LUMIGEN INSTRUMENT CENTER X-RAY CRYSTALLOGRAPHIC LABORATORY: WAYNE STATE UNIVERSITY

Transcription

1 Standard Operating Procedure for the Bruker X8 APEX II Single-Crystal X- Ray Diffractometer Contact Manager: Dr. Cassie Ward Office room 061 Chemistry (313) LIC Lab: (313) Location Lumigen Instrument Center X-Ray Crystallographic Laboratory Department of Chemistry Wayne State University Chemistry Building 5101 Cass Avenue Detroit, MI Safety Requirements Access to the lab will be revoked if you do not follow these safety procedures. 1. While working in the X-Ray Crystallographic Laboratory, researchers are always required to wear Personal Protective Equipment (PPE). The appropriate PPE include dosimetry ring, safety glasses, long pants/skirt covering the legs completely, closed-toe shoes, and gloves. Do not wear gloves while using the computer keyboard or mouse. All required PPE needs to be supplied by the user. Dosimetry rings must not be removed from the lab. 2. Food and beverages cannot enter the lab. Never eat or drink inside the lab. 3. Please keep the area around the instruments and prep stations clean. If you use the lab s sample holders, clean them after use. Properly dispose glass waste in the appropriate container. All samples must be properly disposed of in your research lab. 4. Following a spill, please clean it up with a paper towel or broom. If you are unsure how to clean up the spill, contact the lab manager (Dr. Ward) or the LIC Director (Dr. Westrick). If the spill occurs after hours with personal injury, please contact WSU police department (7-2222) and Dr. Westrick, immediately. 5. The Standard Operating Procedures (SOPs) are next to the instruments. The users are to follow what is written in these procedures. 6. You must log the usage time into the appropriate instrument log book. 7. All researchers working in LIC must complete the EH&S initial course for Laboratory Safety Training and show proof of completion. Users whose safety training has expired will not be permitted access to the laboratory. 8. In case of an instrument malfunction, turn off the instrument and contact Dr. Ward immediately. 9. Be aware that the X-Ray Crystallography Laboratory has a red binder containing the Chemical and Laboratory Safety Information. It is located on the second shelf above the Ward Page 1 of 10

2 dosimetry rings cabinet. Additional Safety Requirements for the SCD 1. In case of an instrument malfunction, turn off the instrument (Figure 1) press one of the two red buttons. Contact Dr. Ward immediately, or the WSU police ( ) if occurrence is after hours. LIC Authorization Information 1. All users must pass the online EH&S Laboratory Safety Training and Radiation (X-ray) Generating Machine training ( Print the certificate after completing the quiz and bring it to Dr. Ward. The two safety trainings must be completed every Janurary and the printed certificate must be brought to Dr. Ward. 2. A dosimetry form must also be filled out ( and signed by Dr. Westrick. Bring the signed form to Dr. Ward. Dosimetry rings must be worn while working in the lab. The rings are stored in the cabinet next to the sink. 3. Contact Dr. Ward for training. 4. Before running, you must have an ilab account with a grant number from your PI. a. Go to if you are a new user or new PI to ilab. 5. After training, you can reserve time for the X8 using the calendar in the lab. 6. Collection needs to be started before 11 a.m. if running for 24 hours. Overview 1. Identify materials and elucidate crystalline structure Sample Preparation 1. Use one of the two microscopes to exam your crystals (Figure 1). It is important to pick out a good single crystal. a. Crystals should be <0.5 mm along each side. b. Crystals should have few imperfections. No cracks, striations, or bubbles. 2. Paratone oil provides protection to the crystals from air and moisture, it makes it easier to cut crystals, and it holds the crystal to the MicroMount. 3. There are a variety of MicroMount sizes (Figure 2). Make sure you do not have too much oil on your mounted crystal. Place the mounted crystal onto the goniometer. 4. The X8 APEX II instrument is pictured in Figure If the doors are locked use the Open Door button to open the cabinet (Figure 4). Open the two doors gently pulling on the door handles and sliding them over. Ward Page 2 of 10

3 Figure 1: For Selecting and mounting crystals, use either the A) light microscope or B) light microscope with a polarizer and camera. Figure 2: MicroMount holder for a single crystal. Ward Page 3 of 10

4 Figure 3: X8 APEX II single-crystal XRD. Ward Page 4 of 10

5 Figure 4: Below the APEX II cabinet on the left (red) and right side (black), respectively, are a serious of buttons and light indicators. The STOP on either side is for emergencies to turn off x- ray generation. The Open Door button on either side needs to be pushed to open the cavity doors. On the left side, the green light bulb button is to turn on/off the light inside the cavity. On the right side, the red button with a O turns off the instrument and the green button with a turns the instrument on. The High Voltage turns on the voltage to the x-ray source. This is used to reset an alarm. When the instrument is operational, the Ready and On lights should be solid and the Alarm may be blinking if the doors are open. Otherwise the Alarm LED should not be on. If the instrument is in use, the Busy light will be solid. Operating the Instrument 1. The APEX3 and Bis programs should already be open. If not, open Bis first, followed by APEX3. If there are any alarms indicated in the Bis or Status menu of APEX3, find Dr. Ward for help. 2. In APEX3, Go to Sample New 3. Give your sample a name and hit OK. When prompt, hit save. The data will be saved into the guest folder on the desktop. 4. Under Setup, click on Center Crystal (Figure 5), then click Center (Figure 6). 5. Put a piece of paper in front of the detector so the crystal can be seen better on the computer monitor (Figure 7). Ward Page 5 of 10

6 Figure 5: Screen shot of APEX3 program. Under Setup, hit Center Crystal to start the alignment process. Use Screen Crystal to check the diffraction. 6. The crystal needs to be centered to the cross-hairs. Use the adjustment tool on the goniometer to move the position of the crystal into the crosshairs (Figure 8). 7. When it is centered in the cross hairs, click Spin Phi 90. This rotates the crystal If the crystal is not centered, make adjustments to the goniometer. 9. Continue until the crystal is centered while rotating a full Next, click on Screen Crystal (Figure 5) on the left side of the program. 11. Click on Zero (Figure 9) and change the Distance to 40. This moves the detector to 40 mm. The detector cannot move any closer than 40 mm. Longer detector distances can help improve resolution if the unit cell is large. Figure 6: Screen shot of the crystal off centered. Ward Page 6 of 10

7 Figure 7: Put a piece of paper in front of the detector to help increase the lighting when doing the alignment. Figure 8: Centering the crystal by using the adjustment tool to move the mounted crystal. Ward Page 7 of 10

8 Figure 9: Screen shot of Simple Scans changing the angles to zero and the detector distance. 12. At the bottom, change the Scan Range to 1 and Image Width to 0.5. The Exposure Time can be left at 10 secs/image. This can be increased if you have a weakly diffracting crystal. 13. Click on the Drive + Scan button at the bottom. This will collect two images. 14. Change the Chi angle to 45 and click Drive + Scan 15. Change the Phi to 90 and click Drive + Scan again. 16. Look at the diffraction pattern. If the spots look bright, single, round, and there are enough of them, click on Evaluate in the left panel, then Determine Unit Cell 17. Most likely you do not need to change any of the settings. You might change the exposure time if you have a weakly diffracting crystal. 18. Click on Collect at the bottom right. 19. After a few minutes of collecting, click on Harvest Spots. 20. Make sure there are at least 100 reflections, then click Index. 21. Accept the Unit Cell dimensions that the program determines. 22. Move the Tolerance slider to More Reflections and hit Accept until the variables stop changing. 23. Next, click on Bravais. Click Accept using the Bravais lattice the software determined. Ward Page 8 of 10

9 a. If you are unsure of the software s choice, you may choose Triclinic. The collection strategy will be based on the unit cell s dimensions and symmetry. 24. Click on Refine. Again, move the Tolerance slider to More Reflections and click on Refine until the variables stop changing. 25. The size of the crystal needs to be reported for publication. Click on Reduce Data, on the left side, and Index Crystal Faces. 26. Click on Acquire New and Save. 27. The program will collect a 360 image of the crystal on the mount. When this is down you should see the image of the mount and crystal. Use the wheel of the mouse to spin the crystal around. Check that the + is in the center of the crystal. If it is not, right click and select Configure overlay. 28. Change the x- and y-centering values until the crystal remains in the center while spinning the crystal image. 29. Whenever you see a dashed line, you can click the mouse at the edge of the crystal on both sides. Then use the wheel of the mouse to spin the crystal until another dashed line appears. Click again at the edge of the crystal. 30. Continue until you are satisfied with the size of the image overlaying the crystal. At the bottom right will have the crystal dimensions. Make sure each side is less than 0.5 mm. 31. Next, click on Collect on the left side panel. Click on Calculate Strategy (Figure 10) Figure 10: Screen shot of the Calculate Strategy menu. The numbering identifies the ordering of the menu options to select. 32. The dimension of the unit cell and Bravais Lattice will be displayed in the Step 1 section, labeled 1 in Figure 10. a. Select the appropriate resolution (label 2 in Figure 10) based on the diffraction of your crystal ( Å). b. Select the appropriate symmetry option (label 3 in Figure 10). Ward Page 9 of 10 7

10 33. Click on Determine strategy (label 4 in Figure 10). a. In the menu, Parameters for the strategy determination, select the detector distance (Figure 11). The program will have a suggested distance, or you can change it to 40 mm. b. Keep the rest of the defaults as shown in Figure 11. You may change the multiplicity, but after the initial strategy determination. c. Click on OK. Figure 11: Determine strategy pop-up menu. 34. The program will determine the optimal strategy based on your unit cell, symmetry, resolution, and detector distance. This will be displayed in the Step 2 section. 35. Pay attention to the average multiplicity (needs to be above 2), missing reflections (should be a small percentage of the total reflections collected), and the completeness (above 99% typically). a. If there are too many missing reflections, you can click Extend strategy (label 5 in Figure 10). Select more of the options available in the menu. The Determine Strategy menu will pop up again. Change the multiplicity to a higher value. This will recalculate the strategy and there should be fewer reflections missing. b. You can also view what reflections are being collection and which ones are missing by clicking on Show reciprocal lattice view (label 6 in Figure 10). 36. Next, click on Select scan parameters. 37. A window will open where you can enter the frame angle. The software will choose one based on your matrix run. You can also change it to Select a Frame time based on the quality of your diffraction spots. The overall time of the experiment will be listed here. 39. To increase the run time, you can increase the frame time and/or the multiplicity in the Determine strategy menu. 40. When you are satisfied with your data collection strategy, go to Run Experiment on the left side (Figure 10). 41. Click on Append Strategy, then click on Validate. a. If an error appears, delete the run that will cause the error. Click on Validate again. 42. Click on Execute to begin the collection. Ward Page 10 of 10