Single crystal X-ray diffraction (XRD) is a crystallographic method for determining atomic structures. A beam of X-rays strikes a crystal and is diffracted. From the angles and intensities of the diffracted rays, a 3D map of electron density of the crystal can be constructed and the arrangement of atoms and bonds within the crystal can be inferred. This technique is one of the least ambiguous and most complete ways of characterizing a compound. In order to take advantage of these benefits, the compound in question has to be rendered as a crystal of suitable size and quality.
Recrystallizations for purification purposes are a well-known and widely applied technique, but growing crystals suitable for single crystal X-ray diffraction is less well known and is more of an art than a science. The most promising crystals for XRD are transparent and have sharp edges. Ideally, crystals are 0.2–0.4 mm in at least two of the three dimensions. There are no hard and fast rules for growing such crystals, but here we present some tips and tricks to maximize your chances of obtaining the perfect crystal.
General Considerations
As with recrystallizations for purification, a saturated or near-saturated solution is prepared and the crystals are allowed to grow slowly. Several variations of this basic technique are possible and there are a few factors to consider that are common to all: disturbances, solvent, purity, nucleation, and time.
Disturbances
Disturbances to the crystals, solution, and vessel should be minimized, or avoided if possible. Mechanical disturbances, such as bumping, swirling, or vibrations, result in smaller crystals. This means that the crystal-growing vessel should be placed in a quiet place in the lab, where it will not be knocked or upset. Do not place the vessel near a vacuum pump or other sources of vibrations. Finally, try to avoid the urge to pick up the vessel and check on the progress of the crystals every day.
Solvent
Solvent choice is crucial. Your compound should be moderately soluble in the solvent you will be using. If the compound is very soluble, the crystals will not be as large. Likewise, if your compound easily forms a super saturated solution in the solvent, the crystals will be smaller.
As a general guideline, approximately 1 mL of solvent is used and typical solvents include toluene, hexane, THF, diethyl ether, and dichloromethane.
The compound should completely dissolve in the solvent and any remaining solid impurities should be filtered off. You may find that binary or tertiary solvent mixtures are required. Other solvent-based parameters to play with are volume/concentration, temperature, and solvent ratio.
Be observant during preparation and work-up of your compound. Noticing the compound’s behavior prior to attempting to grow crystals can help guide you in your choice of solvent. How soluble was it in the solvents used? Did crystals form at any stage during work-up or if the solvent evaporated slightly?
Purity
The purer the compound, the better the chance of getting X-ray quality crystals. Minimum purity of 75–90 % is recommended before attempting to grow XRD crystals.
Nucleation
Nucleation sites promote crystal formation, so the more nucleation sites there are, the more crystals will grow. This sounds promising, but the goal is to get a few crystals of suitable size for XRD rather than many smaller crystals. Fewer nucleation sites will result in fewer crystals each of larger size.
Dust, undissolved material, or extraneous particulate matter can all act as nucleation sites so take care to filter your sample before attempting crystallization and make sure the vessel is as free of dust as possible.
If you have a particularly difficult crystallization, such as if the compound forms an oil at low temperature, it is possible to use nucleation sites to encourage initial crystal formation. One method to introduce a nucleation site in a semi-reproducible manner is to scratch the surface of the vessel with a glass rod or spatula. This is particularly effective if you are using a disposable vial as your vessel. A scratch down the side of the vial can also encourage crystals to grow in a position that makes their retrieval easier.
Time
Growing the perfect crystal requires time and patience. There are many parameters that you can change with each attempt and it can take a week or two for the crystals to grow. Don’t give up if your first few attempts fail. This is one area where experience breeds success.
Again, patience is key. Avoid the urge to check the crystals’ progress, especially if checking them involves lifting or moving the vessel in any way (mechanical disturbance). If you find you really can’t restrain yourself, put the vessel somewhere where you can see the crystals without disturbing them.
The best crystals generally grow when the sample lies forgotten in the back of a fumehood or fridge for a while. If you try this approach, make sure the sample is clearly labeled – the time it takes for a forgotten crystal to grow is surprisingly similar to the time it takes to forget what and why you were growing them.
What To Do Once Crystals Form
Once you have crystals in your vessel, remove the remaining solvent with a syringe or Pasteur pipette. Rinse the crystals with the minimum amount of a cool solvent in which the compound is insoluble and remove this solvent as before. If the compound is air stable, remaining excess solvent can be removed by blotting the crystals on a piece of clean tissue paper. The crystals should be handled carefully with either tweezers or a spatula. Once they are dry, they can be submitted for single crystal X-ray diffraction.
If the compound is air-sensitive, the crystals are cleaned as described above, although a cannula is used to transfer the solvent by using standard air-sensitive techniques. The crystals can then be dried under reduced pressure, although this can also remove any solvent that co-crystallized with the compound – something to keep in mind when analyzing the results.
Summary
Single crystal X-ray diffraction is an excellent way to characterize a compound, but growing crystals that will give you good data is a skill and an art. There are a range of techniques that you can try, all of which require patience and luck. Checking on the progress of your crystals every day is counterproductive – crystals need time and no disturbances to grow.
In part 2 we will look at the best equipment to use to grow the perfect crystal and some techniques suitable for air stable compounds.
Do you have any tip or tricks for growing the perfect crystal? Share them in the comments section …
