2.12: 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.

A single crystal is required for the determination of its structure. The quality of the crystal heavily influences the quality and accuracy of the structural determination.

A single crystal is a solid in which the molecule arrangement repeats in all three dimensions. The spatial arrangement of the atoms within the crystalline solid can be determined using X-ray crystallography. In this technique, a pure crystalline sample is enveloped by a beam of X-rays. The crystal diffracts the X-rays in a distinctive pattern related to the crystals structure and molecular composition. If a crystal is formed too quickly, the molecules may be disordered, impurities may be incorporated into the crystal, or two or more fused crystals may form instead of a single crystal. Therefore, specialized methods with emphasis on slow growth are needed to produce crystals of sufficient quality for X-ray crystallography.

This video will illustrate the desired characteristics of X-ray?quality crystals, demonstrate a procedure for growing them, and introduce a few applications of this technique in chemistry.

Electrons scatter X-rays by emitting a spherical X-ray wave when hit. If the atoms are in an orderly arrangement, constructive interference between the waves produces a characteristic diffraction pattern on an X-ray detector. The crystal is rotated within the beam to collect diffraction patterns from multiple angles. With sufficient diffraction patterns, the molecular structure can be derived.

X-ray-quality crystals generally form symmetric shapes and have smooth, light-reflecting faces. When viewed under a polarizing microscope, they will be transparent, but most should become dark when rotated 90?. This indicates highly-ordered structure. To grow these crystals, liquid-liquid diffusion is often used. This employs two miscible solvents: a low-density solvent, or precipitant, in which the compound to be recrystallized is insoluble; and a high-density solvent in which the compound is soluble. Typically, the volumetric ratio of precipitant to solvent is 2:1.

The low-density precipitant is layered onto a concentrated solution of the compound in the high-density solvent. Over time, the compound becomes less soluble as the precipitant mixes with the solution. A smaller solvent interface results in a slower rate of diffusion, thus yielding larger, purer crystals. Now that you understand the principles of growing X-ray?quality crystals, let's go through a procedure for growing them by liquid-liquid diffusion.

To begin, obtain the necessary equipment found in the text protocol. Acquire a solvent for the compound and a less dense precipitant.

To prepare a pipette filter, place a small piece of Kimwipe into the top of a glass pipette and gently press the paper down to the bottom of the pipette body using a rod or the stem of another pipette, being careful not to puncture the paper. Prepare two pipette filters. Place one into the NMR tube. If necessary, secure the assembly with a laboratory clamp and ring stand. Dissolve about 10 mg of the compound to be recrystallized in 0.75 mL of solvent.

Now, carefully add the sample solution into the pipette filter. Affix a bulb to the top and slowly squeeze to pass the solution into the NMR tube to remove solid impurities. Do not allow the bulb to re-expand while it is attached, as the suction will dislodge the filter paper.

Next, remove the used pipette filter and place the second filter into the NMR tube. Pipette approximately 1.5 mL of precipitant into the tube. Allow the solvent to pass through the filter by gravity. From now on, take care not to disturb the filter during any manipulations. Once all of the precipitant has filtered into the tube, remove the filter and cap the tube. Place it in a cabinet or other easily checked location where it will not be agitated.

After at least one day, inspect the tubes for crystal growth.?If no crystals are present or the crystals are very small, leave the sample tube undisturbed. If crystals are visible, check their size and shape without disturbing the solvent layers.

If the crystals are large, well-defined, and are not clustered together, inspect the crystals under a microscope to verify their potential to be X-ray quality. Do not remove the crystals from the tube until the diffractometer is ready to begin the scan. If solvent molecules are incorporated into the crystal structure, allowing the crystal to dry will degrade the crystal. Using X-ray crystallography, the molecular structure of these dark reddish-purple crystals was verified to be tetraphenylporphyrin.

X-ray crystallography is an essential analytical tool in chemistry and biochemistry.

Recrystallization methods include heating and cooling, liquid-liquid diffusion, vapor diffusion, and slow evaporation. In slow evaporation of a single solvent system, the compound is dissolved in a small amount of solvent and placed in a container with a small hole in the cap. As the solvent evaporates, the concentration increases until the compound begins crystallizing.

The functionality of proteins is often related to their structure. However, proteins can be very difficult to crystallize. Specialized techniques must be developed to grow X-ray-quality crystals of proteins. Here, a drop of protein solution is mixed with a drop of precipitant and this mixture is sealed in a chamber with pure precipitant. As the solvent vapor diffuses out of the drop, the solubility of the protein in the drop decreases, and the protein slowly crystallizes. Another technique mixes the protein solution and precipitant under mineral oil. Using these techniques, a variety of proteins can be crystallized for analysis.

In powder diffraction, each possible spatial orientation is represented in the sample simultaneously. Powder diffraction is not as informative about structure as single crystal X-ray diffraction because of the loss of three-dimensional structure data. Instead, powder diffraction excels in analyzing mixtures of crystalline solids and assessing the crystallinity of amorphous structures.

You've just watched JoVE's introduction to growing crystals for X-ray crystallography. You should now be familiar with the properties of X-ray?quality crystals, a procedure for growing them, and a few applications of this technique in chemistry.

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