X-Band EPR Probe Loop Gap Resonator. (Catalog No. XP-0201) Operator s Manual. Version 5.0-A For the Bruker ELEXSYS E 500 spectrometer

Transcription

1 X-Band EPR Probe Loop Gap Resonator (Catalog No. XP-0201) Operator s Manual Version 5.0-A For the Bruker ELEXSYS E 500 spectrometer Molecular Specialties, Inc Innovation Drive, Suite 301 Milwaukee, WI Phone: Fax: Contact: Richard J. Stevens, President & CEO rich.stevens@molspec.com Web:

2 PAGE 2 Table of Contents SECTION 1 About Molecular Specialties, Inc. 1.1 About Molecular Specialties, Inc Welcome! 6 SECTION 2 Your Loop Gap Resonator 2.1 Your Loop Gap Resonator Technical Data Optimal Experiments The Basics Preventive Maintenance 12 Part 1 Cleaning 12 Part 2 Proper Handling 13 SECTION 3 I n s t a l l a t i o n 3.1 Installation 14 SECTION 4 Preparing Test Samples 4.1 Preparing Test Samples 18 Part 1 Preparing a TPX Capillary 19 Part 2 Preparing a Glass Tube 22

3 PAGE 3 Table of Contents (cont d) SECTION 5 Use with a Bruker ELEXSYS E Tuning a Bruker ELEXSYS E 500 Spectrometer for Your Loop Gap Resonator 25 Part 1 Searching for Resonance 26 Part 2 Tuning 28 Part 3 If Previously Calibrated For Microwave Bridges with Stabilizer Frequency Calibrating a Bruker ELEXSYS E 500 Spectrometer for Your Loop Gap Resonator 35 Part 1 Pre-Calibration 36 Part 2 Calibration 37 Part 3 Confirmation 39 S U P P O R T I N F O R M A T I O N Appendix Quality Control Test 40 Part 1 Preparation 40 Part 2 Measuring Signal 40 Part 3 Measuring Noise 41 Part 4 Calculations 41 Bibliography 42 Index 44 Notes 46 MANUAL TEXT & DESIGN Gregory V. Voss Jr., University of Wisconsin-Milwaukee GRAPHICS Jay Smith, Tadeusz Oles TECHNICAL ASSISTANCE Christopher Felix, Ph.D, Candice S. Klug, Ph.D, Medical College of Wisconsin

4 PAGE 4 Helps and Hints The following conventions are designed to optimize use of this manual and your Loop Gap Resonator. General TROUBLE SHOOTING TIP Trouble Shooting Tip boxes will help you overcome any issues or problems you may encounter while using your Loop Gap Resonator. HANDY TIP Handy Tip boxes are designed to provide tricks of the trade that will help you use your Loop Gap Resonator and its accessories more efficiently. PDF Format Navigation SECTION 1.1 Click on any section header, located in the upper right corner of each page, to return to the Table of Contents. See Page 5. Click on any page reference to go directly to that page.

5 SECTION 1.1 PAGE 5 About Molecular Specialties, Inc. Molecular Specialties, Inc. designs, manufactures, and sells EPR-related products, including modulation coils and probes. Its products serve a worldwide market, and engage the biophysics and molecular/cellular chemistry laboratories of academic institutions. For Further Information, Contact Molecular Specialties, Inc Innovation Drive, Suite 301 Milwaukee, WI Contact: Richard J. Stevens, President & CEO Phone: Fax: rich.stevens@molspec.com Web: If You Are Ordering... If you are ordering a Loop Gap Resonator, you will need to provide several pieces of information to Molecular Specialties so you will receive a unit that is designed specifically for your lab s needs. Required information includes: The exact make/model of the spectrometer(s) with which your Loop Gap Resonator will be used. The exact distance between the tabletop (where the microwave bridge typically rests) and the center of the magnet pole pieces. The exact distance between the magnet pole pieces (or the pole gap). The position of the Hall Probe. The resonant frequency range of your microwave bridge(s). Standard delivery is 60 days after receipt of order with deposit, unless stated otherwise. Shipping dates are approximate and are based on prompt receipt of all necessary information. Pricing does not include sales, use, excise, or similar taxes. All financing plans must be accompanied by a suitable security agreement acceptable to Molecular Specialties, Inc.

6 SECTION 1.2 PAGE 6 Welcome! Congratulations on the purchase of your X-Band Loop Gap Resonator! Its very high sensitivity, strong RF magnetic field, and high resonator efficiency parameter make it the perfect upgrade from the traditional cavity resonators found on most EPR spectrometers. Its features will maximize your research potential: Outstanding sensitivity, making it the choice for small samples. Control and change of oxygen content without removing the sample from the resonator piece, when using Molecular Specialties TPX Capillary, achieved by affixing a hose to the hose barb and blowing gas with different oxygen content past the sample. High filling factor, crucial for EPR pulse experiments and continuouswave saturation studies on transition-metal ions. Low Q Factor 600 unloaded which lessens the susceptibility of the structure to demodulation of source noise, microphonics, and vibrations. Note: The measured Q of a matched resonator is, by definition, the loaded Q. It is half of the Q of a cavity that has no coupling structure. The word loaded is sometimes used to imply that a sample has been introduced. Modification capability, allowing it to operate with a stop-flow apparatus. Temperature control, through hot or cold gas flow via a dewar that fits over the resonator piece but does not interfere with the modulation coil. Warranty Your Loop Gap Resonator is warranted to be free from defects in materials and workmanship for one year, unless otherwise stated, from shipment date. For more information, contact Molecular Specialties. See Page 5.

7 SECTION 2.1 PAGE 7 Your Loop Gap Resonator Figure 2.1 Molecular Specialties X-Band Loop Gap Resonator (Catalog No. XP-0201) and its modulation coil (Catalog No. MC-0001).

8 SECTION 2.2 PAGE 8 Technical Data Resonant Frequency Range GHz (specified by customer) Q (Quality Factor) Value Modulation Coil Capability Overall Length Loop Diameter Loop Length 600 unloaded KHz modulation fields 41.0cm/ inches 1.0mm/0.039 inches 5.0mm/0.196 inches Loop Volume 3.9 µl Recommended Sample Tubes Frequency Shift Optimum diameter/aqueous sample Outside diameter of probe Glass tube, TPX Capillary 80 MHz (0.6mm i.d. aqueous sample) 0.6mm/0.023 inches 23.0mm/0.905 inches Maximum sample tube outside diameter 0.9mm/0.035 inches Distance from resonator to waveguide flange (with 11.5cm waveguide) Temperature range 37.5cm/ inches o Liquid nitrogen to 60 C Minimum performance specifications Signal-to-noise 1,500 (10 µm Tempone sample in 0.6mm i.d. glass tube, 2.5 mw sample arm power, 100 khz modulation; see quality control test on Page 40.)

9 SECTION 2.3 PAGE 9 Optimal Experiments Your X-Band Loop Gap Resonator is a great choice for a wide range of EPR applications, including: Experiments where the sample is in limited quantity, such as sitedirected spin-labeling of a protein where only a few microliters of test material is available. Experiments where you desire higher effective microwave power at the sample, such as EPR pulse experiments. Experiments where you wish to utilize multiple microwave frequencies simultaneously. Experiments where you require a lower Q (Quality) Factor to reduce ringing. Continuous-wave saturation experiments. Stop-flow studies. Note: If you are planning to do stop-flow studies, contact Update Instruments, Inc., to acquire the proper hardware: Update Instruments, Inc Seybold Road Madison, WI Contact: Wayne Mueller Phone:

10 SECTION 2.4 PAGE 10 The Basics The key to your Loop Gap Resonator is the resonator piece itself, which is found at the end of the fiberglass support structure. The resonator piece is constructed of a ceramic material that contains two cylindrical holes (or loops) connected by a slot (or a gap). The internal surface of the ceramic cylinder is coated with a thin layer of silver. The microwave electric field is mainly concentrated within the slot, while the magnetic flux is concentrated within the holes. See Figure 2.2 for a detailed schematic of this process and a photo of the resonator piece. Figure 2.2 The inner operation of your Loop Gap Resonator s resonator piece.

11 SECTION 2.4 PAGE 11 A glass tube or Molecular Specialties TPX Capillary (Catalog No. TPX-2) containing your sample (either aqueous or non-aqueous) is placed within the sample loop via the resonator s sample holder or Molecular Specialties TPX Holder (Catalog No. TPX H-2), respectively. The magnetic flux density to which the sample is exposed in the resonator piece is greater than an air-filled cavity operating at the same frequency.

12 SECTION 2.5 PAGE 12 Preventive Maintenance TROUBLE SHOOTING TIP You should not attempt to repair or disassemble your Loop Gap Resonator. Doing so will void your warranty. Contact Molecular Specialties for service and repair information. See Page 5. Part 1 Cleaning Your Loop Gap Resonator must be kept extremely clean as contamination will greatly limit its performance. Heeding the following tips will greatly enhance its performance and lifespan. Do not brush the interior of the resonator piece. Doing so could scratch or damage its silver coating. Do not use compressed air when cleaning. Compressed air may contain fine particles of dust or droplets of water or oil that could damage the resonator piece. Do not use solvents such as acetone when cleaning. Doing so could damage the finish and materials from which the support structure is fabricated. Pure liquid contaminants may be removed by rinsing with pure water or ethanol, followed by a purge of nitrogen gas. However, evaporation of solvents from a solution will leave a solute residue.

13 SECTION 2.5 PAGE 13 Part 2 Proper Handling It is imperative you exhibit more than ordinary care in handling samples when using your Loop Gap Resonator. Its small dimensions and precision machining make contamination removal difficult and could ultimately require complete disassembly by Molecular Specialties, Inc. Heeding the following tips when handling samples will result in more successful experiments. Do not forcibly insert or remove sample tubes from the resonator piece. Do not turn the coupling adjust knob (the iris) past its limits. See Figure 2.3. Doing so could damage the coupling loop and prevent correct operation. Damage to the coupling loop may appear as excessive baseline noise, or jitter. Figure 2.3 Coupling adjust knob (the iris). Avoid sharp edges on glass sample tubes that could scratch the inner surface of the resonator piece. Silver particles scratched from inside the resonator piece will short out the microwave gap and no microwave absorption will be noted. Do not allow water or other lossy liquids to contaminate the resonator piece. It may cause the resonant dip to disappear. Non-lossy liquid contamination may cause the resonant dip to shift to a different frequency.

14 SECTION 3.1 PAGE 14 Installation The following instructions cover the installation of your Loop Gap Resonator for most Bruker and Varian spectrometers. Figure 3.1 provides a diagram of a properly connected Loop Gap Resonator. Figure 3.1 A Loop Gap Resonator properly connected to a microwave bridge and positioned between the spectrometer s magnet pole pieces.

15 SECTION 3.1 PAGE Remove the cavity resonator from your spectrometer by loosening the thumb screws. HANDY TIP A gasket is typically provided with Bruker and Varian spectrometers and can be used with your Loop Gap Resonator by placing it on the top of the waveguide extension. 2. Position your Loop Gap Resonator at the end of the microwave bridge waveguide so the sample holder faces toward you. 3. Mate your Loop Gap Resonator s waveguide extension to the bridge waveguide with the four included screws/nuts. Install the screws from the top. See Figure 3.2. Figure 3.2 Mating the waveguide extension to the bridge waveguide. Note the screw-jack stabilizers.

16 SECTION 3.1 PAGE 16 HANDY TIP Place screw-jack stabilizers on either side of the waveguide extension and tighten. This helps to reduce vibration. See Figure 3.2 for an example. 4. Adjust the position of the microwave bridge so the resonator piece is centered between the magnet pole pieces. See Figure 3.3. TROUBLE SHOOTING TIP The Hall Probe is a sensor for the magnetic field. Your modulation coil should not be touching it. Contact Molecular Specialties for further instructions if the Hall Probe is too close to the center of the pole face and interferes with securing the modulation coil. See Page 5. Figure 3.3 Centering the resonator piece between the magnet pole pieces. Note the position of the Hall Probe on the left magnet pole.

17 SECTION 3.1 PAGE Slide your Loop Gap Resonator s included modulation coil over the resonator piece from the bottom, with the screws pointing up and its coaxial cable aligned toward you. Note: The resonator piece must be centered inside the modulation coil and should not be touching the sides. 6. Tighten the screws atop the modulation coil to secure it to the magnet pole pieces. 7. Attach the coaxial cable on the modulation coil to your spectrometer s coaxial cable. See Figure 3.4. Figure 3.4 Positioning of the modulation coil over the resonator piece and attachment of coaxial cables. Note: If you are planning to use a dewar with your Loop Gap Resonator, contact the Medical College of Wisconsin Department of Biophysics for information and insight on the part fabrication and attachment process. Medical College of Wisconsin Department of Biophysics Contact: Christopher Felix, Ph.D, Scientific Administrator Phone: cfelix@mcw.edu

18 SECTION 4.1 PAGE 18 Preparing Test Samples Your test sample (both liquids and solids) will be placed in either a glass tube or Molecular Specialties TPX Capillary (Catalog No. TPX-2). A sealant typically a clay or wax must be applied to the end of the tube or TPX Capillary to prevent escape of the sample during testing. Popular sealants include: Fisherbrand Hemato-Seal Tube Sealing Compound (Catalog No ) Bruker X-Sealant (Catalog No ) Oxford Labware Critoseal (Catalog No ) Table 4.1 shows frequency shifts observed in a Loop Gap Resonator with a variety of common sample types in both a glass tube and a TPX Capillary. Resonator Loading Frequency (GHz) Q (loaded) Frequency Shift (MHz) EMPTY Glass tube (0.6mm x 0.84mm) Glass tube/water TPX Capillary/empty TPX Capillary/water TPX Capillary/glycerol Table 4.1 Frequency shifts observed in a Loop Gap Resonator with a variety of common sample types in both a glass tube and a TPX Capillary.

19 SECTION 4.1 PAGE 19 Part 1 Preparing a TPX Capillary Molecular Specialties TPX Capillary (Catalog No. TPX-2) is specially shaped to optimize sample acquisition. Note: TPX Capillaries should not be disposed of due to their high cost. Sealant should be removed after each use. A TPX Capillary threads seamlessly into Molecular Specialties TPX Holder (Catalog No. TPX H-2). See Figure 4.1. TPX Holder TPX Capillary Figure 4.1 Molecular Specialties TPX Capillary and TPX Holder.

20 SECTION 4.1 PAGE Draw the sample to be tested into the TPX Capillary. 2. Place the end of the TPX Capillary into a sealant pad straight down, leaving a small pocket of space between the sample and sealant. TROUBLE SHOOTING TIP Many sealants have a weak EPR signal. If the sealant is in the active region of the resonator piece, a weak EPR signal may be observed. The exception is Bruker XSealant (Catalog No ), which does not contain an EPR signal. 4. Carefully wipe the outside of the tube with a delicate task wipe. 5. Thread the TPX Capillary into the thin end of the TPX Holder. See Figure 4.2. Figure 4.2 Threading a TPX Capillary into the thin end of a TPX Holder. 6. Thread the TPX Holder into the resonator piece and conduct your experiment.

21 SECTION 4.1 PAGE Dispose of your test sample following the experiment. 8. Acquire a small piece of 26AWG copper-coated wire and create a small kink at the end with your thumb and forefinger. 9. Slide the wire kink-first through the TPX Capillary and rotate, which will eliminate leftover sealant. TROUBLE SHOOTING TIP Slide the wire completely through the TPX Capillary. Do not pull it back because you will spread sealant throughout the inner cavity. 10. Acquire a squeeze bottle of distilled water with a tapered end. Place the tapered end on the larger end of the TPX Capillary and squeeze water through it. 11. Repeat Step 10 with ethanol. HANDY TIP To dry more quickly, squeeze the end of a nitrogen gas tube around the larger end of the TPX Capillary so it forces the gas through it.

22 SECTION 4.1 PAGE 22 Part 2 Preparing a Glass Tube Glass tubes have sharp ends that could scratch or damage the silver coating of the interior of the resonator piece. Thus, it is imperative you polish the end of the sample tube to make it smooth before placing it in the resonator piece. Molecular Specialties recommends VitroCom Round Capillary Tubing (Catalog No. CV6084) for use with your Loop Gap Resonator. 1. Ignite a Meeker burner or a natural gas/oxygen torch. 2. Place the tip of the glass tube into the flame for a few seconds. This smooths the end of the tube and limits the chance of scratching the inside of the resonator piece. TROUBLE SHOOTING TIP If you leave the glass tube in the flame too long, the tip may close, bend, or develop a glob so it will no longer correctly fit into the resonator piece. 3. Place the fire-polished end of the sample tube into the sample material. Note: An aqueous sample will automatically be drawn in via capillary action. A hydrophobic sample may require suction to be drawn into the tube. For solids, place the glass tube on top of the sample and lightly tap the top to induce an amount to enter the tube.

23 SECTION 4.1 PAGE Place the fire-polished end of the tube into the sealant pad on a slight angle. The sealant will plug the inside of the tube. See Figure 4.3. Note: Avoid pressing the glass tube straight down onto the sealant pad. Fire-polishing the end of the tube collapses its sides, making it more difficult to place sealant that way. Figure 4.3 Plugging a glass tube with sealant. Note the slight angle. 5. Carefully wipe the outside of the tube with a delicate task wipe after applying sealant. TROUBLE SHOOTING TIP Many sealants have a weak EPR signal. If the sealant is in the active region of the resonator piece, a weak EPR signal may be observed. The exception is Bruker XSealant (Catalog No ), which does not contain an EPR signal.

24 SECTION 4.1 PAGE Place the tube in the Loop Gap Resonator s included sample holder and conduct your experiment. 7. Dispose of the tube following the experiment.

25 SECTION 5.1 PAGE 25 Tuning a Bruker ELEXSYS E 500 Spectrometer for Your Loop Gap Resonator Tuning any spectrometer for use with your Loop Gap Resonator is a process that you will master in time through repetition. The following instructions are a useful guide for beginning that process with the Bruker ELEXSYS E 500 spectrometer. See Figure 5.1 to view Molecular Specialties Loop Gap Resonator attached to a Bruker ELEXSYS E 500 spectrometer. (Refer to your Bruker ELEXSYS E 500 operator s manual for further tuning information.) Figure 5.1 Molecular Specialties Loop Gap Resonator attached to a Bruker ELEXSYS E 500 spectrometer.

26 SECTION 5.1 PAGE 26 Part 1 Searching for Resonance 1. Carefully place your sample tube or TPX Capillary/Holder in the resonator piece. 2. Confirm you are beginning at the Bruker ELEXSYS E 500 Xepr Main Panel default screen. TROUBLE SHOOTING TIP You will utilize the bottom toolbar on the Bruker ELEXSYS E 500 Xepr Main Panel default screen often when tuning and calibrating the spectrometer for use with your Loop Gap Resonator. 3. Click the Tuning button, located third from left on the bottom toolbar. The Microwave Bridge Tuning dialog box will pop up. 4. Confirm the Attenuation is between db. Use the arrow functions to adjust, if necessary.

27 SECTION 5.1 PAGE The signal will appear as a green line on the signal screen within the Microwave Bridge Tuning dialog box. See Figure 5.2. Figure 5.2 Example of a signal line. 6. Search for the resonant dip using the Frequency slider in the upper right corner of the Microwave Bridge Tuning dialog box.

28 SECTION 5.1 PAGE 28 Part 2 Tuning 1. Adjust the Frequency slider so the resonant dip is centered on the vertical line that divides the center of the screen. See Figure 5.3. Figure 5.3 Example of a centered resonant dip in the Microwave Bridge Tuning dialog box. Figure 5.4 Example of a centered and maximized resonant dip in the Microwave Bridge Tuning dialog box. 2. Gently turn the coupling adjust knob (the iris) on your Loop Gap Resonator to maximize the dip. See Figure 5.4. HANDY TIP As a result of turning the coupling adjust knob (the iris), the resonant dip may shift. Adjust the Frequency slider so it is once again centered.

29 SECTION 5.1 PAGE Click the Reference Arm to On. 4. Adjust the Bias slider so it is near the center of its range. Then adjust the Signal Phase slider to minimize the resonant dip. Both sliders are located under the Frequency slider. TROUBLE SHOOTING TIP There is interdependence between the Frequency, Bias, and Signal Phase sliders within the Microwave Bridge Tuning dialog box. Adjusting one may affect the others. 5. Click Operate, located next to the signal screen in the Microwave Bridge Tuning dialog box. 6. Adjust the Frequency slider so the Lock Offset meter is at zero. 7. Click Reference Arm to Off. 8. Decrease the Attenuation by clicking the down-arrow function to Adjust the Frequency slider if necessary so the Lock Offset meter is at zero. 9. Gently turn the coupling adjust knob (the iris) of your Loop Gap Resonator so it minimizes the Diode Current as close to zero as possible. Note: See Figure 5.5 on the next page to view the ELEXSYS Diode Current and Lock Offset meters and their relationship to the inner workings of a microwave bridge.

30 SECTION 5.1 PAGE 30 Figure 5.5 Schematic of the inner operation of a microwave bridge. Note the Diode Current and Lock Offset meters seen on the Bruker ELEXSYS E 500 Xepr Main Panel default screen. Meters from a Varian E-Line Century Series spectrometer are shown for comparison.

31 SECTION 5.1 PAGE 31 HANDY TIP If your sample contains water, it will heat as you increase microwave power. The microwave characteristics will change, and may cause the resonant frequency of your Loop Gap Resonator to change, in addition to effecting a rise in the Diode Current meter. To cool your sample and resonator piece, attach a hose to the air nozzle under the resonator piece and blow dry nitrogen gas. 10. Click the Reference Arm to On and adjust the Bias slider so the Diode Current is about 200 µa. 11. Click Close to close the Microwave Bridge Tuning dialog box. 12. If you have not previously calibrated your Bruker ELEXSYS spectrometer for your Loop Gap Resonator and modulation coil, turn to Section 5.3, Calibrating a Bruker ELEXSYS E 500 Spectrometer for Your Loop Gap Resonator. See Page 35. If you have previously calibrated, turn to Part 3 in this section, If Previously Calibrated... See Page 32. HANDY TIP You may use a modulation coil other than the one included with your Loop Gap Resonator.

32 SECTION 5.1 PAGE 32 Part 3 - If Previously Calibrated If your Bruker ELEXSYS spectrometer has already been calibrated for the modulation coil you are using, click Acquisition on the top Menu Bar. Click Spectrometer Configuration, and the Spectrometer Configuration dialog box will pop up. 2. Click the Signal Channel button. Under the field Standard Calibration Data, choose the data set corresponding to the modulation coil you are using. 3. Click Close. 4. Acquire a spectrum. (Refer to your Bruker ELEXSYS E 500 operator s manual for information on this process.) 5. When you have finished or are ready to change samples, click the Tuning button, located third from the left on the bottom toolbar. The Microwave Bridge Tuning dialog box will pop up. 6. Confirm the Attenuation is between db. Use the Arrow functions to adjust, if necessary. 7. Confirm you are in the Tune mode. 8. Remove your sample. TROUBLE SHOOTING TIP Always confirm the Attenuation is between db and you are in the Tune mode when inserting or removing a sample.

33 SECTION 5.2 PAGE 33 For Microwave Bridges with Stabilizer Frequency Some microwave bridges are equipped with a Stabilizer Frequency dial on the upper left side of the front face of the bridge. See Figure 5.6. It is used when the researcher desires operation at a power level where the AFC is not able to lock onto resonator mode. The following procedure allows you to use this option when tuning a Bruker ELEXSYS E 500 for your Loop Gap Resonator. Figure 5.6 Microwave bridge with Stabilizer Frequency dial. 1. Click the Tuning button, located third from left on the bottom toolbar. The Microwave Bridge Tuning dialog box will pop up. 2. Confirm you are in the Tune mode in the Microwave Bridge Tuning dialog box. 3. Click the box next to Dual Trace. 4. Click the On button under the Stabilizer Frequency dial on the microwave bridge.

34 SECTION 5.2 PAGE Adjust the Stabilizer Frequency dial on the microwave bridge by hand so the yellow stabilizer mode line curves up and aligns with the green resonator dip line, which curves down. See Figure Click the box next to Dual Trace. 7. Click Close. Figure 5.7 Alignment of stabilizer mode and resonator dip lines. In this display, the stabilizer frequency is yellow and the resonant dip is green. The colors may be reversed on some spectrometers.

35 SECTION 5.3 PAGE 35 Calibrating a Bruker ELEXSYS E 500 Spectrometer for Your Loop Gap Resonator You must calibrate your Bruker ELEXSYS E 500 spectrometer for use with your Loop Gap Resonator s modulation coil prior to its initial use. The calibration information is stored on the ELEXSYS E 500 s computer, and you will refer to it whenever you use your Loop Gap Resonator with it. If you do not calibrate, you will not know your modulation amplitude and phase, and you could get less than maximum signal, or potentially no signal. (Refer to your Bruker ELEXSYS E 500 operator s manual for further calibration information.)

36 SECTION 5.3 PAGE 36 Part 1 Pre-calibration TROUBLE SHOOTING TIP To calibrate your Loop Gap Resonator s modulation coil for the first time, use a sample that will provide a singleline spectrum of moderate width. Molecular Specialties recommends a solution of 1g of sodium dithionite per 20mL of water, or DPPH crystals. 1. Place your calibration sample in the resonator piece and follow the tuning procedure described in Section 5.1, Tuning a Bruker ELEXSYS E 500 Spectrometer for a Loop Gap Resonator. See Page Click the Tuning button, located third from left on the bottom toolbar. The Microwave Bridge Tuning dialog box will pop up. 3. Confirm you are in the Operate mode in the Microwave Bridge Tuning dialog box. 4. Click the Create an Experiment button, located on the extreme left of the bottom toolbar. The Build Experiment dialog box will pop up. 5. Type calibration in the Experiment Name field. 6. Click the Calib tab. Then click Standard in the middle of the dialog box. 7. Click Create.

37 SECTION 5.3 PAGE 37 Part 2 Calibration 1. Click the Parameters button, located fourth from left on the bottom toolbar. The Calibration Parameters dialog box will pop up. TROUBLE SHOOTING TIP In the Calibration Parameters dialog box, you may calibrate for many frequencies, or you may calibrate for the default settings displayed. The following configuration is recommended for your Loop Gap Resonator: Mod. Frequency Start [khz]: 100 khz Mod. Frequency End [khz]: 10 khz Mod. Frequency Incr. [khz]: 10 khz 2. Click the Setup Scan button, and then the Setup Scan box. You should see an EPR signal. See Figure 5.8. Figure 5.8 An EPR signal viewed after clicking the Setup Scan box. If you do not see a signal at all, click on the Parameters button. The Calibration Parameters dialog box will pop up. Click the Absc. 1: Field tab. Under Abscissa 1 Sweep Quantity: Field, type in 3300 in the Center Field field. Click Setup Scan.

38 SECTION 5.3 PAGE 38 Note: Depending on the resonator frequency, you may have to modify the Center Field value. If you still do not see a signal, click on the Calibration tab and type 12 in the Tuning C field. Then complete the following procedure: A. Decrease the Receiver Gain under Signal Channel until the signal in the Setup Scan box is less than half the height of the window. B. Click on the Results tab and type in a new calibration name in the Calibration Data field (Example: MolSpecCalib ). Click Setup Scan. TROUBLE SHOOTING TIP Be sure to type in a new name in the Calibration Data field. If you do not, you will erase the previous calibration and replace it with the Loop Gap Resonator s calibration values. 3. Click the Run/Abort Experiment button located in the extreme lower left corner of the Bruker ELEXSYS E 500 Xepr Main Panel default screen. See Figure 5.9. The spectrometer will then take several minutes to sweep the field. Figure 5.9 The Run/Abort Experiment button. 4. Click the Results tab in the Calibration Parameters dialog box. Data should be entered in the cells of the six-column grid under the Calibration Results field. 5. Click Close when calibration is completed.

39 SECTION 5.3 PAGE 39 Part 3 Confirmation 1. Click Acquisition on the top Menu Bar. 2. Click Spectrometer Configuration. The Spectrometer Configuration dialog box will pop up. 3. Click the Signal Channel tab. 4. Click Calibration Data Set under Standard Calibration Data. The name of your calibration should be there. TROUBLE SHOOTING TIP Whenever you perform an experiment using your Loop Gap Resonator and its modulation coil, you will choose the calibration data set you created in Section 5.3 before you attempt to acquire a spectrum. Follow the directions in Section 5.1, Part 3, clicking on the name of your calibration before attempting to acquire a spectrum. See Page 32.

40 S U P P O R T PAGE 40 Appendix Quality Control Test Part 1 Preparation 1. Prepare a 10 µm Tempone sample in a 0.6mm i.d. glass tube. 2. Install your Loop Gap Resonator on your spectrometer and after placing the glass tube with the sample material into the resonator piece, tune the spectrometer. See Section 5.1, Tuning a Bruker ELEXSYS E 500 Spectrometer for Your Loop Gap Resonator, for information on this process. See Page 25. Part 2 Measuring Signal 1. Set your spectrometer to the settings below and acquire a spectrum. Center field As necessary Scan range 50 Gauss Power 2.5 mw Modulation frequency 100 khz Modulation field intensity settings 1 Gauss Receiver gain 5 x 10 2 Receiver time constant 1 second Scan time 2 minutes Note: Adjust the receiver gain to maximize the signal on the plot.

41 S U P P O R T PAGE 41 Appendix (cont d) Part 3 Measuring Noise 1. Change the following settings on your spectrometer and acquire a spectrum: Center field 3,500 Gauss Scan range 0 Gauss Receiver gain 5 x 10 4 Receiver time constant 1 second Scan time 2 minutes Part 4 Calculations 1. Calculate the signal-to-noise ratio using the following equation: S h 1 x G 2 N h 2 x G 1 Equation values: h 1 the peak-to-peak height of the largest spectra from Part 2 in centimeters h 2 the peak-to-peak height of the largest noise amplitude from Part 3 in centimeters divided by 2.5 to arrive at an estimate of RMS noise. G 1 Receiver gain setting in Part 2 G 2 Receiver gain setting in Part 3 2. The equation should result in a signal-to-noise calculation of 1,500 or greater.

42 S U P P O R T PAGE 42 Bibliography R.D. Allendoerfer, W. Froncisz, C.C. Felix, and J.S. Hyde, Electrochemical Generation of Free Radicals in an EPR Loop-Gap Resonator, J. Magn. Reson. 76, (1988). R. Basosi, W. E. Antholine, W. Froncisz, and J.S. Hyde, Spin-Hamiltonian Input Parameters in the EPR Analysis of Liquid Phase Copper Complexes, J. Chem. Phys. 81, (1984). T. Christides, W. Froncisz, T. Oles, and J.S. Hyde, Probehead with Interchangeable Loop-Gap Resonators and rf Coils for Multifrequency EPR/ENDOR, Rev. Sci. Instrum. 65, (1994). W. Froncisz and J.S. Hyde, The Loop-Gap Resonator: A New Microwave Lumped Circuit ESR Sample Structure, J. Magn. Reson. 47, (1982). W. Froncisz, T. Oles, and J.S. Hyde, Murine L-Band ESR Loop-Gap Resonator, J. Magn. Reson. 82, (1989). W. Froncisz, T. Oles, and J.S. Hyde, Q-Band Loop-Gap Resonator, Rev. Sci. Instrum. 57, (1986). W.L. Hubbell, W. Froncisz, and J.S. Hyde, Continuous and Stopped-Flow EPR Spectrometer Based on a Loop-Gap Resonator, Rev. Sci. Instrum. 58, (1987). J.S. Hyde and W. Froncisz, Loop Gap Resonators, in: Advanced EPR: Applications in Biology and Biochemistry, A.J. Hoff, ed. (Elsevier: Amsterdam, 1989), pp J.S. Hyde and W. Froncisz, Loop-Gap Resonators, in: Electron Spin Resonance, M.C.R. Symons, ed. (Specialist Periodical Reports, The Royal Society of Chemistry: London, 1986), Vol. 10, pp J.S. Hyde, W. Froncisz, and A. Kusumi, Dispersion Electron Spin Resonance with the Loop- Gap Resonator, Rev. Sci. Instrum. 53, (1982). J.S. Hyde, W. Froncisz, and T. Oles, Multipurpose Loop-Gap Resonator, J. Magn. Reson. 82, (1989). J.S. Hyde and J. Gajdzinski, EPR Automatic Frequency Control Circuit with Field Effect Transistor (FET) Microwave Amplification, Rev. Sci. Instrum. 59, (1988).

43 S U P P O R T PAGE 43 Bibliography (cont d) J.S. Hyde, J.-J. Yin, W. Froncisz, and J.B. Feix, Electron-Electron Double Resonance (ELDOR) with a Loop-Gap Resonator, J. Magn. Reson. 63, (1985). C. S. Klug, J.F. Feix, SDSL: A Survey of Biological Applications in Biological Magnetic Resonance, Volume 24: Biomedical EPR Part B: Methodology and Instrumentation, S.S. Eaton, G.R. Eaton, L.J. Berliner, eds. (Kluwer Academic/Plenum Publishers, New York 2004), pp M. Mehdizadeh, T.K. Ishii, J.S. Hyde and W. Froncisz, Loop-Gap Resonator: A Lumped Mode Microwave Resonant Structure, IEEE Trans. Microwave Theory Tech. MTT31, (1983). T. Oles, J.S. Hyde, and W. Froncisz, Gordon Coupler for K-Band EPR Loop Gap Resonator, Rev. Sci. Instrum. 60, (1989). S. Pfenninger, W. Froncisz, J. Forrer, J. Luglio, and J.S. Hyde, General Method for Adjusting the Quality Factor of EPR Resonators, Rev. Sci. Instrum. 66, (1995). W. Piasecki, W. Froncisz, and J.S. Hyde, Bimodal Loop-Gap Resonator, Rev. Sci. Instrum. 67, (1996). K.S. Rothenberger, M.J. Nilges, T.E. Altman, K. Glab, R.L. Belford, W. Froncisz, and J.S. Hyde, L-Band Parallel Mode EPR Measurement of Quadrupole Coupling Through Direct Observation of Secondary Transitions, Chem. Phys. Lett. 124, (1986). W.K. Subczynski, S. Lukiewicz, and J.S. Hyde, Murine in Vivo L-Band ESR Spin-Label Oximetry with a Loop-Gap Resonator, Magn. Reson. Med. 3, (1986). D.D. Thomas, C.H. Wendt, W. Froncisz, and J.S. Hyde, Saturation Transfer EPR Spectroscopy on Spin-labeled Muscle Fibers Using a Loop-Gap Resonator, Biophys. J. 43, (1983). A.I. Tsapin, J.S. Hyde, and W. Froncisz, Bimodal Loop-Gap Resonator, J. Magn. Reson. 100, (1982). R.L. Wood, W. Froncisz, and J.S. Hyde, The Loop-Gap Resonator. II. Controlled Return Flux Three-Loop, Two-Gap Microwave Resonators for ENDOR and ESR Spectroscopy, J. Magn. Reson. 58, (1984).

44 S U P P O R T PAGE 44 Index Acetone 12 AFC 33 Bruker 14-15, 18, 20, 23, 25-26, 30-33, 35-36, 38, 40 X-Sealant 18, 20, 23 ELEXSYS E 500 Spectrometer 25-26, 29-33, 35-36, 40 Diode Current meter Lock Offset meter 29, 30 Stabilizer Frequency Xepr Main Panel default screen 26, 30, 38 Dewar 6, 17 DPPH crystals 36 EPR (Electron Paramagnetic Resonance) 5-6, 9, 20, 23, 37, Ethanol 12, 21 Gasket 15 Glass tube 8, 11, 18, 22-23, 40 VitroCom 22 Glycerol 18 Hall Probe 5, 16 Iris (see Loop Gap Resonator, Coupling adjust knob) Loop Gap Resonator 4-7, 9-10, 12-15, 17-18, 22, 24-28, 29, 31, 33, 35-37, 39-40, Cleaning 12 Coupling adjust knob 7, 13, Hose barb 6, 7 Installation 14 Modification capability 6 Modulation coil 5-8, 16-17, 31-32, 35-36, 39 Optimal experiments 9 Ordering 5 Preventive maintenance 12 Proper handling 13 Quality control testing 8, Q (Quality) Factor 6, 8-9, 18 Resonator piece 6, 10-13, 16-17, 20, 22-23, 26, 31, 36, 40 Sensitivity 6 Technical data 8 Temperature control 6 Warranty 6, 12 Waveguide extension 7-8, Meeker burner 22 Medical College of Wisconsin Department of Biophysics 3, 17 Microwave bridge 5, 14-16, 29-30, Molecular Specialties, Inc. 5-7, 11-13, 16, 18-19, 22, 25, 36

45 S U P P O R T PAGE 45 Index (cont d) Natural gas/oxygen torch 22 Nitrogen gas 12, 21, 31 Quartz Tube (see Glass Tube) Screw-jack stabilizers Sealant 18-21, 23 Bruker X-Sealant 18, 20, 23 Fisherbrand Hemato-Seal Tube Compound 18 Oxford Labware Critoseal 18 Sodium dithionite 36 Spectrum acquisition 32, 36, Stop-flow studies 6, 9 Tempone 8, 40 TPX Capillary 6, 8, 11, 18-19, 20-21, 26 TPX Holder 11, 19-20, 26 Update Instruments 9 Varian 14-15, 30 AFC meter 30 Current detector 30

46 S U P P O R T PAGE 46 Notes

47 S U P P O R T PAGE 47 Notes