Recrystallizations for purification purposes are a well-known and widely applied technique, but growing crystals suitable for single crystal X-ray diffraction (XRD) 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.
As we saw in part 1, the best crystals grow when there are no disturbances. A few large, good quality crystals are better for XRD than lots of small crystals and this means few nucleation sites, and allowing the crystals plenty of time to grow.
Equipment (For Air Stable Compounds)
The equipment needed to grow crystals is very basic – you can use anything that is suitable to hold liquid, from beakers to NMR tubes.
As beakers are often needed for other tasks in the lab, and it can take a couple of weeks for crystals to grow before you can recover your beaker, beakers tend to be the last choice. NMR tubes are also a last choice for similar reasons.
Petri dishes and watch glasses can also be used to good effect, although care should be taken with watch glasses as the curved bottom makes them easy to knock and disturb, which can inhibit crystal growth.
The best options are crystallization dishes or disposable glass vials.
Figure 1. Possible vessels to use for growing crystals. Clockwise from top left: Crystallization dish, disposable vials, petri dish, and watch glass.
To give your crystals lots of time to grow, the rate of evaporation should be slow (see below). The rate of evaporation from beakers, crystallization dishes, petri dishes, and watch glasses can be controlled in part by covering the dish with aluminium foil and piercing holes in the foil. The foil slows evaporation compared with an open vessel, while the holes allow some solvent vapor to escape, avoiding a completely closed system. The number of holes in the foil can be increased for less volatile solvents or fewer holes can be made to slow the evaporation of a highly volatile solvent.
Disposable vials allow some control over the rate of evaporation as the polyethylene lids are easy to pierce with a needle. The lid should be pierced prior to being placed on the vial. This avoids the risk of pieces of the lid falling into the sample and acting as a nucleation site.
Vials also have naturally slower rates of evaporation than crystallization dishes as the surface area of the solvent is smaller. Conversely, as vials are taller and narrower than crystallization dishes, they have a higher center of mass and are easier to accidently knock over.
Growing crystals is an art – sometimes referred to as the “dark art” of the lab. Every person who regularly grows crystals will have their own technique, procedure, and good-luck rituals. While each person may do small things differently, the procedure used will be loosely based on one of the techniques described here.
Every molecule has a specific saturation point in each solvent. This is the concentration at which no additional solid (solute) will dissolve in the solvent at a given temperature. Each technique for growing crystals relies on creating a situation where the solute can no longer dissolve in the solvent. This usually involves preparing a saturated solution then altering the conditions, such as temperature and volume of solvent, so that the solvent is unable to contain the material in it. If the change is slow enough, the solute molecules have time to arrange themselves and pack closely, creating a well-defined crystal.
Which technique you choose is mostly personal preference, however, some techniques are more suitable for some compounds. For example, the techniques described here are most suitable for air-stable compounds. They can be adapted for use with air-sensitive compounds – those that react with the air or moisture in the atmosphere and must therefore be kept under an inert gas such as N2 or Ar – but there are better techniques that can be used for growing crystals of air sensitive compounds. These will be described in part 3. Likewise, the air-sensitive techniques in part 3 can also be used for air-stable compounds, but the techniques described here are usually the first choice due to the simplicity of the experimental set-up.
This is one of the simplest methods and is generally the first attempted. It is only suitable for compounds that are air and moisture stable at room temperature. As the name implies, it involves the slow evaporation of the solvent from the solution containing the compound until saturation is reached and crystals begin to form. It works best when there is enough material to make 1–3 mL of saturated or near-saturated solution.
A saturated solution is prepared and transferred to a vial or crystallization dish. The dish is covered with a piece of pierced aluminium foil or a pierced lid is placed on the vial and the sample is left in a safe place while the solvent evaporates.
If using a vial, it can be placed at an angle in a beaker (Fig. 2). This will encourage the crystals to grow on the side of the vial as more solvent is in contact with the side and the angle prevents newly-formed crystals falling straight to the bottom of the vial. Due to the narrowness of the vial, crystals on the side are easier to remove from the vial without damaging them. The beaker will also protect the vial from accidently being knocked over. A disposable needle can be left in the lid if required to stabilize the vial at the right angle.
Figure 2. Slow evaporation technique using a vial.
It is theoretically possible to use the slow evaporation method for air-sensitive compounds, however, there are several practical limitations that mean it is almost never used.
The main limitation is that as it involves the slow evaporation of solvent, you need to have an open vessel in order for the solvent to escape. In a closed vessel, the solvent will evaporate into the space available, but evaporation will stop once the vapor saturation point – where the accumulated vapor molecules are in equilibrium exchange with those in the solvent – is reached. This limited amount of evaporation will mean that the volume of solvent will not decrease enough for the saturation point to be past and for crystals of suitable size to form.
However, an open vessel will always exchange with the atmosphere and it is impossible to prevent air and water from entering the vessel and the resulting decomposition.
There are two possible ways round this. The first is to place the sample in a glovebox and allow it to evaporate under the inert atmosphere in there. This is not recommended. The evaporated solvent will remain in the glovebox and may contaminate future work performed in the glovebox. Also, excess solvent in a glovebox can play havoc with its filters which are expensive to replace.
A better method is to use a Schlenk flask attached to a nitrogen line and with a rubber septum pierced with a disposable needle in the top. A positive pressure or flow of nitrogen over the solvent will encourage evaporation and carry the evaporated solvent out of the flask through the needle. This results in faster evaporation than with an open vessel, which may, in turn, result in small or poor quality crystals.
This is also a very simple, but successful, technique. Most substances are more soluble at higher temperatures than lower temperatures and almost any solvent can be used. The technique involves cooling a saturated solution. As the temperature drops, the solvent’s ability to dissolve the solute decreases and excess solute precipitates out. If the rate of cooling is slow enough, crystals should form.
As with preparative recrystallizations, the solvent can be warmed to just below its boiling point before dissolving your compound in it. Slowly cooling a hot solution to room temperature generally only works with the tried and tested compounds in undergraduate labs – but then placing the sample in a fridge or freezer often produces good results. When attempting crystallization from hot solvent, it is important to cool the sample in a stepwise manner – hot → room temperature → cool – and not place the hot solution directly into the fridge or freezer. Samples can also be placed in a fridge first and then a freezer to slow the rate of cooling compared with placing the sample directly into the freezer.
Ideal solvents for this technique are those in which your compound displays high solubility at high temperature and low solubility at low temperature. Unsuitable solvents are water and benzene if the sample is going to be placed in the freezer.
A variation on this method uses a Dewar flask and a water bath (Fig. 3). This set-up is designed to allow the solvent to cool as slowly as possible so it will take several days or weeks for crystals to form. It is suitable for solvents with boiling points in the range 30–90°C and should only be used if you are sure that your compound is thermally stable.
Figure 3. Slow cooling of a sample with a Dewar flask.
A saturated solution of the product is heated to just below the solvent’s boiling point and transferred to a stoppered tube. The tube is placed in a Dewar flask and the flask filled with water 2–3 degrees cooler than the solvent. The water level should be above that of the solvent but below the stopper of the tube. The Dewar flask is then left in a safe place until the solvent has cooled to room temperature and crystals have formed.
This simple technique also works well with air-sensitive compounds and is easily adapted for them. Rather than preparing the sample in a vial, it can be prepared in a Schlenk flask and placed in the fridge or freezer. The only downside with this is when Schlenk flasks are in short supply and you’ve just put your last one in the fridge for at least a week.
Variations on Slow Evaporation and Cooling
If you find that the slow evaporation or cooling technique gives crystals but the crystals are not of suitable size or shape for XRD, both techniques can be extended to include a binary or tertiary solvent system. The benefit of using two or three solvents is that it can promote or inhibit the growth of certain crystal faces. Solvent molecules get incorporated into the crystal lattice and adding a second or third solvent means more than one type of solvent molecule can be incorporated into the lattice. This can affect the crystal packing and change the morphology of the crystals, ultimately leading to better crystallography results.
Solvents should have similar boiling points so that they evaporate at approximately the same rate and the difference in polarity between them should not be too large so as to avoid phase separation when the compound is added. Commonly used solvent mixtures are shown in Tab. 1.
Table 1. Common combinations of solvents for binary solutions for growing crystals.
A lot of trial and error will be required to find the perfect solvent system and careful notes should be kept so that the crystallization can be reproduced if needed.
A thermal gradient can be useful when attempting to grow crystals. The warmer portion of the solution is more saturated than the cool part. Convection currents carry the saturated solution to the cooler part where crystal growth occurs. A thermal gradient can be introduced by local cooling or heating of one part of the vessel. The speed of the convection current is determined by the thermal gradient across the dish. A small gradient will induce a slow current which will not inhibit crystal growth as much as a large current.
A simple and effective method of local cooling is using a heat sink. A crystallization dish is ideal for this and a cold outside window is usually sufficient as a heat sink. The crystallization dish (Fig. 4) must be in contact with the window to induce convection.
Figure 4. Convection method with localized cooling for growing crystals.
An alternative method involves localized heating. A Thiele tube (Fig. 5) is filled with a solvent in which the solute has limited solubility at room temperature. A container with your sample is suspended in the larger part of the tube. The top of the container should be below that of the side arm. Heat is applied to the bottom section of the smaller side arm using a heating element. This creates a thermal gradient ranging from the temperature of the heating element to near room temperature at the bottom of the larger side of the tube. Convection currents will therefore carry hot solvent into the compound container and slowly increase the solute concentration in the solvent. Crystals should form at the bottom of the larger side of the tube.
Figure 5. Convection method with localized heating for growing crystals.
As with the cooling method, the convection method with localized cooling can be simply adapted for air-sensitive compounds by preparing the sample in a Schlenk flask and placing that against a cold outside window.
This is a less-common technique, in which solutions of reactant are allowed to diffuse into each other. If the product of the reaction is insoluble in the solvent, crystals will form where the reactants meet.
Figure 6. Reaction diffusion technique.
Two vials are placed in a beaker (Fig. 6). Each vial is filled with a solution of a reactant. The solvent should be something in which the product of the reaction is insoluble. The beaker is then filled with this solvent so that the solvent level is higher than the tops of the vials. This will allow the solutions of reactants to diffuse out of the vials and into the beaker where they can react. The insoluble product should then be deposited in crystal form on the bottom of the beaker.
This is not a common technique as many reactions need heating or stirring or additional reagents – it is rare to find a simple A + B → C at room temperature type of reaction. This technique can be useful, however, for equilibria of the type A + B ⇔ C (+ D) which lie to the left-hand side of the equation. As the product C crystallizes out of solution, this can drive to equilibrium to the right-hand side as C is removed from the equation and the equilibrium adjusts to compensate.
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. As we saw in part 1, checking on the progress of your crystals every day is counterproductive – crystals need time and no disturbances to grow. If after two weeks no crystals have formed in your sample, it may be time to reconsider your solvent or crystal growing technique and try another method. There are many parameters that you can change with each attempt so don’t give up if your first few attempts fail.
In part 3, we will look at some more techniques for growing crystals, in particular those that are suitable for air and/or moisture sensitive compounds, yet are still effective for air and moisture stable compounds.
Do you have any tip or tricks for growing the perfect crystal? Share them in the comments section …