeeeeennes 8 2 8 1 2 Weak and Strong Pitch Samples 200 0 cece ececceecceceeeceeeaeceeaceceeeeeeeaeceeaeeensees 8 3 8 2 Calibration of the Signal Channel 0 eee ceesseccesneceseeeeceeeeeeesneeeeeaees 8 4 B 2e1 Basic Theory eaae e E a aene e sc hestes folecuelatedteldlativn EE 8 4 8 2 2 Preparing for Signal Channel Calibration esseeeseseeeereereesesreesesrresresrese 8 8 8 2 3 Calibrating the Signal Channel 0 0 eee ceeceseceseceseceseeceseeseeeseeeseeseneeees 8 15 9 System Performance Tests a nns enneren erenneren 9 1 9 1 Signal to Noise Ratio TeStaccinecinne inne in ei A es 9 2 9 1 1 Preparing for the S N Test oo eceeceseeseeeeceseeeecesecaeeeceesecaaeeeeeseceaeeeeaeeaeees 9 4 9 1 2 Measuring the Signal to Noise Ratio 0 cece eesecesecesseceseceseceeeeeeeceneeeeeeens 9 6 9 2 Cavity Background Signal Test cece eesscceesneecesceceseeeeceeeeeeeeseeeees 9
168. seline Correction Goniometer Angle Flyback Off x X automatic Scaling Figure 4 22 Selection of the Automatic Baseline Cor rection option Acquire the time scan by clicking the RUN button in the tool bar See Figure 4 23 There may be a slight offset particularly if your signal is very weak Acquire another spectrum off reso nance and subtract it from the first spectrum to get an accurate result SPECTR13 PAR joi 10 3 150 100 sec Figure 4 23 A time scan of a decaying radical EMX User s Manual 4 21 BpGsen Interactive Spectrometer Control Interactive Spectrometer Control 4 5 Immediate active visual feedback when parameters are changed helps you to optimize your spectrometer parameters The Experiment Parameter dialog box does not change the param eters on the instrument until you run a spectrum but the Interac tive Spectrometer Control dialog box supplies you with the tools needed for interactive optimizations Click the Interactive Spectrometer Control button in the tool bar Figure 4 24 and the dialog box will appear See Figure 4 25 Diela ox A pr ay EY ory Interactive Spectrometer Control Button Figure 4 24 The Interactive Spectrometer Control button in the tool bar Hall Signal Channel Center Field 3480 00 G Calibrated SweepWidth poo FE ReceiverGain f24 6 64 10 p Sweep Address pa o M
169. sium Springer Verlag Berlin 1988 Feher G Electron Paramagnetic Resonance with Applications to Selected Problems in Biology Les Houches Lectures 1969 Gordon and Breach New York 1970 Forester A R J M Hay R H Thomson Organic Chemistry of Stable Free Radicals Academic Press New York 1968 Foster M A Magnetic Resonance in Medicine and Biolodgy Pergamon Press Oxford 1984 Fraenkel G K Ann New York Acad Science 67 546 1957 Fraissard J P H A Resing Editors Magnetic Resonance in Colloid and Interface Science Reidel Hingham MA 1980 Franconi C Magnetic Resonance of Biological Systems Gor don and Breach New York 1971 EMX User s Manual 10 6 BRUSER Bibliography Freed J H Electron Spin Resonance in Annual Review of Physi cal Chemistry H Erying C J Christensen H S Johnston Editors Annual Reviews Inc Palo Alto CA 1972 Vol 23 pp 265 310 Freeman A J R B Frankel Hyperfine Interactions Academic Press New York 1967 Fujiwara S Editor Recent Developments of Magnetic Reso nance in Biological Systems Hirokawa Tokyo 1968 Gaffney B J C M McNamee Spin Label Measurements in Membranes Methods Enzymol 32 161 198 1974 Gerson F High Resolution E S R Spectroscopy J Wiley and Sons London Chemical Topics for Students 1 1970 Geschwind S Editor Electron Paramagnetic Resonance Ple num Press New York 1972 Goldberg I B A J
170. sssesseeseeseesessesresrrsressesressrssirsresresreesessressss 4 6 4 1 5 ZOOMING Spectra ves Nsw ens bene dehaes aici odes ul dacs Sinton 4 6 4 2 Starting and Stopping Acquisitions ceeeeseceesececeeneeeceeeeeceeeeeeenaeeeeeas 4 9 AD AE LL VE POS seeders aad E EE E EE 4 10 4 3 1 Setting Parameters Via ZOOMING cee eeeceseceseceseceseeeeeeeeeeeeeeeeeeeeeneeeaes 4 10 4 3 2 Setting Center Fieldsi man aor thn notte aed eh tie ae aed 4 12 4353 Sonal AVETASIN ees ces eee oie eds lace bet ante tess evar i E E e eE 4 14 434 ResolutOn is eneee states teers AKA cea ee cted seine tio 4 16 EMX User s Manual viii shen Table of Contents 44 Time SCANS ennn a a a a a aastas 4 18 4 5 Interactive Spectrometer Control sssssesssesssesseseeessseeesseessseessresseeesse 4 22 4 6 Controlling the Microwave Bridge c ccessccesesteccenneeceeeeeeeeeeeeseneeeees 4 27 4 6 1 Auto Tune vs Fine Tune cece ceececeesseceeneeceeececsaecceeaeeceeeeecsaeeeeaeceeaneeeeas 4 27 4 6 2 Setting the Microwave POWED ceeceesceseceseceseceeeeeseeeeaeesseeeeaeesseeeseeeaaes 4 29 4 1 Spectr m Piles erona a E R AEE E RARE 4 31 A elSavane Pleserau a E E E E E 4 31 4 1 2 Disk Housekeeping isorine eii a ET nA E AER E 4 32 4 8 Sending Spectra for Processing esessesessessssereseseessrersseesseesssresseessse 4 34 4 8 1 Sending Spectra to WIN EPR and SimFonia 0 eeeeeeeeseeeeeeeeeeeneeenee 4 34 5 Additional Tec
171. st to wait several hours because the spec trometer is most sensitive and stable after it has achieved thermal equilibrium EMX User s Manual 9 14 BpGsen Cavity Background Signal Test 4 Create a new spectrum window if needed If there is no empty spectrum window create one by clicking on the Create New Spectrum button in the tool bar Performing the Background Signal Test 9 2 2 EMX User s Manual 9 15 BROER Cavity Background Signal Test 1 Open the parameter option dialog box Set the parameters for Weak Pitch Measurement as indicated in Table 9 2 See Figure 9 10 Weak Pitch Background Parameter Measurement Measurement Modulation 8 0 G 8 0 G Amplitude Modulation 100 kHz 100 kHz Frequency Receiver Gain adjust adjust Phase 0 0 Time Constant 1310 72 ms 1310 72 ms Conversion Time 163 84 ms 163 84 ms Center Field 3480 G 2600 G Sweep Width 50G 5000 G Resolution of 1024 points 1024 points Field Axis Microwave 3 dB 3 dB Attenuation Table 9 2 Parameters for Background Signal Measurement EMX User s Manual 9 16 BpGsen Cavity Background Signal Test Standard Parameter BACKWKPPAR OO L U O O Experiment X BETETE Y no Y Sweep Fl Hall Signal Channel 1 Z 6 FY Center Field 3490 63 G Receiver Gain 00 10 Sweep Width 0 00 G Modulation Frequency fro0 00 H kHz Static Field 3459 540 G Modulation Amplitude e c
172. structions of Sections 3 2 through 3 5 of this manual You should have the spectrometer turned on the cavity properly installed with a Bruker stan dard DPPH sample in it and the microwave bridge and cavity tuned Remove cryostats from the cavity because it is easier to position the DPPH sample properly in the cav ity Except for the FlexLine resonators it is necessary to use the ER 4118CF cryostat when calibrating FlexLine resonators In particular the ER 4112HV and ER 4113HV helium cryostats prevent the correct position ing of the sample Another advantage is that the resonant Collet Nut Fiduciary lt TT Mark ie JA L Irradiation Grid Cover Sd Pedestal Figure 8 4 Proper positioning of the DPPH sample frequency of the cavity will be approximately 9 8 GHz without the helium cryostat and the field for the DPPH signal will be known approximately 3480 Gauss The EMX User s Manual 8 8 BRUSER Calibration of the Signal Channel It is not possible to change the actual Sweep Width while the Set Up Scan is enabled Change the Sweep Width before the Set Up Scan is enabled DPPH sample is a small point sample and therefore has a fiduciary mark that indicates the position of the DPPH crystal in the sample tube Center the DPPH sample verti cally in the cavity The center of the black irradiation grid cover cor
173. sues and fluids Antioxidants radical scavengers Contrast agents Oximetry Drug detection metabolism and toxicity Enzyme reactions Photosynthesis Structure and identification of metal binding sites Photochemical and radiolytic generation of radicals Oxygen based radicals NO in biological systems Carcinogenic reactions EMX User s Manual 1 4 Cos BRUKER The Spectrometer The Spectrometer 1 2 The Bruker EMX EPR spectrometer is a research grade scien tific instrument It is capable of routine measurements as well as sophisticated and advanced experiments when equipped with the proper accessories The modular design makes the spectrometer easy to upgrade or expand For information and assistance in choosing and ordering accessories for your specific application contact your local Bruker EPR sales representative EMX User s Manual 1 5 Cos BRUKER Using this Manual Using this Manual 1 3 How to Find Things 1 3 1 Preface Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 First you should read the safety guide in the preface of the man ual Microwaves can be dangerous particularly to your eyes With normal precautions the risk for injury can be minimized Users who are not familiar with EPR should start by reading Chapter 2 which is a concise introduction to the theory and practice of EPR spectroscopy It is by no means exhaustive it gives the necessary information to fo
174. t Strong Pitch Spectrum for EMX Manual Figure 3 23 The Comment dialog box 15 Save the spectrum to a disc After you have acquired a spectrum you may save it in any folder Click on the Save button in the tool bar A dialog box appears that lets you choose a filename a destination folder and a destination disk drive See Figure 3 24 The spectrum to be saved is the spectrum that is presently active To select the appropriate disk drive click on the arrow on the Drives selector To select the appropriate folder click on the appropriate paths in the Folders selector The spec trum filename is selected by typing the filename in the File Name selector Clicking OK saves the spectrum on the hard disk or diskette Cancel exits the dialog box without saving the spectrum EMX User s Manual 3 26 ph ER Acquiring Spectra File Save As ixi File name Folders st_pitch par c winepr data john_spc Save file as type Drives Parameter par J c micron z Figure 3 24 The File Save As dialog box If the chosen filename were already used by another file a warning box gives you the opportunity to decide whether to replace the existing file with the present spectrum See Figure 3 25 Pressing No cancels the save process and allows you to select another name or folder File Save As CAWINEPR DATA JOHN_SPC st_pitch par This file already exists Do you wantto replace it Figure 3 25 Wa
175. t parameters for signal or noise measurement Click either one of the windows with the left mouse button to activate that window The parameters shown on the right will be assigned to that measurement E WINEPR ACQUISITION Spectr1 M E Eile Parameter ZX tim Processing Yiew Options Window Info CIS Ioe AE StertAcquiston Run Taf 12 Lele Heo Iz Calibrate Signal Channel AFC OPERATE Interactive Receiver Level GHz Receiver Level es e a5 nal Noise nt ew Diode Hall Field MUCAUBRATED Q value 3600 LHI 3480 000G Spectr 10 3 2 1 3460 3500 Calculate the Signal Noise ratio of two spectra Figure 9 3 Open Signal Noise Measurement window EMX User s Manual 9 6 Cos BRUKER Signal to Noise Ratio Test 2 Input the calibration factor for the weak pitch sample Enter the calibration factor you copied in Step 2 of the previous section into the Weak Pitch fac tor box See Figure 9 4 M8 WINEPR ACQUISITION Signal Noise Measurement File Parameter Acquisition Processing View Options Window Info Dos far ta 3 ee iia SSA wE eja 2 oag AE e OPERATE Frequency 9 78 Power 0 200 AERATED Q vaiue 2700 Signal Noise Measurement Indicator of Signal the Active Measurement Window Window 3460 3470 3480 3490 x10 3 50 Noise Measurement Window Spectri a
176. t the value in the box It is used for the input and display of quantities that are not restricted to spe cific values but may have a continuum of values such as the cen ter field After a click with the left mouse button in the text of the box an insertion marker appears a vertical line Any text or numbers you type are inserted after the insertion marker Several characters may be selected or highlighted simulta neously by clicking and dragging over the desired text Any typed text replaces the highlighted text The selected text may also be deleted by pressing the Del key The left and right arrow keys of the keyboard moves the insertion marker left and right Keeping the keys pressed repeats the action automatically This input method is used for parameters that have a limited number of options or choices After clicking on the downward pointing arrow next to the box the allowed values appear in a drop down list The presently active option is highlighted The highlighted choice is changed by pressing the up and down arrow keys of the keyboard You may also select the desired choice by clicking the value with the left mouse button The drop down list then disappears with the newly selected value or option displayed in the box The check box acts like a toggle When clicked the action turns the option on or off A cross mark in the box indicates an on or active state A push button will execute a command when you click it with the
177. tandard cavity you are using High Pass Filter and AFC Trap Filter See EMX User s Manual Signal to Noise Ratio Test Figure 9 2 are checked in the default settings In case the default settings have been changed set them back ACCESSORY DEIAL oeg ii OTE power CONTROL A 7 a S AERONAVE O YS L Figure 9 1 Location of the AFC MOD LEVEL potentiometer MB WINEPR ACQUISITION Signal Channel Options File Parameter Acquisition Processing View Options Window Info Dees Fel oe ese ees GE i978 Ctt AFC Frequency Attenuation Power Q Value Signal Channel Options 30 dB 0 201 mS HE Receiver Level 9 78 GHz Diode Hall Field mW 2700 Resonator Tuning Caps X High Pass Filter X AFC Trap Filter F external Acquisition Trigger F Lock tn Integrator Spectr1 enix m ogia T SCT Selftest Ooo E i Lock in Modulation Signal Input Internal External Internal External Internal C External EMX User s Manual ph ER Signal to Noise Ratio Test Figure 9 2 Signal Channel Options Measuring the Signal to Noise Ratio 9 1 2 1 Open the Signal Noise Ratio Test window Open the Signal Noise Ratio Test window under the Acquisi tion drop down menu See Figure 9 3 The window has two empty spectra and each one contains a set of defaul
178. the Microwave Power parameter box Close the Experiment Parameter dialog box Click on the OK button and the dialog box will then dis appear Cancel exits the dialog box without saving the changes EMX User s Manual 3 21 ph ER Acquiring Spectra 9 Open the Experiment Options dialog box in order to check the options If this window is not already open click its button in the Tool Bar See Figure 3 15 The button toggles the dialog box open and closed The Experiment Options dialog box will then appear See Figure 3 16 LCOS i el Eleg Experiment Options Button Figure 3 15 The Experiment Options button 10 Check the Microwave Settings Click and select the Set option With this option the microwave power we entered in Step 7 will be the one used to acquire the spectrum Experiment Options Spectr1 Lx Field Controller Microwave Settings Sweep Direction Up Power Set x Flyback On M Power Flyback Off x Settling Condition Wait LED off Mi icrowave Field Offset Correction 0 00 G s tti b etuings Temperature Unit Tolerance K No Time Delay z Temperature Flyback Off F Delay Time 0 00 sec Condition Don t wait Display Settings Goniometer automatic Baseline Correction Angle Elyback Off 7 X automatic Scaling Figure 3 16 The Experiment Options dialog box EMX User s Manual 3 22 BRgER Acquiring Spectra 11 Close the Expe
179. the procedure for calibrating cavities and signal channels so that you may obtain reproducible quantita tive and high sensitivity spectra In order to maintain your spectrometer in optimal working con dition it is a good idea to periodically test your system s perfor mance Chapter 9 describes how to measure the sensitivity of the spectrometer and how to measure the background signal of the cavity An extensive bibliography of EPR references is given in this chapter It includes many different EPR applications as well as educational texts This is a good place to start a literature search All the answers to all EPR questions can not possibly fit in one manual If you can not find the answer to your question contact your nearest Bruker EPR representative We have a team of skilled application scientists with diverse expertises One of us will probably come up with an answer Typographical Conventions 1 3 2 Times Helvetica Special fonts are used in the text to differentiate between normal manual text and text displayed in the program This is the font used for the normal text in the manual This is the font used for text that is displayed by the program or must be entered into the program by you EMX User s Manual 1 7 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 w
180. the reference phase and the 90 phase difference is extrapolated from the signal intensi ties The phase angle resulting in maximum signal intensity for that particular frequency is recorded and saved with the calibra tion file The phase difference between the modulated EPR signal and the reference signal depends on several experimental conditions The length of the cable leading to the modulation coils the inductance of the coils in the particular cavity the gain setting of the modulation amplifier the tuning capacitors and the signal channel used can all change the phase difference However the reference phase calibration is performed automatically during the routine described in this section The two editions of the book by C P Poole that are mentioned at the beginning of this chapter are very good references for the details on the theory of phase sensitive detection and the calibra EMX User s Manual 8 6 ph ER Calibration of the Signal Channel tion of signal channels We encourage you to explore this topic further to learn more about calibration Figure 8 3 Signal intensity as a function of the reference phase angle EMX User s Manual 8 7 Cos BRUKER Calibration of the Signal Channel Preparing for Signal Channel Calibration 8 2 2 A Do not attempt to cali brate a cavity with an ER 4112HV or ER 4113HV helium cryostat installed in the cavity 1 Follow the in
181. tic field position of the marker To acquire the spectrum with the new center field click on the RUN button in the EMX User s Manual 3 24 BpGsen Acquiring Spectra tool bar The newly acquired spectrum will then be nicely centered See Figure 3 21 Spectr Jor 10 3 2 1 0 3400 3425 3450 3475 3500 3525 3550 Figure 3 21 A centered spectrum 14 Enter a comment If this window is not already open DERG EARN EEEE S NA Save Comment Button Button Figure 3 22 Buttons for saving files and comments click its button in the tool bar See Figure 3 22 The but ton toggles the dialog box open and closed The Com ment dialog box will then appear See Figure 3 23 Commen zy use ful bec at Enter a comment regarding your spectrum The comments keen track of what vou supplement the information which is included in the spec trum parameters If you wish you may enter your name in Comments are very useful because you can the operator box Operator names are useful for keeping keep track of such track of who acquired the spectrum Click on the OK but things as sample prepa ton and the dialog box will then disappear Cancel exits ration details the dialog box without saving the changes EMX User s Manual 3 25 BRUSER Acquiring Spectra Comment ST_PITCH PAR xl Operator J Q Spectroscopist Date 1997 04 29 Time 10 50 Commen
182. tically The value of the parameter is indicated graphi cally by the position of the square in the slider bar You can also vary the parameter by clicking and dragging the square The dip corresponds to the microwave power absorbed by the cavity and thus is not reflected back to the detector diode By centering the dip on the display monitor the microwave source is set to oscillate at the same frequency as the cavity resonant frequency Clean the sample tube to be inserted into the cavity Wiping the outside of the sample tube with tissue paper is usually adequate It is vital to avoid contaminat ing the microwave cavity as paramagnetic contaminants may result in spurious EPR signals or distorted base lines in your EPR spectra EMX User s Manual 5 8 BRUSER Manually Tuning a Microwave Bridge Insert the sample tube carefully into the cavity See Figure 5 4 Make sure you have the appropriate collet size for your sample tube size The tube should be slightly loose before you tighten the collet nut The bot tom of your sample should rest in the indentation on the pedestal This ensures that your sample is centered hori zontally If you have a small sample less than 2 cm in length you should visually judge how far the tube should go into the cavity in order to vertically center the sample in the cavity You can adjust the sample position by loos ening the bottom collet nut and moving the pedestal up and down
183. ting is pre sented in the next chapter Scan over the correct magnetic field range If you do not sweep over the correct magnetic field range you will miss your signals This mistake occurs quite often when using a cryostat in the EPR cavity Consult literature refer ences to determine approximate g values for the species in your sample You can then choose the appropriate magnetic field for your sample Most organic radicals will have a g value of approximately 2 This corresponds to a field for resonance of approximately 3480 Gauss at a microwave fre quency of 9 8 GHz Metal ions can have large departures from g 2 as well as large zero field splittings making it dif ficult to guess where the resonance might occur Performing a wide scan in your initial experiment will maximizes your probability of finding the EPR signal EMX User s Manual Cos BRUKER Hints for Finding EPR Signals Finding an EPR signal Sometimes you may have diffi culty finding the EPR signal from an unknown sample or a sample you are not familiar with Here we provide two exam ples of parameter sets that are useful for finding EPR signals from unknown samples that you suspect will consist of either an organic radical See Figure 6 1 or a transition metal ion See Figure 6 2 respectively These parameters are by no means optimized but they will serve to help you find the sig nal After you find the EPR signal you need to reset the field center an
184. transferring parameters 4 5 troubleshooting 7 1 to 7 17 tuning capacitors 8 5 to 8 6 tuning errors 7 3 tuning mode 5 3 absence of 7 4 tuning of cavity and bridge automatic 3 17 to 3 19 manual 5 1 to 5 10 turning off spectrometer 3 31 to 3 35 turning on spectrometer 3 8 to 3 12 tutorial 4 1 to 4 34 typographical conventions 1 7 W warming up the spectrometer 6 6 7 14 warning noises 7 17 EMX User s Manual Index water condensation 7 12 waveguide gasket installation 5 16 waveguide stabilizers installation 5 15 removal 5 15 window application 3 3 border 3 4 creating new spectrum windows 4 5 exit 3 4 spectrum 3 3 4 2 to 4 8 Windows brief tips 3 2 to 3 7 Z Zeeman effect 2 3 to 2 6 zooming spectra 4 6 to 4 8 EMX User s Manual
185. uence the calibration values of the signal channel By default they are both selected Ensure that both options are checked Only under very rare circum stances would you acquire spectra without these filters 9 Adjust some parameters After centering the DPPH sample most of the parameters should be fairly close to what is needed for the calibration routine Check the val ues in the Standard Acquisition Parameter dialog box and modify them so that they correspond to the values in Figure 8 8 The Center Field value may be somewhat different from what is displayed in Figure 8 8 but the Sweep Width must be 100 Gauss Standard Parameter Spectr2 Experiment X Field Sweep z Y no Y Sweep M Hall Signal Channel Center Field Bagas EHe Receiver Gain fioo 10 e E Sweep Width 100 00 H G Modulation Frequency 100 00 kHz Static Field 3483 116 G Modulation Amplitude 1 00 G Microwave Bridge Modulation Phase 0 00 deg Frequency 9 766000 GHz Offset 0 00 Power 0 64 mW Time Constant 0 64 msec Step F db Conversion Time 5 12 msec Temperature unit S2cey Ula ad Eea Temperature H K Harmonic 1 Ste 1 00 K aaa x F EI Resolution in X 1024 Sees Number of X Scans 1 magjie ie pan Resolution in Y 1 z Step kk geg Repetitive Mode B Save as Default Comment Figure 8 8 Parameters for a signal channel calibration 10 Acquire a spectrum Click the RUN button
186. ulation Amplitude 4 00 H G Microwave Bridge Modulation Phase 0 00 kK deg Frequency 9 766000 GHz Offset 0 00 Power 10 0 mW Time Constant 327 68 msec Step 1 S db Conversion Time 81 92 H msec Temperature unit Sweap Time 335 54 Sec Temperature me K Harmonic 1 Step 1 00 Ek Resolution in X 4096 Ke Soniometer Number of X Scans 1 E Angle deg Resolution in Y 1 Ste ooo Hae P g Repetitive Mode r Figure 6 2 Parameters for finding an EPR signal from a transition metal ion Make sure your sample is positioned correctly in the cavity Only the central region of the cavity contributes significantly to the EPR signal If you place the sample suffi ciently out of this region you may not detect a signal Optimize the sensitivity You may have a very weak sig nal in which case you will need to optimize your parameter settings for sensitivity The chart on the following page sum marizes common factors that are important for getting the optimum sensitivity from your EPR measurements The pages that follow the chart provide a more in depth discus sion of these factors EMX User s Manual 6 3 Cos BRUKER Hints for Finding EPR Signals Sample related factors Adjust sample Optimize Parameters for Make sure the cavity is critically Figure 6 3
187. ulation amplitude of the signal chan nel of an EPR spectrometer DPPH has been studied extensively by e M bius K and R Biehl Multiple Electron Resonance Spec troscopy Plenum Press 1979 e Dalal N S D E Kennedy and C A McDowell J Chem Phys 59 3403 1979 EMX User s Manual 8 2 BROKER Standard Samples e Hyde H S R C Sweed Jr and G H Rist J Chem Phys 51 1404 1969 e Dalal N S D E Kennedy and C A McDowell J Chem Phys 61 1989 1974 e Dalal N S D E Kennedy and C A McDowell Chem Phys Lett 30 186 1975 Weak and Strong Pitch Samples 8 1 2 Pitch in KCl has emerged as a standard because of its long lived paramagnetic radicals and low dielectric loss Because of the long life of the radicals it is unsurpassed as a test of spectrome ter sensitivity The pitch is added to a powder of KCI and the mixture is carefully mechanically mixed to obtain a homoge neous sample After mixing the sample is heated pumped and sealed under vacuum Pitch is generally prepared in two concen trations strong pitch which is 0 11 pitch in KCl and weak pitch which is 0 0003 pitch in KCI To correct for variations in spin concentration each weak pitch sample is compared to a standard and assigned a correction factor The peak to peak line width is typically 1 7 G with a g factor of 2 0028 The size very weak of the signal renders pitch ill suited for modulation amplitude calibrat
188. ulation coils on the cavity are tuned or made resonant by adding a tuning capacitor in series with the modulation coil we Tuning co SX Capacitor _ Modulation wiy Coil Figure 8 2 The LC resonant circuit for high frequencies EMX User s Manual 8 5 BRUSER Calibration of the Signal Channel The calibration routine switches various tuning capacitors in and out of the circuit until the modulation amplitude is maximized The optimal capacitor for that particular frequency as well as the modulation amplitude for full gain of the modulation amplifier are recorded and saved with the calibration file Once this data is available the signal channel will then vary the input signal to the modulation amplifier to produce the modulation amplitude that you have selected Once the modulation amplitude has been calibrated the refer ence phase is easily calibrated by studying the phase angle dependence of the signal intensity The intensity of the output signal is proportional to the cosine of the phase difference between the reference signal and the modulated EPR signal See Figure 8 3 It is most convenient to determine where the 90 phase difference occurs because first the absence of a signal cos 90 0 is easy to detect and second the cosine function and hence the intensity changes rapidly with respect to the phase angle at 90 In the calibration routine spectra are acquired at several different values of
189. ure 3 33 ih ih Console Power Switch nm C Figure 3 33 The location of the console power switch 12 Turn off the power for the system How you do this depends on how the electric power was hooked up when the spectrometer was installed Most likely you will deac tivate the switch on the breaker box for the spectrometer Breaker boxes are usually mounted on the wall Consult EMX User s Manual 3 35 BRUSER Turning the Spectrometer Off the local instrument or facilities manager if you are not sure where the breaker box is EMX User s Manual 3 36 A Brief EMX Tutorial 4 This chapter contains useful and helpful hints to get the most out of the WIN EPR Acquisition software In the previous chapter we blindly followed many instructions to acquire a spectrum Here is the opportunity to explore some of the features in a bit more depth The tutorial is not meant to be an exhaustive treatise on all details of the spectrometer Instead it is a starting point from which you can explore the capabilities of the instrument on your own The first topic covers advice on spectrum windows such as how to keep things neat transferring parameters and zooming in on specific areas of a spectrum The second topic describes stop ping and starting acquisitions Field sweep experiments and the adjustment of parameters are covered in the third topic The fourth topic deals wit
190. ve Svein Bridge eee Control Button Tune Button rs Frequency ideal lel 4 Bias 43 0 4 f l Operate Signal Phas Attenuation Auto Tune a isplay 2 Fine Tune Auto Tune Stop Tuning I Dual Trace Buttons Figure 3 12 The Microwave Bridge Control dialog box EMX User s Manual 3 17 Cos BRUKER Tuning the Microwave Cavity and Bridge If a klystron bridge does not switch to Tune and the Stand By indicator is red wait a minute There is a time delay of approximately three minutes between the time the console is turned on and the time the klystron can be turned on This allows the klystron to warm up sufficiently It is vital to avoid con taminating the micro wave cavity as a paramagnetic contami nant may produce spu rious EPR signals or distorted base lines Switch the microwave bridge to Tune There are three states or modes for the microwave bridge Stand By Tune and Operate When you turn on your spectrometer it should be in Stand By which is indicated by Stand By appearing in the Microwave Bridge Con trol menu See Figure 3 12 If you have been acquiring spectra already your bridge will probably be in Operate Click the Tune button in the dialog box to change to Tune Set the microwave attenuator to 25 dB The micro wave attenuation is set by clicking the arrows on either side of the attenuation display
191. w The appearance of the icon depends on the window or program you open A single mouse click opens a drop down menu Consult your Microsoft Windows documentation for further information regarding the commands in the menu The horizontal bar near the top of the application window is the menu bar It displays the names of the available pull down menus Choose the desired menu by clicking on it with the left mouse button The menu consists of a collection of commands You choose a command by clicking on it with the left mouse button The horizontal bar below the menu bar is the tool bar It displays buttons to execute the most commonly used commands Click ing on a button performs the command The info line is a bar at the bottom of the application window The left corner displays messages regarding the presently selected command or the status of the program The two boxes next to NUM display values of cursor positions when the appro priate options are active The perimeter of the window is the window border When the cursor is placed anywhere on the window border a double arrow replaces the regular cursor If you click and drag the window may be resized to the desired size If you drag a corner the two sides that form the corner are resized simultaneously EMX User s Manual Cos BRUKER Brief Tips on Windows 95 Dialog Boxes 3 1 1 Many commands open a dialog box See Figure 3 3 The dia log box allows you
192. when expanding the stabilizers Reconnect the nitrogen purge line and adjust the flow rate for a light flow Installing the waveguide gasket properly EMX User s Manual 5 18 BRUSER Changing EPR Cavities EACE iz 18 Reconnect the iris motor shaft to the iris screw The procedure here is like Step 10 performed in reverse Reposition the iris screw motor Screw the lock nut onto the iris screw Click and hold the up iris button in the Microwave Bridge Control dialog box until the iris screw is approximately half way out Experiment Options Button Figure 5 13 The Experiment Options button 19 Read in the calibration file for the cavity Open the Experiment Options dialog box in order to read in the calibration information If this window is not already open click its button See Figure 5 13 in the tool bar The button toggles the dialog box open and closed The Experiment Options dialog box will then appear Click on the Change File button A new dialog box Open Calibration File will appear Select the appropriate cali bration file for your cavity and click OK This will auto matically load the calibration data you have selected Confirm that the calibration file is the correct one for the cavity The calibration file name usually consists of two or three letters that identify the type of cavity ST for ER 4102ST or TM for ER 4103TM followed by the serial number of t
193. with respect to the magnetic field Bo EMX User s Manual 2 3 BRUSER Basic EPR Theory From quantum mechanics we obtain the most basic equations of EPR 1 E 8 Ug BoM 75 g Hpg 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 ug 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 EMX User s Manual 2 4 BRUSER 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 we could keep the electromagnetic radiation frequency constant and scan the magnetic field See Figu
194. y Background Signal Test 9 2 Cavity background signals can sometimes be disturbing particu larly when they overlap with your EPR signals or with the area you need to integrate They can distort the EPR signals of your sample and make quantification difficult The best way to avoid these problems is to keep your cavity clean Here we provide a standard procedure to test your cavity background signal The standard cavity background signal test compares the weak pitch signal with the spectrum acquired with an empty cavity over a wide scan range The parameter setting for a standard test is shown in Table 9 2 The ratio of the cavity background signal over the peak to peak height of the weak pitch signal should be less than 1 4 to meet the specifications Preparing for the Background Signal Test 9 2 1 1 Install an ER 4102ST standard cavity See Section 5 2 for instructions The specification for the background signal is based on an ER 4102ST standard cavity and using the weak pitch sample We strongly sug gest that you keep a record and verify the specification periodically 2 Insert the weak pitch sample The weak pitch sample should be inserted in the cavity until the bottom of the label and tape on the sample tube is flush with the collet You also should use the pedestal to hold the weak pitch rigidly 3 Turn on the instrument and tune Turn on the instru ment if it is not on yet Tune the microwave bridge and the cavity It is be
195. y changes the size of the iris See Figure 2 13 Iris Screw W wem n Wave guide AN ee Cavity Figure 2 13 The matching of a microwave cavity to waveguide 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 2 17 BROKER 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
196. y out of phase i e where the magnetic field is maximum the electric field is mini mum and vice versa The spatial distribution 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 EMX User s Manual 2 16 BRUSER 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 tivel
197. zing spectrum windows 4 6 resolution 4 16 to 4 17 S safety chemical iv to vi electrical iv microwave vi samples removing and inserting 3 13 to 3 16 saving spectra 3 26 to 3 27 4 31 to 4 32 sending spectra to SimFonia 4 34 WIN EPR 4 34 setting center fields interactively 4 12 to 4 13 parameters via zooming 4 10 to 4 12 setup scan 4 23 to 4 24 signal averaging 4 14 to 4 15 6 14 signal channel 2 18 to 2 21 EMX User s Manual Index signal distortion 7 15 to 7 16 asymmetric g matrix 7 16 background signal 7 16 baseline 7 7 to 7 8 background signals 7 8 linear 7 7 variable temperature operation 7 8 varying 7 7 conducting samples 7 16 excessive field modulation amplitude 2 19 7 15 microwave power 7 15 modulation frequency 7 15 time constant 2 20 4 23 7 15 lossy samples 7 16 magnetic field drifts 7 16 magnetic field inhomogeneity 7 15 microwave reference phase 7 16 signal intensity 2 9 signal phase adjustment 5 8 signal to noise measurement 9 2 to 9 13 spectrometers 2 10 to 2 11 spectroscopy 2 1 to 2 2 spectrum 2 23 spectrum files 4 31 to 4 33 standard samples 8 2 to 8 3 starting acquisitions 4 9 starting WIN EPR Acquisition 3 11 stopping acquisitions 4 9 system performance tests 9 1 to 9 24 T tiling 4 2 to 4 3 time constant optimization 6 9 time scans 4 18 to 4 21 setting static the field 4 18 to 4 19 turning off automatic baseline correction 4 20

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