Department of Chemistry

X-ray Diffraction Laboratory


Crystallization

Preparation of Single Crystal
For structure determination from single-crystal X-ray diffraction experiment, the sample being studied must (obviously =_=) be a single crystal, in which only one continuous crystal lattice presents in the entire sample.

The following equation is an elaborate version of the Fundamental Equation derived by Dr. Peter Müller from MIT.1

bad crystal quality
= poor diffraction data = inaccurate molecular model = unpublishable results2

Classical Nucleation Theory3 is beyond the scope of this webpage, but keeping in mind that crystallization is a thermodynamic and kinetic controlled process. Other tabs of this page describe common methods to prepare good quality single crystals.


CLASS - A quick judgment on your crystal quality

Clarity
Yes, it is 'crystal clear' that most of the single crystals are transparent indeed, unless the compound has extremely strong absorption in the visible light region.
Considering the crystal lattice as slit, if there is only one slit, some polarized light is able to pass through the crystal.
When many slits stack together randomly, light of all polarization directions are blocked and thus, resulting in a dull opaque solid.

Shape
If your crystals have regular three-dimensional shapes (or crystal habits) such as cube, block, prism, the chance is high.
If the samples look like needle or plate, it is easy to be twin, but still worth to try.
If the samples are non-rigid, such as star-like, radiating outward from a central point, dendritic (tree-like), fibrous, don't waste your time and try other crystallization solvents/methods.

Size
Depending on the configurations (e.g. X-ray beam size, collimator aperture) of different diffractometers, the size requirements of single crystal samples are vary, but for common organic and organometallic compounds, it fall in the range of 0.1 to 0.3 mm for at least two out of the three dimensions.
If the maximum dimension of the crystal is less than 0.1 mm, longer exposure time may help. But if the dimensions are even smaller (e.g. tiny needle crystal), the diffraction data at higher resolution4 may be too weak for further analysis.

Note 1: The original equation: "Garbage in = Garbage out", Crystallography Reviews, 2009, 15, 57 – 83.
Note 2: = nightmare of your supervisor = unhappy life
Note 3: De Yoreo, James J., and Peter G. Vekilov. "Principles of crystal nucleation and growth." Reviews in mineralogy and geochemistry, 2003, 54, 57 – 93.
Note 4: extend to 0.84 Å for a good dataset
Solvent Evaporation
The simplest crystallization method is to increase the concentration of the substrate in solution by evaporation of the solvent. Simple container such as 1.5-mL HPLC sample vial can be used and usually about 0.1 mg of sample is enough for the process. Different solvents can be test at the same time and this procedure provides initial observation of crystal habits from different solvents quickly.

Sample Requirements:
1. Stable in air1
2. At least slightly soluble in the solvents being tested
3. Has no reaction with the solvent

Procedure:
1. Transfer small amount of solid into a 1.5-mL vial. Dipping a glass pasteur pipette into the solid and the amount of material in the tip is already enough.
2. Add solvent into the vial to dissolve the solid. Gentle heating may be required if dissolution is slow at room temperature.
3. Repeat procedure 1 and 2 for 6 ‑ 8 different solvents:
Type Example Remarks
hydrocarbons hexane for non-polar organic compounds
chlorinated hydrocarbons2 DCM, chloroform,
1,2-dichloroethane
for organometallic compounds;
DCM: fast evaporation rate;
disorder in co-crystal
ethers diethyl ether, THF coordinative solvent;
disorder in co-crystal;
slightly hydroscopic
alcohols methanol, ethanol, IPA for polar organic compounds;
hydroscopic;
hydrogen bonding
nitriles acetonitrile, benzonitrile for organometallic compounds;
for some aromatic compounds;
coordinative solvent
aromatics3 toluene, xylene,
1,2-dichlorobenzene
for aromatic compounds
ketones acetone, methyl ethyl ketone disorder in co-crystal;
hydroscopic
miscellaneous ethyl acetate, DMSO,
pyridine
may have reaction with the substrate;
DMSO is difficult to evaporate and is very hydroscopic
4. Cover the vial openings and tightness of the screw cap can control the rate of evaporation.
5. Store the sample vials at a location that is far away from any vibrating objects.
6. Crystals may form overnight, or after days or even weeks, depends on different substances.

Tips:
1. A mixture of solvents may be used.
2. One may even let the solvent evaporates completely in the first trial without covering the vial. Crystal habits in different solvents can be observed quickly and thus the most suitable solvent can be determined by the next day morning.
3. After the best solvent is found, prepare crystallization samples with the same solvent in several vials to increase your chances of success.
solvent evaporation
Note 1: For air-sensitive substance, the procedure shall be done under nitrogen or other inert gas.
Note 2: 1,1,2,2-tetrachloroethane is a good solvent and has high boiling point, but it is not recommended due to concerns about its toxicity
Note 3: Use of benzene is not recommended due to due to concerns about its toxicity
Slow Cooling
Solubility of a substrate increases with temperature usually. Single crystal may grow from a solution by lowering the temperature. Sample Requirements:
1. Stable in air1
2. Stable upon heating
3. At least slightly soluble in the solvents being tested
4. Has no reaction with the solvent at room temperature and at solvent's boiling temperature

Procedure:
1. Transfer small amount of solid into a 20-mL vial. Dipping a glass pasteur pipette into the solid and the amount of material in the tip is already enough.
2. Add about 2 ‑ 4 mL solvent into the vial.2 Observe and record the solubility of the substrate at room temperature.
3. Heat the mixture on a hotplate with swirling until the solution is boiling. Observe and record the solubility of the substrate at boiling temperature. If some undissovlved materials present, transfer the solution, without the undissolved solid, to another clean sample vial.
4. Let the solution cool down to room temperature. Observe and record the solubility of the substrate at room temperature again. You may construct a solubility table like this:
Solvent at RT at boiling back to RT
hexane ** ** **
ethyl acetate * ***** *****
ethanol * ***** **
DCM * ***** **
acetone *** ***** *****
diethylether ***** ***** *****
insoluble *
completely soluble *****
In the above example, ethanol will be chosen for crystal growing.3
5. Repeat step 1 to 3 again. Split 1/3 of the boiling solution into a clean vial (Vial B), and then 1/3 into another clean vial (Vial C).
6. Add 10% and 20% volume of the solvent into Vial B and Vial C respectively while keeping the solution boiling. Label the original vial as Vial A.
7. Cover the vials and cool down the solution to room temperature slowly by surrounding the vials with insulating warps, such as cotton or tissue papers, and then put them into a styrofoam box.
8. The resulting three solutions with different concentrations can be stored at a location which is far away from any vibrating objects.
9. Crystals may form overnight, or after days or even weeks, depends on different substances.

Tips:
1. A mixture of solvents may be used if a satisfactory single solvent cannot be found.
2. This method is more suitable for substrate with poor solubility, higher b.p. solvents usually give better results. Concentration may increase too quickly after boiling if low b.p. solvent is used.
3. Make sure that there is enough room in the vial for heating up the solvent. If you feel that this mini-scale of reflux setup is difficult to control or the mixture is bumping, use a 50-mL round-bottom flask, a magnetic stirring bar, an oil bath and a stirrer hotplate instead.
4. In some cases, a constant temperature environment above (e.g. oil bath or oven) or lower (e.g. refrigerator) then RT may required to grow good quality single crystals.

Note 1: For air-sensitive substance, the procedure shall be done under nitrogen or other inert gas and all solvents used should be degassed.
Note 2: Depends on the boiling point of the solvent being used: the lower the b.p. of the solvent, the smaller the amount.
Note 3: Although DCM and ethanol give the same observation, DCM has a lower b.p. and easy to evaporate.
Note 4: Observation at room temperature after heating is very important, an incorrect judgement on solubility could be made if missing this step.
Vapour Diffusion
In this technique, the substrate is dissolved in a good solvent and a poor solvent vapor diffuse into the solution, resulting a decrease in solubility of the substrate and thus, initiating the growth of crystals.

Sample Requirements:
1. Has no reaction with the solvents
2. Completely soluble in the good solvent and insoluble in the poor solvent

Other Requirements:
1. There is no reaction between the good and poor solvents
2. Perform a solubility test as described in the "Slow Cooling" section

Procedure:
1. Transfer small amount of solid into a 5-mL vial. Dipping a glass pasteur pipette into the solid and the amount of material in the tip is already enough.
2. Add the good solvent into the vial to dissolve the solid.1 Gentle heating may be required if dissolution is slow at room temperature. The solvent volume should be less than 1/3 of the vial.
3. Add about 1 mL of the poor solvent into a 25-mL vial.
4. Place the 5-mL vial into the 25-mL vial carefully with a forceps. Make sure that the good and poor solvents are not mixed accidentally.
5. Cap the 25-mL vial and the setting should be similar to the picture below. Store the setup at a location which is far away from any vibrating objects.
6. Crystals may form overnight, or after days or even weeks, depends on different substances and the diffusion rate of the poor solvent.
Vapour Diffusion


Tips:
1. The total volume of good solvent plus poor solvent should be less then the volume of the inner vial. Otherwise, the inner vial content will overflow into the outer vial.
2. B.p. of poor solvent affects the diffusion rate and the time required for crystal formation. For example, if diethyl ether or DCM is used as poor solvent, crystal formation may observed overnight.
3. Satisfactory result can be expected if the poor solvent is more volatile than the good solvent.

Note 1: Crystal quality may be vary by the initial concentration of substrate.
Layering
Similar to vapour diffusion method, diffusion can also occur between the boundary of the two solvent layers. The poor solvent molecules diffuse into the solution of substrate in good solvent and the solubility decrease slowly, and thus, initiating the growth of crystals.

Sample Requirements:
1. Has no reaction with the solvents
2. Completely soluble in the good solvent and insoluble in the poor solvent

Other Requirements:
1. There is no reaction between the good and poor solvents
2. The poor solvent has a lower density than the good solvent
3. The two solvents are miscible

Procedure:
A. Solubility Test
1. Prepare a satuated solution of the substrate in the good solvent (about 2 mL) in a 5-mL vial.
2. Separate the solution into three 5-mL vials, 0.5 mL each. Label the vials with A, B, and C.
3. Add poor solvent into each vials, with good-to-poor solvent volume ratio 1:0.5 for A, 1:1 for B and 1:1.5 for C.1
4. Mix well and observe the turbidity of the solution mixtures after 15 min. The suitable good-to-poor solvent volume ratio is suggested by that of the mixture shows slightly turbid (or 10 to 40 NTU).2
B. Sample preparation
1. Add 0.1 to 0.2 mL of good solvent into the saturated solution in the original 5-mL vial.
2. Add the poor solvent with the ratio determined in Part A slowly by using a pasteur pipette. Touch the inner wall of the vial with the tip of the pipette 3 to 4 mm above the solution level. Let the solution down slowly along the wall without disturbing the surface of the lower layer.
3. Cap the vial and store the setup at a location which is far away from any vibrating objects.
4. Crystals may form overnight, or after days or even weeks, at the interlayer of the solvent depends on different substances and solvent combination.
Layering

Tips:
1. In Part A, if the mixture is still clear even with good-to-poor solvent volume ratio smaller than 0.5, other solvent combination should be tried.
2. If the density of poor solvent is much higher than the good solvent, the diffusion process may be too fast for growing good quality crystal.
3. This technique may apply on co-crystallization or in situ crystallization, in which two starting materials are dissolved in two different solvents separately.

Note 1: Ratio is vary for different substrate, other ratio should be checked based on observation.
Note 2: If precipitation form immediately, the growing rate will be too fast and the resulting materials are usually not single crystals.
CVD
Chemical vapour deposition (CVD) is a technique involving sublimation and deposition of a substrate. Sublimation process is a phase transition from solid to gas, under specific temperature and pressure conditions. When the conditions change in favor of the reverse process (usually a decrease in temperature), gas molecules deposite to form solid again. Rate of deposition process can be controlled to allow crystal formation. This technique is widely used for the growth of inorganic or metallic materials. By carefully control on the deposition conditions, layer-by-layer growth of materials can be acheived. Some Organic compounds could sublime under atmospheric pressure (e.g. camphor) while some requires high vacuum and temperature. Due to the difficulties to acheive the sublimation conditions and control the deposition process, CVD growth of single crystals are used when other methods are failed only.

Sample Requirements:
1. Able to sublime under practical laboratory conditions
2. Stable under the sublimation condition

Procedure:
1. Put about 100 mg of sample into a vacuum sublimation apparatus.1
2. Connect the apparatus to cooling water to provide low temperature surface for substance deposition.
3. Connect the apparatus to vacuum to adjust the pressure of the system.
4. Heat the apparatus with suitable equipment, for example an oil bath on hotplate.
5. Crystals may form on the cool surface depends on different substances, sublimation and deposition conditions.
CVD

Tips:
1. Small deposition area, such as a needle tip, can favour the growth of single crysal.
2. Rotary vane pump or turbomolecular pump may be needed, depends on the required level of vacuum.
3. Suitable rate of deposition is very important to the quality of the resulting crystals. Sublimation rate and deposition temperature will affect the deposition quality.

Note 1: Examples for vacuum sublimation apparatus can be found in here. Examples for more delicate CVD system, can be found in here
Melt
Crystallization may occur when a material is cooled down slowly from its molten phase. In Bridgman method, material is heated above its melting point in a container and cooling is applied at one end of the container. In the presence of a seed crystal, crystal growth along the container. In Czochralski method, after the starting material melted, crystal is pulling from the molten material to produce large single crystal. Czochralski method is often used in industry to produce defect-free single crystals of semiconducting materials such as silicon, germanim and gallium arsenide.

Crystallization of organic compounds from molten state is not common, due to the fact that it is easiler to obtain single crystal from other methods and the size of the crystal formed it not suitable for X-ray diffraction experiment. A review on the theory of single crystal growth from the melt can be found in here.