Growing Crystals for X-ray Diffraction Analysis

1. Preparation of the Crystal Tube and Filter

1. Place an NMR tube in an Erlenmeyer flask.
2. Prepare a pipette filter.
   1. Construct the filter by placing a piece of lint-free wipe (1 in. by 1 in.) in the pipette, then use a rod to firmly wedge the wipe into the bottleneck portion of the pipette (Figure 1).
   2. Make two pipette filters for every crystal tube needed.

2. Adding the Sample to the Crystal Tube

1. Dissolve the compound (tetraphenylporphyrin, 10 mg) in 0.75 mL of solvent (dichloromethane).
2. With a pipette, gently add the mixture to the top of the tube, by passing it through the filter.
   1. The particles are filtered out to avoid the creation of nucleation sites, which can lead to small multiple crystals instead of the desired larger single crystals.
3. Once the sample has been placed in the crystal tube, very slowly and gently, add the anti-solvent (1.5 mL of methanol) to the tube through a new filter pipette. Allow the anti-solvent to slowly layer on the previously added solution (Figure 2). Do not use a bulb to push the solvent through the pipette, instead allow the solvent to flow through the filter by itself.
   1. Make sure that solvent of higher density is added to the crystal tube first.
   2. Check that the two solvents are miscible with each other. This is done prior to addition of the solvent.
4. Seal the tube with an NMR cap.

3. Crystal Growth

1. Without causing the two solvents to mix, place the crystal tube(s) in a cabinet where they will not be disturbed.
2. Crystallization time will vary with each compound- typically the crystal tubes should be left undisturbed for a week.
3. After a week, inspect the tubes for crystal growth.
   1. Crystal growth typically occurs at the interface of the two solvents.
   2. Visually inspect the tubes for evidence of crystal growth. Be careful not to facilitate mixing of the solvents, in case the compound requires additional time for crystal formation.
   3. If it appears that crystal growth has occurred, further inspect the tubes using a microscope.

4. Crystal Selection

1. X-ray diffraction crystals should have well defined faces.
2. Crystals that have clustered together should be avoided if possible.
3. Leave the crystals in the crystal tube until ready to harvest the crystal, immediately prior to placing the crystal on the diffractometer.
   1. Keeping the crystals in the tube will ensure that the crystals remain solvated. De-solvation can cause the crystals to crack, and hinder the diffraction of the crystal.

Figure 1. An image of the pipette filter. A small piece of lint-free wipe has been firmly wedged at the bottleneck of the pipette. The solutions are passed though these pipette filters prior to being introduced to the crystal tube.

Figure 2. Once the solution containing targeted compound is placed in the crystal tube, the anti-solvent is slowly layered on top by passing it through a new pipette filter.