Successful single crystal X-ray diffraction depends on being able to grow a high quality single crystal which suitably diffracts. Whilst single crystals can occasional be isolated from the product of a recrystallisation, generally this does not result in the isolation of single crystals which would be suitable for diffraction.

Growing single crystals takes patience and a good deal of luck. Crystals usually take between several days to many months to grow, and sometimes it may prove impossible to obtain suitable crystals. Several different crystal growing methods are given below. Generally it is best to work through the methods in order, in each case setting up multiple crystallisations to maximise the chances of obtaining a good quality single crystal. Whilst it is generally helpful for the crystallographer to be provided with a selection of potential crystals, it is perfectly possible to obtain high quality structures where only one single crystal has been obtained.

This guides below should be considered in conjunction with the guidance around identifying suitable crystals. Remember the key is to grow high quality crystals, they do not need to be large. In all cases, starting with compound which is as pure as possible really helps with growing suitable crystals. Your compound should have been purified, usually by recrystallisation (at least once, if not multiple times), to ensure you are starting with a good compound purity in the crystallisations.

Slow solvent evaporation

By far the simplest method to grow crystals is via slow evaporation of a solvent from a solution of the desired compound. This is by far the most common method used to obtain suitable crystals. Usually a small quantity of compound, typically 20 - 50 mg, is dissolved in a solvent in a sample vial, often using hot solvent to aid the initial dissolution. Unlike in recrystallisation, the resulting should not be a saturated solution, but have an excess of solvent when the solvent is at room temperature. Crystals are then obtained by allowing the solvent to slowly evaporate, typically over days or weeks, which is made possible by only loosely fitting the sample vial caps (usually screw caps), so that solvent can be lost via evaporation from the vial over time.

As the solvent evaporates, the solutions in the vials will become saturated, and this will result in the compound slowly coming out of solution. As this process should be happening very slowly, this should result in formation of very regular crystals, providing the vials are left undisturbed.

Many solvents can be used to attempt to grow single crystals. The compound should be readily soluble in the chosen solvent, and as the solvent wants to evaporate slowly, very volatile solvents (e.g. diethyl ether, dichloromethane, pentane etc) are generally less suitable. The most commonly used solvent for growing single crystals is ethanol. If this proves unsuitable, other alcohol solvents (e.g. isopropanol or 1-propanol) are often employed. Other commonly used solvents include ethyl acetate, water, acetone, toluene and acetonitrile.

Vials should be placed somewhere safe where the solvent can evaporate slowly without the vials being disturbed. Inspection of the vials is typically performed every few days to see if crystals have grown, ideally minimising movement of the vial in the process to avoid disrupting the crystal growth.

Where material allows, setting up multiple vials will maximise the likelihood of obtaining suitable crystals, and it may be necessary to experiment with how loosely the lids are fitted to obtain a suitable rate of solvent evaporation. Around 4-6 vials per solvent would be a typical setup.

Step by step instructions

Vapour diffusion

Where slow solvent evaporation hasn't been successful, the next approach to try is a vapour diffusion method. Here, a solvent-antisolvent system is employed, in an analogous manner to solvent-antisolvent recrystallisations.

This setup requires two vials per crystallisation, with the smaller vial able to be placed inside the larger vial. The inner vial does not require a lid, but the outer vial does.

Step by step instructions

Interface growth

Crystals can be grown at the interface of two solvents, one being a solvent containing a fairly saturated solution of the compound, and the other acting as an antisolvent. It is possible to grow crystals at the interface with both miscible and non-miscible solvent pairings.

Note: Some groups like to prepare interface growth crystals in NMR tubes clamped at a 45° angle. Crystals grown in this manner are strongly discouraged due to the difficulties caused to the crystallographer when it comes to isolating the crystal. Isolation from this approach requires the breaking of the NMR tube, and this process can easily damage or destroy the crystal. Sample vials are strongly preferred, as it is much easier to isolate the crystal, with no negative effects on the crystal growth.

Seed crystal growth

Crystals can be grown

Step by step instructions

Alternatives to sample vials

Many of the methods above, particularly slow solvent evaporation can be utilised to grow crystals in alternative vessels to vials.

Well plates

Well plates are especially useful where amounts of compound are limited. These allow smaller scale crystallisations to be set up compared to using a sample vial. Usually multiple well plate holes are used to have multiple crystallisation attempts happening on a single plate. It is also particualrly convinient to screen well plates on a microscope, as they have been designed with this purpose in mind.

Solutions of the desired compound are usually prepared in a sample vial, and then transferred into wells using a micropipette. For slow evaporation, these are then loosely fitted with the lid and left undisturbed to allow crystals to form.

Vapour diffusion methods can also be acheived using well plates. This is most commonly carried out by placing the antisolvent into some of the wells, and the compound solution in other wells, usually in regular patterns so that the solution is surrounded by antisolvent.