Growing Crystals for X-ray Diffraction Analysis

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.

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. 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.

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