1 Single Crystal X-Ray Crystallography 1,2 You will use the program APEX2 to determine the structure of an organic compound (to be referred to as Ylid) from the data supplied by our x-ray diffractometer. The data will be placed in the file ylid_Ja.hkl , which consists of the x-ray intensities observed for reflections from many planes, identified by three index numbers, h, k, and l. The diffractometer automatically orients the crystal and the detector to pick up reflections from each plane, and records the number of counts per second as the intensity. The empirical formula of Ylid is C 11 H10 O2S. Before you start, you should use MOE (or MM3) to construct several small molecules to give you an idea of what bond lengths to expect for C-C, C- O, C-S, O-S, C-H, O-H, and S-H single and (where feasible) double bonds, because you will have to try to recognize them. Try to get an idea of the variability of the bond lengths by building different environments. There are four main programs in the APEX2 suite. Saint+ integrates the X-ray intensity for each of the diffraction spots, to give an overall intensity for each observed h,k,l plane in the diffraction patterns. XPREP helps you make an initial decision about the space group and sets up the files to be used by the other programs. X-ray diffraction doesn’t directly determine the positions of the nuclei, rather the X-rays scatter from the electron density in the crystal. XS tries to find the positions of atoms directly from the X-ray diffraction data without using Patterson maps. XS often finds more atoms than there actually are and may identify only a few of the real atoms. Finding the true atom positions is an iterative process. In early iterations, the atom positions of the real atoms XS finds are often displaced from the true positions. XShell allows you to visualize the structure at any point in the analysis. XShell refines the structure to find the best fit to the X-ray intensity data using iterative calls to XS. The “best fit” is produced by placing atoms to generate an electron density map that best approximates the experimentally determined electron density map. XShell allows you to determine distances and angles between atoms, to rename provisional atoms to real atoms, and to delete bad atoms from the list. APEX2 handles the coordination of the input and output files to Saint+, XPREP, XS, and XShell. Defining a New Project and Opening the First Data File 1. Open a Terminal window, type ssh -Y laue then type “apex &” at the $ prompt (include a space between apex &). Login as guest with the password guest. 2. In the top menu bar pull down the Sample menu and choose New. 3. Specify a project Name. The destination folder should be entered for you in the guest subdirectory. You may need to enter “/export” at the beginning of the Folder path 2 4. In the top menu bar, pull down the Sample menu, slide right on Import and choose P4P/SPIN file… Navigate to the raw data directory given to you by your instructor. Double click on the ylid_Ja1.p4p file entry. Click on the Import all check box and click OK. Saint+ The Integration routine first needs to know where to find the diffraction spots, which are determined by the lattice type and the unit cell parameters. Then the total intensity in each detected diffraction spot is determined by integration from the raw exposure frames. 5. Click on the Evaluate tab in the left column and then click on Determine Unit Cell icon. If you are satisfied with the unit cell determined by the data acquisition software, then skip this step and proceed directly to Integration, step 16 below. 6. Call up the first frame in your first set of frames, by clicking the file browser icon that is just to right of the file name box. Set the file type to “All files (*).” The images are in the original raw data directory (you may need to use the up folder icon, , to navigate back to this folder, which you opened to define your new sample). First frame is called up here File browser icon In the file browser, pull down the File Type menu and choose SMART images for the file type: 3 Click on the file name for the first frame, ylid_Ja1.001 in this case. Click Open. The first raw diffraction pattern image will be displayed. The spots on these images must be integrated to find the intensity for each diffraction plane hkl. 7. Click on the Harvest Spots button on the right under manual mode. 8. Put 3 for the number of runs and 150 for images per run. For example, if you have three runs of 600 frames and a fourth run of 50 frames, you will be using 150 frames from each of the first three runs to determine the unit cell. Using all the acquired frames would be time consuming. Slide the minimum I/sigma(I) to about 10. Click on the Harvest button near the bottom right of the screen. 9. After the spots are harvested, click on the Index button. 10. Accept the default values and click the Index… button at the bottom right of the window. 11. Several indexing methods are used and the one that gives the best result is highlighted in blue. Click the Accept… button at the bottom right. The indexed spots are circled in the raw diffraction pattern. 12. You will then see a window, part of which looks like the following. Click the Refine button to refine the indexing again. You will see the standard deviations of the unit cell parameters appear in the Parameters box. Keep clicking the refine button until the changes in the uncertainties are negligible. Then click on Histograms…. In the HKL graphs, the tall bars should be near 0. Click Close on the histogram plot window. Click on the Accept button near the bottom right. You can switch from one screen to another by clicking on the yellow buttons in the top icon bar. 4 13. After the spots are indexed, click on the Bravais button. 14. You will then see a window, part of which looks as shown below. Here the program has already picked out the Bravais lattice. The Ylid lattice should be Orthorhombic P at this point. Unless you know a change is warranted, accept the default. 15. Click on the Refine button. Refine and accept as discussed in Step 12. 5 Click on the Reciprocal Lattice Viewer icon in the left column. The calculated equivalent of the precession photographs will be shown. Set the Editing tools to None (rotate) in the upper right, if not already selected. After 15 or so to allow the spots to be transferred, try reorienting the reciprocal lattice with the mouse. Observe the reciprocal lattice from the three orthogonal directions. 16. Click on the Integrate tab in the left column. Then click on the Integrate Images icon. 17. Change the Resolution limit to 0.8 angstroms. 18. Click on Find Runs… near the bottom right of the screen. 19. Navigate to your file. In the Run file selection box at the right of the dialog, only select the four sets of non-MATRIX data and click OK. 20. The spreadsheet on the main window will be populated by the runs you selected. Now click the Refinement Options… button near the bottom part of the right column. Accept the default values on the screen that appears. Click OK. 21. In the main window, click the Integration Options… button in the column on the right. 22. In the window that appears, click More Options. 6 23. A part of the screen that appears is shown below. In the Fractional Lower Limit of Average Intensity box enter 0.5, to account for the beam stop. Click OK. 24. In the main window, click Start Integration… near the bottom right of the main window on the right column. 25.After integration, click Close near the bottom right of the screen. 26. Next click the Scale tab in the left column, and then click the yellow Scale ion. 27. In the screen that appears, accept the default values and click Next. 28. You will now be in the Parameter Refinement window. Accept the default values and click Refine. 29. If you are satisfied with the refinement, click Next. Otherwise refine again. 30. You will now be in the Error Model window. Accept the default values and click Determine Error Model. You will see the error statistics for each of your four data sets. 31. You can continue to cycle through steps 29 and 30 and,when done, click Finish. 32. Click Exit AXScale. XPREP In XPREP you will be shown some information about lattice exceptions in order to choose the type of crystal lattice. The lattice types correspond to the positions of lattice points, repeating elements centered on some defined position in the unit cell. The simplest lattice (P, for primitive) has lattice points only on the corners. If the center is also a lattice point, it is designated I (body- centered cubic). If two opposite faces are lattice points, the designation is A if they are along the x-axis, B if the y-axis, and C if the z-axis. F designates a face-centered cubic lattice in which all faces are occupied. The abbreviations Rev and Obv (reverse and obverse) in XPREP refer to hexagonal unit cells, which we will not have to worry about.