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.

Solvent choices

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.

Note that some solvents (e.g. toxic solvents) may restrict where samples can be left.

Solvent incorporation into molecules

Sometimes solvents are incorporated into a crystal structure, where a solvent typically fills a void in the crystal lattice. Water is a particularly common solvent to be incorporated, and crystal structures are regularly found which incorporate one or more solvent molecules from the solvent in which they have been grown. 

The incorporation of water is rarely problematic, as this is a simple molecule which doesn't usually present a problem for crystallography. However, some solvents are much more problematic if they are incorporated into crystal structures, as the solvent can cause disorder within the lattice. This can make solving the crystal structure problematic and may be very time-consuming for a crystallographer, if indeed the structure can be solved. The solvents listed below are known to be extremely problematic and should be avoided for crystal growing, unless all other routes have been explored:

• Dichloromethane

• Chloroform

Crystal growing methods

A number of methods for growing crystals are presented below. However, the vast majority of crystals are grown using the first method (99+%), and it is strongly recommended to have a number of attempts at this method, before considering other routes. Similarly ethanol is by far the most commonly used solvent (90+%).

Slow solvent evaporation

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.

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 3-6 vials per solvent would be a typical setup.

Step by step instructions

1. Place a small quantity of purified material into a sample vial. The precise quantity is unimportant, and if setting up multiple vials it may be advantageous to vary the quantities in each vial.

2. Add a few mL of solvent into the vial. The quantity should be sufficient to be able to fully dissolve the quantity of sample added. Dissolution can be aided by redrawing the solvent from the vial into the Pasteur pippette and reexpelling the liquid.

3. If the sample hasn't fully dissolved, the vials can be gently warmed on a hotplate to aid solubilising.

4. Place the labelled vials in a sample vial rack and loosely fit lids. The vials want to be left undisturbed to slowly evaporate the solvent. Seek advice from a technician for where vials can be left. 

As the solvent slowly evaporates, the solution will become saturated. As further solvent is lost through evaporation, this will result in the compound coming out of solution. As this process happens slowly, hopefully this will result in the formation of good quality crystals. The vials should be periodically inspected for the formation of crystals, typically every few days. See the pages on identifying suitable crystals for details on this stage.

Slow solvent evaporation with antisolvent

This is a very similar process to slow solvent evaporation, but with the addition of some antisolvent into the solution. The choice of solvents is important, as this method is really only suitable where the solvent is more volatile than the antisolvent. Over time the solvent-antisolvent volume and ratio will change, as proportionally more of the solvent evaporates, causing the compound to slowly come out of solution. This can be a useful method to attempt when attempts with slow evaportation of a single solvent have been exhausted. Where the desired antisolvent is more volatile than the solvent, use of a vapour diffusion method is more effective.

Vapour diffusion

Where slow solvent evaporation hasn't been successful, solvent-antisolvent methods are usually the next approach, including the 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, as the intention is for no overall loss of vapour from the setup. The idea is that the smaller vial contains a solution of the desired compound, and this vial is placed inside the outer vial, and antisolvent is added into the space between the inner and outer vial. Over time, the antisolvent will evaporate with the vapours entering the inner solvent vial, reducing the compound solublity, and hopefully resulting in crystallisation. This method is most effective where the antisolvent is more volatile than the solvent, but it is possible to run these setups where this isn't the case, as solvent vapour can also be lost from the inner vial, but this is often harder to achieve in practice.

As with all solvent-antisolvent systems, the chosen solvent-antisolvent pair need to be misicible with each other. Common choices include ethanol-diethyl ether, ethyl acetate-petrols or water-ethanol.

Step by step instructions

1. Obtain a two sample vials which will fit one inside the other, and check this is the case with the outer vial lid being fitted.

2. In the smaller vial prepare a solution of the compound in the same manner as for slow solvent evaporation. A common example would be an ethanol solution where the compound is known to be soluble in ethanol.

3. Place this vial into the larger vial. Carefully pippette some antisolvent into the space between the inner and outer vial, then place the cap onto the outer vial. The cap should be tight to avoid antisolvent loss from the setup.

4. Label and leave the vial somewhere undisturbed. Over time the antisolvent will evaporate from between the vials, with some entering the solution in the inner vial, reducing the solubility and slowly causing the compound to drop out of solution, hopefully forming crystals.

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. Common pairings include ethanol/water, ethanol/diethyl ether and ethyl acetate/cyclohexane. Avoid combinations such as dichloromethane/hexane (see notes about solvents earlier in the page).

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.

Step by step instructions

1. Identify a suitable solvent and antisolvent to use for the crystallisation. Solvents can be miscible or immiscible.

2. Prepare a solution of the compound in the solvent. If preparing multiple samples, it is worth varying the concentrations for this solution.

3. Set up the crystallisation vials. The more dense solvent should be placed in the vial first, then the less dense solvent can be carefully layered on top using a Pasteur pipette. If these are miscible solvents, care needs to be taken to minimise mixing and avoid disturbing the sample vial, eg by moving.

4. For miscible solvents, over time the solutions will begin to mix into each other. For immiscible solvents the interface will remain. Hopefully over time, crystals will begin to grow in the solution. These often grow at the interface, but may later sink in the vial.

5. Periodically inspect the vial for crystal growth, but be careful to minimise disturbance.

Seed crystal growth

It is also possible to grow crystals which are suitable for X-ray diffraction via seeding, although this can be rather capricious. A solution of the sample is prepared in the same way as for slow solvent evaporation. Once this solution is in place, an additional crystal or two of the compound are then added into the vial, without being dissolved. The theory is that these will form a nucleation site for crystal growth from which single crystals might grow.

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.

Conical flasks

Conical flasks can be used to grow single crystals, but these are generally only suitable when large quantities of compound are available.