Growing Crystals for X-ray Diffraction Analysis

Organic Chemistry

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Overview

Source: Laboratory of Dr. Jimmy Franco - Merrimack College

X-ray crystallography is a method commonly used to determine the spatial arrangement of atoms in a crystalline solid, which allows for the determination of the three-dimensional shape of a molecule or complex. Determining the three-dimensional structure of a compound is of particular importance, since a compound's structure and function are intimately related. Information about a compound's structure is often used to explain its behavior or reactivity. This is one of the most useful techniques for solving the three-dimensional structure of a compound or complex, and in some cases it may be the only viable method for determining the structure. Growing X-ray quality crystals is the key component of X-ray crystallography. The size and quality of the crystal is often highly dependent on the composition of the compound being examined by X-ray crystallography. Typically compounds containing heavier atoms produce a greater diffraction pattern, thus require smaller crystals. Generally, single crystals with well-defined faces are optimal, and typically for organic compounds, the crystals need to be larger than those containing heavy atoms. Without viable crystals, X-ray crystallography is not feasible. Some molecules are inherently more crystalline than others, thus the difficulty of obtaining X-ray quality crystals can vary between compounds. The growth of X-ray crystals is similar to the process of recrystallization that is commonly used for purifying compounds, but with an emphasis on producing higher quality crystals. Often, higher quality crystals can be obtained by allowing the crystallization process to proceed slowly, which may occur over the course of day or months.

Cite this Video

JoVE Science Education Database. Organic Chemistry. Growing Crystals for X-ray Diffraction Analysis. JoVE, Cambridge, MA, (2017).

Principles

There are a number of methods for growing X-ray crystals, such as heating and cooling, evaporation, and vapor diffusion, each with its' own advantages and limitations.1 Described herein is one of the most useful methods for growing X-ray quality crystals, liquid-liquid diffusion.2 Successful X-ray crystal growth depends on the proper choice of solvents. The compound must be soluble in one solvent but insoluble in another. Liquid-liquid diffusion involves carefully layering a low-density solvent on top of a higher-density solvent in a thin tube, such as an NMR tube. The rate of diffusion can greatly influence the size and quality of the crystals- rapid diffusion favors smaller crystals, while slow diffusion favors the growth of larger and higher quality crystals. The utilization of thin tubes, such as NMR tubes, slows down the diffusion of the solvents, thus creating an environment that facilitates the growth of higher quality crystals. Commonly used solvents for the lower layer, which the compound is dissolved in, are methylene chloride or chloroform. The compound is dissolved in the less dense solvent, but this can prove problematic as the top solvent can start to evaporate prior to crystal formation. The optimal condition is to have the compounds dissolved in the more dense solvent. The top layer is the anti-solvent or precipitant. Frequently used anti-solvents are hexane, pentane, diethyl ether, or methanol. Once the two solvents have been carefully layered, they are allowed to slowly diffuse into one another. The compound becomes less soluble in the binary solution, facilitating the formation of X-ray crystals.

Procedure

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
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
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.

Thanks for watching!

Results

The technique of liquid-liquid diffusion was used to create X-ray quality crystals of tetraphenylporphyrin. Using dichloromethane as the solvent and methanol as the anti-solvent, the liquids were allowed to slowly diffuse over the course of a week without being disturbed. Large, well-defined, dark purple-reddish crystals formed at the interface of the two solvents (Figure 3). The growth of the crystals can be visually observed. The crystals grew with very well defined faces, which can be seen with a microscope.

Figure 3
Figure 3. X-ray diffraction quality crystals of TPP. Crystals that are clumped together or that are growing out of one another should be avoided. Large single crystals with well-defined faces typically yield better results.

Applications and Summary

X-ray quality crystals can be grown by liquid-liquid diffusion. The slow diffusion of the binary solvent system allows for the creation of crystals suitable for X-ray diffraction.This method allows the crystal lattice to form slowly, often leading to larger and more well defined crystals. The use of NMR tubes facilitates the slow diffusion of the solvents, allowing for optimal crystal growth. This process can take anywhere from a few days to several months. Often during the crystallization process solvent molecules are incorporated into the crystal lattice. Thus it is important to avoid allowing the crystals to "dry out". Thus, one of the advantages of liquid-liquid diffusion is that the crystals typically grow at the interface of the two solvents, which circumvents this phenomenon.

Liquid-liquid diffusion is one of the most useful techniques for producing X-ray quality crystals, which is the most essential component of X-ray crystallography. Obtaining X-ray quality crystals is typically the limiting factor on conducting X-ray crystallography experiments. X-ray crystallography essentially creates a three-dimensional picture of a molecule's structure, making it the least ambiguous method for determining the complete configuration of a compound. Since structure and function of molecules are intimately related, the ability to decipher a compound's three-dimensional structure is extremely useful for a variety of chemical and pharmaceutical applications. Researchers and pharmaceutical companies use X-ray crystallography to determine the structure of proteins to examine how small molecules interact with enzymes for the purpose of drug discovery and design.3-5 X-ray crystallography is also one of the most useful methods for evaluating metal complexes. This technique divulges valuable insight on how metals interact with each other and its ligands. The first ever identified quintuple bond between two chromium atoms was identified using X-ray crystallography.6 This technique can also be used to explain the luminescent properties of metal complexes.7 Crystallography has also been widely used in host-guest chemistry, as this method has been instrumental in revealing valuable information about non-covalent interactions between molecules.8

References

  1. Gilman, J. J., The art and science of growing crystals. Wiley: (1963).
  2. Orvig, C., A simple method to perform a liquid diffusion crystallization. Journal of Chemical Education 62 (1), 84 (1985).
  3. Brown, C. S.; Lee, M. S.; Leung, D. W.; Wang, T.; Xu, W.; Luthra, P. et. al. In silico derived small molecules bind the filovirus VP35 protein and inhibit its polymerase co-factor activity. Journal of molecular biology426 (10), 2045-2058 (2014)
  4. Batt, S. M.; Jabeen, T.; Bhowruth, V.; Quill, L.; Lund, P. A.; Eggeling et. al. Structural basis of inhibition of Mycobacterium tuberculosis DprE1 by benzothiazinone inhibitors. Proceedings of the National Academy of Sciences of the United States of America,109 (28), 11354-9 (2012)
  5. Mortensen, D. S.; Perrin-Ninkovic, S. M.; Shevlin, G.; Elsner, J.; Zhao, J.; Whitefield et al. Optimization of a Series of Triazole Containing Mammalian Target of Rapamycin (mTOR) Kinase Inhibitors and the Discovery of CC-115. Journal of Medicinal Chemistry 58 (14), 5599-5608 (2015)
  6. Nguyen, T.; Sutton, A. D.; Brynda, M.; Fettinger, J. C.; Long, G. J.; Power, P. P., Synthesis of a Stable Compound with Fivefold Bonding Between Two Chromium(I) Centers. Science310 (5749), 844-847 (2005).
  7. Chen, K.; Nenzel, M. M.; Brown, T. M.; Catalano, V. J., Luminescent Mechanochromism in a Gold(I)-Copper(I) N-Heterocyclic Carbene Complex. Inorganic Chemistry 54 (14), 6900-6909.(2015).
  8. Franco, J. U.; Hammons, J. C.; Rios, D.; Olmstead, M. M., New Tetraazaannulene Hosts for Fullerenes. Inorganic Chemistry49 (11), 5120-5125 (2010).

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
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
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.

Thanks for watching!

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